Microxpress

TM

Microbiology Manual
Second Edition, 2010
 Accumix
Mueller Hinton Agar
The medium was very good and gave very clear results when compared to the standard medium.
USER FEEDBACK*
Plate Count Agar
Quantitative enumeration was carried out using Accumix medium, which gave satisfactory results as compared with other
reputed brand.

TCBS Agar
The Accumix medium appears to show better retrievability for Vibrio like organisms with respect to the other compared
medium.

Sabouraud Dextrose Agar, Endo Agar, Tryptone Glucose Extract Agar

Test results are at par with the brand currently used.

Violet Red Bile Agar

Quantitative examination was carried out using the Accumix medium, which gave satisfactory results as compared to the
other media used.

Nutrient Agar, Sabouraud Chloramphenicol Yeast Glucose Agar, Violet Red Bile Agar
With reference to your dehydrated culture media samples under the brand name Accumix, I would like to congratulate for
developing dehydrated culture media. The performance of Accumix media is excellent compared to others.

Brilliant Green bile Broth 2%, Fluid Lactose Medium

The medium powder is very well dehydrated. Good growth and gas production within 24 hours were observed.

EMB Agar, Levine

The medium allowed excellent, luxuriant growth of coliforms with characteristic metallic sheen within 24 hours.

Nutrient Agar
The growth of coliforms on the plates was luxuriant.

MacConkey Agar
Total coliform counts are at par with the standard brand used.

Nutrient Agar with 1% Peptone, Potato Dextrose Agar, Plate Count Agar
Quantitative examination was carried out using the above Accumix medium, which gave satisfactory results.
* Actual user comments across spectrum of laboratories; Data on file: Microxpress - A Division of Tulip Diagnostics (P) Ltd.

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
FOREWORD
Microxpress, a division of Tulip Diagnostics is a part of the
Tulip Group of Companies renowned world over for its
reliable immunodiagnostic products and platforms for
clinical laboratory diagnostics.

Accumix Dehydrated Culture Media, Bases and

Supplements are manufactured out of proven and well
characterized ingredients in state of the art facilities under
stringent product and process control specifications.

Microxpress Second Edition Manual also contains range of

plant tissue culture medias and plant tissue culture
chemicals. Also we are pleased to introduce accessories
range required in microbiology laboratory for our
esteemed customers.

This consolidated Microxpress Manual, lists out 740

products representing the Accumix Dehydrated Culture
Media, Bases, Supplements, Ready to Use, Plant Tissue
Culture Media, Plant Tissue Culture Chemicals and
Accessories for Microbiology laboratory.

The company would add many more products to expand

and consolidate its portfolio of excellent products with
consistent quality and performance.

!
Quality policy... Microxpress

The Quality Policy of Tulip Group of Companies is:

● To develop, manufacture and market state of the art, high
quality, user friendly products.
● To design and manufacture devices in such a way that when
used under the conditions and for the purpose intended,
they will not compromise, directly or indirectly, the clinical
conditions and safety of the products, the safety or health of
the users or where applicable, other persons, or the safety of
the property.
● To meet customer requirements and achieve customer
satisfaction.
● To meet regulatory requirements.
● To be market leader and trend setter in diagnostic and
laboratory testing.

Objectives:
● By periodically assessing customer and regulatory
requirements and up grading products, processes and
services.
● By adopting solutions for design and construction of device
conforming to safety principles taking into account the
generally acknowledged state of art.
● By emphasis on Research an Development of innovative
and new products.
● By implementing Good Manufacturing Practices.
● By adopting and implementing Quality Management
System adhering to international standards.
● By employing the best available personnel and training
them to update the skill and knowledge.
Contents... Microxpress
Contents A Window To Tulip Group

Corporate Flowchart
v
vi
Our Certification vii-viii
A Glance At Microxpress Manual 1

Essentials....... 2-13
Microorganism Growth Requirement 2
Basic Requirements Of Culture Media 2
Factors That Influence Culture Media And Their Growth Promoting Properties 2-3
Dehydrated Culture Media 3
Media Formulation 4-6
Environmental Factors In Culture Media 6
Types of Media 6 -7
Culture Media Ingredients 7-11
Agars 7-9
Peptones and Hydrolysates 9-11
Culture Media Quality Assurance 11-13

User Know How....... 16-25

General Guidelines To Users 16-21
Factors That Influence The Quality Of Dehydrated Culture Media 22-23
APHA Products vis-à-vis Accumix Products 24-25

Exploring....... 27-394
Dehydrated Culture Media 27-367
Media Bases 369-378
Veg Media Bases 379-382
Supplements 382-394
Contents... Microxpress
Contents Ready to Use .......
Water Testing Solutions
One Step Presumptive Identification Tests
395-462
396-397
397-399
Ready Prepared Media 399-402
Blood Culturing Systems 402-403
Mycobacteriology 404-405
Gallery...... 406-412

Microorganism Identification 413

General Biochemical Identification Tests 413-419
Readymade Analytical Reagents 420-423
Staining Solutions 423-425

Applications...... 426-462
Food And Beverage Analysis 426-432
Dairy Analysis 433-437
Pharmaceutical Analysis 438-440
Water And Wastewater Analysis 441-445
Veterinary Testing 446-448
Products for Agriculture 449
Brewery And Fermentation Analysis 450
Products For Medical And Research Institutes 451-455
Products For Biochemical Tests 456-457
Cosmetic Analysis 458-460
Product for Environmental and sanitary testing 461
Textile, Petroleum and Molecular Biology 462

Plant Tissue Culture ....... 463-476

Basic Requirement of a Plant Tissue Culture Media 464-466
Plant Tissue Culture Media 467-474
Plant Tissue Culture Chemicals 475-476

Accessories & Bibliography....... 477-490

Accessories 478-479
Bibliography 481-487
A Window to Tulip Group... Microxpress

INTRODUCTION QUALITY ASSURANCE

Since its inception in 1988, Tulip Group of Companies comprising of eight The companies apply cGMP and GLP in force from time to time and all the
independent diagnostic companies, has emerged as a leading companies are ISO 9001:2000, ISO 13485 (2003), NF EN ISO 13485
manufacturer and marketer of in vitro diagnostic reagents and kits, (2004) compliant. Most products are already CE marked.
dehydrated culture media and high technology disinfectant products HUMAN RESOURCES
nationally and internationally. The company places great importance to talent garnering and skill
Well known for its innovative approach, the companies are owned, development. Inhouse training programmes are conducted at desired
managed and run by highly involved professionals. frequency to develop functional proficiency, understanding processes and
imparting knowledge. Tulip Group team is constantly motivated to be
The individual group companies specialize in research, development and
responsible and responsive to its customers and business.
designing of specific systems and platforms in diverse technological areas
NATIONAL SALES
covering almost all areas of diagnostic relevance.
The Company’s national business is built around twelve branch locations,
The Group believes in creating ‘better systems for diagnosis and
nationwide with product flow all over the country through a diverse and
prevention’ and sets trends by innovating continuously.
efficient distributor network that guarantees product availability,
PRODUCT DEVELOPMENT
maintenance of cool chain and customer responsiveness.
While Tulip Diagnostics (P) Ltd. focuses on assay systems for
The Company has a professional sales team of around 325 sales / service
Immunohaematology, Haematology, Rheumatology, Infectious Diseases
professionals headquartered all over the country to carry forward its
and Haemostasis, its division Microxpress focuses on dehydrated culture
customer contact and sales programme; with a customer base of over
media, bases, supplements, reagents and tests kits for microbiology and
15000 customers and 300 distributors.
mycobacteriology.
INTERNATIONAL PRESENCE
Orchid Biomedical Systems, Qualpro Diagnostics, Zephyr Biomedicals,
Internationally the company channelizes its products and technology
focus on rapid membrane & ELISA based immunodiagnostic platforms for
through distributors, NGO’s and arrangements with other international
Fertility, Infectious Diseases, Parasitology, Cancer and Cardiac Markers.
companies globally. The company also offers bulk, OEM and contract
Coral Clinical Systems focuses on Clinical Biochemistry while its division
manufacturing facilities to various international companies. Currently, the
BioShields focuses on high technology disinfectants.
company exports its products to over 50 countries worldwide, representing
MANUFACTURING
over 45% of its turnover.
The products are manufactured in professionally set up modern facilities OPPORTUNITIES FOR COLLABORATION
complying to relevant FDA guidelines.
The company is constantly seeking distribution partners in un-represented
The innovativeness is fuelled by an inventive streak with an accent on countries. It also seeks competent vendors for various biomaterials,
indigenous technology as a fundamental basis for product development chemicals and instrumentation used in its manufacturing processes.
and designing of viable technological platforms for diagnosis.
The company also seeks collaboration with like-minded companies who
Production systems have been devised around process flows to achieve are looking to commercialize their products and technologies in India
consistent product performance, batch to batch and stringent in coming, in utilizing its deep resources and understanding of the Indian &
process QA ensure adherence to expected performance parameters International business environment.
whereas finished QC benchmarked to standard reference materials
ensures accuracy of products.

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits V
 About us... Microxpress

Corporate Flowchart

Group

TULIP DIAGNOSTICS ORCHID BIOMEDICAL ZEPHYR QUALPRO CORAL CLINICAL

(P) LTD. SYSTEMS BIOMEDICALS DIAGNOSTICS SYSTEMS
·
Immunohaematology ·
Parasitology ·
Parasitology ·
Infectious Diseases ·
Clinical Biochemistry
·
Haematology ·
Fertility ·
Infectious Diseases ·
ELISA based and rapid ·
Analytical Reagents
·
Rheumatology ·
Infectious Diseases ·
Cancer Markers membrane tests ·
Stains
·
Infectious Diseases ·
Cardiac Markers
·
Haemostasis
·
Instruments

MICROXPRESS BIOSHIELDS
- A DIVISION OFTULIP - A DIVISION OFCORAL
DIAGNOSTICS (P) LTD. CLINICAL SYSTEMS

·
Disinfection products

READY TO USE ACCUMIX ACCESSORIES

·
General biochemical ·
Dehydrated
identification tests culture media
·
Analytical reagents ·
Bases
·
Water testing solutions ·
Selective
·
Stains supplements,
·
One step presumptive agents and
identification tests enrichments
·
Ready prepared media ·
Plant tissue
·
Blood culture systems culture media
·
Mycobacteriology ·
Plant tissue
Mycobacteria culture-
identification, chemicals
isolation, staining and
sensitivity testing.
·
Instraprep range of
ready to pour,
sterilized pouched
media

VI Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
Certification... Microxpress

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits VII
 Certification... Microxpress

VIII Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
 A Glance at... Microxpress

A Glance at Microxpress Manual

The Accumix Manual presents in one volume descriptions of Dehydrated Culture Media, Media Bases, Selective Supplements, Ready to use microbiology product and Plant
Tissue Culture Media and Plant Tissue Culture Chemicals currently offered by Microxpress and is intended to provide information about other essential products used in clinical
microbiology.
It includes both classical and modern media used for identification, cultivation and maintenance of diverse bacteria. The media are organized alphabetically. Each media
includes the composition, directions for preparation, uses, etc. The composition section of each medium describes the ingredients that make up the medium, their amounts
and their pH.

Microbiological testing with these products should be performed by a trained or professional microbiologist or under the supervision of microbiologists and staff qualified by
training and experience to handle pathogenic microorganisms, specimens and samples suspected to contain them. It is also expected that the user will be thoroughly familiar
with the intended use of the individual formulations and will follow test procedures outlined in the standard methods and official compendia or the procedure manual of the
using laboratory.
The Microxpress Manual is organized into 7 sections
Section I : Microxpress Accumix Dehydrated Culture Media, Bases and Supplements-Monographs pertaining to the development, quality control and utilization of
dehydrated culture media (Essentials...)
Section II : Microxpress Accumix Dehydrated Culture Media, Bases and Supplements-General technical information (User know how...)
Section III : Microxpress Accumix Dehydrated Culture Media, Bases and Supplements-Product description (Exploring...)
Section IV : Microxpress Accumix Dehydrated Culture Media, Bases and Supplements-Tables summarizing industrial and clinical applications (Applying...)
Section V : Microxpress Accumix Dehydrated Culture Media, Bases and Supplements-Picture Gallery
Section VI : Microxpress Ready to use Microbiology Products
·General biochemical identification tests
·Analytical reagents
·Water testing solutions
·Stains
·One step presumptive identification tests
·Ready prepared media
·Blood culture systems
·Mycobacteriology
Mycobacteria identification, isolation, staining and sensitivity testing.
Section VII : Microxpress Plant Tissue Culture Media
Section VIII : Microxpress Plant Tissue Culture Chemicals
Section IX : Microxpress Accessories
Section X : Microxpress Bibliography

Great care has been taken so that the information and recommendations contained herein are accurate and compatible with the standards generally accepted at the time of
publishing. However, it is difficult to ensure that all the information given is entirely accurate for all circumstances. Therefore Microxpress, a Division of Tulip Diagnostics (P)
Ltd. makes no warranty with respect to its accuracy or its completeness nor assumes any liability resulting from its use.
References for media listed in "official" and "standard methods" are provided. However, since procedures specified in these publications may differ from one another and
from those included in this manual, these publications should be consulted when adherence to procedures is required.
For complete information regarding processing and inoculation of samples, procedures, results, detection of particular organisms, colony morphology and biochemical tests,
counting of colonies and precautions and limitations, refer to appropriate references and official test procedures recommended in standard methods.

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 1
Essentials
Dehydrated Culture Media ■ Bases ■ Supplements

“Inspiration plus perspiration produces a sensation.”

 Essentials... Accumix
Microorganism Growth Requirement
Cultivation of microorganisms depends on a number of important factors: ·
Prevalence of proper temperature relations and appropriate pH
·Availability of proper nutrients ·Medium free from interfering bioburden
·Moisture for growth ·Prevention of contamination
·Availability of oxygen or other gases, as required

Basic Requirements of a Culture Media

Every organism must find in its environment all of the substances required for incorporated in the culture media.
energy generation and cellular biosynthesis and therefore media used in the Inorganic mineral salts and metals (macro and trace elements)
laboratory for the cultivation of microorganisms must supply all the necessary Inorganic salts and metals like sodium, potassium, magnesium, calcium, iron,
nutrients for cellular growth and maintenance of these organisms. phosphorus and traces of zinc, cobalt, manganese, copper, etc. required for
A satisfactory culture medium, must therefore, contain: bacterial growth are derived from various culture media ingredients. Inorganic
A source of carbon and energy salts are generally required in larger concentrations than trace elements.
Carbon required for synthesizing cellular components and energy required for A formulation may not have specific metals and minerals so listed. In such cases,
metabolism, are both derived mainly from carbohydrates like glucose, maltose, it is assumed that all the factors required are present in the hydrolysates, buffers
lactose, sucrose, xylose, cellulose, glycogen etc and incorporated in the culture and agar components incorporated in the medium.
media. Water
A source of nitrogen Water is an essential ingredient of bacterial protoplasm and hence drying is lethal
Nitrogen required for synthesizing cellular components like nucleic acids is to cells. Free water must be available for transfer of nutrients and toxic waste
derived from peptones and other extracts and incorporated in the culture media. products.
A source of phosphate and sulphur Enrichments and growth factors
Sulphur needed for synthesis of certain amino acids like cystine, methionine and Growth factors like vitamins, amino acids, purines, pyrimidines are supplied by
other molecules while phosphorus required for synthesis of nucleotides, nucleic the various ingredients incorporated in the culture media.
acids and phospholipids are both obtained from peptones and other extracts

Factors that Influence Culture Media and

their Growth Promoting Properties
The survival of microorganisms in the laboratory, as well as in nature, depends on It is therefore essential to adjust the pH of the medium suitable for the optimum
their ability to grow under certain physical and chemical conditions. Growth is growth of the organism. Various types of pH indicators are incorporated in culture
based on the replication of microbial cells, and corresponding increase in the media to ascertain the pH of the medium e.g. thymol blue, methyl red, phenol red
density of these cells in the culture. Therefore, in addition to the use of an etc.
appropriate medium containing the nutrients and other growth factors required Furthermore, pH of a medium should not only be adjusted but also kept within the
for replication and survival of microbes, a number of other parameters influence same range. Most microorganisms produce acids or alkalies, as a result of their
the growth of cultures. metabolic activities and these must be prevented from altering the pH of the
1) pH medium too radically. For example, bacteria when grown on a medium
Microorganisms are susceptible to changes in acidity or alkalinity of the containing sugar generally produce acid intermediates or end products (formic,
surrounding medium. This is true with regard to both, growth and survival. Whilst acetic, butyric and lactic acids). This is particularly true of fermentation under
many bacteria show vigorous growth within a fairly wide range of acidity or relatively anaerobic conditions. Accumulation of acid alters the pH and therefore
alkalinity, there are others that require this to be adjusted within narrow limits becomes detrimental to the growth of the organism. It is therefore preferable and
before multiplication takes place. All organisms have a particular alkaline, often essential to include buffers in culture media to resist the changes in pH of the
acidic or neutral pH at which growth is optimum. medium. A buffer is a mixture of a weak acid and its conjugate base. e.g. citrate

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 3
 Essentials... Accumix
buffer, acetate buffer, phosphate buffer. temperature). Hence, it is important to maintain the exact temperature required
2) Oxidation Reduction Potential (Eh) for the optimum growth of the respective organism. An unsatisfactory
In the medium, oxidation-reduction conditions are very important. Strict aerobes temperature may inhibit or even kill the desired organisms.
need oxygen while strict anaerobes require reducing conditions and hence 4) Osmotic pressure
absence of dissolved oxygen. This may be related to the metabolic characteristics Osmotic pressure refers to the unbalanced pressure that gives rise to the
of the organism. Strict aerobes obtain energy only through oxidation involving phenomena of diffusion and osmosis, as in a solution where there are differences
oxygen as the ultimate hydrogen acceptor; anaerobes utilize hydrogen acceptor in concentration. It is related to the concentration of dissolved molecules and ions
other than oxygen while facultative anaerobes can act in both ways. In fact, in a solution. Hypotonic solutions are low solute concentrated and the cells placed
anaerobes may be poisoned by the presence of oxygen because of the formation in these solutions may swell and burst. Hypertonic solutions have high solute
of toxic hydrogen peroxide which cannot be removed by catalase or superoxide concentration and the plasma membrane of cells placed in these solutions will
free radicals which cannot be removed by superoxide dismutase or due to shrink causing plasmolysis.
oxidation of certain essential groupings in the organism, e.g. the sulphydryl A variety of culture media may be used for the cultivation of organisms and these
groups in proteins. It is possible to determine the intensity level of oxidizing or media should preserve, as far as possible, the cells in their original condition.
reducing conditions in a system by the net readiness of all the components in that Many a times, blood has to be incorporated in the media for culture of fastidious
system to take up, or part with electrons. This ability is usually expressed as the organisms and to exhibit activities like haemolysis. The media therefore should
oxidation-reduction (redox) potential of the system. The redox potential can be preserve the cells in their original condition and hence have an osmotic pressure
obtained by adding dyes (oxidation-reduction indicators) and by observing the nearly isotonic with the cells to be suspended in it to avoid lysis. Sodium chloride
colour changes, to note how much they are reduced. Such changes are in intensity is many a times added to the culture medium to maintain osmotic balance.
of colour, not changes from one colour to another, as in the case with the 5) Water availability
indicators used for the measurement of pH e.g. methylene blue, resazurin etc. Water is the solvent in which the molecules of life are dissolved, and the
3) Temperature availability of water is therefore a critical factor that affects the growth of all cells.
Since all processes of growth are dependent on chemical reactions and the rate of The availability of water for a cell depends on its presence in the atmosphere
these reactions are influenced by temperature, the pattern of bacterial growth is (relative humidity) or its presence in a solution (water activity). Water activity is
profoundly influenced by temperature. For each species there is a temperature affected by the presence of solutes such as salts or sugar that are dissolved in the
range. The temperature that allows for most rapid growth in a short span of time is water. The higher the solute concentration of a solution the lower is the water
known as optimum growth temperature. activity (Aw) and vice versa. Organisms live over a range of Aw from 1.0 to 0.7.
Most organisms of medical importance grow best at 370C (incubation Organisms require an aqueous environment and must have “free” water.

Dehydrated Culture Media

One of the basic characteristics of an organism is its nutritional requirement. Any Constituents Source
microbiological medium must provide everything the species under cultivation Amino-Nitrogen Peptones, protein hydrolysates, infusions and extracts
requires like sources of oxygen, nitrogen, carbon, water, energy, inorganic salts, Energy Sources Sugars, carbohydrates and alcohols
trace elements and other growth factors. A dehydrated culture medium is Growth Factors Blood, serum, amino acids, egg yolk, yeast extract or
described as a substance or a group of substances that satisfies these nutritional vitamins, NAD, hemin
requirements. These media are available in the form of powder and are easy to Buffer Salts Phosphates, acetates and citrates
reconstitute and labour saving. Inorganic Mineral Phosphates, sulphate, iron
Common media constituents
Salts and Metals Magnesium, potassium and calcium
Media formulations are developed on the ability of bacteria to use media
Selective Agents Antimicrobials, dyes and chemicals
components. Some of the media constituents and their sources are outlined in the
Indicator Dyes Phenol red, neutral red and bromothymol blue
next table.
Gelling Agent Agar

4 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
 Essentials... Accumix
Media Formulation
Media for the cultivation of microorganisms contain the substances necessary to Dextrose. Others may be used as required. (Carbohydrates incorporated in culture
support the growth of microorganisms. Due to the diversity of microorganisms and media as energy sources may also be used for differentiating genera and
their diverse metabolic pathways and requirement of certain physical and identifying species. Carbohydrates added to the media at 5-10 grams per liter are
chemical conditions there are numerous media components. The components of usually present as biochemical substrates to detect the production of specific
a medium can be divided into various roles or functions. enzymes in the identification of organisms). It is also usual to add pH indicators to
1) Nutrients (Peptones, Protein Hydrolysates, Infusions and Extracts) such formulations.
Peptones are the major source of nitrogen and vitamins in culture media. They are 3) Buffering agents
water-soluble ingredients derived from proteins by hydrolysis or digestion of the Buffering agents are added to maintain the pH of culture media. It is important
source material; e.g. meat, milk, soya, etc. with enzymes or acids. Depending on that the pH of a culture medium is maintained around the optimum necessary for
the protein substrate and the enzyme used, the resulting nitrogen containing growth, of the desired organism. The use of buffering compounds at specific pK
compounds exist in different qualitative and quantitative relations. values is especially necessary when fermentable carbohydrates are added as
energy sources.
Naegali is credited with the earliest publications describing the requirements of
microorganisms for a protein component which he called 'peptone'. Peptones However, a side effect of such compounds is their ability to chelate (bind) divalent
contain mixtures of polypeptides, oligopeptides, amino acids, organic nitrogen cations (Ca++, Mg++). Polyphosphate salts, sometimes present in sodium
bases, salts and trace elements to support the growth of a variety of bacteria. The phosphate, are compounds, which can bind essential cations so firmly that they
variety of peptones produced reflects the differing demands of microorganisms for are not available to the organisms. The effect of these binding agents will be seen
amino acids and peptides. in culture media as diminished growth, unless care has been taken to supplement
the essential cations in the formulation. Opacity forming in a medium, after
Tryptone (Casein hydrolysate) with its pale colour and high tryptophan content
heating or on standing at 500C for several hours, is commonly caused by
and soya peptone with its high carbohydrate content are valuable vegetable
phosphate interaction with metals. Such phosphate precipitates can very
peptones in addition to meat peptones. Meat infusions contain water-soluble
effectively bind ironand lower the available amount of this essential metal in the
fractions of protein (amino acids and small peptides) along with other water-
medium.
soluble products such as vitamins, trace elements, minerals and carbohydrates
4) pH and Oxidation Reduction Indicators
(glycogen). Chemoorganotrophs generally require amino-nitrogen compounds as
essential growth factors. The pH indicator has the task of revealing the formation of acids from
carbohydrates and the formation of bases from peptones, single amino acids or
Infusions or extracts have low amino-nitrogen content to sustain the growth of
amines by changing colour. This is a very effective way of detecting fermentation
large numbers of bacteria. (The infusions of materials such as muscle, liver, yeast
of specific carbohydrates in a culture medium. Such compounds are expected to
cells and malt are usually low in peptides but contain valuable extractives such as
change colour distinctly and rapidly at critical pH values.
vitamins, trace metals and complex carbohydrates). Proteins hydrolyzed with
acids or enzymes (peptones) generally have high concentrations of water-soluble The oxidation-reduction indicators take on particular colourations in their
protein fractions (peptides) to support large bacterial growth. Therefore, it is a oxidized state, when there is oxygen present in the medium. For e.g. methylene
common practice to combine infusions and peptones to obtain the best of both blue changes from colourless to blue, and resazurin, from colourless to pink.
5) Selective agents and Supplements
nutrition sources.
Chemicals and antimicrobials are added to culture media to make them selective
The nutrient components of culture media are carefully selected to recover the
for certain organisms. The selective agents are chosen and added at specific
required spectrum of organisms in the sample. General purpose media such as
concentrations to suppress the growth of unwanted microorganisms in a
Blood Agar in its various forms will often contain mixtures of peptone to ensure
polymicrobial sample. It is, of course, essential to have established that the
that peptides of sufficient variety are available for the great majority of organisms
selective agents, at the appropriate concentration, will allow uninhibited growth
likely to be present. However, more fastidious organisms will require additional
of the desired organisms. A few selective agents are listed below: -
growth factors to be added.
2) Energy (Carbohydrates)
The most common carbohydrate added to culture media as a source of energy is

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 5
 Essentials... Accumix
a) Bile salts b) Antimicrobials
Among the substances of biological origin, bile salts are the most widely Antimicrobials are used in culture media as selective agents to restrict the
used selective agents. They are present as both, bile salt mixtures or bile growth of certain organisms and promote the growth of others. For e.g.
salts, and as pure substances (sodium deoxycholate, sodium taurocholate). chloramphenicol and cycloheximide are incorporated to inhibit a wide range
Bile derivatives like bile salts and bile salt mixtures are used as selective of bacteria and fungi respectively.
agents to differentiate between those organisms that are adapted for c) Organic and inorganic salts
survival in the gut and those, which cannot live in that environment. (Mainly The organic and inorganic salts may also be used as selective agents.
to inhibit gram-positive organisms, non-intestinal organisms and spore Sodium chloride at a high concentration inhibits both the gram-positive and
formers). gram-negative flora, with the exception of staphylococci and some other
Bile is derived from the liver and its composition varies according to its halophilic organisms. Sodium azide at different concentrations is used for
animal source and its state of preservation. It contains bile pigments, bile the selective isolation of streptococci and enterococci. Sodium selenite and
acids in free and conjugate form, fatty acids, cholesterol, mucin, lecithin, tetrathionate stimulates the growth of Salmonella while inhibiting the
inorganic salts, glycuronic acids, urea and porphyrins. The liver detoxifies growth of most gram-positive organisms and restricting the growth of
bile salts by conjugating them to glycine or taurine. A bile salt is the sodium normal intestinal flora. Sodium citrate, sodium tellurite and sodium lauryl
salt of a conjugated bile acid. Bile Salts and Bile Salt Mixture contain sulphate also belong to this group of ingredients.
extracts standardized to provide inhibitory properties for selective media. d) Dyes
Bile Salts consist mainly of sodium glycocholate and sodium taurocholate. Dyes are essential in the preparation of differential and selective culture
Bile salt mixture is a modified fraction of bile acid salts, providing a refined media. In these formulations, dyes act as bacteriostatic agents, inhibitors of
bile salt (increased selectivity) and is generally effective at less than one- growth and sometimes also as pH or Eh indicators. Dyes when used as
third concentration of bile salts. Bile salt mixture incorporated into certain selective agents may interact with other components in the formulation.
media (SS Agar, Violet Red Bile Agar) gives a very sharp differentiation Thus, the same sample of brilliant green may show different inhibition titres
between lactose fermenters and non-lactose fermenters of enteric origin, against the same sample of E.coli when tested in different peptone
permitting the detection of scanty non-lactose fermenters in the presence of solutions. Bile salts interact with brilliant green and reduce the toxicity of the
numerous coliforms. Sodium deoxycholate is the sodium salt of deoxycholic dye and therefore the toxicity levels of such dyes must be ascertained. Most
acid and sodium taurocholate is the sodium salt of taurocholic acid. Since of the compounds used e.g. phenol red, neutral red, fuchsin, etc; are toxic
Sodium Deoxycholate is a salt of a highly purified bile acid, it is used in and therefore it is essential to use low concentrations of pre-screened
culture media in lower concentrations than naturally occurring bile. Sodium material.
Taurocholate contains about 75% sodium taurocholate in addition to other e) Readymade selective supplements
naturally occurring salts of bile acids. (Although bile containing media are Readymade selective supplements are added to the basal media for
expected to suppress gram-positive organisms and allow only bile tolerant selective isolation of organisms. They are freeze dried, accurate preparations
gram-negative organisms to grow, some bile salts (free from bile acids) will of antimicrobials which are added, normally one vial per 500 ml. of sterile
allow staphylococci and streptococci to grow. Particular advantage can be cooled medium, to base nutrient media to create specific, selective media.
taken of this characteristic where for e.g. MacConkey Agar without Crystal The result of the use of these supplements is that it is possible for non-
Violet, NaCl and with 0.5% Sodium Taurocholate (or 0.5% bile salts) is specialized laboratories to isolate unusual but important organisms from
used as a general-purpose medium for clinical bacteriology. The growth of contaminated clinical samples or food raw materials. Legionella, Listeria,
staphylococci and faecal streptococci would be looked for on this medium Campylobacter, Haemophilus, Yersinia, Brucella, Bordetella, Neisseria
when it is used for the culture of urine, faeces and purulent material obtained species as well as many other difficult organisms can be isolated and
from patients. The suppression of swarming growth of Proteus in such confident statements made about their presence or absence in the material
specimens is a particular advantage of this medium). examined. They are more specific in their selective action than the chemical
When incorporated into culture media, bile salts should not affect the colour agents. Hence, they demand special care and post-sterilization addition.
of the indicator dyes or their subsequent change in colour. It is a normal However, the wide variety of organisms and their almost infinite ability to
practice to titrate the level of bile salts into culture media by measuring the adapt to changing conditions makes a truly selective medium unlikely.
growth of appropriate test organisms as an indicator.

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6) Gelling or solidifying agents Milled to a powder. Agar is not an inert gelling agent; it contributes nutrients
Gelling or solidifying agents are added to a liquid medium in order to change the and/or toxic substances to culture media, depending on the chemical process
consistency to a solid or semi solid state. The outstanding gel forming substance adopted in its manufacture.
used in culture media is agar. Its inertness to microbial action, the unique setting 7) Enrichments and growth factors
and melting temperatures, the high gel strength, the ability to allow diffusion of To improve the fertility properties of the culture media for the cultivation of
compounds while interlocking water in a rigid gel, its clarity and low toxicity has auxotrophs (fastidious organisms like Neisseria, Haemophilus, etc.) various
made it the substance of choice. Its ability to retain its gel structure at 600C makes enrichments are added normally after autoclaving and cooling the base medium.
agar of special value to culture media, which have to be incubated at this Blood, serum, haemoglobin, albumin, egg yolk, whole eggs, NAD, hemin, etc. are
temperature to isolate thermophilic organisms. Agar is obtained from agarophyte some of the enrichments added. Growth factors like vitamins, amino acids,
sea-weeds mainly Gelidium, Gracilaria and Pterocladia species and extracted as purines, pyrimidines are supplied by the various ingredients of culture media.
an aqueous solution at greater than 1000C, decolourized, filtered, dried and

Environmental Factors in Culture Media

1) Atmosphere vary in their susceptibility to form toxic oxidation products if exposed to light and
Most bacteria are capable of growth under ordinary conditions of oxygen tension. air.
Obligate aerobes require oxygen, while anaerobes grow only in the absence of 2) Water Activity
oxygen. The microaerophiles develop best under partial anaerobic conditions, Proper moisture conditions are necessary for continued luxuriant growth of
and the facultative anaerobes can grow in either. microorganisms. Organisms require an aqueous environment and must have
Anaerobic conditions for growth of organisms are obtained in a number of ways: “free” water not bound in complex structure necessary for transfer of nutrients and
·
Absorption of oxygen by chemicals. toxic waste products. Evaporation during incubation or storage, results in loss of
free water and reduction of colony size or total inhibition of organism growth.
·
Displacement of the air by hydrogen or nitrogen.
3) Protective Agents
·
Addition of small amounts of agar to liquid media. Calcium carbonate, sodium thioglycollate, starch and charcoal are a few of the
·
Addition of fresh tissue to the medium. protective agents used in culture media to neutralize and absorb toxic metabolites
·
Addition of a reducing substance to the medium; for example sodium produced by bacterial growth. Surfactants, such as polysorbate 80, lower the
thioglycollate, L-cystine. interfacial tension around bacteria suspended in the medium. This permits more
Many organisms require an environment of 5-10% CO2. Levels greater than 10% rapid entry of desired compounds into the bacterial cell to increase growth.
are often inhibitory due to decrease in pH as carbonic acid forms. Culture media

Types of Media
The composition of a particular culture medium formulation determines the exacting bacteria (auxotrophs) like Neisseria and Haemophilus by addition of
classification of a medium as basal (general-purpose), enriched, selective, growth promoting substances like blood, serum, egg, NAD, hemin and other
differential or indicator or a combination of these types. growth factors. e.g. Soya Bean Casein Digest Medium enriched by the addition of
1) Basal media 5% blood, Blood Agar, Loeffler Serum Agar.
A basal medium is the one that supports the growth of a wide variety of 3) Differential media
microorganism types and lacks inhibitory properties. Such a medium may be Differential media incorporate certain chemical constituents or substances that
enriched with a supplement, usually animal blood, so as to cultivate fastidious enable presumptive identification of a specific genus or species either from a pure
microbial species e.g. Nutrient Agar which can be enriched by the addition of or mixed culture. This helps to characterize different bacteria, by their special
blood. colonial appearance. e.g. TSI Agar is a differential medium for gram-negative
2) Enriched media enteric organisms on the basis of their ability to ferment dextrose, lactose and
An enriched media is devised to meet the nutritional requirements of more sucrose and to produce hydrogen sulphide.

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4) Selective media Broth selectively stimulates the growth of Salmonella while inhibiting gram-
Selective media are the ones that inhibit or poison all but a few types of bacteria positive and normal intestinal organisms.
either by containing enrichment that selectively favours it or by containing 5) Indicator media
inhibitory substances that suppress others. Selectivity is usually achieved with a These media incorporate certain substances, which are changed visibly as a result
chemical agent, dye or an antibiotic e.g. MacConkey Agar with bile salts and of the metabolic activities of the particular organisms e.g. the incorporation of
crystal violet is used to inhibit gram-positive bacteria while stimulating the ferrous salts like ferric citrate and a source of sulphite like sodium thiosulphate in
growth of gram-negative enterics. Mannitol Salt Agar with a high concentration of SS Agar helps in the detection of H2S production and thereby helps to differentiate
salt is selective for staphylococci while inhibiting most other bacteria.
between Salmonella (H2S production is indicated as a black dot in the center of
Deoxycholate Citrate Agar containing deoxycholate and citrate selectively inhibits
the colony) and Shigella (without black center).
gram-positive organisms and normal intestinal flora while allowing the growth of
enteric pathogens Salmonella and Shigella. Bile Esculin Azide Agar containing Many culture media formulations combine the properties of selectivity and
azide inhibits most of the gram-negative organisms allowing the growth of differentiation due to the inclusion of a variety of chemicals. The isolation of
streptococci. Cetrimide Agar Base containing cetrimide inhibits most of the gram- microorganisms from clinical and industrial materials frequently requires the use
positive and gram-negative organisms while allowing only Pseudomonas species of enriched broth media in addition to the selective, differential and non-selective
to grow. Sodium selenite in Selenite F Broth and tetrathionate in Tetrathionate plated media.

Culture Media Ingredients

AGAR must be neutralized before autoclaving. Heat sensitive ingredients, which are
INTRODUCTION: damaged by autoclaving, should be prepared sterile separately and then added.
The revolution in the microbiology industry has created a demand for increasingly HISTORICAL BACKGROUND:
innovative and valuable media for growth of cultures. As microbiologists Agar is known to be the phycocolloid of most ancient origin. According to a
endeavor to meet this demand, the need for effective use of hydrocolloids, one of Japanese legend the original manufacturing method of agar was first discovered
the industry's most significant raw materials, is of prime importance. in 1658 by Minora Tarazaemon. It was made and sold as an extract in solution
Agar is one such hydrophilic colloid extracted from certain seaweeds. Agar has (hot) or in gel form (cold), to be used in areas near factories and the product was
been defined in various ways namely; a gelatinous solidifying agent used in then called "tokoroten". Later in the beginning of the 18th century, as the product
culture media to grow microorganisms, a complex polysaccharide derived from underwent industrialization to a dry and stable form, it came to be known as
certain marine algae, a vegetable gelatin made from various kinds of algae or "kanten" meaning "cold weather". The word "agar-agar", however, has a
seaweed, a tasteless dried seaweed that is used as a thickening agent… the list Malayan origin. "Agar" became the most commonly accepted term, although in
is endless. French and Portuguese speaking countries it is called "gelosa".
Bacteriological agar is incorporated into culture media as a solidifying agent for Agar production by modern techniques of industrial freezing was initiated in
the isolation of bacteria and fungi as well as the differentiation of strains and the California. Until World War II, America and Japan were the only producers of this
study of their susceptibility to chemotherapeutic agents and antibiotics. Today, phycocolloid. "Gelidium Pacificum" known as the genuine agar was used by the
agar is utilized around the world in bacteriological culture media as the only Japanese industry. During the Second World War, shortage of available agar
gelling agent of choice. ignited the production opportunity for those countries with coastal resources of
Agar is mainly used as a solidifying agent and by itself has no nutritional value. It "Gelidium Sesquipedale" an agaroid similar to "Gelidium Pacificum" used by the
is soluble in hot water, a 1% neutral solution of which sets at 450C to a firm gel, Japanese industry. In succession to this, Portugal, Spain and other European
melting at 950C. The amount of agar needed to gel broths varies but 10 to 20 countries, which did not have agarophyte seaweeds, followed suit.
grams (1-2%) per liter of water gives a good gel consistency and is commonly The significant use of agar in bacteriology was introduced by a woman named
used. Smaller quantities of agar (0.05-0.5%) are used for motility studies and Fanny Angelina Eilshemius, the wife of a German doctor Walther Hesse who was
growth of anaerobes and (0.1%) microaerophiles. involved in the study of public health and bacterial metabolism. One hot summer,
Agar usually does not alter the pH of the medium but if it contains free acids they when Dr. Walther wasattempting to do counts on bacterial air contaminants, he
was having trouble with his gelatin plates melting in the heat and being digested

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by many of the bacteria he was trying to grow on them. In frustration, he asked his PROPERTIES OF AGAR:
wife as to how her puddings and jellies stayed solid in warm weather. She ·
Gelation
explained to him that she used agar-agar, a complex polysaccharide extracted Agar has a great gelling power due to which it can form gels, which are more
from seaweed, to keep them solid in hot weather. Dr. Hesse evaluated the resistant (stronger) than those of any other gel-forming agent. A 1.5%
possibility of using agar-agar to prepare microbial media and found it to be a aqueous solution gels at 450 C and does not melt below 950 C, a property
perfect medium for the growth of microorganisms on solid medium as it was non- solely unique to agar.
toxic to most microbes, melts only at 950 C, but solidifies at 450 C (a temperature
·
Gel Strength
most bacteria can survive), was non-toxic to other forms of life, stable to
Agar has the ability to form gels upon cooling of a hot solution at 450 C and to
sterilization temperatures and physiologically inert. In 1882, Robert Koch was the
melt to solution upon heating at 950 C. At temperatures above the melting
first to use agar-agar as a culture medium in his experiments on tuberculous
point of the gel, thermal agitation overcomes the tendency to form helices and
bacteria. By the early 1900’s agar became the agent of choice instead of gelatin.
the polymer exists in solution as a random coil. On cooling, these helices form
Agar was found more suitable because it remained solid at the temperature
a three-dimensional network of junction points of polymer chains. Further
required for growth of human pathogens and was resistant to breakdown by
cooling leads to aggregation of these junction points.
bacterial enzymes.
SOURCES OF AGAR: ·
Gelling and Melting Temperatures

Agar is a phycocolloid, water-soluble polysaccharide extracted from a group of The gelling and melting temperatures of agars correlate with their methoxyl
marine algae. Agarophytes, the red seaweeds used as the raw material for content. The higher the methoxyl content in agarose, the higher is the gelling
manufacturing agar, are mainly the genera Gelidium (Gelidiaceae), Gracilaria temperature. For example, gelling temperatures of agars from Gelidium
(Gracilariaceae), Pterocladia (Gelidiaceae), and Ahnfeltia (Phyllophoraceae) in species range from 28 to 310 C and melting temperatures from 80 to 900 C,
different countries. Gelidium yields the best quality of agar, but its cultivation is the difference between the two temperatures being 51 to 600 C. The difference
difficult and its natural resource is less than Gracilaria, which is being cultivated in between the gelling and melting temperatures is called as ‘hysteresis'. Higher
several countries and regions on a commercial scale. Pterocladia and Ahnfeltia the hysteresis better is the agar-agar quality.
grow only in a few regions and are utilized only in New Zealand and U.S.S.R., ·
Viscosity and Molecular Weight
respectively. The viscosity of an agar solution at constant temperature and concentration is
CHEMISTRY OF AGAR:
a direct function of the average molecular weight. Usually, the viscosity is
Chemically, agar consists of D-galactose, 3, 6-anhydro-L-galactose and sulfate. lower as the gel strength is greater for the agar solution. The average
Agar can be separated into two polysaccharides, agarose (70%) and agaropectin molecular weight of agar ranges from 8,000 to greater than 100,000.
(30%) using the acetylation method. The agarose is a neutral polymer, while the
·
pH and Sterilization
agaropectin is an acidic polymer. Acid hydrolysis and enzymic degradation of agar
Agar can be used over a wide range of pH, from 5 to 8. In some cases, it can
isolated the agarobiose and neoagarobiose respectively. This revealed that
also be used beyond these limits. Agar withstands thermal treatments very
agarose is composed of agarobiose repeating disaccharide units alternating with
well above 1000 C, which allows for good sterilization.
1,3-linked-b -D-galactopyranose and 1,4-linked-3, 6-anhydro-b -L-
galactopyranose. Agaropectin has the same background structure as agarose, but ·
Compatibility
contains considerable amount of acid groups such as sulfate, pyruvate and Agar is usually compatible with most other polysaccharides and with proteins
glucuronate groups. in neutral conditions.
Recent fractionation studies by Yaphe et. al. (1971) indicated that agar is not only APPLICATIONS OF AGAR :

made up of one neutral and one charged polysaccharide, but is composed of a ·

Bacteriological agar has been developed to address the needs of large-scale
complex series of related polysaccharides which range from a virtually neutral commercial and industrial applications, where good quality agar is required
molecule to a highly charged (sulfated) galactan. The neutral polysaccharide has a at a competitive price. It is incorporated into culture media for the isolation of
gelling ability and approaches the structure of an ideal agarose, which contains a bacteria and fungi as well as in the differentiation of strains and the study of
trace of polar residues such as sulfate (0.1 to 0.5%) and pyruvic acid (0.02%). their susceptibility to chemotherapeutic agents and antibiotics.
These sulfate and pyruvate groups remain linked in small quantities to the agarose
structure, depending on the seaweed used in agar production.

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·It is also used as a solidifying agent in microbiological culture media where ·Consistent gelling and melting temperatures, called hysteresis
good clarity and toxic inhibition is required for the growth of bacteria, yeasts ·Consistent gel strength that withstands the rigors of streaking
and moulds.
·Freedom from metabolically useful substances such as peptides, proteins and
·Bacteriological agar is widely employed in immunology, cell culture, marine fermentable hydrocarbons
biology, clinical microbiology, mycology and protozoology. It is used in
·Freedom from toxic substances (bacterial inhibitors) and not degraded by
culture media for isolation of pathogens that cause infections. The growth of
bacterial enzymes
microorganisms in specific media helps to identify and diagnose the cause of
diseases such as urinary infection, throat infection etc. The bacteria do not use ·Freedom from haemolytic substances that might interfere with normal
the agar for growth; in fact the agar merely provides a semi- solid surface to haemolytic reactions of bacteria
grow the bacteria on. ·Freedom from contamination by thermophilic spores
·Agar is the first phycocolloid to be used in food industry. In food industry, agar ·Ability to allow diffusion of compounds although locking water into a rigid
is mainly used as a gelling agent and a stabilizing agent for controlling gel, a property exploited in microbiological assays.
viscosity. Its satiating and regulating characteristics make it an ideal fiber PEPTONES AND HYDROLYSATES
additive in preparation of low calorie food products. Peptone was originally formulated in 1880 by Naegeli. Peptones are rich in
·Agar has proved to be most valuable in microbiology and is being widely used amino acids and nitrogen compounds readily utilized by bacteria and are one of
as a solidifying agent in culture medium for growing practically all the most important constituents of culture media.
pathogenic and non- pathogenic bacteria, yeasts and moulds. Protein Hydrolysates (Peptones), Infusions and Extracts
Protein Biochemistry
·In prosthetic dentistry, it is necessary to make accurate casts of intricate
objects. Agar, mixed with other substances, serves as the ideal dental Proteins consist of amino acids joined together by means of the covalent peptide
impression material. bond linkage. When the bonds are hydrolyzed, proteins yield polypeptides of
varying molecular sizes, proteoses and peptides down to the level of simple amino
·Agar is used as a laxative in the prevention of constipation. Agar is a
acids. Bacterial peptones are mixtures of various products of protein hydrolysis,
hygroscopic substance and can absorb water and expand considerably. This
organic nitrogen bases, inorganic salts and trace elements.
increases the bulk and stimulating recovery of the intestine, facilitating waste
Preparation of Peptones
elimination.
The composition of peptones varies with the origin and the method of
·Agar is used in cosmetics, lotions, shaving creams, fertilizers, rubber, shoe
preparation. Some common sources of peptone are meat, beef, fish, casein,
polish and paints. It is also a major ingredient in dairy products, ice creams,
gelatin, soyabean meal, etc. Proteins may be broken down into amino acids and
chocolates, ointments, bread, pet food, air-fresheners, soft drinks and such
peptides by hydrolysis using strong acids or proteolytic enzymes such as pepsin,
other industrial products.
papain or pancreatin, which contains trypsin and chemotrypsin. These protein
·Agarose is an ideal gel matrix for diffusion and electrokinetic movement of hydrolysates are called peptones. Microbial proteoses and proteolytic enzymes
biopolymers and its gel is biologically inert with controlled ionic properties. secreted by microorganisms are becoming more widely used in peptone
This property of agarose has a wide usage in biomedicine and biotechnology. production.
·Agar gel electrophoretic media have been used for many years to separate Manufacture
and identify serum and spinal fluid proteins and other biological mixtures, Peptone manufacture includes hydrolysis / digestion, centrifugation, filtration,
facilitating the diagnosis of illnesses of patients. Separation of nucleic acids, concentration and drying. Animal tissues are chopped in order to prepare for
lipoproteins, lactic dehydrogenase isoenzymes, serum proteins, digestion and demineralized water is added to the starting material to form a
glycoproteins, heparin, bacterial proteins, plant viruses, etc. are done using thick suspension. The digestion process follows with an acid or an enzyme
agarose gels. Agarose gel particles are used for separations of molecular hydrolysis. Acid and alkaline hydrolysis are performed by boiling the protein with
weight and artificial mixtures of ribosomes, proteins and viruses. acids or strong alkalies at increased pressure to raise the temperature of the
Agar is suitable in Bacteriology because of its properties like reaction. This procedure can however, decrease the vitamin content of the protein
·Good transparency in solid and gel forms which allows identification of colony and a portion of the amino acid content. (Tryptophan is usually totally lost;
type cystine, serine and threonine are partially broken down and asparagine and

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reaction. This procedure can however, decrease the vitamin content of the protein A variety of proteolytic enzymes, or proteoses may be used to accomplish
and a portion of the amino acid content. (Tryptophan is usually totally lost; enzymatic hydrolysis of animal protein. Pepsin and trypsin are widely used for
cystine, serine and threonine are partially broken down and asparagine and animal peptone manufacture. Pepsin is isolated from porcine or other animal
glutamine are converted to their acidic form). stomach. Trypsin, along with chemotrypsin, carboxypeptidase A,
Digestion with proteolytic enzymes is performed at lower temperatures and carboxypeptidase B, and elastase, are enzymes isolated from animal pancreas.
normal atmospheric pressure. The resulting material from a proteolytic digestion CASEIN PEPTONES

is a mixture of amino acids and polypeptides of varying lengths. This process is Casein peptones are derived from milk. Milk contains water, carbohydrates
often less harmful to the protein and amino acids. The proteolytic enzymes are (lactose), proteins, lipids and salts. The protein portion of the milk is the starting
specific to the peptide bonds they will break. The enzyme pepsin will cut an amino material for casein peptones. It is separated out by hydrolysis after the removal of
acid chain where there is a phenylalanine or leucine bond. Papain will cut the cream. The soluble protein (whey protein) is discarded. The insoluble portion,
chain adjacent to arginine, lysine and phenylalanine and pancreatin will show which precipitates out, consists of a group of heterogeneous phospho-proteins
activity at arginine, lysine, tyrosine, tryptophan, phenylalanine and leucine (micelle) collectively called as casein. Casein is one of the most nutritive of milk
bonds. Microbial proteoses and proteolytic enzymes secreted by microorganisms proteins, as it contains all of the common amino acids and is rich in the essential
are becoming more widely used in peptone production. Before digestion with ones. Hydrolysis is affected either with hydrochloric acid or with a proteolytic
enzymes, a base or acid is added to bring the pH of the protein suspension to the enzyme.
optimum for the specific enzyme being used. For e.g. pepsin is most effective at Casein Acid Hydrolysate (Acid Hydrolysate) is poor nutritionally because
pH 2.0 and trypsin at pH 8.5. The enzyme is added when the pH and temperature tryptophan is destroyed during the hydrolysis and some other amino acids are
are optimal. The amount of enzyme necessary, the time for digestion, and control reduced in amount (cystine is destroyed, serine and threonine are reduced,
of pH and temperature are dependent on the degree of hydrolysis intended. aspargine and glutamine undergo hydrolysis to yield aspartic and glutamic acid)
Once protein digestion is complete, the suspension may be heated to inactivate Tryptophan must therefore be added to the medium to make it suitable for
the enzymes. The protein / enzyme slurry is then centrifuged and filtered to tryptophan requiring bacteria.

remove the insoluble materials and to clarify and concentrate the material. The In the enzymatic hydrolysis of casein, enzymes from the pancreas are utilized to
suspension is then concentrated to approximately 67% total solids and the manufacture these peptones. The proteoses found in the pancreas consist of
product now appears as a syrup, which is spray dried and packaged. Peptones trypsin, chymotrypsin, carboxypeptidase A, carboxypeptidase B and elastase and
could be of different types based on the source it is derived from. are used for this purpose. One of the characteristics of pancreatic digest of casein,
Peptone Performance in contrast to the acid hydrolysis of casein, is a peptone that contains greater
Ingredients used for peptone manufacture, agents of hydrolysis, buffering agent amount of peptide and tryptophan.
used, conditions of hydrolysis (amount of enzyme used, time of digestion, pH and Tryptone (enzymic hydrolysate) contains abundant tryptophan and the full
temperature), determine the degree of hydrolysis and the quality of the range of amino acids and does not require such supplementation. Casein
hydrolysate and therefore must be carefully controlled. Purification, concentration hydrolysate may be substituted for peptone in broth and other media. Different
and drying steps must also be carefully regulated due to their effect on the grades of milk peptones are available.
characteristics of the peptone. The essential requirements of a good peptone SOY PEPTONES
include the ability to support the growth of moderately exacting bacteria from Soy peptones are derived from enzymatic digestion of soy flour. Before digestion,
small inocula, the absence of fermentable carbohydrate and a very low content of the soy flour is thoroughly washed to eliminate the tryptic inhibitor present in it.
copper. Soy flour is rich in high quality protein, carbohydrates, calcium and B vitamins.
MEAT PEPTONES The enzymes used in the digestion process must be from animal free sources or
Meat peptones are proteins from animal sources that have been hydrolyzed, or obtained from organisms that have been grown in animal free media. Sometimes
broken down to amino acids and peptides, to provide nitrogen for organisms. the organism from which the enzyme has been sourced has been stored with
Sources of animal protein include meat from muscular tissue or offal and gelatin. bovine serum albumin (BSA). All these factors should be taken into consideration
Muscular tissue and offal are utilized fresh, frozen or dried. Gelatin is extracted by when selecting for animal free peptones.
boiling collagen, the fibrous protein found in connective tissue, bone and
cartilage.

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FISH PEPTONES by several filtration steps. The final filtration product is concentrated and then
Fish Peptone is a non-mammalian, non-animal peptone used as a nitrogen spray dried, or is left in the concentrated paste form, which contains
source in microbiological culture media. This is a non-bovine origin peptone, to approximately 60-80% solids.
reduce Bovine Spongiform Encephalopathy (BSE) risk. It is suitable for INFUSIONS AND EXTRACTS
pharmaceutical and vaccine production and can replace any peptone, depending
(Liver extract, Malt extract, Yeast extract, Beef Heart for infusion, Beef extract, etc)
on the organism and production application.
YEAST PRODUCTS
The water-soluble fractions of materials such as muscle, liver, yeast cells and malt
are usually low in peptides but contain valuable extractives such as vitamins,
The main sources of yeast extract are “brewer's” yeast and “baker's” yeast.
trace metals and complex carbohydrates. It is a common practice to combine
Brewers yeast is a byproduct from the brewing industry, and requires de-bittering infusions and peptones to obtain the best of both nutrients.
before it is suitable for fermentation use. The process of manufacturing bakers It is our policy to minimize the theoretical risk of transmitting agents
yeast to obtain yeast extract is different compared to the manufacture of other responsible for causing animal disease when selecting starting
peptones. Yeast extract is an autolysate. Cell hydrolysis is performed by the materials to manufacture our microbial culture media. The culture media
range does not contain, and are not derived from, specified risk
endogenous enzymes of the Sacchoromyces organisms. Autolysis is done either materials. The countries of origin of raw materials and enzymes are not
by controlled temperature shock or osmotic shock. After autolysis, soluble included by USDA on the list of countries where Bovine Spongiform
material is separated from insoluble material by means of centrifugation followed Encephalitis (BSE)is reported.

Culture Media Quality Assurance Programme

Culture Media Quality Assurance plus literature, labels and pack inserts is carried out under the aegis of
The development of dehydrated culture media is a process leading to the large- R&D/Marketing. Further production lots are manufactured under surveillance
scale manufacture of a reproducible, stable product. At Microxpress, the which include GMP monitoring and end-product testing by the Quality Control
individual ingredients and the final dehydrated product are carefully quality- Department.
controlled to ensure consistent, high quality performance and obviate the need for Quality Assurance Programme pertains to Raw Material Testing, In-process
media to be prepared in the laboratory from raw materials. Sample Testing and Finished Product Testing.
Quality Assurance Programme Raw Materials
All manufacturing operations are conducted according to protocols and standards It is very important that the quality of products is maintained at the highest level
which describe such procedures as the monitoring, maintenance, cleaning and and lot-to-lot variation is minimal. Raw materials may vary considerably in
calibration of equipment; plant sanitation, warehouse control of incoming composition and the extent to which the protein components have been
materials and materials under test; labeling control and handling, storage and denatured during any processing procedures, therefore the conditions of
distribution of finished goods. The master formula and accompanying documents manufacturing should be carefully controlled to minimize the variations inherent
for each lot/ batch of product includes manufacturing control and packaging in biological materials so as to maintain quality.
information pertaining to the product.
Quality tests on raw materials include identity tests, tests for performance and
The finished product requires statistical evaluation of each ingredient and compatibility with other ingredients in a pre production laboratory mix of the
measurement of the interaction of each ingredient on the other. Various quality medium components. Bought-in components have buying specifications (each
control measures are adopted prior to and during manufacturing processes so that raw material is analyzed for grade, quality and purity as per standards) and in-
desired end products with predictable reproducible results are obtained. All the house components have manufacturing specifications and standard-operating
components of the medium, including special protein hydrolysates, which may processes produced. Peptones are examined physically, chemically and
have to be specially manufactured, are assembled and a laboratory mix tested to microbiologically, agar is tested for clarity, gel strength, diffusion characteristics,
see that it meets the performance specification. Finally, the components are etc. Additional tests are also done on other media ingredients like carbohydrates,
milled, mixed and blended to produce a fine homogeneous, free flowing powder inorganic minerals, selective agents, dyes and pH indicators, growth factors, etc.
which is held in containers for further testing before release. Stability trials begin
Dehydrated media mixtures are examined for appearance, homogenicity, and
only after ascertaining that the desired end product has been achieved. All this,
moisture content.

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Peptones Glycerol
Protein hydrolysates or peptones are a major component of culture media. Glycogen
Besides in-house specifications, peptones are analyzed as specified in U.S.
Inositol
Pharmacopoeia under the title “Peptic Digests of Animal Tissue” and “Pancreatic
Lactose
Digest of Casein” (107). To ensure that the product confirms to predetermined
specifications, tests are carried out routinely to analyze peptones and the Maltose
following criteria monitored : Mannitol
1. Degree or extent of digestion Mannose
2. Loss at 1000C Rafinose
3. Total nitrogen content Rhamnose
4. Alpha amino nitrogen content Salicin
5. Residue on ignition Sorbitol
6. Salt content Starch
7. Phosphate content Sucrose
8. Freedom from nitrites Trehalose
9. Trace metals Xylose
10. Lipids Organisms can be identified and differentiated by their oxidative or fermentative
11. Fermentable carbohydrates assimilation of test carbohydrates added to the medium. Growth supportive
12. Vitamins properties of carbohydrates are tested by preparing liquid and solid culture media
13. Incompatibility with other ingredients at 1210C for 15 minutes along with known standard carbohydrates. After sufficient incubation,
characteristics like size, shape, colour and enzyme reactions with indicators are
14. Microbiological evaluation
compared between reference and test carbohydrate on solid media. Carbohydrates
(a) Growth Response
are also tested to ensure their identity and purity.
Microbial growth is measured in liquid media by change in absorbance and Selective Agents (Bile Salts and Bile Salt Mixture)
solid media are used to study colony characteristics.
Bile salts are analyzed chemically as described in National Formulary for Sodium
(b) Production of desired results
cholate, Sodium taurocholate and Sodium deoxycholate (107). Bile salts when
(1) Indole production incorporated in culture media should not affect the colour of the indicator dye(s) or
(2) Acetyl methyl carbinol production their subsequent change in colour as desired in the medium. Bile salts should not
(3) Hydrogen sulphide production form a surface scum or deposit in the medium after storage. Bile functional test is
Carbohydrates carried out using standard bile salts as control. Growth and recovery test is carried
Carbohydrates commonly used in culture media are: out with a standard test organism when a bile salt of known performance is used as
Adonitol a control.
Dyes and Indicators
Aesculin
Dyes are used in the preparation of differential and selective culture media. In
Cellulose
these formulations dyes act as bacteriostatic agents, inhibitors of growth, and
Dextrin sometimes as pH or Eh indicators. When used as selective agents, interaction can
Dextrose occur between the dye and other components of the medium. Thus, the same
Fructose sample of a particular dye may show different inhibition titers against an organism
Galactose like E. coli when tested in different peptone solutions. Bile salts interact with the
dyes and reduce their toxicity. The toxic levels of the dyes are therefore

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 13
 Essentials... Accumix
ascertained by adding to bile salts medium. Aniline dyes are more toxic when fully 15. Total microbial count
oxidized and therefore a change in its toxicity is possible as reduction of the dye Toxicity test is also carried out to ascertain the presence of toxic and inhibitory
takes place when it is sterilized in the presence of other media components like substances by growing fastidious organisms on culture media containing agar
peptone or tryptone. Hence, the dyes are analyzed as specified by H. J. Conn's and various parameters like growth, colony size, shape, pigments, etc. are
(Biological stains, 9th edition). Microbiological suitability is also carried out by compared with known standard agar.
adding dyes in culture media tested with various test organisms compared with Vitamins
standard known dyes, besides ascertaining chemical purity of dyes as per Vitamins and growth factors are analyzed as per the U.S. Pharmacopeia (107) for
biological stains. chemical purity and other tests. Besides, potency is also confirmed by
Solidifying Agent microbiological methods.
The clarity of agar gel in culture media is of utmost importance. Ideally, the Inprocess Sample
molten solution should be crystal clear without any haziness or deposit. The agar A sample batch is prepared from pre-tested and approved components and
should also allow the diffusion of compounds although locking water in a rigid analyzed. All tests are performed in parallel with a previously approved reference
gel. All agars must be free from toxicity to microorganisms and free from batch of the medium. This use of a control standard medium with each test
impurities such as nitrogenous compounds, insoluble salts, non-agar gums, free ensures uniformity in reading the results. Specifications like colour, clarity,
sugar compounds, dead microorganisms and live thermophilic organisms. solubility, pH, gelling and cultural characteristics are ascertained before large
Microbiological agar is specially processed to yield a low toxicity, high clarity and scale batch is approved for manufacturing. Samples are withdrawn at
high diffusion gel. intermediate stages to ascertain purity. The lot in process is continuously recorded
Agar is routinely analyzed for the following : under controlled conditions of moisture, humidity, temperature, etc.
1. Ash content Quality Control of Final Medium

2. Acid insoluble ash Representative samples are reconstituted and examined for appearance, odour,
colour, clarity, moisture, solubility, gelling temperature, pH, gel strength and
3. Sulphate
compatibility with post sterilization additives. Microbiological performance is
4. Chloride
accessed by testing with small inocula of well defined cultures to measure
5. Calcium recovery of growth, colony size and morphology, colour reactions, biochemical
6. Magnesium performance, differentiation and selectivity to ascertain intended or desired
7. Iron results of the product. Special procedures such as antimicrobial succeptibility tests
8. Total nitrogen are performed where appropriate for the recommended use of the medium. All
tests are performed in parallel with a previously approved reference batch of the
9. Gelling strength
medium.
10. Setting temperature Stability Study to Evaluate the Shelf Life of the Final Product
11. Moisture content Samples of each manufactured lot/ batch are retained for the total shelf life of the
12. pH at 1.2% product. Stability tests and lot-to-lot uniformity tests are carried out using these
13. Agar gel retained samples.
14. Melting point

14 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
user know how
Dehydrated Culture Media ■ Bases ■ Supplements

“It does not take great men to do great things,

just those who are greatly dedicated to doing them.”
 User know how... Accumix
General Guidelines to Users
APPROPRIATE USE OF DEHYDRATED CULTURE MEDIA Fermentation tubes and vials
Dehydrated culture media are subject to strict physical, chemical and Use fermentation tubes of any type, if their design permits conforming to medium
bacteriological quality control measures on both the raw materials and finished and volume requirements for concentration of nutritive ingredients. Where tubes
products. Generally, dehydrated media should be sterile, able to support the are used for testing gas production, enclose a vial, inverted. Use tube and vial of
growth of the respective organism and produce appropriate biochemical such size that the vial will be completely filled with the medium, at least partly
reactions. The preparation of culture media from dehydrated media requires submerged in the tube, and large enough to make gas bubbles easily visible.
accuracy and attention to preparation. The following points help in successful and Sample bottles
reproducible preparation of culture media. For bacteriological samples, use sterilizable bottles of glass or plastic of any
DEHYDRATED MEDIA AND INGREDIENTS suitable size and shape. Use bottles capable of holding a sufficient volume of
Dehydrated media ingredients are hygroscopic and sensitive to moisture and sample for all required tests and an adequate air space permitting proper
therefore the storage and other conditions mentioned must be strictly followed. washing, and maintaining samples uncontaminated until examinations are
The media must be stored in a cool (15-300C), dark and dry area unless otherwise completed. Metal or plastic screw cap closures with liners can be used on sample
specified. The date of opening the container must be noted. Expiry must be bottles provided no toxic compounds are produced on sterilization.
checked prior to opening the container (applies to intact container). It requires to Before sterilization, cover tops and necks of sample bottles having glass closures
be verified that the physical characteristics of the powder are typical. with aluminium foil or heavy craft paper.
Culture media have a tendency to form lumps due to the following conditions: - Water

1. High humidity during storage Distilled or deionized water that meets the specifications for purified water, pH
2. Container left open for too long 5.5-7.5 must be used. It must be free from dissolved metals and bactericidal or
inhibitory compounds. Distilled water should also be free of contaminating
3. Container not tightly sealed every time after opening
nutrients. Store distilled water out of direct sunlight to prevent growth of algae
4. Medium is too old and turn supplies over as rapidly as possible. Aged distilled water may contain
Glassware and Plastic ware (Media preparation utensils)
toxic volatile organic compounds absorbed from the atmosphere if stored for
High quality, low alkali borosilicate glass must preferably be used. Detergent prolonged periods in unsealed containers. Good housekeeping practices usually
residues must be avoided. Large enough vessels, at least 2-3 times the volume of eliminate nutrient contamination.
the medium must be used to allow proper mixing. Chipped glassware must not be Equipment
used. Measuring devices, pH meters, scales, optical counting devices and other
Dilution bottles or tubes
equipment that is frequently used need to be accurately calibrated.
Use glass bottles or tubes of resistant glass, preferably borosilicate glass, closed Incubators
with glass stoppers or screw caps equipped with liners that do not produce toxic or Incubators must contain a uniform and constant temperature at all times in all
bacteriostatic compounds on sterilization. Try and avoid cotton plugs as closures. areas, that is they must not vary more than 0.5 in the areas used. Obtain such
Plastic bottles of nontoxic material and acceptable size may be used provided they accuracy by using a water jacketed or anhydric-type incubator with
are sterilized properly. thermostatically controlled low-temperature electric heating units properly
Petri dishes
insulated and located in or adjacent to the walls or floor of the chamber and
For the plate count, use glass or plastic petri dishes about 100x15 mm. Use preferably equipped with mechanical means of circulating air. Maintain an
dishes, the bottoms of which are free from bubbles and scratches and flat so that accurate thermometer. It is also desirable, in addition, to maintain a maximum
the medium will be of uniform thickness throughout the plate. For membrane and minimum-registering thermometer within the incubator on the middle shelf
filter technique, use loose-lid glass or plastic dishes, 60x15 mm, or tight-lid to record the gross temperature range over a 24-hour period. Install a recording
dishes, 50x12 mm. Sterilize petri dishes and store in metal cans (aluminum or thermometer whenever possible, to maintain a continuous and permanent record
stainless steel), or wrap in paper-preferably best quality sulfate pulp. of temperature.

16 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
 User know how... Accumix
Autoclaves Place approximately weighed amount of the medium in a clean dry flask, 2-3
Use autoclaves of sufficient size to avoid internal crowding; constructed to provide times larger than the final volume of the prepared medium to allow adequate
uniform temperatures within the chambers (up to and including the sterilization mixing. Add part of the required amount of distilled water and swirl to dissolve.
temperature of 1210C); equipped with an accurate thermometer the bulb of which After thorough mixing, add the remainder of the water with care being taken to
is located properly on the exhaust line so as to register minimum temperature wash down the sides of the container. Allowing agar preparations to stand for 5
within the sterilizing chambers, equipped with pressure gauge and properly minutes with occasional agitation prior to boiling enhances dissolution. Majority
adjusted safety valves connected directly with saturated-steam supply lines of broth media are clear and dissolve easily at this stage. To completely dissolve
equipped with filters to remove particulates and oil droplets. the powder, it is heated using an open flame, hot plate or boiling water bath.
pH equipment When heating is required, heat should be applied gently and the preparation
Use electronic pH meters, accurate to at least 0.1 pH units, for determining pH agitated as required to prevent scorching. However, care must be taken to avoid
values of media. media eruptions. Boil as briefly as possible to obtain solution; 1 minute is usually
Balances sufficient. Care must be taken not to over boil the culture media as this could result
Use balances providing a sensitivity of at least 0.1 g at a load of 150 g, with in pH drift, darkening, precipitation, poor gel strength and reduced
appropriate weights. Use an analytical balance having a sensitivity of 1 mg under bacteriological performance.
a load of 10 g for weighing small quantities of materials. Single pan rapid-weight Complete dissolution of the medium is indicated by perfect transparency of the
balances are most convenient. solution that runs down the walls of the flask. In no case should powdered
Temperature monitoring devices medium be added to water and immediately placed into an autoclave since
Use glass or metal thermometers graduated to 0.50C to monitor most incubators layering and separation of ingredients, precipitate formation and darkening are
and refrigerators. Use thermometers graduated to 0.10C for incubators operated likely to occur with diminition of performance. The culture media should not be
above 400C. used at a temperature higher than 500C and should not be remelted as far as
Inoculating equipment possible.
Use wire loops made of 22- or 24- gauge nickel alloy or platinum-iridium for Sterilization
flame sterilization. Use loops of at least 3 mm in diameter. Sterilize by dry heat or After preparation, most culture media require sterilization in an autoclave.
steam. Although sterilization of culture media is best carried out in a steam autoclave at
Optical counting equipment temperatures around 1210C it has to be recognized that damage is caused to the
1. Pour and spread plates: Use Quebec-type colony counter, dark field model medium by the heating process. Heat- treatment of complex culture media, which
preferred, or one providing equivalent magnification (1.5 diameters) and contain peptides, sugars, minerals and metals results in nutrient destruction,
satisfactory visibility. either by direct thermal degradation or by reaction between the medium
2. Membrane filters: Use a binocular microscope with magnification of 10X to components. Toxic products caused by chemo oxidation can also be formed during
15X provide day light fluorescent light source at angle of 60-800 above the heat treatment.
colonies; use low-angle lighting for non-pigmented colonies. It is important therefore, to optimize the heating process so that the medium is
Membrane filtration equipment sterile after heating but minimal damage is caused to the ingredients. As a
Use filter funnel and membrane holder made of seamless stainless steel, glass or general rule, it is accepted that short duration, high temperature processes are
autoclavable plastic that does not leak or corrode. more lethal to organisms and less chemically damaging than longer, lower
Reconstitution / Rehydration temperature processes.
The procedure employed for dissolving dehydrated culture media very often The completely dissolved medium is dispensed as desired and sterilized as
determines the clarity and performance of the finished product. Homogenicity of directed on the label. Generally sterilization is done at 1210C for 15 minutes in an
the solution and minimal direct heat exposure are important factors. Complete autoclave. The recommended 15-minute sterilization assumes a volume of 1
instructions for the preparation of culture media are given on the label of each liter or less. For larger volumes, the sterilization time should be extended but the
bottle. For rehydration, as mentioned; clean, undamaged glassware and distilled temperature should not be raised. When larger volumes are used, validation
or deionized water must be used. studies should be performed to determine the optimum sterilization cycle for each

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 17
 User know how... Accumix
unique container size / volume combination. Autoclave media containing to 35-370C before adding to the medium, which has been cooled to 40-450C.
carbohydrates at a temperature not exceeding 116-1180C to avoid pH Adjustment
caramalization of the carbohydrate. Do not autoclave media that should not be Commercial dehydrated media are designed to fall within the specified pH range
heat- sterilized. after steam sterilization. (The pH value of reconstituted dehydrated culture media
Temperature pressure relation is listed below. prepared with distilled or deionized water shall produce the equivalent value as
prescribed on the label at a temperature of 250C). The pH tends to fall
Pressure of saturated steam Temperature Temperature
P.S.I (inside the autoclave) attained in 0C attained in 0F approximately 0.2 units during steam sterilization. For filter sterilization, the pH
must be adjusted if necessary, prior to filtering. pH measurement and correction
5 lbs 108 226 of the pH should be carried out at 250C. For liquid culture media, pH measurement
10 lbs 116 240 after sterilization should be carried out by cooling the medium to 250C. Take an
12 lbs 118 244 aliquot of liquid medium aseptically and measure pH. For solid culture media, pH
15 lbs 121 250 should be measured after cooling the medium to 400C. Take an aliquot of the
20 lbs 127 260 cooled medium aseptically and measure pH. The pH should be adjusted to the
value specified and it is corrected by adding 1N or 0.1 N HCl or NaOH solution to a
25 lbs 131 267
sample of known volume taken from the reconstituted culture medium. Finally
30 lbs 134 274 after calculation, the required acid or alkali is added in the remaining culture
Efficiency of autoclaving should be ascertained from time to time. Temperature medium.
conditions inside autoclave may not be uniform. This can be tested by putting 2 Dispensing Culture Media
bottles containing 2% w/v glucose in 2% w/v disodium phosphate in the The medium is generally dispensed into petri plates or tubes in a laminar flow
autoclave at different places. Heat produces browning of solution. Bottle near cabinet with the surface disinfected. Heat labile supplements are generally added
steam inlet will be browner than the one away from it. at this stage. Gentle mixing during dispensing is required. Sterile media should be
Overheating Effects poured into plates at about 45-500C to reduce water evaporation and to avoid the
Overheating is a common cause of drift in pH, darkening, precipitation, poor gel formation of condensed water in the lids of the petri plates.
strength and reduced bacteriological performance. These effects can also be Dispensing should be done quickly without bubble formation and can be done
produced if a concentrated pool of ingredients at the bottom of the container is manually or automatically. The tubes should be recapped immediately to avoid
heated. All culture media should be in solution before sterilization. This will chances of contamination. Petri dish covers are left slightly open for 1-2 hrs to
reduce the occurrence of Maillard-type reactions (non enzymatic browning) obtain a dry surface, (before inoculation, agar surface should be dried at 30-400C
taking place in the medium. to avoid microbial swarming).
Overheating effects will occur if agar media are allowed to gel in bottles and later Storage of Prepared Media
steamed to melt the agar. It will also occur if molten medium is held at 500C for If the medium is not used on the same day it is prepared, it should be stored
more than three hours before use. Agar media, with pH values below 5.0 are very properly in moisture proof containers to prevent drying of the medium. In general,
sensitive to overheating in any form because the agar is hydrolyzed and the gel store steam sterilized plated media inverted in a plastic bag or other container in a
strength fails. dark refrigerator at 2-80C(remains for several weeks). However, media should not
Adding Enrichments and Supplements be stored below 00C as this destroys their gel structure. It is recommended to use
Enrichments and supplements tend to be heat sensitive and therefore must be fresh media than stored media. Agar containing media should not be stored at
sterilized separately and then added. Heat labile supplements must be added to higher temperatures (40-500C) for longer time, as agar tends to clump. Media
the medium that has been cooled to 45-500C. The sterile supplement must be containing labile beta-lactum selective agents have a very short active life and
brought to room temperature before it is added to the medium. Adequate mixing therefore should be used within a few days of preparation. Loss of water can result
of the supplements with the basal medium by thorough mixing must be ensured. in precipitation and crystallization of certain substances in culture media.
Sterile broths may be cooled to room temperature before adding enrichment. Prepared broth media can be stored at 2-80C, without allowing to freeze. Liquid
To prepare Blood Agar, defibrinated blood is recommended rather than blood media in test tubes or flasks should also be provided with airtight seals. Prepared
containing an anticoagulant. Sterile blood should be stored at 2-80C, and brought plates are often vulnerable to contamination, dehydration and chemical

18 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
 User know how... Accumix
degradation. Proper storage at 2-80C prevents water loss of the medium. Most Biosafety Level 4 practices are applicable for work with highly dangerous
media, and especially those containing dyes or indicators, should be protected agents, which may be transmitted via the aerosol route, for which there is no
from light during storage. Prepared media that have been refrigerated should be available vaccine or therapy and for which specialized equipments and facilities
removed from refrigeration and brought to room temperature prior to inoculation are required.
to allow water of condensation to evaporate and to avoid temperature shock to Consult the reference for specific recommendations on the practices, equipment
the inoculum. and facilities of the four-biosafety levels.
However, before inoculation the plates should be carefully examined for Handling of Dehydrated Culture Media
contamination, uneven filling or bubbles on agar surface, colour changes, Most of the products supplied have no known risks except those usually associated
haemolysis and cracks due to shrinking and loss of volume. with fine powder. These products are meant for carrying out bacteriological work
Precautions in the use and disposal of culture media in the laboratory as specified under the use of individual medium and should not
Inoculation of culture media with bacteria, deliberately or accidentally, leads to a be used for human or animal consumption. Powder may cause irritation of the
very great number of organisms being produced. High concentrations of any upper respiratory tract. To avoid mild skin rashes, prevent prolonged contact with
organisms are potentially hazardous and must be disposed off safely. Therefore, the powder and ensure that excessive dust is not produced. Any residue should be
after use, prepared plates, samples, sample containers or other contaminated washed off with ample cold water. To prevent the risk of inhaling fine dust it is
material must be sterilized or incinerated before discarding. All autoclaved recommended that approved masks should be worn while handling dehydrated
biohazards should be disposed off in accordance with state and local media.
environmental regulations. Hazardous and Toxic Products

Only qualified personnel who have been trained in microbiological procedures There are a number of products, which contain toxic substances. These must be
should handle all infected specimens and inoculated culture media. User should used in accordance with the product specific “material safety data sheet”. All
ensure that any machinery or apparatus used and by chance contaminated must relevant risk and safety phrases are on the product label e.g. media containing
be safely disinfected or sterilized. The environment in which microbiological sodium azide, bismuth, cycloheximide, lithium chloride, basic fuchsin, sodium
cultures are handled must also be taken into account. Most countries have hydrogen selenite, etc. The material safety data sheet is available for all products,
categories of organisms, which are divided into those, which may be handled in a hard copy of which can be obtained from the company on request.
the general microbiological laboratory, those which require special laboratory Sodium azide
conditions and for the most dangerous organisms a totally contained and highly Generally, the products contain less than 1% of sodium azide and have low
protected environment. toxicity. However, some individuals have enhanced sensitivity to azide and
The United States Department of Health and Human Services has published therefore precaution must be taken to prevent ingestion or inhalation of the dust.
guidelines for handling infectious agents and materials. The guidelines describe Sodium azide has a tendency to react with many metals, particularly copper and
four-biosafety levels. lead which produce explosive metal azides. It is recommended to observe strict
regulation of local and national legislation of disposal. Flush using sufficient
Biosafety Level 1 is applicable when work is done with defined and
water to prevent the powder remaining in contact with pipeline and drains.
characterized strains of viable organisms known to consistently cause disease in
Basic fuchsin
healthy adult humans.
Basic fuchsin is a potential carcinogen. Care should be taken to avoid inhalation
Biosafety Level 2 practices are applicable to laboratories where work is done
of the powdered dye and contact with skin.
with the broad spectrum indigenous moderate risk agents that are associated with
Lithium chloride
human diseases; activities can be performed on the open bench provided the
Lithium chloride is harmful hence all bodily contacts and inhalation of vapours
potential for producing splashes or aerosol is low.
must be avoided.
Biosafety Level 3 practices are applicable to laboratories working with agents
Sodium hydrogen selenite
with a potential for respiratory transmission and which may cause potential and
Sodium hydrogen selenite is highly toxic, terratogenic and corrosive. It must be
seriously lethal infection. All laboratory manipulations must be performed in a
handled with great care. On contact with skin, it is necessary to wash with plenty
biological safety cabinet or other enclosed equipment to protect personnel and the
of water.
environment from the exposure to potentially infectious aerosol.

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 19
 User know how... Accumix
Chloramphenicol label of the container. Generally products should be used in order of their lot /
Chloramphenicol is a potent carcinogen and must be handled with care. Avoid batch numbers. (Provision should be made for rotating the stock of dehydrated
inhalation. On contact with skin, it is necessary to wash with plenty of water. media to ensure product freshness).
Potassium Hydroxide Environmental conditions like light, humidity and temperature are the frequent
Potassium Hydroxide is corrosive. It must be handled with care. On contact with causes of instability of the product
skin, wash immediately with plenty of water. Light
The following hazard symbols are mentioned on the respective product labels as All the dehydrated culture media should be stored away from light and exposure
well as warnings in the manual. to direct sunlight.
Humidity and Temperature
It is important not to leave bottles open to atmosphere since dehydrated powder
will be affected by humidity. Hot or media preparation rooms are not suitable to
Xn T store containers of culture media, particularly containers that are frequently
Harmful Toxic opened and closed. They should be stored in a dry, dark place at room
temperature.
Recommended Storage Temperature and Time
Dehydrated Culture Media
N C Sealed, unopened containers Temperature (RT) below 300C, for a few storage at
Dangerous for the environment Corrosive 2-80C is required. Unless mentioned, media described in this manual should be
stored at Room Temperature (RT) below 300C. Such media stored under optimal
First Aid Measures
conditions have a shelf life of 2-5 years. When the container is opened for the first
The following first aid precautions should be taken in case of accidents while time, the date should be noted on the container. The lid should be replaced
handling any of the toxic or hazardous products: - quickly after the media has been taken out and the cap is to be closed tightly to
Inhalation
protect from hydration.
Remove person from area of exposure. Rest and keep warm and seek medical Media Bases
advice. Sealed unopened containers should be stored at Room Temperature (RT) below
Skin Contact
300C and they generally have a shelf life of 3-5 years.
Remove all contaminated clothing, wash the effected area thoroughly and seek Selective Supplements
medical advice.
Selective Supplements should be stored at 2-80C. They generally have a shelf life
Ingestion
of 1-3 years. Horse serum should be stored at -200C.
Wash out mouth with water, give water to drink immediately and seek medical Packing
advice.
Dehydrated Culture Media are filled in HDPE, double sealed containers in 100
Eye Contact
grams and 500 grams pack with respective package inserts for instructions,
Irrigate thoroughly with water and seek medical advice. individually encased in shrink film packs. The catalogue numbers AM1XXX and
Spillage
AM5XXX indicates 100 gm and 500 gm packaging respectively where X denotes a
Wash away with large volumes of running water; wear protective overalls, gloves, number.
eye protection and facemask. Collect the material in a suitable container, seal and
All Microxpress products are compliant and the following symbols are
dispose off.
mentioned in the respective packaging.
Storage of Dehydrated Culture Media
It is essential to store all the products at specified conditions to achieve optimum
performance. Products should not be stored beyond the allocated shelf life. The
storage conditions and expiry dates of each product are shown on the respective

20 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
 User know how... Accumix
300C 80C
Store at 2-80C
0
0
22 C Store at 22-30 C 0
2C

REF Catalogue Number LOT Batch Number

IVD In vitro Diagnostic Medical Device DCB Dehydrated Culture Bases

Consult Instructions for use DCS Dehydrated Culture Supplements

Manufacturer
RO Received on

H Hygroscopic keep container tightly closed OO Opened on

DCM Dehydrated Culture Media

Use by
EC REP Authorised Representative

Expiry Product Deterioration

The expiration date applies to the products in their intact containers when stored Verify that the physical characteristics of the powder are typical. Hydration can
as directed. Do not use a product if it fails to meet specifications for identity and lead to deterioration, which render the medium unusable.
performance. Use the product before expiry date on the label.

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 21
 User know how... Accumix
Factors that Influence the Quality of Dehydrated Culture Media
Effects of the media on the colony growth may be too poor or too strong because of requires testing. Atypical growth of organisms may be observed if the dehydrated
residues of growth inhibiting substances present in improperly washed glassware, culture media is old or if residues of foreign substances are present in the
overheating of the medium and shift in pH. Also, if the surface of the media glassware or water used. Pouring of plates correctly and aseptic culturing methods
remains moist as a result of improper media preparation, the colonies of certain are very essential so as to avoid contamination on plates.
microorganisms tend to liquefy or swarm inhibiting the growth of bacteria that
Faults Possible Causes
Clumping of the media Container was not tightly sealed after use
Container was left open for a long time
Humidity was high during storage
Culture medium was too old
Drift in pH Overheating due to excessive sterilization, heterogeneous mixing, repeated remelting
and inadequate storage
Impure water and glassware used
Hydrolysis of ingredients
pH determined when medium was hot
Incomplete solubility Inadequate water usage
Incomplete mixing which causes overheating
Turbidity, precipitation, contamination, toxicity Water not adequately demineralized
Lab ware used for preparation not clean
Incorrect value of pH, insufficient sterilization
Overheating
Loss of water due to evaporation
Poor technique in adding enrichments and pouring plates
Solidification point too high Too much dehydrated media weighed out
Unsuitable agar used
Darkening of the medium or colour change Overheating or repeated remelting
Incorrect pH
Incomplete or soft gelling Glassware not clean
Incorrect proportions of product to volume of water
Agar not in solution
Incomplete mixing
Repeated remelting
Acid hydrolysis of agar
Loss of growth promoting properties or differentiating properties Repeated remelting or overheating
Incomplete mixing
Disturbance in the formula by the inoculum carriers

22 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
 User know how... Accumix
Limitations of the Procedure The selective agents may inhibit some strains of the organisms to be cultivated
Detergent residues in lab ware / glassware can compromise quality of the media. and permit the growth of organisms they were supposed to inhibit if the unwanted
Quantities of media in excess of one litre may require an extended autoclave time organisms are present in large numbers in the specimen. Culture of specimens
to achieve sterilization. Longer sterilization cycles can cause nutrient grown on selective media should, therefore, be compared with specimens
concentration changes and generation of inhibitory substances. Since the cultured on non-selective media to obtain additional information and help
nutritional requirements of organisms vary, a single medium is rarely adequate recovery of potential pathogens and other significant organisms.
for detecting all organisms of potential significance in a clinical specimen or Though some of the media formulations are developed for isolation of specific
industrial sample. Colour differentiation and the growth of colonies may not be organisms, it is recommended that biochemical and / or serological tests be
optimal if the heat sensitive carbohydrates and other enrichments are added to performed on colonies from pure cultures for complete identification.
the medium when hot.

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 23
 User know how... Accumix
Comparative Chart
APHA PRODUCTS VIS A VIS ACCUMIX PRODUCTS
APHA ACCUMIX CAT. NO. ACCUMIX FOOD DAIRY WATER

Baird Parker Medium AM1011/AM 5011 Baird Parker Agar Base

AS010 Egg Yolk Emulsion
AS011 Egg Yolk Tellurite Emulsion * *

Bile Esculin Agar AM1012/AM5012 Bile Esculin Agar *

Bismuth Sulphite Agar AM1013/AM5013 Bismuth Sulphite Agar * * *
Brain Heart Infusion Agar AM1017/AM5017 Brain Heart Infusion Agar * * *
Brain Heart Infusion Broth AM1018/AM5018 Brain Heart Infusion Broth * * *
Brilliant Green Agar AM1018/AM5018 Brilliant Green Agar, Modified * * *
Brilliant Green Lactose Bile Agar AM1019/AM5019 Brilliant Green Bile Agar * *
Brilliant Green Lactose Bile Broth 2% AM1020/AM5020 Brilliant Green Bile Broth 2% * * *
Cefsulodin Irgasan- Novobiocin (CIN) Agar AM1116/AM5116 Yersinia Selective Agar Base
AS029 Yersinia Selective Supplement *

Christensen's Urea Agar AM1105/AM5105 Urea Agar Base, Christensen's

AS028 Urea, 40% * *
Citrate Agar, Christensen AM1025/AM5025 Christensen Citrate Agar *
Columbia Blood Agar Base AM1029/AM5029 Columbia Blood Agar Base *
Cooked Meat Medium AM1030/AM5030 Cooked Meat Medium *
Deoxycholate Citrate Agar AM1031/AM5031 Deoxycholate Citrate Agar * *

Dextrose Tryptone Bromo Cresol Purple Agar AM1036/AM5036 Dextrose Tryptone Agar * *
Dextrose Tryptone Brom Cresol Purple Broth AM1037/AM5037 Dextrose Tryptone Broth *
EC Broth AM1039/AM5039 EC Broth * * *
Eosin Methylene Blue Agar, Levine AM1040/AM5040 EMB Agar, Levine * * *
Lactose Broth AM1042/AM5042 Fluid Lactose Medium * * *

Kligler Iron Agar (KIA) AM1050/AM5050 Kligler Iron Agar *

Lactose Broth AM1052/AM5052 Lactose Broth * * *
Lauryl Sulphate Tryptose Broth (LST) AM1053/AM5053 Lauryl Sulphate Broth * * *

24 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
User know how... Accumix
APHA ACCUMIX CAT. NO. ACCUMIX FOOD DAIRY WATER

MR-VP Medium AM1070/AM5070 MR-VP Medium * * *

MacConkey Agar AM1059/AM5059 MacConkey Agar with Crystal Violet, NaCl and
0.15% Bile Salts * * *

MacConkey Broth AM1063/AM5063 MacConkey Broth Purple with

Bromocresol Purple * * *

Malonate Broth, Modified AM1065/AM5065 Malonate Broth, Ewing Modified * * *

Mueller Hinton Agar AM1071/AM5071 Mueller Hinton Agar *
Nutrient Agar AM1075/AM5075 Nutrient Agar pH 6.8 * *
Nutrient Broth AM1077/AM5077 Nutrient Broth *
Plate Count Agar AM1081/AM5081 Plate Count Agar * * *
Potato Dextrose Agar (Acidified) AM1082/AM5082 Potato Dextrose Agar * *

Salmonella Shigella Agar AM1093/AM5093 SS Agar * * *

Selenite Cystine Broth AM1044/AM5044 Fluid Selenite Cystine Medium * * *
Simmons Citrate Agar AM1090/AM5090 Simmons Citrate Agar * * *
Tetrathionate Broth AM1096/AM5096 Tetrathionate Broth Base, Hajna * *
Thiosulphate Citrate Bile Salt Sucrose Agar AM1095/AM5095 TCBS Agar * *
Triple Sugar Iron Agar AM1099/AM5099 Triple Sugar Iron Agar * * *
Trypticase Soya Agar AM1091/AM5091 Soyabean Casein Digest Agar * *
Trypticase Soya Broth AM1092/AM5092 Soyabean Casein Digest Broth * * *
Tryptone (Tryptophan) Broth AM1104/AM5104 Tryptone Water * *
Tryptone Phosphate Broth AM1103/AM5103 Tryptone Phosphate Broth *
Tryptone Glucose Extract Agar AM1101/AM5101 Tryptone Glucose Yeast Extract Agar * * *

Urea Broth AM1106/AM5106 Urea Broth Base

AS028 Urea 40% * *
Violet Red Bile Agar AM1107/AM5107 Violet Red Bile Agar * *
XLD Agar AM1112/AM5112 Xylose Lysine Deoxycholate Agar * * *
Yeast Malt Agar AM1114/AM5114 Yeast Malt Agar * *

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 25
Exploring Dehydrated Culture Media ■ Bases ■ Supplements

“Before you discover you must explore.”

 Exploring... Accumix
Acetate Differential Agar AM5908
Use 5. Cool in slanted position for use as slants.
Acetate Differential Agar is used to differentiate species of Shigella from Quality Control
Escherichia coli and non-fermentative gram-negative bacilli. Dehydrated Appearance
Summary Greenish yellow, homogeneous, free flowing powder.
Prepared Appearance
Bacteria that are capable of growing on a simple, chemically defined medium can
Emerald green coloured, clear to slightly opalescent gel form in tubes as slants.
readily be tested for their ability to use a given compound as their sole source of
Cultural Response
carbon and energy. A defined medium is prepared with the test compound Cultural characteristics up to 1-7 days at 35-370C.
substituted for the normal carbon and energy source. Acetate Differential Agar is Organisms (ATCC) Growth Acetate utilization RGI
used to test the ability of an organism to utilize acetate as the sole carbon and Citrobacter freundii Good-luxuriant + More than 70%
energy source. Organic acids have been widely used in media containing organic (8090)
nitrogen sources as a means of differentiating Enterobacteriaceae. Most bacteria Enterobacter cloacae Good-luxuriant + More than 70%
can use citrate and acetate in the presence of organic nitrogen. Trabulsi and (23355)
Ewing developing Acetate Differential Agar by replacing sodium citrate by sodium Escherichia coli Good-luxuriant + More than 70%
(25922)
acetate made a new approach. This medium differentiates the members of the
Klebsiella Good-luxuriant + More than 70%
genus of Shigella species from Escherichia coli. Differentiation is based on the
pneumoniae (13883)
organism's ability to utilize acetate. Salmonella serotype Good-luxuriant + More than 70%
Principle Arizonae (13314)
Acetate Differential Agar consists of a mixture of salts and sodium acetate, as a Salmonella serotype Poor _ 0%
sole source of carbon. Bromothymol blue acts as an indicator. Shigella species Typhi (19430)
are unable to utilize acetate and fail to grow; Shigella sonnei None _ 0%
(25931)
and therefore the medium remains unchanged. Most strains of Escherichia coli
Proteus vulgaris None _ 0%
grow well within 24-48 hours, but some strains grow more slowly. Growth (13315)
indicates the utilization of acetate, which results in For growth RGI should be more than 70%
the production of alkaline products. The colour of the indicator changes to blue For Inhibition RGI should be 0%
due to the increase in pH. RGI- Relative Growth Index
Formula* Key: + = Colour change of the medium from green to blue.
– = No change, medium remains green.
Ingredients in grams per liter
Sodium acetate 2.00 Procedure
Magnesium sulphate 0.10 1. Touch only the center of an isolated colony on an enteric plated medium with
Sodium chloride 5.00 a cool and sterile needle and streak on the surface of the agar slant.
Mono-ammonium phosphate 1.00
2. Incubate aerobically at 35-37ºC for at least 7 days with intermittent
Dipotassium phosphate 1.00
observation.
Bromothymol blue 0.08
Agar 20.00 3. Observe for a colour change from green to blue along the slant.
Final pH (at 250C) 6.7 ±0.2 Interpretation of Results
* Formula adjusted to suit performance parameters 1. Bacteria capable of utilizing acetate as the sole carbon source will grow on
Directions the medium and produce an alkaline reaction
1. Suspend the 29.18 gms of powder in 1000 ml distilled water. 2. Colour of the medium changes to blue. There is no change of colour in
2. Mix thoroughly. negative reaction.
3. Warm slightly with frequent agitation to dissolve the powder completely. Precautions / Limitations

4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes. 1. Some strains of E. coli utilize acetate slowly or not at all and may give a

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 27
 Exploring... Accumix
false-negative reaction. Storage

2. Some biotypes of Shigella flexneri utilize acetate and thereby may give Store at 22-300C and prepared medium at 2-80C.
Shelf Life
false- positive result.
Use before expiry date as mentioned on the label

Acetamide Agar AM59081

Use Directions
Acetamide Agar is recommended for confirmation Pseudomonas aeruginosa in 1. Suspend 22.63 grams of part B in 1000 ml distilled water.
water samples.
2. Add 10.0 grams of Part A.
Summary
3 Heat to boiling to dissolve the medium completely.
Assimilation studies by Gilardi and others using basal mineral media showed that 4. Dispense in tubes or as desired.
acetamide was utilized by a wide variety of nonfermenting organisms (34.1 & 5. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
104.1). However, few organisms are reported to deaminate acetamide. A variety 6. Cool the tubes in a slanted position.
of media formulations have been developed to determine the ability of various Quality Control
nonfermenting gram-negative organisms to deaminate acetamide for purposes Dehydrated Appearance
of identification (42.3 & 85.1.2). The formulation of this medium is the one Part A :Colourless deliquescent crystals Part B : Light yellow to light pink
homogeneous free flowing powder
recommended in Standard Methods for the Examination of Water and
Prepared Appearance
Wastewater
Orange coloured clear to slightly opalescent gel forms in tubes as slants.
Principle
Cultural Response
The ability to deaminate acetamide (acylamidase activity) has been found to be Cultural characteristics after 4-7 days at 35-370C.
most actively accomplished by P. aeruginosa, Comamonas (Pseudomonas) Organisms (ATCC) Growth Deamination RGI
acidovorans, Achromobacter xylosoxidans subsp. xylosoxidans (Alcaligenes Pseudoomonas Luxuriant Negative reaction, More than 70%
maltophila (13637) no purplish red
xylosoxidans) and Alcaligenes faecalis (odorans). Deamination of acetamide colour within 7 days
produces ammonia which increases the pH of the medium causing a Pseudomonas Luxuriant Positive reaction, More than 70%
corresponding color change from yellow-orange to purplish-red. aeruginosa (27853) purplish red
Formula colour within 7 days
Ingredients in Grams/Litre For growth RGI should be more than 70%
Part A RGI- Relative Growth Index
Acetamide 10.00 Limitations of the procedure
Part B 1. Some strains deaminate acetamide slowly and may require as long as 7
Sodium chloride 5.00 days to yield a positive test result.
Dipotassium hydrogen phosphate 1.39
Potassium dihydrogen phosphat 0.73 2. Only about 37% of apyocyanogenic strains of P. aeruginosa will produce a
Magnesium sulphate 0.50 positive reaction. Therefore, this test should not be relied upon as a sole
Phenol red 0.012 criterion for identification.
Agar 15.00 Storage
Final pH (at 250C) 6.9 ± 0.2 Store at 22-300C and prepared medium at 2-80C.
* Formula adjusted to suit performance parameters Shelf Life
Use before expiry date as mentioned on the label

Acetamide Broth AM59082

Use Summary
Acetamide Broth is recommended for confirmation of non-fermentative gram- Assimilation studies by Gilardi and others using basal mineral media showed that
negative bacteria, particularly Pseudomonas aeruginosa . acetamide was utilized by a wide variety of nonfermenting organisms (34.1 &

28 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
 Exploring... Accumix
104.1). However, few organisms are reported to deaminate acetamide. A variety 2. Add 10grams of Part A.
of media formulations have been developed to determine the ability of various 3. Heat to boiling to dissolve the medium completely.
nonfermenting gram-negative organisms to deaminate acetamide for purposes of 4. Dispense in tubes or as desired.
identification (42.3 & 85.1.2). The formulation of this medium is the one
5. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes
recommended in Standard Methods for the Examination of Water and Wastewater
Quality Control
Principle
Dehydrated Appearance
The ability to deaminate acetamide (acylamidase activity) has been found to be Part A :Colourless deliquescent crystals Part B : Light yellow to light pink
most actively accomplished by P. aeruginosa, Comamonas (Pseudomonas) homogeneous free flowing powder
acidovorans, Achromobacter xylosoxidans subsp. xylosoxidans (Alcaligenes Prepared Appearance
xylosoxidans) and Alcaligenes faecalis (odorans). Deamination of acetamide Orange coloured, clear to slightly opalescent gel forms in the tube as slants
solution
produces ammonia which increases the pH of the medium causing a
Cultural Response
corresponding color change from yellow-orange to purplish-red. Cultural characteristics after 4-7 day at 35-370C.
Formula* Organisms (ATCC) Growth Deamination
Ingredients in Grams/Litre Pseudomonas maltophila Luxuriant Negative, no reaction,
Part A (13637) no purplish red colour
Acetamide 10.00 within 7 days.
Part B Pseudomonas aeruginosa Luxuriant Positive reaction,
Sodium chloride 5.00 (27853) purplish red colour
Dipotassium hydrogen phosphate 1.39 within 7 days.
Potassium dihydrogen phosphat 0.73 Storage
Magnesium sulphate 0.50
Store at 22-300C and prepared medium at 2-80C.
Phenol red 0.012
Shelf Life
Final pH (at 250C) 7.0 ± 0.2
* Formula adjusted to suit performance parameters Use before expiry date as mentioned on the label
Directions
1. Suspend 7.63 grams of part B in 1000 ml distilled water.

AK Agar No. 2 (Sporulating Agar) (Arret and Kirshbaum Medium) AM5909

Use important role in the sporulation process.
A K agar No. 2 is a culture medium recommended for the production of spores of Formula
Bacillus subtilis ATCC 6633, which are used for detection of penicillin and other Ingredients Grams/Litre
antibiotic residues in milk and diary products. Pancreatic digest of Gelatin 6.0
Casein enzymic hydrolysate 4.0
Summary
Yeast extract 3.0
A K Agar No. 2 was devised by Arret and Kirshbaum for specific use in the Beef extract 1.50
production of spores of Bacillus subtilis ATCC 6633 for use in the Penicillin Milk Dextrose 1.0
Test Procedure. This medium was formerly specified in the spore preparation Manganous sulphate 0.30
phase of the American Public Health Association disc assay procedure for the Agar 15.00
detection of sulfa drugs and antibiotics in milk. Final pH ( at 250C) 6.6 + 0.2
Principle Directions

Pancreatic digest of Gelatin, casein enzymic hydrolysate and beef extract are 1. Suspend the 30.8 gms of powder in 1000 ml distilled water.
sources of nitrogen, sulfur, amino acids and essential trace ingredients. Yeast 2. Boil with stirring to dissolve the medium completely.
extract is a rich source for bacterial replication. Manganous sulfate plays an 3. Dispense in 300 ml amounts in Roux on other suitable bottles.

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4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes. 4. Centrifuge the suspension and decant and discard the supernatant fluid.
NOTE: Do not autoclave until the medium has been completely dissolved. 5. Resuspend the sediment in sterile saline and heat shock the suspension at
Quality Control 700c for 30 min.
Dehydrated Appearance
6. The resultant spore suspension can be stored for several months.
Light yellow coloured, homogeneous, free flowing powder.
Prepared Appearance For preparation of spore suspensions for use in the APHA procedure for detection
Light amber coloured, clear to slightly opalescent gel forms in petriplates. of sulfa drugs and antibiotics in milk.
Cultural Response 1. Transfer cells of Bacillus megaterium ATCC 9855 by streaking the entire
Cultural characteristics after 5 days at 350C. surface of sterile AK agar NO. 2 contained in a prescription (180 ml
Organisms(ATCC) Growth RGI
capacity) or Roux bottle.
Bacillus subtilius (6633) Luxuriant with sporulation More than 70%
Bacillus megaterium (9855) Luxuriant with sporulation More than 70% 2. Incubate the bottles at 35+ 20C for 48 hours.
For growth RGI should be more than 70% 3. After incubation, wash the spores and vegetative cells from the agar surface
RGI- Relative Growth Index with buffered MS (microbiologically suitable) water.
Procedure
4. Sediment the spores and cells by centrifugation at 5,000x g for 15 minutes
For preparation of spore suspensions for use in the FDA procedure for the Penicillin at 30C.
Milk Test.
5. Store the spore suspension in buffered MS water under refrigeration.
1. transfer cultures of Bacillus subtilis ATCC 6633 monthly to fresh slants. Storage
2. Wash the growth from the fresh slant culture with sterile physiological saline Store at 22-300C and prepared medium at 2-80C.
onto the surface of a Roux bottle containing 300 ml of AK agar NO. 2. Shelf Life
0
3. Incubate the bottles for 5 days at 35+ 2 C and wash off the resulting Use before expiry date as mentioned on the label
growth into 50 ml of sterile physiological saline.

Alkaline Peptone Water AM1910/AM5910

Alkaline Peptone Water ISO AM5911
Alkaline Peptone Water BIS AM1912/AM5912
Use Formula*
Alkaline Peptone water is used as an enrichment medium for the cultivation of Ingredients in Alkaline Alkaline Alkaline
grams/ liter Peptone Peptone Peptone
vibrio s pecies from clinical and nonclinical specimen.
Water Water ISO Water BIS
Summary
Peptic digest of animal tissue 10.0 20.0 10.0
Alkaline peptone water is recommended by APHA for isolation of vibrio species Sodium Chloride 10.0 30.0 5.0
from food. Vibrio species grow best in alkaline conditions and this medium Final pH (at 250C) 8.4 ±0.2 8.6 ±0.2 8.2 ±0.2
provides a favorable environment for the growth of vibrios (1). is recommended * Formula adjusted to suit performance parameters
by ISO committee under the specifications ISO 8914: 1990. It is also useful for Directions
preliminarily enrichment of vibrios from faeces or other contaminated materials 1. Suspend then powder in 1000 ml distilled water.
(89.2). Alkaline Peptone Water - 20 gms
Principle
Alkaline Peptone Water ISO - 50 gms
Peptic digest of animal tissue provide carbon, nitrogen and essential nutrients
Alkaline Peptone Water BIS - 15 gms
necessary for growth. Sodium chloride provides sodium ions for the membrane
transport and maintains osmotic equilibrium of the medium. 2. Mix thoroughly.

30 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
 Exploring... Accumix
3. Boil with frequent agitation to dissolve the powder completely. 4. Inoculate a loopful from the top of the peptone water on to a selective
0
4. Sterilize by autoclaving at 121 C(15lbs pressure) for 15 minutes. plating medium such as TCBS Agar (Cat. No.AM5095).
Quality Control For Food Sample
Dehydrated Appearance 1. Add 10gms of seafood to 90 ml of APW.
Light yellow coloured, homogeneous, free flowing powder.
Prepared Appearance 2. Incubate at 370 C for 18-20 hrs.
Light yellow coloured, clear solution. 3. Inoculate a loopful from the peptone water on to a selective plating medium
Cultural Response such as TCBS Agar (Cat. No.AM5095).
Cultural characteristics after 18-24 hours at 35-370C Interpretation of Results
Organisms(ATCC) Growth
Growth in the medium is indicated by the presence of turbidity compared to an
Vibrio parahaemolyticus (17802) Luxuriant
uninoculated control.
Vibrio cholerae (15748) Luxuriant
Precautions / Limitations
Vibrio fischeri (1738) Luxuriant
Procedure 1. It is preferable that biochemical and / or serological tests be performed on
For Clinical Sample colonies from pure culture for complete identification.
Storage
1. Swab specimen inserts directly into medium.
Store at 22-300C and prepared medium at 2-80C.
2. For faecal specimen inoculate1g faeces into 10ml APW, or about 2g into 20 Shelf Life
ml. Use before expiry date as mentioned on the label
3. Incubate at 370C for 18-12 hrs.

Alternative Thioglycollate Medium AM1900/AM5900

Use for sterility testing of viscous materials and devices having tubes with small
Alternative Thioglycollate Medium (NIH Thioglycollate Broth) is recommended for lumina.
sterility testing of certain biological products, which may be turbid or viscous. Formula*
Summary Ingredients in grams per liter
Alternative Thioglycollate Medium (NIH Thioglycollate Broth) recommended by Tryptone 15.0
Dextrose 5.5
USP is the Fluid Thioglycollate Medium without agar and indicator components. It
Yeast Extract 5.0
is used for the same sterility tests as that of Fluid Thioglycollate Medium except
Sodium Chloride 2.5
that anaerobic incubation is recommended rather than aerobic incubation. This Sodium Thioglycollate 0.5
medium also meets the requirements of the USP growth promotion test (111). L-Cystine 0.5
Final pH (at 250 C) 7.1 ± 0.2
Principle * Formula adjusted to suit performance parameters
Directions
Tryptone, Yeast extract and L-cystine provide sources of nitrogen, carbon and
other growth factors while dextrose is the carbohydrate source. Sodium chloride 1. Suspend 29 grams of the powder in 1000 ml distilled water.
provides essential ions and maintains the osmotic balance. Sodium thioglycollate 2. Mix thoroughly.
is a reducing agent, which prevents the accumulation of peroxides that is lethal to 3. Heat with frequent agitation and boil for one minute to dissolve the powder
bacterial growth and neutralizes the antibacterial effect of mercurial completely.
preservatives. L-cystine is also a reducing agent, since it contains sulphydryl 4. Dispense as desired into containers.
groups that inactivate heavy metal compounds, which exert a bacteriostatic effect
5. Sterilize by autoclaving at 1210 C (15 lbs pressure) for 15 minutes.
in the materials under examination, and also maintains a low redox potential,
thereby maintaining anaerobiosis. Absence of agar makes it a suitable medium 6. Tighten lids of the containers immediately (while still warm) to reduce
oxidation.

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7. Cool to 250 C and store in a cool dark place preferably below 250 C. Interpretation of Results

Note: If more than the upper one third of the medium is pink prior to use, reheat
1. Upon incubation, growth is indicated by the presence of turbidity compared to
once (1000C) in a water bath to drive off absorbed oxygen (till pink colour an uninoculated control.
disappears). 2. Subcultures to appropriate solid media should be made to obtain pure
Quality Control cultures of isolates, which can then be further tested and identified.
Dehydrated Appearance Note: Inspect the tubes for clarity before inoculation. Ensure that the tubes are
Yellow coloured, homogeneous, free flowing powder. labeled correctly with the respective sample.
Prepared Appearance Precautions / Limitations
Yellow coloured, clear solution without any precipitate. 1. Some dextrose fermenting organisms, which are able to reduce the pH of the
Cultural Response medium to a critical level, may not survive in this medium. Early subculture is
Cultural characteristics after 24 to 72 hours at 350 C.
required to isolate these organisms.
Organisms (ATCC) Growth
*Bacillus subtilis (6633) Luxuriant
2. In test samples, the proper surface to volume ratio of the medium must be
*Bacteroides vulgatus (8482) Luxuriant maintained to avoid oxidation of the medium, which is unsuitable for
*Candida albicans (10231) Luxuriant microaerophilic and anaerobic growth.
*Clostridium sporogenes (11437) Luxuriant 3. Anaerobes can be overgrown by more rapidly growing facultative organisms.
*Micrococcus luteus (9341) Luxuriant Gram stain and examine the broth if plating medium reveals no growth.
Streptococcus pyogenes (19615) Luxuriant
4. The growth of some anaerobes may be inhibited by metabolic products or
Staphylococcus aureus (25923) Luxuriant
acids produced from more rapidly growing facultative anaerobes.
Neisseria meningitidis (13090) Luxuriant
Bacteroides fragilis (25285) Luxuriant 5. Do not rely on broth cultures exclusively for isolation of anaerobes.
*These cultures may be incubated at 25-300 C for 2-7 days. 6. Do not reheat the medium more than once as it may give rise to toxicity.
Procedure Storage
1. Aseptically inoculate each tube with the sample for testing. Store at 22-300C and prepared medium at 2-80C.
2. Incubate the tubes anaerobically at 35-370 C. Shelf Life
3. Observe for growth after 24 hours and up to 72 hours. Use before expiry date as mentioned on the label

Anaerobic Agar AM1000/AM5000

Use Principle
Anaerobic Agar is recommended for the cultivation of anaerobic microorganisms. Tryptone in the medium provides nitrogen, vitamins, minerals and amino acids
Summary essential for growth. Dextrose is the fermentable carbohydrate providing a source
Anaerobic bacteria cause a variety of infections in humans including ottis media, of carbon and energy. Sodium Chloride maintains the osmotic equilibrium.
oral infections, endocarditis, meningitis, wound infections following bowel Sodium Thioglycollate and Sodium Formaldehyde Sulfoxylate are reducing agents
surgery or trauma and bacteremia. Anaerobic bacteria are the predominant flora generating a low oxidation-reduction potential, thus maintaining good anaerobic
colonizing the skin and mucous membranes of the body. Anaerobes vary in their conditions. Methylene Blue serves as an indicator of anaerobiosis with a blue
sensitivity to oxygen and nutritional requirements. Anaerobic bacteria lack colour indicating the presence of oxygen.
cytochromes and thus are unable to use oxygen as a terminal electron acceptor. Formula*
Ingredients in grams per liter
Brewer described a special petri dish cover that allows surface growth of anaerobes
Tryptone 20.0
and microaerophiles without anaerobic equipment. The microorganisms were
Dextrose 10.0
grown on an agar-based medium having a low oxidation-reduction potential. Sodium Chloride 5.0
Anaerobic Agar is a modification of brewer's original formula. The medium is Sodium Thioglycollate 2.0
suitable for standard plating procedures used in cultivating anaerobic bacteria. Sodium Formaldehyde Sulfoxylate 1.0
Methylene Blue 0.002

32 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
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Agar 20.0 4. The incubation period may be extended if necessary to recover some
Final pH (at 250 C) 7.2 ± 0.2 anaerobes.
* Formula adjusted to suit performance parameters
Directions
For Brewer Anaerobic Agar Plates
1. Suspend 58 grams of powder in 1000 ml distilled water. 1. Dispense 50-60 ml of Anaerobic Agar into a standard petri dish. For best
results use porous tops during solidification to obtain a dry surface.
2. Mix thoroughly.
2. Inoculate the surface of the medium by streaking or smearing.
3. Heat with frequent agitation and boil for one minute to dissolve the powder
completely. 3. Replace the petridish lid with a sterile Brewer petri dish cover. Note: The
cover should not rest on the petri dish bottom. The inner glass ridge should
4. Sterilize by autoclaving at 1210 C (15 lbs pressure) for 15 minutes.
seal against the uninoculated periphery of the agar. The sealing ring inside
Quality Control
Dehydrated Appearance
the cover must be in contact with the medium. The seal must not be broken
Light beige coloured, homogeneous free flowing powder. before the end of the incubation period. A small amount of air may be
Prepared Appearance caught over the surface of the medium, which will react with the reducing
Light green, slightly opalescent. agents to form an anaerobic environment.
Cultural Response 4. Incubate aerobically as desired.
Cultural characteristics after 48-72 hours at 350C when incubated Interpretation of Results
anaerobically.
Organisms (ATCC) Growth RGI 1. Refer to U.S.P. and other appropriate references for interpretation of
Clostridium butyricum (9690) Luxuriant More than 70% results.
Clostridium perfringens (12914) Luxuriant More than 70% Precautions / Limitations
Clostridium sporogenes (11437) Luxuriant More than 70% 1. Methylene Blue is inhibitory to some anaerobic microorganisms.
Bacteroides fragilis (25285) Good More than 70%
2. Clinical specimens must be obtained properly and transported to the
Fusobacterium mortiferum (9817) Good More than 70%
For growth RGI should be more than 70%
laboratory in suitable anaerobic transport container.
RGI- Relative Growth Index 3. It is essential to determine the environment of the medium so as to
Procedure ascertain whether is anaerobic.
For Standard Petri Dishes 4. To ascertain whether an organism is an anaerobe, it is essential to perform
1. Inoculate a properly obtained specimen onto the medium using the pour aerotolerance testing on each isolate recovered.
plate technique. Storage

2. Incubate anaerobically at 35 C.0

Store below 300C and prepared medium at 2-80C.
Shelf Life
3. If the plates are incubated in an anaerobic chamber, examine at 24 hours. If
the plates are incubated in an anaerobic jar or anaerobic pouch, examine at Use before expiry date as mentioned on the label.
48 hours.

Andrade Peptone Water AM1001/AM5001

Andrade Peptone Water BIS AM10011
Use Principle
Andrade Peptone Water is a base to which various carbohydrates may be added to Peptone used in this medium is free from fermentable carbohydrates and nitrates,
study fermentation reactions, particularly of members of the Enterobacteriaceae. which may interfere with gas production. Andrade indicator is a solution of acid
Summary fuchsin which when titrated with sodium hydroxide, changes colour from pink to
Andrade Peptone Water is used for studying the various carbohydrate yellow. As the pH decreases, the colour changes from yellow to pink. The medium
fermentation patterns of different organisms. is pink when hot but becomes straw coloured on cooling.

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 Exploring... Accumix
The biochemical identification of organisms capable of growing in this medium is Procedure
made by observing the catabolism of various carbohydrates added to separate 1. Aseptically inoculate each tube of Andrade Peptone Water containing an
tubes of peptone water. To detect fermentation, which is the production of acid inverted Durham's tube and the respective carbohydrate with a single, well
and gas, a small inverted Durham's tube is inserted into the peptone water to trap separated colony or with a colony from a plate.
any gas produced by the organism. A single bubble in the tube is sufficient to label 2. Incubate the tubes at 35-370C aerobically. If anaerobic incubation is
the organism positive for gas formation. Some organisms will utilize the sugar to required then fresh indicator should be added to the tubes after incubation at
produce acid only, without gas formation. the time of examination.
Formula*
3. Observe for bubbles or gas and any colour change in the medium. Most
Ingredients in grams per liter
reactions are complete within 18-24 hours, however it may be necessary to
Peptone 10.00
Sodium Chloride 5.00
look for late reactions after prolonged incubation.
Andrade Indicator 0.10 Interpretation of Results
0
Final pH (at 25 C) 7.4 ± 0.2 1. Acid is produced when the carbohydrate is fermented and this is indicated by
* Formula adjusted to suit performance parameters a yellow color change in the medium.
Directions
2. Gas production is indicated by the presence of gas bubbles in inverted
1. Suspend 15.1 gms of the powder in 1000 ml distilled water. Durham’s tube.
2. Mix thoroughly. Precautions / Limitations
3. Boil with frequent agitation to dissolve the powder completely. 1. Inspect the tubes for clarity before inoculation. Ascertain that there is no
4. Dispense desired quantity in test tubes containing inverted Durham's tubes. colour or only a slight pinkness in the medium and that the tubes contain no
air trapped in the inverted Durham’s tube.
5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
2. Check whether each individual tube is correctly labeled for the contained
6. Cool to room temperature. Aseptically add sterile stock solution of
sugar.
carbohydrate to a final concentration of 0.5% to 1.0% (w/v).
Quality Control 3. Andrade Peptone Water is reddish-pink when hot, it should return to a
Dehydrated Appearance colourless or slightly pink coloured solution when cooled to room
Light yellow coloured with pink tinge, homogeneous, free flowing powder. temperature.
Prepared Appearance 4. Some sugar solutions may affect the pH of the medium. Care should be taken
Light pink coloured, clear solution without any precipitate.
to avoid alterations in pH.
Cultural Response
Cultural characteristics after 18-24 hours at 350C. 5. Mixed or contaminated cultures may give false reactions.
Organisms (ATCC) Growth Acid* Acid** 6. Andrade indicator may fade on prolonged storage and should not be used
Escherichia coli (25922) Luxuriant - + beyond the expiry date.
Salmonella serotype typhi (6539) Luxuriant - + Storage
Shigella sonnei (25931) Luxuriant - +
Store at 22-300C and prepared medium at 2-80C.
Key:
Shelf Life
*= Acid in absence of added Dextrose.
Use before expiry date as mentioned on the label.
**= Acid in the presence of added Dextrose.

Antibiotic Assay Medium A (No. 1, Seed Agar) AM1002/AM5002

Antibiotic Assay Medium C (No. 3, Assay Broth) AM1003/AM5003
Antibiotic Assay Medium No. 11 AM1004/AM5004
(Neomycin, Erythromycin Assay Agar)
Use microbiological assay techniques.
Antibiotic Assay Media are used for determining antibiotic potency by

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Summary Medium A (No. 1) coloured, coloured, characteristics
Antibiotic Assay Media are used in the performance of antibiotic assays and homogeneous, slightly after 18-24
free-flowing opalescent hours at
conforms to the specifications of USP (113) and FDA. The activity or potency of an
powder gel 35-370C
antibiotic can be demonstrated under suitable conditions by its inhibitory effect
Antibiotic Assay Light yellow Light yellow Cultural
on microorganisms. Reduction in antimicrobial activity may reveal changes not Medium C (No. 3) coloured, coloured, characteristics
demonstrated by chemical methods. Antibiotic assays are performed by the homogeneous, clear solution, after 18-24
cylinder plate method, punched-hole method, paper disc method, serial dilution free-flowing without any hours at
method and the turbidimetric assay methods. Antibiotic Assay Media are powder precipitate 35-370C
identified numerically with names assigned by Grove and Randall in the Assay Antibiotic Assay Light yellow Light yellow, Cultural
Methods of Antibiotics. Medium No. 11 coloured, very slightly characteristics
Principle homogeneous, to slightly after 18-24
free-flowing opalescent gel hours at
Peptone, tryptone, yeast extract and beef extract provide nutrients and growth
powder 35-370C
factors. Sodium chloride maintains the osmotic balance. Dipotassium phosphate
Antibiotic Assay Medium A (No. 1)
and potassium dihydrogen phosphate provide buffering action. Dextrose is the Organisms (ATCC) Growth
carbon and energy source. Bacillus subtilis (6633) Good to luxuriant
Formulae* Escherichia coli (25922) Good to luxuriant
Ingredients in Antibiotic Assay Antibiotic Assay Antibiotic Assay Micrococcus luteus (10240) Good to luxuriant
grams per liter Medium A (No.1) Medium C (No.3) Medium No.11 Staphylococcus aureus (29737) Good to luxuriant
Peptone 6.0 5.0 6.0 Staphylococcus epidermidis (12228) Good to luxuriant
Tryptone 4.0 - 4.0 Klebsiella pneumoniae (10031) Good to luxuriant
Yeast Extract 3.0 1.5 3.0 Antibiotic Assay Medium C (No. 3)
Beef Extract 1.5 1.5 1.5 Organisms (ATCC) Growth Serial Dilution test with
Dextrose 1.0 1.0 1.0 Escherichia coli (10536) Luxuriant Chloramphenicol,
Sodium Chloride - 3.5 - Spectinomycin
Dipotassium Phosphate - 3.68 - Staphylococcus aureus (29737) Luxuriant Amikacin,
Potassium Dihydrogen - 1.32 - Chlortetracycline
Phosphate
Agar 15.0 - 15.0 Organisms (ATCC) Growth Serial Dilution test with
Final pH (at 250C) 6.6 ± 0.2 7.0 ± 0.2 8.3 ± 0.1 Klebsiella pneumoniae (10031) Luxuriant Streptomycin,
* Formula adjusted to suit performance parameters Capreomycin
Directions Streptococcus faecium (10541) Luxuriant Gramicidin, Tyrothricin
1. Suspend the powder in 1000 ml distilled water.
Antibiotic Assay Medium No. 11
Antibiotic Assay Medium A (No. 1) 30.5 gms.
Organisms (ATCC) Growth Inhibition Zones with
Antibiotic Assay Medium C (No. 3) 17.5 gms. Bacillus pumilus (14884) Luxuriant Neomycin, Framycetin
Antibiotic Assay Medium No. 11 30.5 gms. Micrococcus luteus (10240) Luxuriant Ampicillin, Clindamycin,
Erythromycin
2. Mix thoroughly.
Staphylococcus aureus (25923) Luxuriant Kanamycin, Neomycin
3. Boil with frequent agitation to dissolve the powder completely. Staphylococcus epidermidis (12228) Luxuriant Paromomycin,
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. Neomycin, Netilmycin
Procedure
5. Cool to 45-500C. Pour into sterile petri plates as desired.
1. Maintain stock cultures of test organisms on agar slants and make transfers
Quality Control
Antibiotic Assay Dehydrated Prepared Cultural
at 1 or 2 week intervals.
Media Appearance Appearance Response 2. Prepare the inoculum for assay by suspending growth from a fresh 24-48
Antibiotic Assay Light yellow Yellow Cultural hour agar slant using sterile purified water or saline.

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3. Dilute the culture to obtain desired concentration of the test organism. Paper discs with a diameter of 9 mm are impregnated with the antibiotic solution
Note: When Bacillus subtilis is used as the test organism; and placed on the culture medium. Antibiotic can also be applied to the disc after it
· Inoculate it on Antibiotic Assay Medium A (No. 1). has been placed on the medium. Use plates containing a single layer of medium
· Incubate at 370C for 1 week. with 2 mm thickness for these tests. Rest of the procedure is similar to the cylinder
plate method.
· Wash spores from the agar surface and heat the spores at 560C for 30
Serial Dilution Method
minutes.
Minimum Inhibitory Concentration (MIC) of an antibiotic can be expressed by
· Wash the spores three times in purified water and heat again at 650C for
determining the antibiotic activity quantitatively. It is done using the known
30 minutes.
sensitivity of a test organism towards a particular antibiotic. First, serial dilutions
· Dilute to the required optimal concentration. of the antibiotic to be tested are pipetted out into the antibiotic broth. A defined
Storage
quantity of the test organism is then inoculated in this medium. The last tube that
Store at 22-300C and prepared medium at 2-80C. does not show any turbidity due to suppression of microbial growth indicates the
Shelf Life
presence of active antibiotic at a concentration corresponding to the minimum
Use before expiry date as mentioned on the label.
inhibitory concentration.
Assay Methods
Turbidimetric Assay Method
Cylinder Plate Method
The turbidimetric method depends upon the inhibition of the test organism in a
This method depends upon diffusion of the antibiotic from vertical steel cylinders medium containing uniform solution of an antibiotic. The advantage of this
placed on the surface of the inoculated agar medium. This produces a zone of method over that of cylinder plate method is that it requires a shorter incubation
inhibition around the cylinder containing antibiotic solution depending upon the period of 3-4 hours. Use glass or plastic test tubes (16 x 125 mm or 18 x 150 mm)
concentration of the antibiotic. that are relatively uniform in length, diameter and thickness and substantially free
For the assay, use 20 x 100 mm glass or plastic petri plates with sufficient depth from surface blemishes. Tubes that will be placed in the spectrophotometer should
so that the cylinders used in the assay will not be pushed into the medium by the be matched and free from scratches or blemishes. Clean the tubes thoroughly to
cover. Use stainless steel or porcelain assay cylinders having the following remove all antibiotic residues and traces of cleaning solution. Sterilize previously
dimensions : 8 mm outside diameter, 6 mm inside diameter and 10 mm long used tubes prior to subsequent use.
(0.1 mm). Carefully clean the cylinders to remove all residues, using an Prepare working dilutions of the antibiotic reference standards in specific
occasional acid bath (approximately 2N nitric acid or chromic acid). Four or six concentrations. To a 1 ml quantity of each solution, add 9 ml of inoculated broth,
cylinders are generally used per plate, evenly spaced on a 2.8 cm radius. To assure as required. Prepare similar solutions of the sample containing approximately the
accurate assays, work on a level surface to obtain uniformly thick base and seed same amount of antibiotic activity.
layers in the petri plate. Allow the base layer to solidify and then overlay the seed
Incubate the tubes in a water bath for 3-4 hours at the required temperature. At
layer containing a proper concentration of the test organism. The amount of
the end of the incubation period add 0.5 ml of 1:3 formalin to stop growth.
medium in the layers varies for different antibiotics with most assays specifying a
Determine the amount of growth by measuring light transmittance with a suitable
21 ml base layer and a 4 ml seed layer. In any case, plates with flat bottoms are
spectrophotometer. Determine the concentration of the antibiotic by comparing
required to assure complete coverage of the bottom of the plate when a small
the growth obtained with that given by reference standard solutions.
amount of basal medium is used. Tilt the plate to obtain even coverage of the base
Precautions /Limitations
layer by the seed layer and allow it to solidify in a level position. Plates should be
1. The use of standardized culture media and careful control of all test conditions
used the same day as prepared.
are fundamental requisites in the microbiological assay of antibiotics in order to
Punched-Hole Method
achieve satisfactory test results.
In this method, holes are punched out of the inoculated culture medium and the
antibiotic solutions are then pipetted into them. Rest of the procedure is similar to 2. The assay of antibiotics is a highly skilled process, which requires very close
that of the cylinder plate method. attention to the details specified in the official publications, and these must be
Paper-Disc Method
consulted.

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Selection of Method, Media and Test Organism for Antibiotic Assay

Antibiotic Assay Method Organism (ATCC) Antibiotic Medium for

Maintenance Inoculum Cylinder Plate Turbidimetric
Base Seed Assay
Layer Layer
Amikacin Turbidimetric Staphylococcus aureus (29737) 1 1 - - 3
Ampicillin Cylinder plate Micrococcus luteus (9341) 1 1 11 11 -
Bacitracin Cylinder plate Micrococcus luteus (10240) 1 1 2 1 -
Capreomycin Turbidimetric Klebsiella pneumoniae (10031) 1 1 - - 3
Cephalexin Cylinder plate Staphylococcus aureus (29737) 1 1 2 1 -
Cephalothin Cylinder plate Staphylococcus aureus (29737) 1 1 2 1 -
Cephapirin Cylinder plate Staphylococcus aureus (29737) 1 1 2 1 -
Cephradine Cylinder plate Staphylococcus aureus (29737) 1 1 2 1 -
Chloramphenicol Turbidimetric Escherichia coli (10536) 1 1 - - 3
Chlortetracycline Turbidimetric Staphylococcus aureus (29737) 1 1 - - 3
Clindamycin Cylinder plate Micrococcus luteus (9341) 1 1 11 11 -
Cloxacillin Cylinder plate Staphylococcus aureus (29737) 1 1 2 1 -
Cycloserine Turbidimetric Staphylococcus aureus (29737) 1 1 - - 3
Demeclocycline Turbidimetric Staphylococcus aureus (29737) 1 1 - - 3
Demethylchlortetracycline Turbidimetric Staphylococcus aureus (29737) 1 1 - - 3
Dicloxacillin Cylinder plate Staphylococcus aureus (29737) 1 1 2 1 -
Dihydrostreptomycin Turbidimetric Bacillus subtilis (6633) 1 1 - - 3
Doxycycline Turbidimetric Staphylococcus aureus (29737) 1 1 - - 3
Erythromycin Cylinder plate Micrococcus luteus (9341) 1 1 11 11 -
Gentamicin Cylinder plate Staphylococcus epidermidis (12228) 1 1 11 11 -
Gramicidin Turbidimetric Streptococcus faecium (10541) 3 3 - - 3
Kanamycin Turbidimetric Staphylococcus aureus (29737) 1 1 - - 3
Lincomycin Turbidimetric Staphylococcus aureus (29737) 1 1 - - 3
Methacycline Turbidimetric Staphylococcus aureus (29737) 1 1 - - 3
Methicillin Cylinder plate Staphylococcus aureus (29737) 1 1 2 1 -
Minocycline Turbidimetric Staphylococcus aureus (29737) 1 1 - - 3
Nafcillin Cylinder plate Staphylococcus aureus (29737) 1 1 2 1 -
Neomycin Turbidimetric Klebsiella pneumoniae (10031) 1 1 11 11 -
Netilmycin Cylinder plate Staphylococcus epidermidis (12228) 1 1 11 11 -
Novobiocin Cylinder plate Staphylococcus epidermidis (12228) 1 1 2 1 -
Oxacillin Cylinder plate Staphylococcus aureus (29737) 1 1 2 1 -
Oxytetracycline Turbidimetric Staphylococcus aureus (29737) 1 1 - - 3
Paromomycin Cylinder plate Staphylococcus epidermidis (12228) 1 1 11 11 -
Penicillin-G Cylinder plate Staphylococcus aureus (29737) 1 1 2 1 -
Rolitetracycline Turbidimetric Staphylococcus aureus (29737) 1 1 - - 3
Sisomycin Cylinder plate Staphylococcus epidermidis (12228) 1 1 11 11 -
Spectinomycin Turbidimetric Escherichia coli (10536) 1 1 - - 3
Streptomycin Turbidimetric Klebsiella pneumoniae (10031) 1 1 - - 3
Tetracycline Turbidimetric Staphylococcus aureus (29737) 1 1 - - 3
Tobramycin Turbidimetric Staphylococcus aureus (29737) 1 1 - - 3
Troleandomycin Turbidimetric Klebsiella pneumoniae (10031) 1 1 - - 3
Tyrothricin Turbidimetric Streptococcus faecium (10541) 3 3 - - 3
Viomycin Turbidimetric Klebsiella pneumoniae (10031) 1 1 - - 3

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Antibiotic Assay Medium No. 4 (Yeast Beef Agar) AM50013
Use Beef extract 1.50
Antibiotic Assay Medium No. 4 (Yeast Beef Agar) is used for detection of Penicillin- Yeast extract 3.00
Dextrose 1.00
G in milk samples.
Agar 15.00
Summary
Final pH ( at 25°C) 6.6±0.2
Antibiotic Assay Medium No. 4 (Yeast Beef Agar) is suitable for plate counts in * Formula adjusted to suit performance parameters
pharmaceutical and related products and for the microbial assay and detection of Directions
antibiotics like penicillin in milk. These medium is formulated in accordance to 1. Suspend 26.5 grams in 1000 ml of distilled water.
the specifications and procedures listed by the Food and Drug Administration
2. Heat to boiling to dissolve the medium completely.
(110.3).This medium is identical numerically with name assigned by Grove and
Randall (36.2). 3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
Quality Control
Principle
Dehydrated Appearance
Peptic digest of animal tissue, yeast and beef extract provides nutritional Cream to yellow homogeneous free flowing powder
requirement for growth of the indicator organisms like Bacillus Prepared Appearance
stearothermophilus , Micrococcus luteus . This medium is similar to Antibiotic Yellow coloured clear to slightly opalescent gel forms in Petri plates
assay medium no. 2 except for the additional ingredient dextrose. Dextrose in the Cultural Response
medium serves as easily available source of carbon stimulating luxuriant growth Cultural characteristics observed after an incubation at 55°C for 18-24 hours.
of the test organims. Generally presence of penicillin in milk is detected by the Organisms (ATCC) Growth
cylinder plate method, using Micrococcus luteus as the test organism, and a paper Bacillus stearothermophilus (7953) Luxuriant
Micrococcus luteus (10240) Luxuriant
disk method, using Bacillus stearothermophilus .
Storage
Freshly prepared plates should be used for antibiotic assays. The use of this
Store at 22-300C and prepared medium at 2-80C.
medium assures well defined zones of the test organism. Shelf Life
Formula* Use before expiry date as mentioned on the label.
Ingredients in Grams/Litre
Peptic digest of animal tissue (Peptone) 6.00

Antibiotic Assay Medium E (No. 5) AM50031

(Streptomycin Assay Agar with Yeast Extract)
Antibiotic Assay Medium F (No. 8) (Base Agar with low pH) AM50032
Antibiotic Assay Medium G (No. 19) AM500411
Antibiotic Assay Medium No. 32 AM500412
Use cylinder plate method, punched hole method, paper disc method, serial dilution
Antibiotic Assay Media are used for determining antibiotic potency by method and the turbidimetric assay methods. Antibiotic Assay Media are
microbiological assay techniques. identified numerically with names assigned by Grove and Randall in the Assay
Summary Methods of Antibiotics.
Antibiotic Assay Media are used in the performance of antibiotic assays and Principle
conforms to the specifications of USP (113) and FDA. The activity or potency of an Peptone, yeast extract and beef extract provide nutrients and growth factors.
antibiotic can be demonstrated under suitable conditions by its inhibitory effect Sodium chloride maintains the osmotic balance. Dextrose is the carbon and
on microorganisms. Reduction in antimicrobial activity may reveal changes not energy source.
demonstrated by chemical methods. Antibiotic assays are performed by the

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Formula* Antibiotic Assay Light yellow Yellow Cultural
Ingredients Antibiotic Antibiotic Antibiotic Antibiotic Medium (No. 32) coloured, coloured, characteristics
in grams Assay Assay Assay Assay homogeneous, slightly, after 18-24
per liter Medium E Medium F Medium G Medium free-flowing opalescent hours at
(No.5) (No.8) (No.19) (No.32) powder gel 35-370C
Peptic digest of Antibiotic Assay Medium E(No. 5)
animal tissue 6.0 6.0 9.4 6.0 Organisms (ATCC) Growth Inhibition zone with
Yeast extract 3.0 3.0 4.7 3.0 Bacillus subtilis (6633) Good to Dactinomycin,
Beef extract 1.5 1.5 2.4 1.5 luxuriant Dihydrostreptomycin,
Dextrose - - 10.0 1.0 Kanamycin B,
Sodium chloride - - 10.0 - Rifampicin, Streptomycin
Manganese sulphate - - - 0.3 Antibiotic Assay Medium F(No. 8)
Casein enzymic Organisms (ATCC) Growth Inhibition zone with
hydrolysate - - - 4.0 Bacillus subtilis (6633) Luxuriant Mitomycin, Vancomycin,
Agar 15.0 15.0 23.5 15.0 Plicamycin
Final pH (at 250C) 7.9 ±0.2 5.9 ±0.2 6.1 ±0.2 6.6 ±0.2 Staphylococcus aureus Luxuriant
* Formula adjusted to suit performance parameters (29737)
Directions Antibiotic Assay Medium G (No. 19)
1. Suspend the powder in 1000 ml distilled water. Organisms (ATCC) Growth Inhibition zone with
Saccharomyces Luxuriant Amphotericin B, Netamycin,
Antibiotic Assay Medium E (No. 5) 25.5 gms.
cerevisiae (9763) Nystatin
Antibiotic Assay Medium F (No. 8) 25.5 gms. Saccharomyces Luxuriant
Antibiotic Assay Medium G(No. 19) 60.0 gms. cerevisiae (2601)
Antibiotic Assay Medium (No. 32)
Antibiotic Assay Medium (No. 32) 30.8gms.
Organisms (ATCC) Growth Inhibition zone with
2. Mix thoroughly. Bacillus subtilis (6633) Good Dihydrostreptomycin,
3. Boil with frequent agitation to dissolve the powder completely. to luxuriant Vancomycin
Procedure
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
1. Maintain stock cultures of test organisms on agar slants and make transfers
5. Cool to 45-500C. Pour into sterile petri plates as desired.
at 1 or 2 week intervals.
Quality Control
Antibiotic Assay Dehydrated Prepared Cultural 2. Prepare the inoculum for assay by suspending growth from a fresh 24-48
Media Appearance Appearance Response hour agar slant using sterile purified water or saline.
Antibiotic Assay Light yellow Medium amber Cultural 3. Dilute the culture to obtain desired concentration of the test organism.
Medium E (No. 5) coloured, coloured, characteristics
Note: When Bacillus subtilis is used as the test organism;
homogeneous, slightly after 18-24
free-flowing opalescent hours at ?
Inoculate it on Antibiotic Assay Medium (No. 5 & 32).
powder gel 35-370C ?
Incubate at 37 for 1 week.
Antibiotic Assay Light yellow Light amber Cultural
?
Wash spores from the agar surface and heat the spores at 56 for 30
Medium F (No. 8) coloured, coloured, characteristics
minutes.
homogeneous, slightly, after 18-24
free-flowing opalescent hours at ?
Wash the spores three times in purified water and heat again at 65
powder gel 35-370C for 30 minutes.
Antibiotic Assay Light yellow Yellow Cultural ?
Dilute to the required optimal concentration.
Medium G (No. 19) coloured, coloured, clear characteristics
Storage
homogeneous, to slightly, after 18-24
free-flowing opalescent hours at
Store at 22-300C and prepared medium at 2-80C.
Shelf Life
powder gel 25-300C

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Antibiotic Assay Medium No. 10 AM50034
Use Directions
Antibiotic Assay Medium No. 10 is used as seed layer medium for assaying the 1. Suspend 42 gm the powder in 1000 ml distilled water containing 10 ml of
products containing Polymyxin B, Carbenicillin, Colistin and Colistimethate Polysorbate 80..
sodium. 2. Mix thoroughly.
Summary
3. Boil with frequent agitation to dissolve the powder completely.
Antibiotic Assay Media are used in the performance of antibiotic assays and
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
conforms to the specifications of USP (113) and FDA.
Quality Control
Principle
Dehydrated Appearance
Papaic digest of soyabean meal and casein enzymic hydrolysate in this medium Cream to yellow homogeneous free flowing powder
provide the essential nutrient for growth of test organisms. Sodium chloride Prepared Appearance
maintains the osmotic balance. Dipotassium phosphate provide buffering action. Medium amber coloured clear to slightly opalescent gel forms in Petri plates
Dextrose is the carbon and energy source and agar provide solid substratum. Cultural Response
Formula* Cultural characteristics upto 18-48 Hour at 36-37.50C.
Ingredients in Grams/Litre Organisms (ATCC) Growth Inhibition zone with
Casein enzymic hydrolysate 17.00 Pseudomonas aeruginosa (27853) Luxuriant Carbenicillin
Papaic digest of soyabean meal 3.00 Bordetella bronchiseptica Luxuriant Polymixin B,
Colistimethate
Sodium chloride 5.00
sodium, Colistin
Dextrose 2.50
Storage
Dipotassium phosphate 2.50
Agar 12.00 Store at 22-300C and prepared medium at 2-80C.
Final pH ( at 25°C) 7.2±0.2 Shelf Life
* Formula adjusted to suit performance parameters Use before expiry date as mentioned on the label.

Antibiotic Assay Medium No.12 (Nystatin Assay Agar) AM50014

Use Beef extract 2.50
Antibiotic Assay Medium No.12 (Nystatin Assay Agar) is used for microbiological Yeast extract 5.00
Agar 25.00
assay of Amphotericin B and Nystatin using Saccharomyces cerevisiae .
Final pH ( at 25°C) 6.1±0.2
Summary
* Formula adjusted to suit performance parameters
This medium is prepared from the Groove and Randall formula (36.2)Antifungal Directions
antibiotics like Amphotericin B and Nystatin can be assayed using this medium.
1. Suspend 62.5 grams in 1000 ml distilled water.
Principle
2. Heat to boiling to dissolve the medium completely.
Peptic digest of animal tissue, yeast and beef extract are essential nutrients,
minerals and growth factors for the growth of test organism. Dextrose in the 3. Sterilize by autoclaving at 15 lbs pressu re (121°C) for 15 minutes.
Quality Control
medium provides enhanced source of carbon and energy. Osmotic equilibrium in
Dehydrated Appearance
the medium is by sodium chloride which maintain the cell intergrity and viability.
Cream to yellow homogeneous free flowing powder
Freshly prepared plates should be used for antibiotic assays. Prepared Appearance
Formula* Yellow coloured clear to slightly opalescent gel forms in Petri plates
Ingredients in Grams/Litre Cultural Response
Peptic digest of animal tissue 10.00 Cultural characteristics observed after an incubation at 25-30°C for 18-24
Sodium chloride 10.00 hours.
Dextrose 10.00

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Organisms (ATCC) Growth Inhibition zone with Storage
Saccharomyces cerevisiae (2601) Luxuriant Amphotericin B, Store at 22-300C and prepared medium at 2-80C.
Nystatin
Shelf Life
Use before expiry date as mentioned on the label.

Antibiotic Assay Medium No. 35 AM500414

Use Final pH ( at 25°C) 7.0±0.2
* Formula adjusted to suit performance parameters
Antibiotic Assay Medium No. 35 is used for the microbiological assay of
Directions
Bleomycin using Mycobacterium smegmatis
Summary
1. Suspend 40 gm the powder in 1000 ml distilled water containing 10 ml
glycerol.
This medium is formulated in accordance to CFR (110.3). This medium is
employed widely as base agar for agar diffusion assay of Bleomycin using 2. Mix thoroughly.
Mycobacterium smegmatis. 3. Boil with frequent agitation to dissolve the powder completely.
Principle 4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Peptic digest of animal tissue and beef extract in this medium provide the 5. Cool to 45-500C. Pour into sterile petri plates as desired.
essential nutrient for growth of test organisms. Agar provides excellent solid Quality Control
substratum for support and over layering of seed agar, for the assay of Bleomycin. Dehydrated Appearance
Addition of glycerol is important for provision of carbon to the test organism. To Cream to yellow homogeneous free flowing powder
perform the antibiotic assay the Base Agar should be prepared on the same day as Prepared Appearance
the test. For the cylinder method, a base layer of 21 ml is required. Once the base Medium amber coloured clear to slightly opalescent gel forms in Petri plates
Cultural Response
medium has solidified, seed layer inoculated with the standardized culture can be
Cultural characteristics upto 18-48 Hour at 36-37.50C.
overlaid. Even distribution of the layer is important.
Organisms (ATCC) Growth Inhibition zone with
Formula*
Mycobacterium smegmatis (607) Luxuriant Bleomycin
Ingredients in grams per liter
Storage
Peptone 10.000
Beef extract 10.000 Store at 22-300C and prepared medium at 2-80C.
Shelf Life
Sodium chloride 3.000
Agar 17.000 Use before expiry date as mentioned on the label.

Antifungal Assay Agar AM10041/AM50041

Use organisms. The appearance of a zone of inhibition surrounding the disc is
Antifungal Assay Agar is recommended for assaying the antifungal activity. indicative of sensitivity. By comparing the diameter of the zones to the standard,
Summary it is possible to determine whether the test organisms are susceptible or resistant
Antifungal Assay Agar is recommended for assaying the antifungal activity of to the respective antibiotic.
pharmaceutical and other products using the cylinder plate or paper disc method. Formula*
Principle Ingredients in grams per liter
Dextrose 50.0
Antifungal Assay Agar was formulated by Berger and Lazecka to conveniently
Tryptone 4.0
assay the antifungal activity of of pharmaceutical and other products by both base
Sodium Citrate 4.5
and seed layers using the cylinder plate or paper disc methods. The principle used Potassium Phosphate 0.55
here is that antibiotics of varying concentrations are impregnated on the paper Citric Acid 1.0
disc or small cylinder, which will diffuse into the medium containing the test Pyridoxine hydrochloride 0.00025

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Thiamine 0.00025 Prepared Appearance
Inositol 0.025 Light yellow coloured clear gel.
Calcium Pantothenate 0.0025 Cultural Response
Niacin 0.0025 Cultural characteristics after 18-48 hours at 300 C.
Potassium Chloride 0.425 Organisms (ATCC) Growth RGI
Calcium Chloride 0.125 Saccharomyces cerevisiae (9763) Luxuriant More than 70%
Magnesium Sulphate 0.125 Asperigillus niger (16404) Luxuriant More than 70%
Ferric Chloride 0.0025 For growth RGI should be more than 70%
Manganese Sulphate 0.0025 RGI- Relative Growth Index
Biotin 0.000008 Procedure
Agar 15.0
1. Refer to U.S.P. and other appropriate references for procedures and
Final pH (at 250 C) 5.5 ± 0.2
interpretation of results.
* Formula adjusted to suit performance parameters
Precautions / Limitations
Directions
1. The assay of antibiotics is a highly skilled process, which requires very close
1. Suspend 75.76 grams of the powder in 1000 ml distilled water.
attention to the details specified in the official publications, and these must
2. Mix thoroughly.
be consulted.
3. Boil with frequent agitation to dissolve the powder completely.
2. Inspect the petriplates before inoculation. Keep the plates at 2-80 C for half an
4. Sterilize by autoclaving at 1210 C (15 lbs pressure) for 15 minutes. hour after placing the antibiotics discs for diffusion to take place.
Quality Control Storage
Dehydrated Appearance
Store at 22-300C and prepared medium at 2-80C.
Pale beige coloured, homogeneous free flowing powder.
Shelf Life
Use before expiry date as mentioned on the label.

Artificial Sea Water AM50042

Use Formula*
Artificial sea-water used for maintaining or cultivating organisms and plants Ingredients in grams per liter
Sodium chloride 24.6
living in the sea-water.
Potassium chloride 0.67
Summary
Calcium chloride. 2H2O 1.36
Artificial seawater is a complex mix of many purified salts formulated to have a Magnesium sulphate. 7H2O 6.29
composition similar to natural seawater when dissolved in distilled water. The Magnesium chloride. 6H2O 4.66
quality of sea water is dependent on the formula, the quality of the raw materials Sodium bicarbonate 0.18
and the uniformity of the blending. The salinity is the sum of all of the dissolved Final pH at 250C 8.0 ±0.2
ions. Natural seawater is generally considered to have a salinity of 35 parts per * Formula adjusted to suit performance parameters
thousand (ppt) or grams of salt per kilogram of water. Directions
Principle 1. Suspend 37.76 gms of the powder in 1000 ml distilled water.
Artificial sea-water is based on the formulation described by Layman and Fleming 2. Mix thoroughly.
(73.1). The high salt content helps to simulate sea water. These salts provide
3. Filter through Whatman Filter paper.
marine environment for maintenance and cultivation of sea living organisms. The
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
mixture of inorganic salts approximating the composition of sea water, is used in
Storage
place of natural sea water for the growth of marine organisms(79.2).
Store at 22-300C and prepared medium at 2-80C.
Shelf Life
Use before expiry date as mentioned on the label.

42 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
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Ashby’s Glucose Agar AM50043
Use 3. Heat gently with frequent agitation to dissolve the powder completely.
Ashby’s Glucose Agar is used for cultivation of Azotobacter species by using 4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
glucose as carbon source. Quality Control
Summary Dehydrated Appearance
Subha Rao has formulated the Ashby’s media(105.1). It is recommended for the Off white coloured, homogeneous free flowing powder.
isolation of Azotobacter species. Prepared Appearance
Principle Whitish opalescent gel form in petridishes.
Cultural Response
Glucose acts as carbon source. Azotobacter species is a non-symbiotic nitrogen
Cultural characteristics upto 5 days at 370C.
fixer. It uses atmospheric nitrogen as nitrogen source. Organisms (ATCC) Growth RGI
Formula* Azotobacter vinelandii (478) Luxuriant More than 70%
Ingredients in grams per liter Azotobacter nigricans (35009) Luxuriant More than 70%
Glucose 20.0 For growth RGI should be more than 70%
Dipotassium phosphate 0.20 RGI- Relative Growth Index
Magnesium sulphate 0.20 Procedure
Sodium chloride 0.20
Refer to appropriate references for specific procedures for the cultivation of
Potassium sulphate 0.10
Calcium carbonate 5.00 Azotobacter species.
Agar 15.00 Interpretation of Results
0
Final pH (at 25 C) 7.4 ± 0.2 Refer to appropriate references and procedures for results.
* Formula adjusted to suit performance parameters Storage
Directions Store below 300C and prepared medium at 2-80C.
1. Suspend the 40.7 gms of powder in 1000 ml distilled water. Shelf Life
2. Mix thoroughly. Use before expiry date as mentioned on the label.

Ashby’s Mannitol Agar AM50044

Use Calcium carbonate 5.00
Ashby’s Mannitol Agar is used for cultivation of Azotobacter species by using Agar 15.00
Final pH (at 250C) 7.4 ± 0.2
mannitol as carbon source.
* Formula adjusted to suit performance parameters
Summary
Directions
Subha Rao (105.1) has formulated the Ashby’s media. It is recommended for the
1. Suspend the 40.7 gms of powder in 1000 ml distilled water.
isolation of Azotobacter species.
Principle 2. Mix thoroughly.
Mannitol acts as carbon source. Azotobacter species is a non-symbiotic nitrogen 3. Heat gently with frequent agitation to dissolve the powder completely.
fixer. It uses atmospheric nitrogen as nitrogen source. 4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Formula* Quality Control
Ingredients in grams per liter Dehydrated Appearance
Mannitol 20.0 Off white coloured, homogeneous free flowing powder.
Dipotassium phosphate 0.20 Prepared Appearance
Magnesium sulphate 0.20 Whitish opalescent gel form in petridishes.
Sodium chloride 0.20 Cultural Response
Potassium sulphate 0.10 Cultural characteristics upto 5 days at 370C.

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Organisms (ATCC) Growth RGI Interpretation of Results
Azotobacter vinelandii (478) Luxuriant More than 70% Refer to appropriate references and procedures for results.
Azotobacter nigricans (35009) Luxuriant More than 70% Storage
For growth RGI should be more than 70%
RGI- Relative Growth Index
Store below 300C and prepared medium at 2-80C.
Procedure Shelf Life

Refer to appropriate references for specific procedures for the cultivation of Use before expiry date as mentioned on the label.
Azotobacter species.

Asparagine Broth AM50045

Use Magnesium sulphate 1.50
Asparagine Broth is used for the preparation of Coccidioidin and Histoplasmin Ferric citrate 0.30
Dextrose 10.00
antigens for immunodiagnostic work.
Final pH ( at 25°C) 6.8±0.2
Summary
* Formula adjusted to suit performance parameters
A dimorphic fungus, Histoplasma capsulatum causes histoplasmosis, a systemic Directions
fungal disease. H. capsulatumis an obligate intracellular organism residing in
1. Suspend 28.01 grams in 1000 ml distilled water containing 25 ml glycerol.
macrophages of the reticuloendothelial system. Of current concern is the
2. Mix thoroughly and then dispense in a wide bottom flask, to give a depth of
increased incidence of histoplasmosis in patients with AIDS (49.3). Coccidiodes
1to 1.5 inches.
immitis , the causative agent of coccidioidomycosis (Valley fever) is endemic in
hot regions with dry climate and alkaline soil. Patients with AIDS are at a risk of 3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
developing coccidioidomycosis. Quality Control
Dehydrated Appearance
Principle
Off-white to yellow homogeneous free flowing powder
Asparagine Broth is a chemically defined medium used for the preparation of Prepared Appearance
Coccidioidin and Histoplasmin antigens. The amino acid asparagine, favours the Light yellow coloured clear solution without any precipitate.
synthesis of antigens from Histoplasma and Coccidiodes . Salts included in the Cultural Response
medium buffer the medium well. Dextrose and slightly acidic pH of the medium Cultural characteristics an incubation at 35-37°C for 1 week
helps for the luxuriant growth of the fungi. Organisms(ATCC) Growth
Formula* Coccidiodes immitis Luxuriant
Ingredients in grams per liter Histoplasma capsulatum (10230) Luxuriant
Ingredients in grams per liter Storage
L-Asparagine 7.00 Store at 22-300C and prepared medium at 2-80C.
Ammonium chloride 7.00 Shelf Life
Dipotassium phosphate 1.31 Use before expiry date as mentioned on the label.
Sodium citrate 0.90

Asparagine Proline Broth AM1005/AM5005

Use beings through use of natural fresh and marine recreational waters that may be
Asparagine Proline Broth is used for the cultivation of Pseudomonas aeruginosa contaminated by wastewater.
using the membrane filter technique. Principle
Summary Asparagine Proline Broth contains the two amino acids DL-asparagine and L-
Pseudomonads are widely distributed in soil and water including sinks and proline required by Pseudomonas for its growth. Phosphates and sulphates
drains. Pseudomonads can multiply in recreational waters in the presence of incorporated in the medium provide ions for growth as well as buffers the
sufficient nutrients, as it is an opportunist, hence it can be transmitted to human medium.

44 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
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Formula* Quality Control
Ingredients in grams per liter Dehydrated Appearance
DL- Asparagine 2.0 White coloured, homogeneous free flowing powder.
L- Proline 1.0 Prepared Appearance
Magnesium Sulphate 0.5 Colourless, clear solution without any precipitate.
Dipotassium Phosphate 1.0 Cultural Response
Potassium Sulphate 10.0 Cultural characteristics after 18- 24 hours at 350C.
Final pH (at 250C) 7.4 ± 0.2 Organisms (ATCC) Growth
* Formula adjusted to suit performance parameters Escherichia coli (25922) None to poor
Directions Pseudomonas aeruginosa (27853) Luxuriant with greenish
yellow pigment
1. Suspend 14.5 gms of the powder in 1000 ml distilled water containing
Storage
25ml ethanol.
Store at 22-300C and prepared medium at 2-80C.
2. Mix thoroughly. Shelf Life
3. Boil with frequent agitation to dissolve the powder completely. Use before expiry date as mentioned on the label.
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
5. Dispense as desired.

Azide Blood Agar Base AM1006/AM5006

Use Formula*
Azide Blood Agar Base is used for the isolation and differentiation of streptococci Ingredients in grams per liter
Peptone Special 10.0
and staphylococci and when supplemented with blood is used for determining
Sodium Chloride 5.0
haemolytic reactions.
Beef Extract 3.0
Summary
Sodium Azide 0.2
Various workers indicated that sodium azide could be used to inhibit gram- Agar 15.0
negative organisms while allowing the growth of gram-positive organisms. This Final pH (at 250C) 7.2 ± 0.2
medium is similar to the one used by Edwards for the isolation of mastitis * Formula adjusted to suit performance parameters
streptococci. Sodium azide has a bacteriostatic effect on gram-negative Directions
organisms but permits growth of gram-positive organisms such as streptococci 1. Suspend 33.2 gms of the powder in 1000 ml distilled water and mix well.
and some strains of staphylococci. Proteus species are slightly more resistant 2. Heat with frequent agitation and boil for 1 minute to dissolve the powder
than other Enterobacteriaceae but swarming is prevented by a concentration of completely.
about 0.01% of sodium azide, thus permitting the isolation of streptococci from
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes
mixed bacterial populations (Synder and Lichstein). At the above concentration
4. To prepare blood agar plates, aseptically add 5% v/v sterile defibrinated
and pH, sodium azide exerts no appreciable effect on haemolysis so that the
blood to the medium cooled to 45-500C and mix well. Dispense as desired.
medium with added blood (5-10% sheep, rabbit or horse blood) is used for
isolating, cultivating and determining haemolytic reactions of fastidious Warning: Sodium azide has a tendency to form explosive metal azides with
pathogens. It is recommended for the isolation of streptococci from cheese (23). plumbing materials and it is advisable to flush off the disposables with
Principle water.
Quality Control
Peptone and beef extract provide nitrogen, carbon, vitamins and amino acids.
Dehydrated Appearance
Sodium azide is the selective agent while sodium chloride maintains the osmotic
Beige to yellow coloured, homogeneous, free flowing powder.
balance. Supplementation with 5-10% blood provides additional growth factors Prepared Appearance
for fastidious organisms. Basal medium - Yellow coloured, slightly opalescent gel.
With addition of 5% v/v sterile defibrinated blood - Cherry red opaque gel,
which darkens on standing.

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Cultural Response a) Alpha haemolysis – reduction of haemoglobin to methemoglobin in
Cultural characteristics after 18-48 hours at 350C. the medium surrounding the colony, causing a greenish discolouration
Organisms(ATCC) Growth Haemolysis RGI
of the medium.
Enterococcus faecalis Luxuriant Alpha / gamma More than 70%
(29212)
b) Beta haemolysis – lysis of the red blood cells, causing a clear zone
Escherichia coli None to poor - 0% surrounding the colony.
(25922) c) Gamma haemolysis – wherein there is no lysis of the red blood cells
Streptococcus Luxuriant Alpha More than 70% and hence no change is observed in the medium.
pneumoniae (6303) d) Alpha-prime haemolysis – a small zone of complete haemolysis that is
Streptococcus Good to Beta More than 70%
surrounded by an area of partial lysis.
pyogenes(19615) luxuriant
Precautions / Limitations
For growth RGI should be more than 70%
For Inhibition RGI should be 0%
1. Proteus and Escherichia species may not always be inhibited on the
RGI- Relative Growth Index Edward's formulation.
Procedure 2. Use a light inoculum for best selective results.
1. Process each specimen appropriately, and inoculate directly onto the surface 3. Anaerobic incubation is shown to enhance haemolytic reactions.
of the medium. 4. Haemolytic pattern of streptococci on this medium are somewhat different
2. Use streak plate method for isolation, stab the agar several times with the than those observed on ordinary Blood Agar.
inoculating wire loop to deposit beta-haemolytic streptococci beneath the 5. Azide Blood Agar Base is recommended for selective use and should be
agar surface as subsurface growth displays the most reliable haemolytic inoculated in parallel with non-selective media.
reactions. 6. Sodium azide enhances haemolysis and alpha and beta haemolytic zones
3. Use light inoculum for best results and incubate anaerobically for may be extended.
enhancement of haemolytic reactions. 7. Haemolytic patterns may vary with the source of animal blood or base
4. Incubate plates aerobically, anaerobically or in the presence of increased medium used.
Storage
carbon dioxide.
Interpretation of Results Store below 300C and prepared medium at 2-80C.
1. Examine plates for growth and haemolytic reactions after 18-24 and 40-48 Shelf Life

hours of incubation. Four different types of haemolysis on Blood Agar media Use before expiry date as mentioned on the label.
can be described.

Azide Dextrose Broth AM50061

Use Formula*
Azide Dextrose Broth is recommended for detection and cultivation of Streptococci Ingredients in grams per liter
Peptone, special 15.0
in water, sewage, milk and other material.
Beef extract 4.5
Summary
Dextrose 7.5
Various workers indicated that sodium azide could be used to inhibit gram- Sodium chloride 7.5
negative organisms while allowing the growth of gram-positive organisms. Sodium azide 0.20
Rothe, Mullmann and Seligmann formulated Azide Dextrose Broth for Final pH (at 25ºC) 7.2 ± 0.2
quantitative determination of enterococci in water, sewage, food and other * Formula adjusted to suit performance parameters
materials, suspected of contamination with sewage (82.1). Directions
Principle 1. Suspend 34.7gms of the powder in 1000 ml distilled water.
Peptone, special and beef extract provide amino acids and other growth factors. 2. Mix thoroughly.
Dextrose is a carbon and energy source. Sodium azide is the selective agent while 3. Boil with frequent agitation to dissolve the powder completely.
sodium chloride maintains the osmotic balance.

46 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
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Organisms(ATCC) Growth
4. Dispense into tubes.
Enterococcus faecalis (29212) Luxuriant
5. Sterilize by autoclaving at 118ºC (12 lbs pressure) for 15 minutes. Escherichia coli (25922) Inhibited
Warning: Sodium azide has a tendency to form explosive metal Interpretation of Results
azides with plumbing materials and it is advisable to flush off the
disposables with water. Refer to appropriate references and procedures for results.
Quality Control Storage
Dehydrated Appearance Store at 22-300C and prepared medium at 2-80C.
Light Yellow coloured, homogeneous, free flowing powder. Shelf Life
Prepared Appearance Use before expiry date as mentioned on the label.
Amber coloured, clear solution without any precipitate.
Cultural Response
Cultural characteristics after 18-24 hours at 35-37ºC.

B12 Assay Agar (using E.coli mutant culture) AM1007

Use
Ferrous Sulphate 0.000054
B12 Assay Agar using E.coli mutant culture is used for the microbiological assay of Manganese Chloride 0.000046
vitamin B12 by the cup plate or disc plate method. Copper Sulphate 0.000025
Summary Agar 15.0
B12 Assay is carried out according to the procedures of the vitamin B12 Activity Final pH (at 250C) 7.2 ± 0.2
Assay as per the USP (114). E.coli mutant 113-3D is the test organism used. * Formula adjusted to suit performance parameters
Principle Directions
Vitamin B12 Assay Agar is a vitamin B12 free dehydrated medium containing all 1. Suspend 51.5 gms of the powder in 1000 ml distilled water.
other nutrients and vitamins essential for the cultivation of the test organism 2. Boil with frequent agitation to dissolve the powder completely. AVOID
Escherichia coli 113-3D ATCC 11105. Incorporation of vitamin B12 in specified OVERHEATING.
increasing amounts gives a growth response curve that can be measured by the 3. Mix well to distribute the slight precipitate evenly.
plate method.
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
5. Generally satisfactory results are obtained with B12 levels ranging from
Formula*
0 to 300 ng per ml.
Ingredients in grams per liter
Quality Control
Dipotassium Phosphate 14.0
Dehydrated Appearance
Monopotassium Phosphate 6.0
Very light to light beige, homogeneous, free flowing powder with a tendency to
Ammonium Chloride 5.0 clump.
Glucose 5.0 Prepared Appearance
DL-Asparagine 3.0 Light amber coloured, clear gel, which may have a slight precipitate.
Ammonium Nitrate 2.0 Cultural Response
Sodium Chloride 1.0 Cultural characteristics after 24 hours at 370C.
Magnesium Sulphate 0.2 Organism (ATCC) Growth RGI
L-Arginine Hydrochloride 0.2 Escherichia coli 113-3D (11105) Luxuriant More than 70%
Ammonium Sulphate 0.1 For growth RGI should be more than 70%
Calcium Chloride 0.001 RGI- Relative Growth Index
Zinc Sulphate 0.00009 Procedure
Ammonium Molybdate 0.00001 1. Refer to U.S.P. and other official publications for assay procedure.
Borax 0.00001

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Interpretation of Results 5. All conditions of the assay must be followed precisely as outlined in the
1. Refer to U.S.P. and other official publications for assay procedure. references.
Precautions /Limitations 6. The use of altered or deficient media may cause mutants having different
1. Care must be taken to avoid contamination of media or glassware in nutritional requirements and will not give satisfactory response.
microbiological assay procedures. 7. Over heating or over sterilization will give unsatisfactory results.
2. Detergents or other chemicals present in the glassware may give erroneous 8. Sometimes lumps may be formed which will not effect the performance of the
results and therefore glassware must be heated to 2500C for at least 1 hour medium.
to burn off any organic residues that might be present. Storage
3. Sterilization and cooling conditions must be kept uniform throughout the Store at 22-300C and prepared medium at 2-80C.
assay. Shelf Life
4. The test organism used for inoculating an assay medium must be cultured Use before expiry date as mentioned on the label.
and maintained on media recommended for this purpose.

B12 Assay Medium AM10071

Use Xanthine 20 mg
B12 Assay Medium is used for determining vitamin B12 concentration by the Riboflavin 1 mg
Thiamine Hydrochloride 1 mg
microbiological assay technique. Biotin 10ìg
Summary
Niacin 2 mg
Three types of Vitamin assay media are generally used in the microbiological p-Aminobenzoic Acid 2 mg
assay of vitamins.: Calcium Pantothenate 1 mg
Pyridoxine Hydrochloride 4 mg
1. Maintenance Media: For carrying the stock culture to preserve the viability
Pyridoxal Hydrochloride 4 mg
and sensitivity of the test organism for its intended purpose. Pyridoxamine Hydrochloride 800 ìg
2. Inoculum Media: To condition the test culture for immediate use. Folic Acid 200 ìg
3. Assay Media: To quantify a vitamin under test. Monopotassium Phosphate 1.0 g
Dipotassium Phosphate 1.0 g
B12 Assay Medium is used for the microbiological assay of vitamin B12 according to Magnesium Sulphate 0.4 g
USP using E.coli mutant 113-3D ATCC 11105 and Lactobacillus leichmannii as Sodium Chloride 20.0 mg
the test organism. Ferrous Sulphate 20.0 mg
Principle Manganese Sulphate 20.0 mg
Polysorbate 80 2.0 g
B12 Assay Medium is a vitamin B12-free dehydrated medium containing all
Final pH (at 250 C) 6.0 ± 0.1
nutrients and vitamins essential for the cultivation of E.coli mutant 113-3D * Formula adjusted to suit performance parameters
ATCC 11105 and Lactobacillus leichmannii. Directions
Formula* 1. Suspend 85 grams of the powder in 1000 ml distilled water.
Ingredients in grams per liter
2. Heat with frequent agitation and boil for 2-3 minutes to dissolve the powder
Vitamin Assay Casamino Acids 15.0 g
Dextrose 40.0 g completely.
Asparagine 0.2 g 3. Dispense in quantities of 5 ml in tubes, evenly dispersing the precipitate.
Sodium Acetate 20.0 g 4. Add to the tubes standard or test samples.
Ascorbic Acid 4.0 g
L-Cystine 0.4 g 5. Adjust the tube volume to 10 ml with distilled water.
DL-Tryptophan 0.4 g 6. Sterilize by autoclaving at 1210 C (15 lbs pressure) for 5 minutes.
Adenine Sulphate 20 mg Quality Control
Guanine Hydrochloride 20 mg Dehydrated Appearance
Uracil 20 mg Very light to light beige, homogeneous, with a tendency to clump.

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Prepared Appearance 2. Heat all glassware at 2500 C for at least one hour to burn off any organic
Very light to light amber, clear may have a slight precipitate. residues that might be present before use.
Cultural Response
3. Ensure that the sterilization and cooling conditions are uniform throughout
Cultural characteristics after 16-24 hours at 35-370 C when tubes incubated
with caps loosened. the assay.
Organisms (ATCC) Growth 4. The test organisms used for inoculating an assay medium must be cultured
E.coli mutant 113-3D (11105) Luxuriant and maintained on media recommended for this purpose. The use of altered
Lactobacillus leichmannii (7830) Luxuriant or deficient media may give rise to mutants having different nutritional
Procedure
requirements and hence will not give a satisfactory response.
1. Refer to U.S.P. and other appropriate references for procedures and
5. Use the aseptic technique throughout the assay procedure.
interpretation of results. Storage
Precautions / Limitations
Store at 22-300C and prepared medium at 2-80C.
1. Ensure that the medium as well as the glassware used in the assay is free from
Shelf Life
contamination. Use before expiry date as mentioned on the label.

BAT (Bacillus acidoterrestris thermophilic) Medium AM100711

Use Directions
BAT Medium is used for the detection of Alicyclobacillus in fruit jucies. 1. Suspend 29.0 gms of the powder in 1000 ml distilled water.
Summary 2. Mix thoroughly.
BAT Medium supports the growth of Alicyclobacilli. Alicyclobacilli are aerobe, 3. Boil with frequent agitation to dissolve the powder completely.
gram-positive spore forming bacteria, whose optimum of growth is at low pH Note: The medium has a spontaneous pH of 5.3 ±0.2 in order to maintain the
value and increased temperatures. Alicyclobacilli are spoilages organisms gel strength during autoclavation. Adjustment of the pH to 4.0 ±0.2 is
especially effecting the quality of fruit jucies(13.1). made after the autoclavation.
Principle
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
The BAT agar supports the growth of Alicyclobacilli. The low pH-value in
5. Cool to 45-50°C. Adjust the pH to 4.0 ±0.2 by adding 1.7 ml 1 NH2SO4.
combination with the high incubation temperature inhibit the contaminating
flora in growth. Mix well and pour into petri dishes.
Quality Control
Formula*
Dehydrated Appearance
Ingredients in grams per liter
Yeast extract 2.0 Yellow coloured, homogeneous, free flowing powder.
Glucose 5.0 Prepared Appearance
Calcium chloride 0.25 Yellow colour clear gel forms in petri plates.
Magnesium sulfate 0.5 Cultural Response
Ammonium sulfate 0.2 Cultural characteristics after 16-24 hours at 35-370 C when tubes incubated
Potassium-dihydrogenphosphate 3.0 with caps loosened.
Zinc sulfate 0.00018 Organisms (ATCC) Growth RGI
Copper sulfate 0.00016 Alicyclobacillus acidocaldarius DSMZ 446 Good More than 70%
Manganese sulfate 0.00015 Alicyclobacillus acidoterrestris DSMZ 2498 Good More than 70%
Cobalt-chloride 0.00018 Alicyclobacillus cycloheptanicus DSMZ 4006 Good More than 70%
Boric acid 0.00010 Alicyclobacillus hesperidium DSMZ 12766 Good More than 70%
Sodium molybdate 0.00030 Staphylococcus aureus ATCC 25923 None 0%
Agar 18.00 Escherichia coli ATCC 25922 None 0%
Final pH (at 250C) 4.0 ± 0.2 Application and interpretation
* Formula adjusted to suit performance parameters 1. Inoculate the medium by spreading 0.1 ml on the surface.

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2. Membranefilter technique can be used with samples being filterable. Storage

3. Incubation for 3-5 days at 45 ±1.0 °C. Store at 22-300C and prepared medium at 2-80C.
Shelf Life
4. Count all colonies growing on the BAT agar as suspicious Alicyclobacilli.
Use before expiry date as mentioned on the label.
5. Confirm the suspicious colonies by further testing.

B12 Maintenance Media (For E.coli Mutant) AM1008

Use Ferrous Sulphate 0.001
B12 Maintenance Media is used for the propagation, cultivation and maintenance Agar 15.0
Final pH (at 250C) 7.0 ± 0.2
of Escherichia coli 113-3D, ATCC 11105, which is the test organism in the
* Formula adjusted to suit performance parameters
vitamin B12 assay.
Directions
Summary
1. Suspend 37.85 gms of the powder in 1000 ml distilled water and mix well.
Escherichia coli mutant species grow poorly on non-selective culture media and
2. Boil with frequent agitation to dissolve the powder completely.
require special nutrients for its growth. B12 Maintenance Media for E.coli mutant
is used for carrying stock cultures to preserve the viability and sensitivity of the test 3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
organism for its intended purpose. 4. Dispense as required.
Principle Quality Control
Dehydrated Appearance
Tryptone is the source of carbon and nitrogen; yeast extract serves as the energy
Light yellow coloured, free flowing homogeneous powder.
source as well as supplies B-complex vitamins. Sucrose is the fermentable
Prepared Appearance
carbohydrate, liver extract provides B-vitamins, potassium phosphate is the Light to medium amber, slightly opalescent gel that may have a slightly
buffer and sodium chloride maintains the osmotic balance. flocculent precipitate.
Formula* Cultural Response
Ingredients in grams per liter Cultural characteristics after 16-24 hours at 350C.
Sucrose 12.0 Organisms (ATCC) Growth
Yeast Extract 5.0 Escherichia coli mutant 113-3D (11105) Luxuriant
Tryptone 5.0 Storage
Monopotassium Phosphate 0.5
Store below 80C and prepared medium at 2-80C.
Magnesium Sulphate 0.2
Shelf Life
Sodium Chloride 0.1
Use before expiry date as mentioned on the label.
Liver Extract 0.05

Bacillus Cereus Agar Base AM1009/AM5009

Use Principle
Bacillus Cereus Agar Base with added supplements is used as a selective medium Peptone level of 0.1% and sodium pyruvate improve egg yolk precipitation and
for the isolation and enumeration of Bacillus cereus. enhance sporulation. Bromothymol blue is added as a pH indicator to detect
Summary mannitol fermentation. Moulds if present in large numbers, can be suppressed by
Bacillus Cereus Agar Base with selective supplements is based on the highly the addition of filter sterilized cycloheximide in a concentration of 40 mcg per ml
specific diagnostic and selective PEMBA medium, developed by Holbrook and of the medium. The primary diagnostic features of the medium are the colonial
Anderson (44) for the isolation and enumeration of B.cereus in foods. It supports appearance, precipitation of hydrolyzed lecithin and the failure of B.cereus to
the growth of even a small number of B.cereus cells and spores in the presence of utilize mannitol. The typical colonies of B.cereus are crenated, about 5 mm in
a large number of other food contaminants. Colonies of B.cereus are readily diameter and have a distinctive turquoise to peacock blue colour surrounded by a
identified and confirmed by microscopic examination. good egg yolk precipitate of the same colour. These features distinguish B.cereus
from other Bacillus species except B.thuringiensis. Other egg yolk-precitipating

50 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
 Exploring... Accumix
organisms, which can grow on the medium, include S.aureus, S.marcescens and Peptone Water.
P.vulgaris, which are distinguished from B.cereus by colony form and colour. 3. Dried foods should first be rehydrated by soaking 20 gms in Tryptone Salt
These organisms also produce an egg yolk-clearing reaction in contrast to the egg solution for 50 minutes at room temperature.
yolk precipitate formed by B.cereus.
4. Add a further 90 ml of Peptone Water to give a final dilution of 10-1 .
Formula*
Ingredients in grams per liter
5. Homogenize for 30 seconds.
Peptone 1.00 6. Further dilutions of the homogenate should be made in 0.1% Peptone
Mannitol 10.00 Water.
Sodium Pyruvate 10.00
7. Inoculate 0.1 ml or 1.0 ml of the 10-1 dilution and higher dilutions on to the
Sodium Chloride 2.00
surface of the medium using spread plate or pour plate method.
Magnesium Sulphate 0.10
Monopotassium Phosphate 0.25 8. Incubate the plates at 350C for 24 hours.
Disodium Phosphate 2.50 For examining clinical specimens inoculate plates using standard procedures
Bromothymol Blue 0.12
like streak plate method.
Agar 15.00
Interpretation of results
Final pH (at 250C) 7.2 ± 0.2
* Formula adjusted to suit performance parameters
1. Typical colonies of B.cereus are crenated, about 5 mm in diameter with a
Directions distinctive turquoise to peacock blue colour surrounded by a good egg
1. Suspend 20.5 gms of the powder in 475 ml distilled water. yolk precipitate of the same colour.
2. Mix thoroughly. 2. The spores stain pale green to mid green, are paracentral or central in
position and do not swell the sporangium.
3. Boil with frequent agitation to dissolve the powder completely.
3. Lipid globules and the vegetative cytoplasm are both red.
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
4. Only B.cereus are capable of possessing lipid globules in their vegetative
5. Cool to 45-500C and aseptically add rehydrated contents of 1 vial of
cells when grown on selective medium and the presence of lipid globules is
Polymixin B Selective Supplement (AS021) and 25 ml sterile Egg Yolk
recommended as a rapid and confirmatory test for B.cereus.
Emulsion (AS010).
For Quantitative test
6. Mix well and pour into sterile petri plates.
Quality Control
1. Leave the plates for a further 24 hours at room temperature in order to detect
Dehydrated Appearance all the Bacillus cereus colonies.
Greenish yellow coloured, homogeneous, free flowing powder. 2. Report the results as the number of B.cereus colonies per gram weight of the
Prepared Appearance food sample.
Basal medium - Green coloured, clear to slightly opalescent gel. Precautions / Limitations
With addition of 5% Egg Yolk Emulsion -Yellowish green coloured, opaque gel.
1. Identify B.cereus by colony form, colour, egg yolk hydrolysis and confirm
Cultural Response
Cultural characteristics after 24 hours at 35-370C.
with cell and spore morphology.
Organisms (ATCC) Growth Colour of colony Egg Yolk 2. Occasional strains of B.cereus show weak or negative egg yolk reactions.
Reaction 3. Confirm the presumptive identification of B.cereus by the rapid confirmatory
Bacillus cereus (10876) Good to luxuriant Blue Precipitation staining procedure.
Proteus vulgaris (13315) Good to luxuriant Green -
Staphylococcus aureus Good to luxuriant Yellow Clearing
4. On this medium B.cereus is indistinguishable from B.thuringiensis. Other
(25923) organisms like S.aureus, P.vulgaris and S.marcescens may also grow on
Procedure this medium.
For Quantitative test Storage

1. Samples should be processed in a manner suitable for the source material, Store at 22-300C and prepared medium at 2-80C.
Shelf Life
liquid, solid, semi-solid or frozen.
Use before expiry date as mentioned on the label.
2. Homogenize 10 gms of the food sample for 30 seconds in 90 ml of 0.1%

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 51
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Bacteroides Bile Esculin Agar (BBE) AM1010/AM5010
Use Quality Control
Bacteroides Bile Esculin Agar is used for the selective isolation, cultivation and Dehydrated appearance
Yellow coloured, homogeneous free flowing powder.
presumptive identification of the Bacteroides fragilis group.
Prepared appearance
Summary
Slightly amber coloured, clear to slightly opalescent gel with a bluish tinge.
Among the most frequently encountered anaerobes in human clinical infections Cultural response
are members of the “Bacteroides fragilis” group. These pathogens frequently Cultural characteristics after 40-48 hours at 350C when incubated anaerobically.
occur in a mixture of microorganisms, which may overgrow the primary isolation
medium. Livingston et al described this as a primary plating medium, which was Organisms (ATCC) Growth Esculin RGI
found to provide selective recovery of the B.fragilis group and also evidence for Hydrolysis
presumptive identification (71). Bacteroides Good to luxuriant + More than 70%
Principle fragilis (25285)
Bacteroides Good to luxuriant - More than 70%
Tryptone, soya peptone and hemin provide essential nutrients. Oxgall is the
vulgatus (8482)
selective agent, which inhibits almost all gram-negative bacilli except B.fragilis. Clostridium Inhibited - More than 70%
The medium is made more selective by the addition of Bacteroides Selective perfringenes (11124)
Supplement containing gentamicin which inhibits most organisms other than the Proteus mirabilis None to poor - More than 70%
esculin positive Bacteroides that can tolerate bile. Differentiation of the (12453)
B.fragilis group is based on esculin hydrolysis, which produces esculetin and For growth RGI should be more than 70%
dextrose. Esculitin reacts with the iron salt (ferric ammonium citrate) contained in For Inhibition RGI should be 0%
RGI- Relative Growth Index
the medium to produce a dark brown to black complex that appears in the
Procedure
medium surrounding colonies of the members of the B. fragilis group.
Formula* 1. As some strains of the B.fragilis group may not grow due to the selective
Ingredients in grams per liter nature of the medium, it is advisable to include a non-selective Blood Agar
Tryptone 15.0 medium simultaneously.
Soya Peptone 5.0 2. All media should be pre-reduced.
Oxgall 20.0
3. Incubate immediately under anaerobic conditions for at least 48 hours at 35-
Sodium Chloride 5.0
Esculin 1.0 370C.
Ferric Ammonium Citrate 0.5 Interpretation of results
Vitamin K1 0.01 1. After incubation, colonies of the B.fragilis group should be greater than
Hemin 0.01 1 mm in diameter and appear grey, circular, entire and raised.
Agar 15.0
2. Most anaerobes other than B.fragilis are generally inhibited.
Final pH (at 250C) 7.0 ± 0.2
* Formula adjusted to suit performance parameters 3. Blackening of the medium around the colonies indicates esculin hydrolysis.
Directions Precautions / Limitations

1. Suspend 61.52 gms of the powder in 1000 ml distilled water and mix well. 1. B.vulgatus may not hydrolyze esculin.
Storage
2. Boil with frequent agitation to dissolve the powder completely.
Store below 80C and prepared medium at 2-80C.
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. Shelf Life
4. Cool to 45-500C and aseptically add 2 vials of Bacteroides Use before expiry date as mentioned on the label.
Selective Supplement (AS001).
5. Mix well and pour into sterile petri plates.

52 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
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Baird Parker Agar Base AM1011/AM5011
Baird Parker Agar Base IP AM101111/AM501111
Baird Parker Agar Base USP AM101112/AM501112
Baird Parker Agar Base (Agar Medium O) EP AM101113/AM501113
Baird Parker Agar Base (Agar Medium O) BP AM101114/AM501114
Use positive colonies are white to grey black surrounded by an opaque zone of
Baird Parker Agar Base with added supplements (Egg Yolk Tellurite Emulsion) is coagulase activity, within 24-40 hours of incubation at 350C. Reduction in
used for the selective isolation and enumeration of coagulase positive tellurite is required because of the absence of egg yolk emulsion, resulting in
staphylococci from clinical and non-clinical specimens. translucent agar and white to grey coloured colonies of staphylococci.
Summary Formula*
Baird Parker Agar was developed by Baird-Parker (3, 4) from the tellurite-glycine Ingredients in grams per liter
formulation of Zebovit et al (124) for the recovery of coagulase positive Tryptone 10.0
Yeast Extract 1.0
staphylococci. It was suggested that this medium be substituted for Vogel and
Beef Extract 5.0
Johnson Agar (VJ) (2,107) because it was less inhibitory than VJ Agar, yet more
Sodium Pyruvate 10.0
selective and also possessed a diagnostic aid (egg yolk reaction) not present in VJ Glycine 12.0
Agar. Subsequently, it was officially accepted by the AOAC and is also Lithium Chloride 5.0
recommended by the USP and IP for use in microbial limit tests (114, 46). Baird Agar 20.0
Parker Agar is recommended by APHA for the examination of milk (39) and foods Final pH (at 250C) 7.0 ± 0.2
(20) and is also included in the Bacteriological Analytical Manual for testing of * Formula adjusted to suit performance parameters
cosmetics (113). Directions
Principle 1. Suspend 63 gms of the powder in 950 ml distilled water.
Tryptone, beef extract and yeast extract provide nitrogenous compounds, carbon, 2. Mix thoroughly.
sulphur and other growth factors. Sodium pyruvate protects the injured cells, 3. Boil with frequent agitation to dissolve the powder completely.
helps recovery and stimulates the growth of S.aureus without destroying the
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
selectivity. Glycine enhances the growth of Staphylococcus. Lithium chloride
5. Cool to 45-500C and aseptically add 50 ml concentrated Egg Yolk Emulsion
inhibits most of the micro flora except Staphylococcus aureus. The tellurite
(AS010) and 3 ml sterile 3.5% Potassium Tellurite Solution (AS023) or
additive inhibits egg-yolk clearing strains other than S.aureus and imparts a
50ml Egg Yolk Tellurite Emulsion (AS011). One vial of BP Sulpha
black colour to the colonies. The egg yolk, apart from being an enrichment, aids in
supplement can be added if desired.
the identification process by demonstrating lecithinase activity (egg-yolk
reaction). Egg yolk makes the medium yellow, opaque. Proteolytic bacteria 6. Mix thoroughly, but gently and pour into sterile petri plates.
produce a clear zone around colonies in egg yolk containing medium. A clear zone Warning: Lithium chloride is harmful, bodily contact and
inhalation of vapours must be avoided. On contact
around grey-black colonies on this medium is diagnostic for coagulase positive with skin, wash with plenty of water immediately.
staphylococci. An opaque zone of lipolytic activity may be developed around the Quality Control
colonies on further incubation. Dehydrated Appearance
Identity of Staphylococcus aureus isolated on Baird Parker Agar must be Yellow coloured, homogeneous, free flowing powder.
confirmed with a coagulase reaction. Coagulase activity can be detected by Prepared Appearance
adding plasma fibrinogen mixture in place of egg yolk emulsion. 375 mg bovine Basal medium - Light amber coloured clear to slightly opalescent gel.
With addition of Egg Yolk Tellurite Emulsion - Yellow coloured opaque gel.
fibrinogen, 2.5 ml plasma, 2.5 mg trypsin inhibitor and 2.5 mg potassium
Cultural Response
tellurite is dissolved in 10 ml sterile distilled water and added to 90 ml sterile,
Cultural characteristics after 24-48 hours at 35-370C.
molten medium stored at 45-500C. On this medium staphylococcal coagulase

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 53
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Organisms Growth Colour Lecithinase RGI 2. If negative, reincubate for additional 24 hours.
(ATCC) of colony production
Bacillus subtilis None Dark brown - 0% Quantitative results
(6633) to poor 1. Count the plates with 20-200 typical Staphylococcus aureus like colonies,
Escherichia coli None Large - 0%
(25922) to poor brown black
express as colony forming units (CFU) per gram or ml of sample, taking into
Micrococcus luteus Poor Very small in - More than 70% account the applicable dilution factor.
(10240) to good shades of
2. Also perform coagulase test.
brown black
Proteus mirabilis (25933) Good Brown to black - More than 70%
Precautions / Limitations
to luxuriant 1. Colonies of some contaminating organisms may digest the coagulase halo
Staphylococcus Good Grey black + More than 70% reaction.
aureus (25923) to luxuriant shiny
Staphylococcus Poor to good Black - More than 70% 2. Regardless of negative reactions, consider all doubtful colonies as S.aureus
epidermidis (12228) and carry out further tests like coagulase reaction because some strains of
For growth RGI should be more than 70% S.aureus give negative egg yolk reactions (in foods, especially cheese).
For Inhibition RGI should be 0%
3. Baird Parker Agar Base with supplements is selective for coagulase positive
RGI- Relative Growth Index
staphylococci, but other bacteria including Proteus species may grow
Procedure
(addition of 50mg/l Sulphamethazine is found to suppress growth and
For Quantitative test
swarming of Proteus species).
1. Food samples are macerated in a suitable broth medium, dilutions are made
4. The majority of contaminating flora that grows produces white to brown
if required and inoculated by spread plate method onto the agar surface.
colonies with no clearing of the egg yolk
2. Incubate plates aerobically for 24 hours at 35-370C.
5. Prepare fresh medium for best results.
Interpretation of Results
Storage
1. Typical colonies of S.aureus are black, shiny, convex and surrounded by
Store at 22-300C and prepared medium at 2-80C.
clear zones (E-Y reaction) of approximately 2-5 mm. Coagulase negative
Shelf Life
staphylococci generally do not grow well; if growth occurs, the typical clear
Use before expiry date as mentioned on the label.
zones are absent.

Baird Parker Agar Base BIS AM101115/AM501115

Use Principle
Baird Parker Agar Base BIS is a medium with added supplements for selective Tryptone, meat extract and yeast extract provide nitrogenous compounds, carbon,
isolation and enumeration of coagulase positive staphylococci from food and sulphur and other growth factors. Sodium pyruvate protects the injured cells,
other materials in compliance with BIS specification IS:5887 (Part III) 1976. helps recovery and stimulates the growth of S.aureus without destroying the
Summary selectivity. Glycine enhances the growth of Staphylococcus. Lithium chloride
Baird Parker Agar was developed by Baird-Parker from the tellurite-glycine inhibits most of the micro flora except Staphylococcus aureus. The tellurite
formulation of Zebovit et al., (124) for the recovery of coagulase positive additive inhibits egg-yolk clearing strains other than S.aureus and imparts a
staphylococci. It was suggested that this medium be substituted for Vogel and black colour to the colonies. The egg yolk, apart from being an enrichment, aids in
Johnson Agar (VJ) because it was less inhibitory than VJ Agar, yet more selective the identification process by demonstrating lecithinase activity (egg-yolk
and also possessed a diagnostic aid (egg yolk reaction) not present in VJ Agar. reaction). Egg yolk makes the medium yellow, opaque. Proteolytic bacteria
Subsequently, it was officially accepted by the AOAC and is also recommended by produce a clear zone around colonies in egg yolk containing medium. A clear zone
the USP and IP for use in microbial limit tests. Baird Parker Agar is recommended around greyblack colonies on this medium is diagnostic for coagulase positive
by APHA for the examination of milk and foods and is also included in the staphylococci. An opaque zone of lipolytic activity may be developed around the
Bacteriological Analytical Manual for testing of cosmetics. colonies on further incubation.

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Identity of Staphylococcus aureus isolated on Baird Parker Agar must be Micrococcus Poor Very small - 0%
luteus (10240) to good shades of
confirmed with a coagulase reaction. Coagulase activity can be detected by brown black
adding plasma fibrinogen mixture in place of egg yolk Proteus mirabilis Good to Brown to - More
(25933) luxuriant black than 70%
emulsion. 375 mg bovine fibrinogen, 2.5 ml plasma, 2.5 mg trypsin inhibitor
Staphylococcus Good to Grey black
and 2.5 mg potassium tellurite is dissolved in 10 ml sterile distilled water and aureus (25923) luxuriant shiny + More
added to 90 ml sterile, molten medium stored at 45- 500C. On this medium than 70%
staphylococcal coagulase positive colonies are white to grey black surrounded by Staphylococcus Poor Black - 0%
epidermidis to good
an opaque zone of coagulase activity, within 24-40 hours of incubation at 350C. (12228)
Reduction in tellurite is required because of the absence of egg yolk emulsion, For growth RGI should be more than 70%
resulting in translucent agar and white to grey coloured colonies of staphylococci. For Inhibition RGI should be 0%
Formula* RGI- Relative Growth Index
Ingredients in grams per liter Warning: Lithium chloride is harmful, bodily contact and inhalation of
Tryptone 10.0 vapours must be avoided. On contact with skin, wash with plenty of
Yeast extract 1.0 water immediately.
Meat extract 5.0 Procedure
Sodium pyruvate 12.0 For Quantitative test
Glycine 12.0
1. Food samples are macerated in a suitable broth medium, dilutions are made
Lithium chloride 5.0
if required and inoculated by spread plate method onto the agar surface.
Agar 20.0
Final pH (at 250C) 7.0 ±0.2 2. Incubate plates aerobically for 24 hours at 35-370C.
* Formula adjusted to suit performance parameters Interpretation of Results
Directions 1. Typical colonies of S.aureus are black, shiny, convex and surrounded by clear
1. Suspend 65 gms of the powder in 950 ml distilled water. zones (E-Y reaction) of approximately 2-5 mm. Coagulase negative
2. Mix thoroughly. staphylococci generally do not grow well; if growth occurs, the typical clear
zones are absent. 2. If negative, reincubate for additional 24 hours.
3. Boil with frequent agitation to dissolve the powder completely.
Quantitative results
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
1. Count the plates with 20-200 typical Staphylococcus aureus like colonies,
5. Cool to 45-500C and aseptically add 50 ml concentrated Egg Yolk Emulsion
express as colony forming
(AS010) and 3 ml sterile 3.5% Potassium Tellurite Solution (AS023) or
50ml Egg Yolk Tellurite Emulsion (AS011). One vial of BP Sulpha units (CFU) per gram or ml of sample, taking into account the applicable
supplement can be added if desired. dilution factor.
6. Mix thoroughly, but gently and pour into sterile petri plates. 2. Also perform coagulase test.
Quality Control Precautions / Limitations
Dehydrated Appearance 1. Colonies of some contaminating organisms may digest the coagulase halo
Yellow coloured, homogeneous, free flowing powder. reaction.
Prepared Appearance
2. Regardless of negative reactions, consider all doubtful colonies as S.aureus
Basal medium - Light amber coloured clear to slightly opalescent gel.
and carry out further tests like coagulase reaction because some strains of
With addition of Egg Yolk Tellurite Emulsion - Yellow coloured opaque gel.
Cultural Response
S.aureus give negative egg yolk reactions (in foods, especially cheese).
Cultural characteristics after 24-48 hours at 35-370C. 3. Baird Parker Agar Base with supplements is selective for coagulase positive
Organisms Growth Colour Lecithinase RGI staphylococci, but other bacteria including Proteus species may grow
(ATCC) of colony production
(addition of 50mg/l Sulphamethazine is found to suppress growth and
Bacillus subtilis None Dark brown - 0%
(6633) to poor swarming of Proteus species).
Escherichia coli None Dark brown - 0% 4. The majority of contaminating flora that grows produces white to brown
(25922) to poor black
colonies with no clearing of the egg yolk

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5. Prepare fresh medium for best results. Shelf Life
Storage Use before expiry date as mentioned on the label.
Store at 22-300C and prepared medium at 2-80C.

Beef Extract Agar AM10111/AM50111

Use Quality Control
Beef Extract Agar is a general-purpose nutrient medium, which supports the Dehydrated Appearance
growth of not particularly fastidious bacteria. Yellow coloured, homogeneous, free flowing powder.
Summary Prepared Appearance
Yellow coloured, clear to slightly opalescent gel.
Beef Extract Agar is a general-purpose nutrient medium, which is also
Cultural Response
recommended for preparation of pure cultures of Candida s pecies before carrying
Cultural characteristics after 24-48 hours at 350 C.
out fermentation studies and for cultivation and maintenance of a wide variety of Organisms (ATCC) Growth
microorganisms including Pseudomonas aeruginosa. Escherichia coli (25922) Luxuriant
Principle Staphylococcus aureus (25923) Luxuriant
Peptone and beef extract provide nutritious sources of nitrogen and carbon. Candida albicans (10231) Luxuriant
Sodium chloride acts as a source of electrolytes. Agar is the solidifying agent. Salmonella serotype Typhi (6539) Luxuriant
Formula* Pseudomonas aeruginosa (27853) Luxuriant
Ingredients in grams per liter Procedure
Peptone 10.0 1. Use standard streaking methods to obtain isolate organisms.
Beef Extract 3.0 Interpretation of Results
Sodium Chloride 5.0 1. Luxuriant growth of non-fastidious organisms is seen.
Agar 15.0
Precautions / Limitations
Final pH (at 250 C) 7.6 ± 0.2
* Formula adjusted to suit performance parameters
1. Inspect the petriplates for contamination before inoculation.
Directions 2. Some strains may fail to grow on this medium due to variation in nutritional
1. Suspend 33 grams of the powder in 1000 ml distilled water. requirements of organisms.
Storage
2. Boil with frequent agitation to dissolve the powder completely.
Store at 22-300C and prepared medium at 2-80C.
3. Dispense in tubes. Shelf Life
4. Sterilize by autoclaving at 1210 C (15 lbs pressure) for 15 minutes. Use before expiry date as mentioned on the label.
5. Allow the tubes to cool in a slanting position if required.

Beef Extract Broth AM50112

Use Principle
Beef Extract Broth is a general-purpose nutrient medium, which supports the Peptic digest of animal tissue and beef extract provide nutritious sources of
growth of not particularly fastidious bacteria. nitrogen and carbon. Sodium chloride acts as a source of electrolytes.
Summary Formula*
Beef Extract Broth is a general-purpose nutrient medium, which is also Ingredients in grams per liter
Peptic digest of animal tissue 10.0
recommended for preparation of pure cultures of Candida species before carrying
Beef extract 3.0
out fermentation studies and for cultivation and maintenance of a wide variety of
Sodium chloride 5.0
microorganisms including Pseudomonas aeruginosa (31.1).
Final pH (at 250 C) 7.2±0.2
* Formula adjusted to suit performance parameters

56 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
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Directions Candida albicans (10231) Luxuriant
1. Suspend 18 grams of the powder in 1000 ml distilled water. Salmonella serotype Typhi (6539) Luxuriant
Pseudomonas aeruginosa (27853) Luxuriant
2. Boil with frequent agitation to dissolve the powder completely.
Procedure
3. Dispense in tubes. 1. Use standard methods to obtain isolate organisms.
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. Interpretation of Results
5. Allow the tubes to cool in a slanting position if required. 1. Luxuriant growth of non-fastidious organisms is seen.
Quality Control Precautions / Limitations
Dehydrated Appearance 1. Inspect the tubes for contamination before inoculation.
Yellow coloured, homogeneous, free flowing powder.
2. Some strains may fail to grow on this medium due to variation in nutritional
Prepared Appearance
Yellow coloured, clear to slightly opalescent solution. requirements of organisms.
Cultural Response Storage
Cultural characteristics after 24-48 hours at 350 C. Store at 22-300C and prepared medium at 2-80C.
Organisms (ATCC) Growth Shelf Life
Escherichia coli (25922) Luxuriant Use before expiry date as mentioned on the label.
Staphylococcus aureus (25923) Luxuriant

Bile Esculin Agar AM1012/AM5012

Bile Esculin Agar ISO AM1012/AM501211
Use genus Klebsiella-Enterobacter-Serratia from other Enterobacteriaceae on the
Bile Esculin Agar is a differential medium used for isolation and presumptive basis of esculin hydrolysis.
identification of group D streptococci / enterococci from foods. Formula*
Summary Ingredients in grams per liter
Swan (106) formulated Bile Esculin Agar for the isolation and identification of Peptone 5.0
group D streptococci from foods. Originally, Bile Esculin Test was used for the Beef Extract 3.0
identification of enterococci. However, since group D streptococci share the test Esculin 1.0
with enterococci, it is advisable that other tests such as salt tolerance be Oxgall 40.0
Ferric Citrate 0.5
performed while identifying enterococci. Meyer and Schonfeld showed that
Agar 15.0
majority of enterococci were able to grow in esculin and split it, while other
Final pH (at 250C) 6.6 ± 0.2
streptococci could not. This medium is used to differentiate enterococci and * Formula adjusted to suit performance parameters
Streptococcus bovis from other streptococci. Rochaix (94) explored the value of Directions
esculin hydrolysis in the identification of enterococci. Bile Esculin Agar is included 1. Suspend 64.5 gms of the powder in 1000 ml distilled water and mix well.
in the Bacteriological Analytical Manual for food and cosmetics testing (113) and
2. Boil with frequent agitation to dissolve the powder completely.
is recommended by APHA for the examination of foods (20) and water and
wastewater (36). 3. Mix and dispense in tubes as desired.
Principle 4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Oxgall inhibits gram-positive bacteria other than group D streptococci / 5. Allow the tubed medium to solidify in slanted position.
enterococci. Ferric citrate is an indicator of esculin hydrolysis and resulting Quality Control
esculetin formation. Enterococci / group D streptococci hydrolyze the glycoside Dehydrated Appearance
esculin to dextrose and esculetin which reacts with ferric citrate producing Brownish yellow coloured, homogeneous, free flowing powder.
brownish black complex. This medium is also shown to aid differentiation of Prepared Appearance
Yellow coloured, clear to slightly opalescent gel with a bluish tinge.

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 Exploring... Accumix
Cultural Response result for esculin hydrolysis.
Cultural characteristics after 18-24 hours at 35-370C in an increased
atmosphere of carbon dioxide.
2. For slants, if more than half of the slant is blackened within 24-48
Organisms (ATCC) Growth Esculin RGI hours, the test is positive; if less than half of it is blackened or no
Hydrolysis blackening occurs within 24-48 hours, the test is negative.
Enterococcus faecalis Luxuriant + More than 70% Precautions / Limitations
(29212)
1. Use a light inoculum. A heavy inoculum may cause difficulty in
Streptococcus bovis Luxuriant + More than 70%
interpretation of results and decrease the ability of the bile to inhibit growth
(27960)
Proteus mirabilis Luxuriant - More than 70%
of other gram-positive organisms that may hydrolyze esculin.
(25933) 2. A few streptococci may grow on the medium in the presence of bile without
Staphylococcus aureus Good - More than 70% hydrolysis of esculin.
(25923)
3. Growth without blackening of the medium is a negative result.
Streptococcus pyogenes Partial to - 0%-70%
(19615) Non poor 4. Gram-negative rods may grow on this medium and hydrolyze esculin.
For growth RGI should be more than 70% 5. Strains of Lactococcus, Leuconostoc and Pediococcus if present (isolated
RGI- Relative Growth Index from human infections) may give a positive bile-esculin reaction.
Key: + = blackening of medium
- = no change
6. Occasionally, strains of Streptococcus viridans blacken the medium or
For growth RGI should be more than 70% shows weakly positive reactions.
For Inhibition RGI should be 0% 7. This medium
RGI- Relative Growth Index Storage
Procedure
Store at 22-300C and prepared medium at 2-80C.
1. Inoculate the medium with two or three colonies Shelf Life
2. Incubate at 370C for 18-24 hours aerobically. Use before expiry date as mentioned on the label.is not recommended for
Interpretation of Results primary isolation of specimens.
1. Blackening of the medium around the colonies indicates a positive

Bile Esculin Azide Agar AM501212

Use Proteose peptone 3.0
A medium for selective isolation and presumptive identification of fecal Beef extract 5.0
Oxgall 10.0
Streptococci.
Sodium chloride 5.0
Summary
Esculin 1.0
Bile Esculin Agar formulated by Swan (106). Bile Esculin Azide Agar is the Ferric ammonium citrate 0.50
modified form of Bile Esculin Agar as described by Isenberg (46.1). Bile Esculin Sodium azide 0.15
Azide Agar is a selective medium for Group D Streptococci. Agar 15.0
Principle Final pH (at 250C) 7.1 ± 0.2
Bile Esculin Azide Agar is a medium riched with casein enzymic hydrolysate, * Formula adjusted to suit performance parameters
Directions
proteose peptone and yeast extract. Sodium azide acts as an inhibitor for Gram-
negative organisms. Oxgall is used to inhibit gram positive bacteria other than 1. Suspend the 56.65 gms of powder in 1000 ml distilled water.
enterococci. Hydrolysis of Esculin helps to detect Group D Streptococci. 2. Mix thoroughly.
Formula* 3. Heat gently with frequent agitation to dissolve the powder completely.
Ingredients in grams per liter
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Casein enzymic hydrolysate 17.0

58 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
 Exploring... Accumix
Quality Control For growth RGI should be more than 70%
Dehydrated Appearance For Inhibition RGI should be 0%
Yellow coloured, homogeneous free flowing powder. RGI- Relative Growth Index
Prepared Appearance Key: + = blackening of medium
Medium amber coloured clear to slightly opalescent gel with a bluish tinge – = no change
forms in petri plate. Procedure
Cultural Response
Refer to appropriate references for specific procedures.
Cultural characteristics after 18-24 hours at 37°C.
Interpretation of Results
Organisms (ATCC) Growth Esculin RGI
Hydrolysis Refer to appropriate references and procedures for results.
Storage
Enterococcus faecalis (29212) Luxuriant + More than 70% Store at 22-300C and prepared medium at 2-80C.
Streptococcus bovis (27960) Luxuriant + More than 70% Shelf Life
Proteus mirabilis (25933) Luxuriant - More than 70% Use before expiry date as mentioned on the label.
Staphylococcus aureus (25923) Good - More than 70%
Streptococcus pyogenes (19615) None-poor - 0% or 70%

Bile Salt Agar AM10121/AM50121

Use 3. Sterilize by autoclaving at 1210 C (15 lbs pressure) for 20 minutes.
Bile Salt Agar is used for the isolation and enumeration of enteric bacilli. 4. Cool to 60-700 C.
Summary 5. Pour into sterile petriplates. Remove air bubbles if any using a flame before
A variety of special differential and selective culture media are used in the solidifying.
isolation of enteric bacteria from clinical specimens. A selective agent Sodium Quality Control
Taurocholate has been incorporated in Bile Salt Agar specifically to enhance the Dehydrated Appearance
growth of enteric bacilli. Yellow coloured, homogeneous, free flowing powder.
Principle Prepared Appearance
Yellow coloured, clear to slightly opalescent gel.
Tryptone and Meat Extract provide sources of nitrogen, minerals and amino acids.
Cultural Response
Sodium Chloride maintains the osmotic equilibrium. Sodium taurocholate is a Cultural characteristics after 18-24 hours at 35-370C.
selective agent that inhibits growth of gram-positive organisms. Agar is the Organisms (ATCC) Growth RGI
solidifying agent. Enterobacter aerogenes (13048) Luxuriant More than 70%
Formula* Enterococcus faecalis (29212) Fair to good More than 70%
Ingredients in grams per liter Escherichia coli (25922) Luxuriant More than 70%
Tryptone 10.0 Proteus vulgaris (13315) Luxuriant More than 70%
Meat Extract 5.0 Salmonellae serotype enteritidis (13076) Luxuriant More than 70%
Sodium Chloride 5.0 Shigella flexneri (12022) Luxuriant More than 70%
Sodium Taurocholate 5.0 Staphylococcus aereus (25923) Fair to good More than 70%
Agar 18.0 For growth RGI should be more than 70%
Final pH (at 250 C) 7.2 ± 0.2 RGI- Relative Growth Index
* Formula adjusted to suit performance parameters Storage
Directions
Store at 22-300C and prepared medium at 2-80C.
1. Suspend 43.0 grams of the powder in 1000 ml distilled water. Shelf Life
2. Boil with frequent agitation to dissolve the powder completely. Use before expiry date as mentioned on the label.

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 59
 Exploring... Accumix
Bile Salt Agar BIS AM10122/AM50122
Use 2. Boil with frequent agitation to dissolve the powder completely.
Bile Salt Agar is used for the isolation and enumeration of bile tolerant enteric 3. Sterilize by autoclaving at 1200 C (15 lbs pressure) for 20 minutes.
bacilli in compliance with BIS specification IS:5887 (Part5) 1976 reaffirmed
4. Cool to 60-700 C.
1986.
Summary
5. Pour into sterile petriplates. Remove air bubbles if any using a flame before
solidifying.
A variety of special differential and selective culture media are used in the
Quality Control
isolation of enteric bacteria from clinical specimens. A selective agent Sodium
Dehydrated Appearance
Taurocholate has been incorporated in Bile Salt Agar specifically to enhance the Yellow coloured, homogeneous, free flowing powder.
growth of enteric bacilli. Prepared Appearance
Principle Yellow coloured, clear to slightly opalescent gel.
Tryptone and Meat Extract provide sources of nitrogen, minerals and amino acids. Cultural Response
Sodium Chloride maintains the osmotic equilibrium. Sodium taurocholate is a Cultural characteristics after 18-24 hours at 35-370C.
selective agent that inhibits growth of gram-positive organisms. Agar is the Organisms (ATCC) Growth RGI
solidifying agent. Enterobacter aerogenes (13048) Luxuriant More than 70%
Enterococcus faecalis (29212) Fair to good More than 70%
Formula*
Ingredients in grams per liter
Escherichia coli (25922) Luxuriant More than 70%
Peptone 10.0 Proteus vulgaris (13315) Luxuriant More than 70%
Meat Extract 5.0 Salmonellae serotype enteritidis (13076) Luxuriant More than 70%
Sodium Chloride 5.0 Shigella flexneri (12022) Luxuriant More than 70%
Sodium Taurocholate 5.0 Staphylococcus aereus (25923) Fair to good More than 70%
Agar 15.0 For growth RGI should be more than 70%
Final pH (at 250 C) 8.5 ± 0.2 RGI- Relative Growth Index
* Formula adjusted to suit performance parameters Storage
Directions Store at 22-300C and prepared medium at 2-80C.
Shelf Life
1. Suspend 40.0 grams of the powder in 1000 ml distilled water.
Use before expiry date as mentioned on the label.

Bismuth Sulphite Agar AM1013/AM5013

Use is also included in the Bacteriological Analytical Manual for food testing (113).
Bismuth Sulphite Agar is a highly selective medium used for preliminary Principle
identification of Salmonella species, particularly S.typhi from clinical and non- Beef extract and peptone provide nitrogen, growth factors and trace elements.
clinical specimens. Dextrose is the energy source. Disodium hydrogen phosphate is the buffer.
Summary Bismuth sulphite and brilliant green are complimentary in inhibiting gram-
Bismuth Sulphite Agar is a modification of the original Wilson and Blair (121, positive bacteria and intestinal gram-negative bacteria (coliform group) while
122) selective medium. It is recommended by various associations (20, 36,39) allowing Salmonella to grow luxuriantly. This inhibitory action permits the use of
for the isolation and preliminary identification of Salmonella typhi and other a much larger inoculum than possible with other media employed for similar
salmonellae from pathological materials, food, sewage, water supplies, etc. More purposes. The use of larger inocula greatly increases the possibility of recovering
positive isolates of S.typhi were obtained on this medium compared to Endo the intestinal pathogens. Ferrous sulphate detects H2S production. S.typhi,
Agar, Eosin Methylene Blue Agar and Deoxycholate Agar. It is recommended by S.enteritidis and S.typhimurium typically grow as black colonies with
USP and IP for use in microbial limit testing (114, 46). For food testing, this surrounding metallic sheen resulting from H2S production and reduction of
medium is specified for the isolation of pathogenic bacteria from raw and sulphite to black ferric sulphide. S.paratyphi A produces light green colonies.
pasteurized milk, cheese products, dry dairy products, cultured milk and butter. It Shigellae species are mostly inhibited on this medium.

60 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
 Exploring... Accumix
Formula* Key: *depends on the inoculum density.
Ingredients in grams per liter Procedure
Beef Extract 5.0 1. Bismuth Sulphite Agar may be used in conjunction with other selective
Peptone 10.0
enteric agars for the isolation of salmonellae by direct plating or from
Dextrose 5.0
enrichment media.
Disodium Hydrogen Phosphate 4.0
Ferrous Sulphate 0.3 2. Inoculate directly on Bismuth Sulphite Agar and one or more of the following
Bismuth Sulphite Indicator 8.0 agars like Deoxycholate Citrate Agar, XLD Agar, Brilliant Green Agar,
Brilliant Green 0.025 MacConkeys Agar and SS Agar.
Agar 20.0
3. Also, inoculate in an enrichment broth such as Selenite F Broth or
Final pH (at 250C) 7.7 ± 0.2
Tetrathionate Broth Base.
* Formula adjusted to suit performance parameters
Directions 4. Subculture onto Bismuth Sulphite Agar and any of the other selective media
1. Suspend 52.33 gms of the powder in 1000 ml distilled water. after 12-18 hours incubation. Examine the plates after 18 hours incubation
and subculture suspect colonies to identification media like TSI agar or
2. Mix thoroughly.
Kligler Iron Agar.
3. Heat with frequent agitation and boil for 1 minute to dissolve the powder
For isolating Salmonella species from food:
completely.
1. Samples must be selectively enriched.
4. DO NOT AUTOCLAVE.
2. Streak 10 microlitres of selective enrichment broth onto Bismuth Sulphite
5. Disperse the precipitate evenly while dispensing (the sensitivity of the
Agar.
medium depends mainly upon uniform dispersion of freshly precipitated
bismuth sulphite in the final gel) and use the medium the same day it is 3. Incubate plates for 24-48 hours at 350C.
prepared. For isolation of Salmonella from clinical specimens:
Quality Control 1. Inoculate fecal specimens or rectal swabs onto a small area of the agar and
Dehydrated Appearance streak using four-quadrant method to give discrete colonies.
Greenish yellow coloured, homogeneous, free flowing powder.
2. Incubate plates at 24-48 hours at 350C and examine for colonies resembling
Prepared Appearance
Greenish yellow coloured opaque gel with a flocculent precipitate. Salmonella species.
Cultural Response Interpretation of Results
0
Cultural characteristics after 40-48 hours at 35 C. 1. The typical discrete S.typhi colonies are black and often surrounded by a
Organisms (ATCC) Growth Colour of colony RGI black or brownish black zone, which may be several times the size of the
Enterobacter None Brown to green* 0% or More colony.
aerogenes (13048) to poor than 70%
2. In reflected light, preferably daylight, the zone exhibits a distinctly
Enterococcus faecalis Inhibited - 0%
(29212)
characteristic metallic sheen. In heavy growth areas the organism frequently
Escherichia coli None Brown to green* 0% or More appears as small light green colonies. This emphasizes the importance of
(25922) to poor than 70% inoculating plates by the four quadrant method so that some areas are
Salmonella serotype Luxuriant Black with metallic More than 70% sparsely populated to give discrete colonies.
Enteritidis (13076) sheen 3. Other strains of Salmonella produce black to green colonies with
Shigella flexneri (12022) None Brown 0% or More
little or no darkening of the surrounding medium.
to poor than 70%
Salmonella serotype Luxuriant Black with More than 70% 4. Generally, Shigella species other than S.flexneri and S.sonnei are
Typhi (19430) metallic sheen inhibited. These two, do grow on this medium to produce brown to green
For growth RGI should be more than 70% raised colonies with depressed centers but show a crater like appearance.
For Inhibition RGI should be 0% 5. E.coli is partially inhibited on this medium. If at all present, it produces
RGI- Relative Growth Index
small brown or greenish glistening colonies. The colour however, is confined
to the colony itself and shows no metallic sheen.

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 61
 Exploring... Accumix
6. Enterobacter colonies if present exhibit a silvery sheen, appreciably lighter S.typhi colonies will appear light green in these circumstances and may thus
in colour than that produced by S.typhi. be misinterpreted as negative growth for S.typhi.
7. To isolate S.typhi for agglutination or fermentation studies, pick 5. S.typhi and S.arizonae are the only enteric organisms to exhibit typical
characteristic colonies from this medium and subculture on MacConkey brown zones on the medium. However, S.arizonae is usually inhibited on this
Agar. The purified colonies from MacConkey Agar may then be inoculated in medium.
differential tubed media like TSI Agar. 6. Some members of the coliform group that produce H2S may grow on this
8. All cultures that give reactions consistent with Salmonella species on this medium, giving colonies similar to S.typhi. However, they may be
medium should be confirmed biochemically as Salmonella species before differentiated because they produce gas from lactose in differential media, for
any serological testing is performed. example, Triple Sugar Iron Agar. Proteus species may be differentiated on
Precautions / Limitations the basis of urea hydrolysis in Urea Broth or on Urea Agar Base.
1. DO NOT AUTOCLAVE or overheat as it may destroy the selectivity of the 7. Colonies on this medium may be contaminated with other viable organisms;
medium. therefore, isolated colonies should be subcultured to a less selective medium
2. Prepared plates should not be stored for longer then two days at 2-80C; after like MacConkey Agar.
which time the dye oxidizes to give a green medium that can be inhibitory to 8. All plates should be incubated for a total of 48 hours to allow growth of all
some salmonellae. typhoid strains.
3. The medium may be inhibitory to some strains of salmonellae and therefore Storage
should not be used as the sole selective medium for these organisms. Store at 22-300C and prepared medium at 2-80C.
S.sendai, S.berta, S.gallinarum, S.abortus-equi and S.cholerae-suis are Shelf Life
markedly inhibited. Use before expiry date as mentioned on the label.
4. It is important to streak for well isolated colonies. The typical colonial
characteristics will not develop if the growth is too heavy or confluent;

Bismuth Sulphite Agar Medium IP (Twin Pack) AM10131/AM50131

Use Disodium hydrogen phosphate is the buffer. Bismuth sulphite and brilliant green
Bismuth Sulphite Agar is a highly selective medium used for preliminary are complimentary in inhibiting gram-positive bacteria and intestinal gram-
identification of Salmonella species, particularly S.typhi from clinical and non- negative bacteria (coliform group) while allowing Salmonella to grow
clinical specimens in compliances with IP. luxuriantly. This inhibitory action permits the use of a much larger inoculum than
Summary possible with other media employed for similar purposes. The use of larger
inocula greatly increases the possibility of recovering the intestinal pathogens.
Bismuth Sulphite Agar is a modification of the original Wilson and Blair (121,
Ferric Citrate detects H2S production. S.typhi, S.enteritidis and S.typhimurium
122) selective medium. It is recommended by various associations (20, 36,39)
typically grow as black colonies with surrounding metallic sheen resulting from
for the isolation and preliminary identification of Salmonella typhi and other
H2S production and reduction of sulphite to black ferric sulphide. S.paratyphi A
salmonellae from pathological materials, food, sewage, water supplies, etc. More
produces light green colonies. Shigellae species are mostly inhibited on this
positive isolates of S.typhi were obtained on this medium compared to Endo
medium.
Agar, Eosin Methylene Blue Agar and Deoxycholate Agar. It is recommended by
Formula*
USP and IP for use in microbial limit testing (114, 46). For food testing, this
Ingredients in grams per liter
medium is specified for the isolation of pathogenic bacteria from raw and Part A
pasteurized milk, cheese products, dry dairy products, cultured milk and butter. It Beef extract 6.0
is also included in the Bacteriological Analytical Manual for food testing (113). Peptone 10.0
Principle Ferric Citrate 0.4
Beef extract and peptone provide nitrogen, growth factors and trace elements. Brilliant green 0.01

62 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
 Exploring... Accumix
Agar 24.0 Kligler Iron Agar.
Part B
For isolating Salmonella species from food:
Ammonium bismuth citrate 3.0
Sodium sulphate 10.0 1. Samples must be selectively enriched.
Anhydrous disodium hydrogen phosphate 5.0 2. Streak 10 microlitres of selective enrichment broth onto Bismuth Sulphite
* Formula adjusted to suit performance parameters Agar.
Directions
3. Incubate plates for 24-48 hours at 350C.
1. Suspend the 40.41 gms of Part A of powder in 1000 ml distilled water.
For isolation of Salmonella from clinical specimens:
2. Mix thoroughly.
1. Inoculate fecal specimens or rectal swabs onto a small area of the agar and
3. Heat gently with frequent agitation to dissolve the powder completely.
streak using four- quadrant method to give discrete colonies.
4. Sterilize by autoclaving at 115°C (10 lbs pressure) for 30 minutes.
2. Incubate plates at 24-48 hours at 350C and examine for colonies resembling
5. Suspend the 22.54 gms of Part B of powder in 100 ml distilled water. Salmonella species.
6. Mix, heat to boiling, cool to room temperature, add 1 volume of Part B to 10 Interpretation of Results
volume of Part A previously melted and cooled to a room temperature of 1. The typical discrete S.typhi colonies are black and often surrounded by a
550C and pour. black or brownish black zone, which may be several times the size of the
Quality Control colony.
Dehydrated Appearance
2. In reflected light, preferably daylight, the zone exhibits a distinctly
Greenish yellow coloured, homogeneous, free flowing powder.
Prepared Appearance
characteristic metallic sheen. In heavy growth areas the organism frequently
Greenish yellow coloured opaque gel with a flocculent precipitate. appears as small light green colonies. This emphasizes the importance of
Cultural Response inoculating plates by the four quadrant method so that some areas are
Cultural characteristics after 40-48 hours at 350C. sparsely populated to give discrete colonies.
Organisms (ATCC) Growth Colour of colony 3. Other strains of Salmonella produce black to green colonies with little or no
Enterobacter aerogenes (13048) None to poor Brown to green*
darkening of the surrounding medium.
Enterococcus faecalis (29212) Inhibited -
Escherichia coli (25922) None to poor Brown to green* 4. Generally, Shigella species other than S.flexneri and S.sonnei are
Salmonella serotype Enteritidis Luxuriant Black with inhibited. These two, do grow on this medium to produce brown to green
(13076) metallicsheen raised colonies with depressed centers but show a crater like appearance.
Shigella flexneri (12022) None to poor Brown 5. E.coli is partially inhibited on this medium. If at all present, it produces
Salmonella serotype Typhi Luxuriant Black with metallic
small brown or greenish glistening colonies. The colour however, is confined
(19430) sheen
to the colony itself and shows no metallic sheen.
Key: *depends on the inoculum density.
Procedure 6. Enterobacter colonies if present exhibit a silvery sheen, appreciably lighter
1. Bismuth Sulphite Agar may be used in conjunction with other selective in colour than that produced by S.typhi.
enteric agars for the isolation of salmonellae by direct plating or from 7. To isolate S.typhi for agglutination or fermentation studies, pick
enrichment media. characteristic colonies from this medium and subculture on MacConkey
2. Inoculate directly on Bismuth Sulphite Agar and one or more of the following Agar. The purified colonies from MacConkey Agar may then be inoculated in
agars like Deoxycholate Citrate Agar, XLD Agar, Brilliant Green Agar, differential tubed media like TSI Agar.
MacConkeys Agar and SS Agar. 8. All cultures that give reactions consistent with Salmonella species on this
3. Also, inoculate in an enrichment broth such as Selenite F Broth or medium should be confirmed biochemically as Salmonella species before
Tetrathionate Broth Base. any serological testing is performed.
Precautions / Limitations
4. Subculture onto Bismuth Sulphite Agar and any of the other selective media
1. DO NOT AUTOCLAVE or overheat as it may destroy the selectivity of the
after 12-18 hours incubation. Examine the plates after 18 hours incubation
medium.
and subculture suspect colonies to identification media like TSI agar or

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 63
 Exploring... Accumix
2. Prepared plates should not be stored for longer than two days at 2- 80C; after very great number of organisms being produced. High concentrations of any
which time the dye oxidizes to give a green medium that can be inhibitory to organisms are potentially hazardous and must be disposed off safely. Therefore,
some salmonellae. after use, prepared plates, samples, sample containers or other contaminated
3. The medium may be inhibitory to some strains of salmonellae and therefore material must be sterilized or incinerated before discarding. All autoclaved
should not be used as the sole selective medium for these organisms. biohazards should be disposed off in accordance with state and local
S.sendai, S.berta, S.gallinarum, S.abortus-equi and S.cholerae-suis are environmental regulations.
markedly inhibited. Only qualified personnel who have been trained in microbiological procedures
4. It is important to streak for well isolated colonies. The typical colonial should handle all infected specimens and inoculated culture media. User should
characteristics will not develop if the growth is too heavy or confluent; ensure that any machinery or apparatus used and by chance contaminated must
S.typhi colonies will appear light green in these circumstances and may be safely disinfected or sterilized. The environment in which microbiological
thus be misinterpreted as negative growth for S.typhi. cultures are handled must also be taken into account.
Bibliography
5. S.typhi and S.arizonae are the only enteric organisms to exhibit typical
brown zones on the medium. However, S.arizonae is usually inhibited on 1. Wilson and Blair, 1926. J. Pathol. Bacteriol. 29:310.
this medium. 2. Wilson and Blair. 1931. J. Hyg. 31:138.
6. Some members of the coliform group that produce H2S may grow on this 3. US Pharmacopeial Convention, Inc. 2001. The United States
medium, giving colonies similar to S.typhi. However, they may be Pharmacopoeia 25/NF 20-2002. The US Pharmacopeial Convention, Inc;
differentiated because they produce gas from lactose in differential media, Rockville, Md.
for example, Triple Sugar Iron Agar. Proteus species may be differentiated 4. IP, 1996, Ministry of Health and Family Welfare, Govt. of India, Vol. 2.
on the basis of urea hydrolysis in Urea Broth or on Urea Agar Base. 5. US Food and Drug Adm; 1998, Bacteriological Analytical Manual, 8th Ed;
7. Colonies on this medium may be contaminated with other viable organisms; Rev. A, AOAC, International, Gaithersburg, Md.
therefore, isolated colonies should be subcultured to a less selective medium 6. Data on file: Microxpress, a division of Tulip Diagnostics (P) Ltd.
like MacConkey Agar. Storage
8. All plates should be incubated for a total of 48 hours to allow growth of all Store at 22-300C and prepared medium at 2-80C.
typhoid strains. Shelf Life
Use and Disposal of Dehydrated Culture Media Use before expiry date as mentioned on the label.
Inoculation of culture media with bacteria, deliberately or accidentally, leads to a

Bismuth Sulphite Agar Medium USP AM10132/AM50132

Use Bacteriological Analytical Manual for food testing.
Bismuth Sulphite Agar is a highly selective medium used for preliminary Principle
identification of Salmonella species, in compliance with USP. Beef extract and peptone provide nitrogen, growth factors and trace elements.
Summary Dextrose is the energy source. Disodium hydrogen phosphate is the buffer.
Bismuth Sulphite Agar is a modification of the original Wilson and Blair selective Bismuth sulphite and brilliant green are complimentary in inhibiting gram-
medium. It is recommended by various associations for the isolation and positive bacteria and intestinal gram-negative bacteria (coliform group) while
preliminary identification of Salmonella typhi and other salmonellae from allowing Salmonella to grow luxuriantly. This inhibitory action permits the use
pathological materials, food, sewage, water supplies, etc. More positive isolates of a much larger inoculum than possible with other media employed for similar
of S.typhi were obtained on this medium compared to Endo Agar, Eosin purposes. The use of larger inocula greatly increases the possibility of recovering
Methylene Blue Agar and Deoxycholate Agar. It is recommended by USP and IP the intestinal pathogens. Ferrous sulphate detects H2S production. S.typhi,
for use in microbial limit testing. For food testing, this medium is specified for the S.enteritidis and S.typhimurium typically grow as black colonies with surrounding
isolation of pathogenic bacteria from raw and pasteurized milk, cheese products, metallic sheen resulting from H2S production and reduction of sulphite to black
dry dairy products, cultured milk and butter. It is also included in the ferric sulphide. S.paratyphi A produces light green colonies. Shigellae species are

64 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
 Exploring... Accumix
mostly inhibited on this medium. 2. Inoculate directly on Bismuth Sulphite Agar and one or more of the following
Formula* agars like Deoxycholate Citrate Agar, XLD Agar, Brilliant Green Agar,
Ingredients in grams per liter MacConkeys Agar and SS Agar.
Beef extract 5.0
3. Also, inoculate in an enrichment broth such as Selenite F Broth or
Pancreatic digest of casein 5.0
Peptic digest of animal tissue 5.0
Tetrathionate Broth Base.
Dextrose 5.0 4. Subculture onto Bismuth Sulphite Agar and any of the other selective media
Sodium phosphate 4.0 after 12-18 hours incubation. Examine the plates after 18 hours incubation
Ferrous sulphate 0.3 and subculture suspect colonies to identification media like TSI agar or
Bismuth sulphite indicator 8.0 Kligler Iron Agar.
Brilliant green 0.025
Agar 20.0
For isolating Salmonella species from food:
0
Final pH (at 25 C) 7.6 ±0.2 1. Samples must be selectively enriched.
* Formula adjusted to suit performance parameters 2. Streak 10 microlitres of selective enrichment broth onto Bismuth Sulphite
Directions Agar.
1. Suspend 52.32 gms of the powder in 1000 ml distilled water. 3. Incubate plates for 24-48 hours at 350C.
2. Mix thoroughly. For isolation of Salmonella from clinical specimens:
3. Heat with frequent agitation and boil for 1 minute to dissolve the powder 1. Inoculate fecal specimens or rectal swabs onto a small area of the agar and
completely. streak using four-quadrant method to give discrete colonies.
4. DO NOT AUTOCLAVE.
2. Incubate plates at 24-48 hours at 350C and examine for colonies resembling
5. Disperse the precipitate evenly while dispensing (the sensitivity of the Salmonella species.
medium depends mainly upon uniform dispersion of freshly precipitated Interpretation of Results
bismuth sulphite in the final gel) and use the medium the same day it is 1. The typical discrete S.typhi colonies are black and often surrounded by a
prepared. black or brownish black zone, which may be several times the size of the
Quality Control
colony.
Dehydrated Appearance
Greenish yellow coloured, homogeneous, free flowing powder. 2. In reflected light, preferably daylight, the zone exhibits a distinctly
Prepared Appearance characteristic metallic sheen. In heavy growth areas the organism frequently
Greenish yellow coloured opaque gel with a flocculent precipitate. appears as small light green colonies. This emphasizes the importance of
Cultural Response inoculating plates by the four quadrant method so that some areas are
Cultural characteristics after 40-48 hours at 350C. sparsely populated to give discrete colonies.
Organisms (ATCC) Growth Colour of colony
3. Other strains of Salmonella produce black to green colonies with little or no
Enterobacter aerogenes (13048) None to poor Brown to green*
darkening of the surrounding medium.
Enterococcus faecalis (29212) Inhibited -
Escherichia coli (25922) None to poor Brown to green* 4. Generally, Shigella species other than S.flexneri and S.sonnei are
Salmonella serotype Enteritidis (13076) Luxuriant Black with metallic inhibited. These two, do grow on this medium to produce brown to green
sheen raised colonies with depressed centers but show a crater like appearance.
Shigella flexneri (12022) None to poor Brown
5. E.coli is partially inhibited on this medium. If at all present, it produces
Salmonella serotype Typhi (19430) Luxuriant Black with metallic
small brown or greenish glistening colonies. The colour however, is confined
sheen
Key: *depends on the inoculum density.
to the colony itself and shows no metallic sheen.
Procedure 6. Enterobacter colonies if present exhibit a silvery sheen, appreciably lighter
1. Bismuth Sulphite Agar may be used in conjunction with other selective in colour than that produced by S.typhi.
enteric agars for the isolation of salmonellae by direct plating or from 7. To isolate S.typhi for agglutination or fermentation studies, pick
enrichment media. characteristic colonies from this medium and subculture on MacConkey

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 65
 Exploring... Accumix
Agar. The purified colonies from MacConkey Agar may then be inoculated in 5. S.typhi and S.arizonae are the only enteric organisms to exhibit typical
differential tubed media like TSI Agar. brown zones on the medium. However, S.arizonae is usually inhibited on
8. All cultures that give reactions consistent with Salmonella species on this this medium.
medium should be confirmed biochemically as Salmonella species before 6. Some members of the coliform group that produce H2S may grow on this
any serological testing is performed. medium, giving colonies similar to S.typhi. However, they may be
Precautions / Limitations differentiated because they produce gas from lactose in differential media,
1. DO NOT AUTOCLAVE or overheat as it may destroy the selectivity of the for example, Triple Sugar Iron Agar. Proteus species may be differentiated
medium. on the basis of urea hydrolysis in Urea Broth or on Urea Agar Base.
2. Prepared plates should not be stored for longer than two days at 2- 80C; after 7. Colonies on this medium may be contaminated with other viable organisms;
which time the dye oxidizes to give a green medium that can be inhibitory to therefore, isolated colonies should be subcultured to a less selective medium
some salmonellae. like MacConkey Agar.
3. The medium may be inhibitory to some strains of salmonellae and therefore 8. All plates should be incubated for a total of 48 hours to allow growth of all
should not be used as the sole selective medium for these organisms. typhoid strains.
S.sendai, S.berta, S.gallinarum, S.abortus-equi and S.cholerae-suis are Storage
markedly inhibited. Store at 22-300C and prepared medium at 2-80C.
4. It is important to streak for well isolated colonies. The typical colonial Shelf Life
characteristics will not develop if the growth is too heavy or confluent; S.typhi Use before expiry date as mentioned on the label.
colonies will appear light green in these circumstances and may thus be
misinterpreted as negative growth for S.typhi.

Blood Agar Base AM1014/AM5014

Use Principle
Blood Agar Base is a non-selective general-purpose medium to which blood may Beef heart infusion and tryptose provide nitrogen, carbon and other growth
be added for use in isolation and cultivation of streptococci, and other fastidious factors. Sodium chloride maintains the osmotic balance. Supplementation with
pathogenic organisms like Neisseria, etc. It is also used for detection of blood provides additional growth factors for fastidious organisms and is the basis
haemolytic activity. for determining haemolytic reactions.
Summary Formula*
Without addition of blood the medium may be employed as a Nutrient Agar, or as Ingredients in grams per liter
Beef Heart, Infusion from 500.0
a medium for the short-term maintenance of stock cultures. With added blood or
Sodium Chloride 5.0
serum, the medium is suitable for the cultivation of many fastidious organisms as
Tryptose 10.0
well as determination of haemolytic reactions, which is an important diagnostic Agar 15.0
criteria for organisms like streptococci, staphylococci, etc. However, haemolytic Final pH (at 250C) 7.3 ± 0.2
reactions depend on the animal blood used. Group A streptococci gives best results * Formula adjusted to suit performance parameters
on sheep blood. Haemophilus haemolyticus colonies produce haemolysis and Directions
mimic Streptococcus pyogenes on horse blood. Norton found that slight acidic pH 1. Suspend 40.0 gms of the powder in 1000 ml distilled water.
(6.8 ± 0.2) shows distinct haemolytic reaction and is ideal for cultivation of 2. Mix thoroughly.
streptococci and pneumococci. The low pH also helps in stabilization of red blood
3. Boil with frequent agitation to dissolve the powder completely.
corpuscles and helps in producing clear haemolytic zone. Blood Agar Base is
specified in standard methods for food testing (20) and is included in the 4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Bacteriological Analytical Manual for testing of cosmetics (113). 5. To prepare Blood Agar, cool the base to 45-500C and aseptically add 5% v/v
sterile, defibrinated blood.

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6. Mix well and pour into sterile petri plates. Interpretation of Results

Quality Control Colony morphology of some organisms on Blood Agar containing 5% sheep
Dehydrated Appearance blood:
Yellow coloured, homogeneous free flowing powder. 1. Haemolytic streptococci may appear as opaque or translucent, greyish,
Prepared Appearance
small or large, matt or mucoid colonies, surrounded by a zone of haemolysis.
Basal Medium - Light amber coloured, slightly opalescent gel.
With addition of 5% v/v sterile defibrinated blood - Cherry red opaque gel. 2. Pneumococci usually appear as very flat, smooth, translucent, greyish and
Cultural Response sometimes mucoid colonies surrounded by a narrow zone of alpha (green)
Cultural characteristics after 18-48 hours at 35-370C. haemolysis.
Organisms (ATCC) Growth Growth Haemolysis 3. Staphylococci appear as opaque, white to golden yellow colonies with or
w/o blood w / blood
without zones of beta haemolysis.
Neisseria meningitidis (13090) Luxuriant Luxuriant None
Staphylococcus aureus (25923) Luxuriant Luxuriant Beta 4. Listeria may form small zones of beta haemolysis.
Staphylococcus epidermidis (12228) Luxuriant Luxuriant None 5. Other organisms of clinical significance may also grow on this medium.
Streptococcus pneumoniae (6303) Fair Luxuriant Alpha Precautions / Limitations
Streptococcus pyogenes (19615) Fair Luxuriant Beta 1. The animal blood used (horse or sheep) and the incubation conditions
Procedure
(aerobic or anaerobic) affect the haemolytic reactions of organisms on this
1. Use standard procedures like the streak plate method to obtain isolated medium.
colonies.
2. Colonies of Haemophilus haemolyticus are beta haemolytic on horse and
2. After streaking, stab the agar several times to deposit beta haemolytic rabbit blood agar and therefore must be distinguished from colonies of beta
streptococci beneath the agar surface. Subsurface growth will display the haemolytic streptococci. The use of sheep blood has been recommended to
most reliable haemolytic reactions owing to the activity of both oxygen obviate this problem since sheep blood does not support the growth of H.
stable and oxygen labile streptolysins. Haemolyticus.
3. Plates may be incubated in an atmosphere containing 3-10% carbon Storage
dioxide since many pathogens require carbon dioxide on primary isolation. Store at 22-300C and prepared medium at 2-80C.
4. Incubate at 35-370C for 18-24 hours. Shelf Life
Use before expiry date as mentioned on the label.

Bordet Gengou Agar Base AM1015/AM5015

Use haemolytic reactions, which help in the identification of B.pertussis. This medium
Bordet Gengou Agar Base is used for detection and isolation of Bordetella supports luxuriant growth of Bordetella and can be used for mass cultivation of
pertussis from clinical specimens. Bordetella pertussis for vaccine production and maintenance of stock cultures.
Summary Enrichment of the basal medium with 15% sheep blood aids in the detection of
Bordet Gengou Agar Base with the addition of sterile blood and glycerol is used in B.pertussis by virtue of its haemolytic reaction and with 25% human blood aids
clinical laboratories for diagnosing whooping cough. Specimens like aspirated in the detection of Mycobacterium species from small sputum inocula and
bronchial or nasopharyngeal secretions, perinasal swabs, etc; are used to isolate streptomycin sensitivity testing.
Bordetella pertussis, the causative agent of whooping cough. The present The medium can be made selective for Bordetella by the addition of Bordetella
formulation is a modification by Kendrich (54) of the Bordet Gengou Agar Selective Supplement, containing cephalexin at a concentration of 40 mg / litre.
originally formulated by Bordet and Gengou (7), and is prepared according to Amphotericin B at a concentration of 10 microgram per ml can be added as an
APHA requirement. antifungal agent.
Principle Formula*
Peptone and potato infusion provide carbon and nitrogen compounds while Ingredients in grams per liter
glycerol and blood provide additional nutrients and enable the detection of Potato, Infusion from 125.0

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Peptone 10.0 Bordetella Good to luxuriant Beta More than 70%
Sodium Chloride 5.5 pertussis (8467)
Agar 20.0 For growth RGI should be more than 70%
Final pH (at 250C) 6.7 ± 0.2 RGI- Relative Growth Index
* Formula adjusted to suit performance parameters Procedure
Directions 1. Use standard procedures like the streak plate method to obtain isolated
1. Suspend 40 gms of the powder in 1000 ml distilled water containing 10 ml colonies.
glycerol and mix thoroughly. 2. Incubate plates in an inverted position in a moist chamber (60% humidity) at
2. Boil with frequent agitation to dissolve the powder completely. 370C for up to 7 days.
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. 3. Examine the plates daily with or without a microscope to detect the presence of
4. Cool to 45-500C and aseptically add 15-20% sterile, fresh defibrinated blood B.pertussis and spreading bacteria or moulds that could mask the presence of
(sheep, rabbit, human or horse). this species.
5. Mix well, (avoid incorporation of air bubbles) and pour into sterile petri plates. 4. Plates may be discarded as negative if no growth occurs after incubation for 7
6. Two vials of Bordetella Selective Supplement (AS004) may be aseptically days.
added if required. Interpretation of Results

Quality Control 1. B.pertussis produce small, domed, glistening colonies that have a 'bisected
Dehydrated Appearance pearl' appearance surrounded by a zone of haemolysis with an indefinite
Light yellow coloured, homogeneous, free flowing powder. periphery. Some strains, however, are non-haemolytic.
Prepared Appearance Precautions / Limitations
Basal medium - Light yellow coloured, clear to slightly opalescent gel. 1. Some Haemophilus species, if present, will grow on this medium and cross-
With addition of sterile 15% defibrinated blood - Cherry red coloured, opaque
gel.
react with B.pertussis antisera. It may be prudent to rule out X and V factor
Cultural Response dependence.
Cultural characteristics after 3-4 days at 35-370C. Storage
Organisms (ATCC) Growth Haemolysis RGI Store at 22-300C and prepared medium at 2-80C.
Bordetella Good to luxuriant Gamma More than 70% Shelf Life
bronchiseptica (4617) Use before expiry date as mentioned on the label.

B. Q. Vaccine Medium AM50151

Use Formula*
B.Q. Vaccine Medium (Thioglycollate Broth w/ liver extract) is recommended for Ingredients in grams per liter

the cultivation of anaerobic organisms on large scale. Dipotassium phosphate 4.0

Summary Liver tissues 250.0
Black Quarter is an infectious bacterial disease of sheep and cattle, caused by Muscle tissues 250.0
Clostridium species. B.Q. Vaccine is used to vaccinate cattle to prevent Black Peptic digest of animal tissue 10.0
Quarter disease. B.Q. Vaccine Medium (Thioglycollate Broth w/ Liver extract) is
Sodium chloride 5.0
used for mass cultivation of anaerobes for the vaccine production.
Principle Sodium thioglycollate 1.0
0
Peptic digest of animal tissue serves as a source of nitrogen and carbon. Sodium Final pH (at 25 C) 8.2± 0.2
chloride maintains the osmotic balance. Liver and muscle tissue support the *Formula adjusted to suit performance parameters
growth of anaerobic bacteria. Sodium thioglycollate acts as reducing agent and Directions
maintains a low oxygen tension in the medium. 1. Suspend the 30 gms of powder in 1000 ml distilled water.

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2. Mix thoroughly. Organisms (ATCC) Growth
Clostridium sporogenes (11437) Luxuriant
3. Boil with frequent agitation to dissolve the powder completely. DO NOT
Clostridium perfringens (12919) Luxuriant
OVERHEAT. Bacillus cereus (10876) Good
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes. S. Pyogenes (19615) Luxuriant
5. Cool to 50°C and aseptically add 0.5% sterile glucose solution. Mix Procedure
thoroughly. Refer to appropriate references for specific procedures.
Quality Control Interpretation of Results
Dehydrated Appearance Refer to appropriate references and procedures for results.
Yellow coloured homogeneous free flowing powder. Storage
Prepared Appearance
Store at 22-300C and prepared medium at 2-80C.
Light amber coloured clear to slightly opalescent solution
Shelf Life
Cultural Response
0
Use before expiry date as mentioned on the label.
Cultural characteristics after 18-48 hours at at 35-37 C under anaerobic
condition.

Brain Heart Infusion Agar AM1016/AM5016

Use of pathogenic systemic fungi. A mixture of captan (0.005gm per liter) and
Brain Heart Infusion Agar is a general-purpose medium used for the cultivation chloramphenicol can also be used for selective isolation of pathogenic fungi
of wide variety of organisms including bacteria, yeasts and moulds. (incubation at 25-300C for 1-2 weeks). For the selective isolation of fungi,
Summary without blood, 0.5 microgram per ml of cycloheximide and 0.05 microgram per
Meat infusions were utilized as growth-supporting ingredients in a large number ml of chloramphenicol may be used. However, some fungi may be inhibited on
of culture media and though were cumbersome to prepare enabled the this medium with gentamicin, chloramphenicol and 10% sheep blood.
cultivation of organisms in both solid and liquid media. Nowadays, peptones Formula*
have largely replaced infusions as nutritional additives in culture media, however Ingredients in grams per liter
Beef Heart, Infusion from 250.0
infusions are still utilized in specific media. Brain Heart Infusion Agar can be used
Calf Brain, Infusion from 200.0
as a general medium for aerobic bacteriology and for the primary recovery of
Proteose Peptone 10.0
fungi from clinical specimens. Brain Heart Infusion Agar slant is used for the Sodium Chloride 5.0
cultivation and maintenance of pure cultures. This medium enriched with 10% Dextrose 2.0
sheep blood can be used to isolate systemic fungi that may grow poorly on non- Disodium Phosphate 2.5
enriched medium. It is also used for the cultivation of streptococci, Neisseria and Agar 15.0
other fastidious organisms. Brain Heart Infusion Agar is recommended by APHA Final pH (at 250C) 7.4 ± 0.2
for the examination of foods (20) and is included in the Bacteriological Analytical * Formula adjusted to suit performance parameters
Manual for testing of cosmetics (113). Directions

Principle 1. Suspend 52.0 gms of the powder in 1000 ml distilled water and mix well.
Peptone and infusion from calf brain and beef heart provide sources of nitrogen, 2. Boil with frequent agitation to dissolve the powder completely.
carbon, sulphur and other growth factors. Dextrose is the fermentable 3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
carbohydrate and disodium phosphate is the buffer. Sodium chloride maintains 4. Shake well to distribute the precipitate uniformly throughout the medium
the osmotic balance. Addition of defibrinated sheep blood provides additional and pour into sterile petri plates.
essential growth factors for more fastidious organisms. Addition of antimicrobials
5. If required, add antimicrobials to make the medium selective.
like 50 mg per liter of chloramphenicol or 40 mg per liter of streptomycin or
Quality Control
mixture of 50 mg of gentamicin and 50 mg chloramphenicol along with 5-10% Dehydrated Appearance
defibrinated blood is often recommended for inhibition of bacteria and isolation Light yellow coloured, homogeneous, free flowing powder.

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Prepared Appearance 4. To isolate fungi causing systemic mycoses, two sets of the medium should be
Basal Medium - Light to medium amber, clear to slightly opalescent gel. inoculated, one set incubated at 25-300C and the other set at 35-370C.
With addition of 5-10% sterile defibrinated blood - Cherry red coloured opaque
Interpretation of Results
gel.
Cultural Response 1. After proper incubation, the plates should show isolated colonies in streaked
0
Cultural characteristics after 18-24 hours at 35- 37 C. areas and confluent growth in areas of heavy inoculation.
Organisms (ATCC) Growth RGI 2. In cultures for fungi, examine plates for fungal colonies exhibiting typical
Candida albicans (26790) Luxuriant More than 70% colour and morphology.
Escherichia coli (25922) Luxuriant More than 70%
Shigella flexneri (12022) Luxuriant More than 70%
3. All cultures must be examined weekly for fungal growth and should be held
Staphylococcus aureus (25923) Luxuriant More than 70% for 4-6 weeks before being reported as negative.
Streptococcus pneumoniae (6303) Luxuriant More than 70% Precautions / Limitations
For Inhibition RGI should be 0% 1. Organisms like H.capsulatum, C.immitis and other pathogenic fungi can
RGI- Relative Growth Index produce free infective spores and therefore extreme care must be taken to
Procedure avoid dissemination of infective particles while culturing.
1. Use standard procedures like streak plate method to obtain isolated colonies. Storage
2. Since many pathogens require CO2 on primary isolation, plates of plain Brain Store at 22-300C and prepared medium at 2-80C.
Heart Infusion Agar may be incubated in an atmosphere containing Shelf Life
approximately 5-10% CO2 , at 35-370C for 24-48 hours. Use before expiry date as mentioned on the label.
3. For isolation of fungi from contaminated specimens, a selective medium may
be inoculated simultaneously and the plates incubated at 25-300C in an
inverted position with increased humidity.

Brain Heart Infusion Broth AM1017/AM5017

Use Brain Heart Infusion Broth containing 6.5% sodium chloride is used to
Brain Heart Infusion Broth is a highly nutritious general-purpose liquid medium differentiate enterococci from group D streptococci by the 6.5% salt tolerance
used for the cultivation of fastidious and non-fastidious microorganisms, test. With the addition of 10% defibrinated sheep blood, it is useful for the
including aerobic and anaerobic bacteria from a variety of clinical and non- isolation and cultivation of Histoplasma capsulatum. Fildes enrichment (peptic
clinical specimens. digest of sheep blood) can be incorporated for the cultivation of fastidious
Summary organisms like H.influenzae. This medium is especially useful as a growth and
Rosenow (96) developed the original medium by adding brain tissue to meat suspension medium for staphylococci, which is to be tested for coagulase
infusion or beef extract dextrose broth. Brain Heart Infusion Broth is used for production and supplemented with yeast extract, hemin and menadione, it was
cultivation of bacteria, yeasts and moulds and propagation of fastidious found to be better in producing heavy growth of five species of Bacteroides than
pathogenic cocci (streptococci, meningococci, pneumococci ) associated with three standard anaerobic broths. This medium without dextrose is used as a basal
blood culture work and allied pathological investigations. Brain Heart Infusion medium to which carbohydrates are added to study fermentation reactions.
Broth, 0.5 ml per tube is used for the cultivation of bacteria employed in the Principle
preparation of inoculum for micro dilution minimal inhibitory concentration (MIC) Calf brain, beef heart infusion and peptone provide carbon, nitrogen and other
and identification test panels. The medium can be used for the preparation of growth factors. Dextrose is the fermentable carbohydrate source and disodium
inoculum in antimicrobial susceptibility test procedures and for the cultivation of phosphate is the buffer. The addition of 0.1% agar helps in the cultivation of
anaerobes with the addition of 0.1% agar. Prereduced medium can be used for microaerophiles and anaerobes because it yields conditions of reduced oxygen
the cultivation of obligate anaerobes. Brain Heart Infusion Broth is included in the tension. In the formulation containing 6.5% sodium chloride, the salt acts as a
Bacteriological Analytical Manual for food and cosmetics testing (113) and is differential and / or selective agent by interfering with membrane permeability
recommended by APHA for the examination of foods (20) and milk (39). and osmotic and electro kinetic equilibrium in salt intolerant organisms.

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Formula* 2. Swab specimens may be inserted into Broth after inoculation of plated
Ingredients in grams per liter media.
Beef Heart, Infusion from 250.0
Calf Brain, Infusion from 200.0
3. For anaerobic incubation, the medium must be reduced prior to inoculation
Proteose Peptone 10.0 by placing the tubes, with caps loosened, under anaerobic conditions for 18-
Sodium Chloride 5.0 24 hours before use. This may be achieved through use of anaerobic
Dextrose 2.0 systems. Alternatively, the medium may be reduced immediately prior to
Disodium Phosphate 2.5 use by boiling with caps loosened and cooling with tightened caps to room
Final pH (at 250C) 7.4 ± 0.2 temperature before inoculation.
* Formula adjusted to suit performance parameters Interpretation of Results
Directions
1. Growth in tubes is indicated by turbidity.
1. Suspend 37.0 gms of the powder in 1000 ml distilled water and mix well.
2. Examine cultures by gram stain method and subculture onto appropriate
2. Boil with frequent agitation to dissolve the powder completely. media like Soyabean Casein Digest Agar with 5% sheep blood or EMB Agar.
3. Dispense in tubes or bottles as desired. 3. Incubate the subcultures anaerobically if anaerobes are suspected.
4. Sterilize at 1210C. 4. Enterococci grow in the medium containing 6.5% sodium chloride within
Quality Control
24-48 hours while non-enterococcal group D streptococci fail to grow.
Dehydrated Appearance
Precautions / Limitations
Light yellow coloured, homogeneous, free flowing powder.
Prepared Appearance 1. Tubes of Brain Heart Infusion Broth not used on the same day, should be
Light to medium amber coloured, clear solution without any precipitate. placed in boiling water bath for a few minutes to remove absorbed oxygen,
Cultural Response and cooled rapidly without shaking, just before use.
Cultural characteristics after 24-48 hours at 35-370C. 2. Brain Heart Infusion Broth with addition of 1.5% agar should not be used for
Organisms (ATCC) Growth detection of haemolytic activity of streptococci, since the presence of
Enterococcus faecalis (29212) Luxuriant
dextrose in it may cause atypical haemolytic reactions when used in blood
Neisseria meningitidis (13090) Luxuriant
containing media.
Streptococcus pneumoniae (6303) Luxuriant
Storage
Streptococcus pyogenes (19615) Luxuriant
Procedure Store at 22-300C and prepared medium at 2-80C.
1. For testing liquid specimens, the bottled or tubed media should be Shelf Life

inoculated with 1-2 drops of the specimen using a sterile pipette. Use before expiry date as mentioned on the label.

Brilliant Green Agar, Modified AM1018/AM5018

Use resemble Salmonella. S.cholerasuis grows well on this medium compared to
Brilliant Green Agar, Modified is used for selective isolation of Salmonella other Deoxycholate Citrate Agar. Brilliant Green Agar, Modified is recommended by
than S.typhi from clinical and non-clinical samples. APHA for food testing (20), USP (114) and IP (46).
Summary Principle
Kristensen et al (62) first described Brilliant Green Agar as a primary plating Proteose peptone provides carbon, nitrogen and other growth factors while yeast
medium for isolation of Salmonella, which was further modified by Kauffmann. extract provides B complex vitamins. Lactose and sucrose are the carbohydrate
Brilliant Green Agar, Modified is recommended for the isolation of Salmonella, sources. Sodium chloride maintains the osmotic balance. Brilliant green inhibits
other than S.typhi from faeces, water, meat and meat products, food and dairy majority of gram-positive and gram-negative bacteria, allowing Salmonella to
products and poultry and poultry products. This medium is more selective than grow. S.typhi, E.coli, Staphylococcus aureus, Shigella, Pseudomonas and
Deoxycholate Citrate Agar and other brilliant green media. The advantages Proteus species are mostly inhibited. In the presence of phenol red, lactose and
claimed for the medium are that it inhibits the growth of E.coli, Pseudomonas sucrose, non-fermenting Salmonella will form white to pinkish red colonies while
aeruginosa and partially inhibits the growth of Proteus species, which may fermenters will form yellow colonies.

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Formula* Salmonella serotype None to poor 0%
Ingredients in grams per liter Typhi (6539) Red
Proteose Peptone 10.0 For growth RGI should be more than 70%
Yeast Extract 3.0 For Inhibition RGI should be 0%
Sucrose 10.0 RGI- Relative Growth Index
Lactose 10.0 Interpretation of Results
Sodium Chloride 5.0 1. Salmonella species produce pinkish-white to red colonies surrounded by
Brilliant Green 0.0125
brilliant red zones in the medium.
Phenol Red 0.08
Agar 20.0 2. Lactose fermenting or sucrose fermenting organisms produce yellow to
0
Final pH (at 25 C) 6.9 ± 0.2 yellow-green colonies surrounded by yellow-green zones in the medium.
* Formula adjusted to suit performance parameters Proteus, Citrobacter and Pseudomonas species, if present may mimic
Directions enteric pathogens by producing small red colonies.
1. Suspend 58.0 gms of the powder in 1000 ml distilled water. Precautions / Limitations

2. Mix thoroughly. 1. Brilliant Green Agar, Modified being highly selective, it is recommended
that this medium be used along with a less inhibitory medium to increase
3. Boil with frequent agitation to dissolve the powder completely. AVOID
the chances of recovery. Often cultures enriched in Selenite Broth or
OVERHEATING.
Tetrathionate Broth Base is plated on this medium along with Bismuth
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Sulphite Agar, SS Agar, MacConkey Agar, DCA and XLD Agar.
5. To increase the selectivity, aseptically add 2 vials of Sulpha Supplement
2. The recovery of many Salmonella species is greatly reduced if the
(AS027) and mix well before pouring into sterile petri plates.
specimens (stool samples) remain unpreserved for more than 3 hours
Quality Control
before processing.
Dehydrated Appearance
Pink coloured, homogeneous, free flowing powder. 3. In cases of delay, inoculate the specimen onto an appropriate transport
Prepared Appearance media to maintain viability of the organisms.
Greenish brown coloured, clear to slightly opalescent gel. 4. Organisms other than Salmonella species, like Morganella morgani and
Cultural Response some Enterobacteriaceae may grow on this medium. Lactose fermenting
Cultural characteristics after 18-24 hours at 35-370C. S.arizona may be present in foods.
Organisms(ATCC) Growth Colony Colour RGI
Escherichia coli (25922) None to poor Yellowish green 0%
5. The medium is not recommended for isolation of S.typhi, S.paratyphi and
Salmonella serotype Luxuriant Pinkish white More than 70% Shigella species.
Enteritidis (13076) 6. Protect the medium from light to avoid discolouration.
Salmonella serotype Luxuriant Pinkish white More than 70% Storage
Typhimurium (14028)
Store at 22-300C and prepared medium at 2-80C.
Staphylococcus Inhibited - 0%
Shelf Life
aureus (25923)
Use before expiry date as mentioned on the label.

Brilliant Green Agar, Modified IP (Agar Medium L) AM10181/AM50181

Use Green Agar, Modified is recommended for the isolation of Salmonella, other than
Brilliant Green Agar, Modified is used for selective isolation of Salmonella other S. typhi from faeces, water, meat and meat products, food and dairy products
than S. typhi from clinical and non-clinical samples in compliance with IP. and poultry and poultry products. This medium is more selective than
Summary Deoxycholate Citrate Agar and other brilliant green media. The advantages
Kristensen et al., first described Brilliant Green Agar as a primary plating medium claimed for the medium are that it inhibits the growth of E. coli, Pseudomonas
for isolation of Salmonella, which was further modified by Kauffmann. Brilliant aeruginosa and partially inhibits the growth of Proteus species, which may

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resemble Salmonella. S. cholerasuis grows well on this medium compared to Prepared Appearance
Deoxycholate Citrate Agar. Brilliant Green Agar, Modified is recommended by Greenish brown coloured, clear to slightly opalescent gel.
Cultural Response
APHA for food testing , USP and IP .
Cultural characteristics after 18-24 hours at 35-37ºC.
Principle
Organisms(ATCC) Growth Colour of colony RGI
Peptone provides carbon, nitrogen and other growth factors while yeast extract Escherichia coli (25922) None to poor Yellowish green 0% or 70%
provides B complex vitamins. Lactose and sucrose are the carbohydrate sources. Salmonella serotype Luxuriant Pinkish white more than 70%
Sodium chloride maintains the osmotic balance. Brilliant green inhibits majority Enteritidis (13076)
Salmonella serotype Luxuriant Pinkish white more than 70%
of gram-positive and gram-negative bacteria, allowing Salmonella to grow. S.
Typhimurium (14028)
typhi, E. coli, Staphylococcus aureus, Shigella, Pseudomonas and Proteus species Staphylococcus Inhibited - 0%
are mostly inhibited. In the presence of phenol red, lactose and sucrose, non- aureus (25923)
fermenting Salmonella will form white to pinkish red colonies while fermenters Salmonella serotype None to poor Red 0% or 70%
will form yellow colonies. Typhi (6539)
Formula* Precautions / Limitations
Ingredients in grams per liter 1. Brilliant Green Agar, Modified being highly selective, it is recommended that
Peptones (meat and casein) 10.0 this medium be used along with a less inhibitory medium to increase the
Yeast extract 3.0 chances of recovery. Often cultures enriched in Selenite Broth or Tetrathionate
Sucrose 10.0 Broth Base is plated on this medium along with Bismuth Sulphite Agar, SS
Lactose monohydrate 10.0
Agar, MacConkey Agar, DCA and XLD Agar.
Sodium chloride 5.0
Brilliant green 0.0125 2. The recovery of many Salmonella species is greatly reduced if the specimens
Phenol red 0.08 (stool samples) remain unpreserved for more than 3 hours before processing.
Agar 12.0 3. In cases of delay, inoculate the specimen onto an appropriate transport
Final pH (at 25ºC) 6.9 ± 0.2 media to maintain viability of the organisms.
* Formula adjusted to suit performance parameters 4. Organisms other than Salmonella species, like Morganella morgani and
Directions some Enterobacteriaceae may grow on this medium. Lactose fermenting S.
1. Suspend 50.09 gms of the powder in 1000 ml distilled water. arizona may be present in foods.
2. Mix thoroughly. 5. The medium is not recommended for isolation of S. typhi, S. paratyphi and
3. Boil with frequent agitation to dissolve the powder completely. AVOID Shigella species.
OVERHEATING. 6. Protect the medium from light to avoid discolouration.
4. Sterilize by autoclaving at 121ºC (15 lbs pressure) for 15 minutes. Storage

5. To increase the selectivity, aseptically add 2 vials of Sulpha Supplement Store at 22-300C and prepared medium at 2-80C.
Shelf Life
(AS027) and mix well before pouring into sterile petri plates.
Use before expiry date as mentioned on the label.
Quality Control
Dehydrated Appearance
Pink coloured, homogeneous, free flowing powder.

Brilliant Green Agar, Modified USP AM10182/AM50182

Brilliant Green Agar, Modified (Agar Medium L) BP AM10184/AM50184
Use Summary
Brilliant Green Agar, Modified is used for selective isolation of Salmonella other Kristensen et al., first described Brilliant Green Agar as a primary plating medium
than S.typhi from clinical and non-clinical samples. for isolation of Salmonella, which was further modified by Kauffmann. Brilliant

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Green Agar, Modified is recommended for the isolation of Salmonella, other than Quality Control
S.typhi from faeces, water, meat and meat products, food and dairy products Dehydrated Appearance
Pink coloured, homogeneous, free flowing powder.
and poultry and poultry products. This medium is more selective than
Prepared Appearance
Deoxycholate Citrate Agar and other brilliant green media. The advantages
Greenish brown coloured, clear to slightly opalescent gel.
claimed for the medium are that it inhibits the growth of E.coli, Pseudomonas Cultural Response
aeruginosa and partially inhibits the growth of Proteus species, which may Cultural characteristics after 18-24 hours at 35-370C.
resemble Salmonella. S.cholerasuis grows well on this medium compared to Organisms(ATCC) Growth Colour of RGI
Deoxycholate Citrate Agar. Brilliant Green Agar, Modified is recommended by colony
APHA for food testing , USP and BP . Escherichia coli None to Yellowish 0% or More than
Principle (25922) poor green 70%
Salmonella serotype
Proteose peptone provides carbon, nitrogen and other growth factors while yeast
Enteritidis (13076) Luxuriant Pinkish white More than 70%
extract provides B complex vitamins. Lactose and sucrose are the carbohydrate
Salmonella serotype
sources. Sodium chloride maintains the osmotic balance. Brilliant green inhibits Typhimurium (14028) Luxuriant Pinkish white 0% or More than
majority of gram-positive and gramnegative bacteria, allowing Salmonella to 70%
grow. S.typhi, E.coli, Staphylococcus aureus, Shigella, Pseudomonas and Staphylococcus Inhibited - 0%
Proteus species are mostly inhibited. In the presence of phenol red, lactose and aureus (25923)
sucrose, non-fermenting Salmonella will form white to pinkish red colonies Salmonella serotype None to Red 0% or More than
while fermenters will form yellow colonies. Typhi (6539) poor 70%
Formula* For growth RGI should be more than 70%
Ingredients in grams per liter USP BP For Inhibition RGI should be 0%
Peptic digest of animal tissue 5.0 - RGI- Relative Growth Index
Pancreatic digest of casein 5.0 - Precautions / Limitations
Peptones (meat and casein) - 10.0 1. Brilliant Green Agar, Modified being highly selective, it is recommended that
Yeast extract - 3.0 this medium be used along with a less inhibitory medium to increase the
Sucrose 10.0 10.0 chances of recovery. Often cultures enriched in Selenite Broth or
Lactose 10.0 - Tetrathionate Broth Base is plated on this medium along with Bismuth
Lactose monohydrate - 10.0
Sulphite Agar, SS Agar, MacConkey Agar, DCA and XLD Agar.
Sodium chloride 5.0 5.0
Brilliant green 0.0125 0.0125 2. The recovery of many Salmonella species is greatly reduced if the specimens
Phenol red 0.08 0.08 (stool samples) remain unpreserved for more than 3 hours before
Agar 20.0 20.0 processing.
Final pH (at 250C) 6.9 ± 0.2 3. In cases of delay, inoculate the specimen onto an appropriate transport
* Formula adjusted to suit performance parameters
media to maintain viability of the organisms.
Directions 4. Organisms other than Salmonella species, like Morganella morgani and
some Enterobacteriaceae may grow on this medium. Lactose fermenting
1. Suspend 58.09 gms of the powder in 1000 ml distilled water.
S.arizona may be present in foods.
2. Mix thoroughly.
5. The medium is not recommended for isolation of S.typhi, S.paratyphi and
3. Boil with frequent agitation to dissolve the powder completely. AVOID
Shigella pecies.
OVERHEATING.
6. Protect the medium from light to avoid discolouration.
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. Storage
5. To increase the selectivity, aseptically add 2 vials of Sulpha Supplement Store at 22-300C and prepared medium at 2-80C.
(AS027)and mix well before pouring into sterile petri plates. Shelf Life
Use before expiry date as mentioned on the label.

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Brilliant Green, Phenol Red, Lactose Monohydrate, AM10183/AM50183
Sucrose Agar (Agar Medium L) EP
Use OVERHEATING.
Brilliant Green Agar, Modified is used for selective isolation of Salmonella other 4. Sterilize by autoclaving at 121ºC (15 lbs pressure) for 15 minutes.
than S. typhi from clinical and non-clinical samples.
5. To increase the selectivity, aseptically add 2 vials of Sulpha Supplement
Summary
(AS027) and mix well before pouring into sterile petri plates.
Kristensen et al., first described Brilliant Green Agar as a primary plating medium Quality Control
for isolation of Salmonella, which was further modified by Kauffmann. Brilliant Dehydrated Appearance
Green Agar, Modified is recommended for the isolation of Salmonella, other than Pink coloured, homogeneous, free flowing powder.
S. typhi from faeces, water, meat and meat products, food and dairy products Prepared Appearance
and poultry and poultry products. This medium is more selective than Greenish brown coloured, clear to slightly opalescent gel.
Deoxycholate Citrate Agar and other brilliant green media. The advantages Cultural Response
Cultural characteristics after 18-24 hours at 35-37ºC.
claimed for the medium are that it inhibits the growth of E. coli, Pseudomonas
Organisms(ATCC) Growth Colour of colony RGI
aeruginosa and partially inhibits the growth of Proteus species, which may
Escherichia coli None to Yellowish green 0% or More
resemble Salmonella. S. cholerasuis grows well on this medium compared to (25922) poor than 70%
Deoxycholate Citrate Agar. Brilliant Green Agar, Modified is recommended by Salmonella serotype
APHA for food testing , USP , BP (9.1) and EP (26.1) . Enteritidis (13076) Luxuriant Pinkish white More than 70%
Principle Salmonella serotype
Peptone provides carbon, nitrogen and other growth factors while yeast extract Typhimurium (14028) Luxuriant Pinkish white More than 70%
provides B complex vitamins. Lactose and sucrose are the carbohydrate sources. Staphylococcus Inhibited - 0%
aureus (25923)
Sodium chloride maintains the osmotic balance. Brilliant green inhibits majority
Salmonella serotype None to Red 0% or More
of gram-positive and gram-negative bacteria, allowing Salmonella to grow. S.
Typhi (6539) poor than 70%
typhi, E. coli, Staphylococcus aureus, Shigella, Pseudomonas and Proteus For growth RGI should be more than 70%
species are mostly inhibited. In the presence of phenol red, lactose and sucrose, For Inhibition RGI should be 0%
non-fermenting Salmonella will form white to pinkish red colonies while RGI- Relative Growth Index
fermenters will form yellow colonies. Precautions / Limitations
Formula* 1. Brilliant Green Agar, Modified being highly selective, it is recommended that
Ingredients in grams per liter this medium be used along with a less inhibitory medium to increase the
Peptones (meat and casein) 10.0
chances of recovery. Often cultures enriched in Selenite Broth or
Yeast extract 3.0
Tetrathionate Broth Base is plated on this medium along with Bismuth
Sucrose 10.0
Lactose monohydrate 10.0
Sulphite Agar, SS Agar, MacConkey Agar, DCA and XLD Agar.
Sodium chloride 5.0 2. The recovery of many Salmonella species is greatly reduced if the specimens
Brilliant green 0.0125 (stool samples) remain unpreserved for more than 3 hours before
Phenol red 0.08 processing.
Agar 20.0
3. In cases of delay, inoculate the specimen onto an appropriate transport
Final pH (at 25ºC) 6.9 ± 0.2
* Formula adjusted to suit performance parameters
media to maintain viability of the organisms.
Directions 4. Organisms other than Salmonella species, like Morganella morgani and
1. Suspend 58.0 gms of the powder in 1000 ml distilled water. some Enterobacteriaceae may grow on this medium. Lactose fermenting S.
2. Mix thoroughly. arizona may be present in foods.

3. Boil with frequent agitation to dissolve the powder completely. AVOID 5. The medium is not recommended for isolation of S. typhi, S. paratyphi and
Shigella species.

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6. Protect the medium from light to avoid discolouration. Shelf Life
Storage Use before expiry date as mentioned on the label.
0 0
Store at 22-30 C and prepared medium at 2-8 C.

Brilliant Green Bile Agar AM1019/AM5019

Use 2. Boil with frequent agitation to dissolve the powder completely.
Brilliant Green Bile Agar is used for isolating, differentiating and enumerating 3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
coliform bacteria.
4. For plating 10 ml quantities of water samples, prepare the medium in
Summary
double strength.
Noble and Tonney (85) described Brilliant Green Bile Agar for determining the Warning: Basic fuchsin is a potent carcinogen and must be handled with
relative density of coliform bacteria in water and sewage and its usefulness in care so as to avoid inhalation or contact with skin.
selectively isolating Salmonella species from other coliforms. APHA has Quality Control
approved this medium for the estimation of number of coliforms in test samples of Dehydrated Appearance
various materials (20). Light purple coloured, homogeneous free flowing powder.
Principle Prepared Appearance
Bluish purple coloured, slightly opalescent gel.
Peptone provides nitrogen, carbon, vitamins and other growth factors. Lactose is
Cultural Response
the fermentable carbohydrate. Basic fuchsin and erioglaucine are the pH Cultural characteristics after 18-24 hours at 35-370C.
indicators while monopotassium phosphate is the buffer. Oxgall and brilliant Organisms(ATCC) Growth Colour of RGI
green combination is highly selective for coliforms, inhibiting most of the gram- colony
positive and gram-negative bacteria. Enterobacter Luxuriant Pink More than 70%
When pH is neutral, colour of the medium is blue, while acid production from aerogenes (13048)
Escherichia coli Luxuriant Deep red with More than 70%
lactose turns the medium pink. Differentiation of coliforms is based on
(25922) bile precipitate
fermentation of lactose. Coliform bacteria typically ferment lactose producing
Salmonella serotype Luxuriant Colourless to More than 70%
acid, and in the presence of basic fuchsin produce deep red colonies surrounded Enteritidis (13076) light pink
by a pink halo against blue background of the medium, while Salmonella which Staphylococcus Inhibited - 0%
do not ferment lactose, produce colourless to faint pink colonies. aureus (25923)
Formula*
Ingredients in grams per liter For growth RGI should be more than 70%
Peptone 8.25 For Inhibition RGI should be 0%
Lactose 1.9 RGI- Relative Growth Index
Sodium Sulphite 0.205 Precautions
Ferric Chloride 0.0295
1. The medium must be prepared just prior to use and when necessary to store
Basic Fuchsin 0.0776
Erioglaucine 0.0649
the medium, it should be kept in the dark.
Monopotassium Phosphate 0.0153 2. The medium is sensitive to light, particularly direct sunlight, which may
Oxgall 0.00295 cause a decrease in the productivity of the medium and a change in colour
Brilliant Green 0.0000295 from deep blue to purple or red.
Agar 10.15
Storage
Final pH (at 250C) 6.9 ± 0.2
* Formula adjusted to suit performance parameters Store at 22-300C and prepared medium at 2-80C.
Directions Shelf Life
Use before expiry date as mentioned on the label.
1. Suspend 20.7 gms of the powder in 1000 ml distilled water and mix well.

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Brilliant Green Bile Broth 2% AM1020/AM5020
Use Cultural Response
Brilliant Green Bile Broth 2% is used for the detection of coliform organisms in Cultural characteristics after 18-48 hours at 35-370C.
water and wastewater, foods, milk and dairy products, as well as in other Organisms (ATCC) Growth Gas
Bacillus cereus (10876) Inhibited -
materials of sanitary importance.
Enterobacter aerogenes (13048) Luxuriant +
Summary
Enterococcus faecalis (19433) None to poor -
Brilliant Green Bile Broth 2% is formulated as per American Public Health Escherichia coli (25922) Luxuriant +
Association specifications for use in presumptive identification and confirmation Staphylococcus aureus (25923) Inhibited -
of coliforms (20, 36, 39). It is included in the Bacteriological Analytical Manual Procedure
for food testing (113). 1. Food macerates are decimally diluted and added to the broth in the
Principle proportion 1:10.
Peptone provides the essential nutrients. Lactose is the fermentable carbohydrate. 2. Double strength broth can be used for large volume samples. Incubation can
Oxgall and brilliant green inhibit most of the gram-positive organisms including be carried out at 440C for 48 hours to detect E.coli, at 320C for 25-48 hours
lactose fermenting clostridia and selected gram-negative organisms. Coliforms, to detect coli-aerogenes and at 40C for 10 days to detect psychrotrophic
which are resistant to the action of inhibitors and which ferment lactose, can coliform organisms.
replicate in this medium. Fermentation is detected by gas production and is seen
3. In water plants, control tests where less than 1 ml to 10 ml volumes is used,
as bubbles in the inverted Durham's tubes. Production of gas in the inverted
it is important not to over dilute the medium. Thus 1 ml or less volumes of
Durham's tube indicates presence of faecal coliforms, since non-faecal coliforms
water can be added to 10 ml of Brilliant Green Bile Broth. For 10 ml volumes
growing in this medium do not produce gas.
of water, double strength Brilliant Green Bile Broth should be used in equal
It is important that the inhibitory agents in the medium are balanced with the volumes.
nutrient and mineral components, so that clostridial and bacillus spores if Interpretation of Results
present will not give false positive reactions in the medium e.g. gas formation. 1. The medium becomes turbid and yellowish-green in colour when bacterial
Formula*
growth occurs, and when accompanied by copious gas formation, it is
Ingredients in grams per liter
presumptive of the presence of coli-aerogenes organisms.
Oxgall 20.0
Lactose 10.0 2. Turbidity and gas production in the Brilliant Green Bile Broth 2% incubated
Peptone 10.0 at 440C is indicative of a positive test for E.coli. To confirm the presence of
Brilliant Green 0.0133 E.coli carry out indole production test at 440C in Tryptone Water.
Final pH (at 250C) 7.2 ± 0.2 Precautions / Limitations
* Formula adjusted to suit performance parameters
1. Do not autoclave double strength broth.
Directions
2. Gram-positive sporing organisms may produce gas to give a false positive
1. Suspend 40.01 gms of the powder in 1000 ml distilled water and mix well.
reaction if the bile / brilliant green inhibition is compromised by the food
2. Warm slightly to dissolve the powder completely. material.
3. Dispense into tubes containing inverted Durham's tubes. 3. Turbidity alone is not a positive test for coliforms.
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. 4. It may be necessary to invert the tube prior to inoculation if bubbles are
5. DO NOT AUTOCLAVE DOUBLE STRENGTH BROTH. trapped in the Durham's tube.
Quality Control Storage
Dehydrated Appearance
Store at 22-300C and prepared medium at 2-80C.
Greenish yellow coloured, homogeneous, free flowing powder.
Shelf Life
Prepared Appearance
Use before expiry date as mentioned on the label.
Emerald green coloured, clear solution, without any precipitate.

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Bromothymol Blue Lactose Agar AM1021/AM5021
Use Quality Control
Bromothymol Blue Lactose Agar is used for the detection and isolation of Dehydrated Appearance
Greenish yellow coloured, homogeneous, free flowing powder.
pathogenic staphylococci.
Prepared Appearance
Summary
Greenish blue coloured, clear to slightly opalescent gel.
Bromothymol Blue Lactose Agar was developed by Chapman et al (15) for the Cultural Response
detection and isolation of pathogenic staphylococci. Cultural characteristics after 24-48 hours at 350C.
Principle Organisms (ATCC) Growth Colour of colony
Beef extract and proteose peptone provide sources of carbon and nitrogen. Escherichia coli (25922) Luxuriant Yellow
Lactose is the carbohydrate source and bromothymol blue is the acid-base Salmonella serotype Enteritidis (13076) Luxuriant Blue / colourless
indicator. Pathogenic saphylococci are differentiated by their ability to grow at a Salmonella serotype Typhi (6539) Luxuriant Blue / colourless
Staphylococcus aureus (6538) Luxuriant Golden yellow
high pH and in the presence of bromothymol blue indicator.
Staphylococcus epidermidis (12228) Luxuriant Blue / colourless
Formula*
Procedure
Ingredients in grams per liter
Beef Extract 3.0 1. Plates should be inoculated preferably by spread plate method and
Proteose Peptone 5.0 incubated for about 36 hours at 350C.
Lactose 10.0 Interpretation of Results
Bromothymol Blue 0.17 1. Typical colonies of staphylococci appear deep / golden yellow about
Agar 15.0 90%, blue grey about 10%.
Final pH (at 250C) 8.6 ± 0.2
Precautions / Limitations
* Formula adjusted to suit performance parameters
Directions
1. Coliforms and other organisms may also grow on this medium but are
differentiated by their appearance.
1. Suspend 33.17 gms of the powder in 1000 ml distilled water.
Storage
2. Mix thoroughly.
Store at 22-300C and prepared medium at 2-80C.
3. Boil with frequent agitation to dissolve the powder completely. Shelf Life
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. Use before expiry date as mentioned on the label.

Buffered Peptone Water AM10211/AM50211

Use
the recovery and growth of cells prior to selective enrichment, BPW buffers the pH
Buffered peptone water used for pre-enrichment of injured Salmonella species. It
of the growth system against pH changes brought about by the growth and
increases recovery of Salmonella species prior to selective enrichment from food.
metabolism of microorganisms during enrichment and those imposed by the food
Summary
sample (6.1).
Buffered peptone water (BPW) is one of the most widely used pre enrichment Principle
broths for Salmonella in a wide range of foods. Moreover, it is often the medium
Proteose peptone serves as a source of carbon, nitrogen, vitamins and minerals.
of choice in many published reference methods, including the current
Sodium chloride provides sodium ions for the membrane transport and maintains
International Organization for Standardization (ISO) method, for detection of
osmotic equilibrium of the medium. Phosphates buffer the medium.
Salmonella in foods. Edel and Kampelmaacher in their comparative study
Formula*
observed that food preservation methods cause subleathal injury to Salmonellae. Ingredients in grams per liter
It is known that freezing and drying may injure Salmonellae so that they are Proteose peptone 10.0
unable to multiply in selective media (23.1). Pre enrichment of sublethally Sodium chloride 5.0
injured Salmonellae in buffered peptone water showed superior results in Disodium phosphate 3.5
comparison with direct selection methods. In addition to providing conditions for Monopotassium phosphate 1.5

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Final pH (at 250C) 7.2 ±0.2 Organisms (ATCC) Growth
* Formula adjusted to suit performance parameters Salmonella serotype Enteritidis (13076) Luxuriant
Directions Salmonella serotype Typhi (19430) Luxuriant
1. Suspend the 20 gms of powder in 1000 ml distilled water. Salmonella serotype Typhimurium (14028) Luxuriant
Procedure
2. Mix thoroughly.
Test specimens as per recommended guidelines.
3. Warm slightly with frequent agitation to dissolve the powder completely.
Interpretation of Results
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes. Growth in the medium is indicated by the presence of turbidity compared to an
Quality Control
uninoculated control.
Dehydrated Appearance
Storage
Light yellow coloured, homogeneous, free flowing powder.
Prepared Appearance Store at 22-300C and prepared medium at 2-80C.
Light yellow coloured, clear solution without any precipitate. Shelf Life
Cultural Response Use before expiry date as mentioned on the label.
Cultural characteristics after 18-24 hours at 35-370C.

Buffered Peptone Water BIS AM10212/AM50212

Buffered Peptone Water ISO AM10213/AM50213
Use Disodium hydrogen phosphate anhydrous 3.5
Buffered Peptone Water used for pre-enrichment of injured Salmonella species. Potassium dihydrogen phosphate (KH2 PO4 ) 1.5

It increases recovery of Salmonella species prior to selective enrichment from Final pH (at 250C) 7.0 ±0.2
* Formula adjusted to suit performance parameters
food.
Directions
Summary
1. Suspend the 20.0 gms of powder in 1000 ml distilled water.
Buffered Peptone Water (BPW) is one of the most widely used pre enrichment
broths for Salmonella in a wide range of foods. Moreover, it is often the medium 2. Mix thoroughly.
of choice in many published reference methods, including the current 3. Warm slightly with frequent agitation to dissolve the powder completely.
International Organisation for Standardisation (ISO) method, for detection of 4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Salmonella in foods. Edel and Kampelmaacher in their comparative study Quality Control
observed that food preservation methods cause subleathal injury to Salmonellae. Dehydrated Appearance
It is known that freezing and drying may injure Salmonellae so that they are Light yellow coloured, homogeneous, free flowing powder.
unable to multiply in selective media. Pre enrichment of sublethally injured Prepared Appearance
Salmonellae in buffered peptone water showed superior results in comparison Light yellow coloured, clear solution without any precipitate.
Cultural Response
with direct selection methods. In addition to providing conditions for the recovery
Cultural characteristics after 16-20 hours at 35-370C.
and growth of cells prior to selective enrichment, BPW buffers the pH of the growth
Organisms (ATCC) Growth
system against pH changes brought about by the growth and metabolism of Salmonella serotype Enteritidis (13076) Luxuriant
microorganisms during enrichment and those imposed by the food sample. Salmonella serotype Typhi (19430) Luxuriant
Principle Salmonella serotype Typhimurium (14028) Luxuriant
Enzymatic digest of casein serves as a source of carbon, nitrogen, vitamins and Procedure
minerals. Sodium chloride provides sodium ions for the membrane transport and Test specimens as per recommended guidelines.
maintains osmotic equilibrium of the medium. Phosphates buffer the medium. Storage
Formula* Store at 22-300C and prepared medium at 2-80C.
Ingredients in grams per liter Shelf Life
Enzymatic digest of casein 10.0 Use before expiry date as mentioned on the label.
Sodium chloride 5.0

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Buffered Sodium Chloride-Peptone Solution pH7.0 AMH50214

(Harmonized)
Use Final pH (at 250C) 7.0 ±0.2
Buffered Sodium Chloride-Peptone Solution pH 7.0 is used for pre-enrichment of * Formula adjusted to suit performance parameters
* (equivalent to 0.067 M phosphate)
injured Salmonella species. It increases recovery of Salmonella species prior to
Directions
selective enrichment from food.
Summary 1. Suspend the 16.1 gms of powder in 1000 ml distilled water.
Buffered peptone water (BPW) is one of the most widely used pre enrichment 2. Mix thoroughly.
broths for Salmonella in a wide range of foods. Moreover, it is often the medium 3. Warm slightly with frequent agitation to dissolve the powder completely.
of choice in many published reference methods, including the current 4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
International Organization for Standardization (ISO) method, for detection of Note: To this solution surface active agents or inactivators of antimicrobial
Salmonella in foods. Edel and Kampelmaacher in their comparative study agents may be added before autoclave, such as: Polysorbate 80 or
observed that food preservation methods cause subleathal injury to Salmonellae. Polysorbate 20 in1-10 gms per liter.
It is known that freezing and drying may injure Salmonellae so that they are Quality Control
unable to multiply in selective media. Pre enrichment of sublethally injured Dehydrated Appearance
Salmonellae in buffered peptone water showed superior results in comparison Light yellow coloured, homogeneous, free flowing powder.
with direct selection methods. In addition to providing conditions for the recovery Prepared Appearance
and growth of cells prior to selective enrichment, BPW buffers the pH of the growth Light yellow coloured, clear solution without any precipitate.
system against pH changes brought about by the growth and metabolism of Cultural Response
microorganisms during enrichment and those imposed by the food sample. Cultural characteristics after 18-24 hours at 35-370C.
Organisms (ATCC) Growth
Principle
Salmonella serotype Enteritidis (13076) Luxuriant
Proteose peptone serves as a source of carbon, nitrogen, vitamins and minerals. Salmonella serotype Typhi (19430) Luxuriant
Sodium chloride provides sodium ions for the membrane transport and maintains Salmonella serotype Typhimurium (14028) Luxuriant
osmotic equilibrium of the medium. Phosphates buffer the medium. Procedure
Formula* Test specimens as per recommended guidelines.
Ingredients in grams per liter
Storage
Peptone (meat and casein) 1.00
Sodium chloride 4.30
Store at 22-300C and prepared medium at 2-80C.
Disodium hydrogen phosphate dihydrate 7.23* Shelf Life
Potassium dihydrogen phosphate 3.6 Use before expiry date as mentioned on the label.

Buffered Peptone Water with NaCl AM50214

Use observed that food preservation methods cause subleathal injury to Salmonellae.
Buffered peptone water used for pre-enrichment of injured Salmonella species. It It is known that freezing and drying may injure Salmonellae so that they are
increases recovery of Salmonella species prior to selective enrichment from food. unable to multiply in selective media. Pre enrichment of sublethally injured
Summary Salmonellae in buffered peptone water showed superior results in comparison
Buffered peptone water (BPW) is one of the most widely used pre enrichment with direct selection methods. In addition to providing conditions for the recovery
broths for Salmonella in a wide range of foods. Moreover, it is often the medium and growth of cells prior to selective enrichment, BPW buffers the pH of the growth
of choice in many published reference methods, including the current system against pH changes brought about by the growth and metabolism of
International Organization for Standardization (ISO) method, for detection of microorganisms during enrichment and those imposed by the food sample.
Salmonella in foods. Edel and Kampelmaacher in their comparative study

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Principle Quality Control
Proteose peptone serves as a source of carbon, nitrogen, vitamins and minerals. Dehydrated Appearance
Light yellow coloured, homogeneous, free flowing powder.
Sodium chloride provides sodium ions for the membrane transport and maintains
Prepared Appearance
osmotic equilibrium of the medium. Phosphates buffer the medium.
Light yellow coloured, clear solution without any precipitate.
Formula*
Cultural Response
Ingredients in grams per liter
Cultural characteristics after 18-24 hours at 35-370C.
Peptone ( meat and casein) 1.00
Organisms (ATCC) Growth
Sodium chloride 4.30
Salmonella serotype Enteritidis (13076) Luxuriant
Disodium hydrogen phosphate 7.23
Salmonella serotype Typhi (19430) Luxuriant
Potassium dihydrogen phosphate 3.56
Salmonella serotype Typhimurium (14028) Luxuriant
Final pH (at 250C) 7.0 ±0.2
Procedure
* Formula adjusted to suit performance parameters
Directions
Test specimens as per recommended guidelines.
1. Suspend the 16.09 gms of powder in 1000 ml distilled water. Interpretation of Results
2. Mix thoroughly. Growth in the medium is indicated by the presence of turbidity compared to an
uninoculated control
3. Warm slightly with frequent agitation to dissolve the powder completely.
Storage
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Store at 22-300C and prepared medium at 2-80C.
Note: To this solution surface active agents or inactivators of antimicrobial Shelf Life
agents may be added before autoclave, such as: Polysorbate 80 or Use before expiry date as mentioned on the label.
Polysorbate 20 in1-10 gms per liter.

Buffered Peptone Water with NaCl IP AM50215

Buffered Peptone Water with NaCl USP AM502151
Buffered Peptone Water with NaCl EP AM50216
Use microorganisms during enrichment and those imposed by the food sample.
Buffered Peptone Water with NaCl used for pre-enrichment of injured Salmonella Principle
species. It increases recovery of Salmonella species prior to selective enrichment Proteose peptone serves as a source of carbon, nitrogen, vitamins and minerals.
from food. Sodium chloride provides sodium ions for the membrane transport and maintains
Summary osmotic equilibrium of the medium. Phosphates buffer the medium.
Buffered Peptone Water with NaCl is one of the most widely used pre enrichment Formula*
broths for Salmonella in a wide range of foods. Moreover, it is often the medium Ingredients in grams per liter
of choice in many published reference methods, including the current Peptone ( meat and casein) 1.0
Sodium chloride 4.3
International Organization for Standardization (ISO) method, for detection of
Disodium hydrogen phosphate 7.2
Salmonella in foods. Edel and Kampelmaacher in their comparative study
Potassium dihydrogen phosphate 3.6
observed that food preservation methods cause subleathal injury to Salmonellae. Final pH (at 250C) 7.0 ±0.2
It is known that freezing and drying may injure Salmonellae so that they are * Formula adjusted to suit performance parameters
unable to multiply in selective media. Pre enrichment of sublethally injured Directions
Salmonellae in buffered peptone water showed superior results in comparison 1. Suspend the 16.1 gms of powder in 1000 ml distilled water.
with direct selection methods. In addition to providing conditions for the recovery
2. Mix thoroughly.
and growth of cells prior to selective enrichment, BPW buffers the pH of the growth
3. Warm slightly with frequent agitation to dissolve the powder completely.
system against pH changes brought about by the growth and metabolism of

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4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes. Organisms (ATCC) Growth
Salmonella serotype Enteritidis (13076) Luxuriant
Note: To this solution surface active agents or inactivators of antimicrobial
Salmonella serotype Typhi (19430) Luxuriant
agents may be added before autoclave, such as: Polysorbate 80 or
Salmonella serotype Typhimurium (14028) Luxuriant
Polysorbate 20 in1-10 gms per liter. Procedure
Quality Control
Test specimens as per recommended guidelines.
Dehydrated Appearance
Storage
Light yellow coloured, homogeneous, free flowing powder.
Prepared Appearance Store at 22-300C and prepared medium at 2-80C.
Light yellow coloured, clear solution without any precipitate. Shelf Life
Cultural Response Use before expiry date as mentioned on the label.
Cultural characteristics after 18-24 hours at 35-370C.

Buffered Peptone Water with NaCl BP AM50217

Use Final pH (at 250C) 7.0 ±0.2
Buffered peptone water used for pre-enrichment of injured Salmonella species. It * Formula adjusted to suit performance parameters
increases recovery of Salmonella species prior to selective enrichment from food. Directions

Summary 1. Suspend the 16.09 gms of powder in 1000 ml distilled water.

Buffered peptone water (BPW) is one of the most widely used pre enrichment 2. Mix thoroughly.
broths for Salmonella in a wide range of foods. Moreover, it is often the medium 3. Warm slightly with frequent agitation to dissolve the powder completely.
of choice in many published reference methods, including the current 4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
International Organization for Standardization (ISO) method, for detection of
Note: To this solution surface active agents or inactivators of antimicrobial
Salmonella in foods. Edel and Kampelmaacher in their comparative study
agents may be added before autoclave, such as: Polysorbate 80 or
observed that food preservation methods cause subleathal injury to Salmonellae.
Polysorbate 20 in1-10 gms per liter.
It is known that freezing and drying may injure Salmonellae so that they are
Quality Control
unable to multiply in selective media. Pre enrichment of sublethally injured Dehydrated Appearance
Salmonellae in buffered peptone water showed superior results in comparison Light yellow coloured, homogeneous, free flowing powder.
with direct selection methods. In addition to providing conditions for the recovery Prepared Appearance
and growth of cells prior to selective enrichment, BPW buffers the pH of the growth Light yellow coloured, clear solution without any precipitate.
system against pH changes brought about by the growth and metabolism of Cultural Response
microorganisms during enrichment and those imposed by the food sample. Cultural characteristics after 18-24 hours at 35-370C.
Principle Organisms (ATCC) Growth
Salmonella serotype Enteritidis (13076) Luxuriant
Proteose peptone serves as a source of carbon, nitrogen, vitamins and minerals.
Salmonella serotype Typhi (19430) Luxuriant
Sodium chloride provides sodium ions for the membrane transport and maintains Salmonella serotype Typhimurium (14028) Luxuriant
osmotic equilibrium of the medium. Phosphates buffer the medium. Procedure
Formula*
Test specimens as per recommended guidelines.
Ingredients in grams per liter
Storage
Peptone ( meat and casein) 1.00
Sodium chloride 4.30 Store at 22-300C and prepared medium at 2-80C.
Disodium hydrogen orthophosphate 7.23 Shelf Life
Potassium dihydrogen orthophosphate 3.6 Use before expiry date as mentioned on the label.

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Campylobacter Agar Base AM50218

Use 6. Add 1 vial of Campylobacter supplement (AS0061) to prepare Blaser’s
Campylobacter agar base is used for selective isolation of Campylobacter species medium or add 1 vial of Campylobacter supplement (AS0071) to prepare
from clinical and non-clinical samples. Skirrow’s medium.
Summary 7. After addition, the medium must be gently but thoroughly mixed to ensure
Campylobacter agar base is based on the formulation described by Bolton and that the antibiotics are uniformly distributed throughout the medium.
Robertson (29.1). Campylobacter agar base is designed to isolate Quality Control
Campylobacter species from fecal specimens, food and environmental Dehydrated Appearance
specimens. It is used as a highly nutritious basal medium, which after Yellow coloured, homogeneous, free flowing powder.
appropriate additions, is used for selective isolation of Campylobacter species Prepared Appearance
from fecal specimens, food and environmental specimens. The antimicrobial Basel medium yield yellow coloured, cleared to slightly opalescent gel.
Addition of 5-7% v/v lysed blood forms reddish brown coloured opalescent gel
agents described by Skirrow and Blaser et al., markedly reduce growth of normal in petri plates.
enteric bacteria (79.1). Cultural Response
Principle Cultural characteristics after 24-48 hours at 35ºC in microaerophilic
atmosphere.
Proteose peptone serves as a nitrogen source for fastidious organism like
Organisms (ATCC) Growth* Growth** RGI
Campylobacter. Liver digest and yeast extract provide essential carbon, nitrogen,
Campylobacter fetus
vitamin and amino acid sources. Sodium chloride provides the osmotic balance. subsp. jejuni (29428) Good - Good - More than 70%
The selective isolation of Campylobacter depends on the incorporation of the Luxuriant Luxuriant
proper antimicrobial agent in the medium. Skirrow’s antimicrobial supplement Candida albicans None to poor Moderate 0% or More than 70%
contains Vancomycin, Polymixin B and Trimethoprim where as antimicrobial (10231)
supplement of Blaser-Wang contains Vancomycin, Polymixin B, Trimethoprim, Escherichia coli None to poor None to poor 0% or More than 70%
Cephalothin and Amphotericin B. inhibits Gram-positive bacteria, Polymyxin B (25922)
inhibits most Gram negative bacilli except Proteus trimethoprim is inhibitory for Streptococcus None to poor None to poor 0% or More than 70%
faecalis (29212)
Proteus species. Cephalothin inhibits Gram-positive organisms and also inhibits
For growth RGI should be more than 70%
enteric bacteria. Amphotericin B is an antifungal agent.
For Inhibition RGI should be 0%
Formula*
RGI- Relative Growth Index
Ingredients in grams per liter
* after addition of Campylobacter supplement I (Blaser- Wang), AS0061
Proteose peptone 15.00
** after addition of Campylobacter supplement II (Skirrow), AS0071
Liver digest 2.50
Procedure
Yeast extract 5.00
Sodium chloride 5.00
1. Use standard procedures to obtain isolated colonies from specimens.
Agar 12.00 2. Inoculate and incubate the plates in an atmosphere consisting of
Final pH (at 250C) 7.4± 0.2 approximately 5-6% oxygen, 10% carbon dioxide and 84-85% nitrogen
*Formula adjusted to suit performance parameters for 24-48 hours at 42°C.
Directions Precautions / Limitations
1. Suspend the 19.75 gms of powder in 500 ml distilled water. Since Campylobacter jejuni thermophilic, it is important to incubate the plates at
2. Mix thoroughly. 42°C; otherwise, growth will be delayed. Also, the higher temperature improves
3. Boil with frequent agitation to dissolve the powder completely. Do not selectivity by inhibiting the normal flora.
overheat. Storage

4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes. Store at 22-300C and prepared medium at 2-80C.
Shelf Life
5. Cool the medium to -50°C. aseptically add 5-7% sterile lysed horse blood
Use before expiry date as mentioned on the label.
or 10% sheep blood. Mix thoroughly.

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Candida Medium AM50219
Use 0
4. Cool to 50-52 C and ascetically add 0.3 units of Penicillin and 25ìg
Candida Medium is used for cultivating Candida species. Streptomycin per ml of sterile medium.
Summary 5. Mix well and pour into sterile petri plates.
Candida Medium used for the selective cultivation and differentiation of Candida Quality Control
species. Originally this medium was developed by Nickerson (84.1). It is also use Dehydrated Appearance
for processing and incubation of specimens like tissues, skin scrapping, nails and Yellow coloured, homogeneous, free flowing powder.
hair (42.1 & 24.1). Differentiation of Candida species is based on the growth Prepared Appearance
patterns and pigmentation of isolated colonies. Light yellow coloured, clear to slightly opalescent gel forms in petri plates.
Cultural Response
Principle
Cultural characteristics after 24-48 hours at 300C.
Mycological peptone provide essential nitrogenous nutrients while dextrose act as Organisms (ATCC) Growth RGI
carbon source and phosphate maintain buffering action of medium. This medium Candida albicans (10231) Good to luxuriant More than 70%
contains sodium sulphite which is reduced by Candida species to form sulphide. Candida tropicalis (1369) Good to luxuriant More than 70%
Bismuth in the medium combine with the sulphide to produce brown to black Escherichia coli (25922) Inhibited 0%
pigmented colonies and zones of dark precipitate in the medium surrounding the For growth RGI should be more than 70%
colonies of some species. Bismuth sulphite also acts as an inhibitor of bacterial For Inhibition RGI should be 0%
growth. Selectivity of medium is increased by incorporation of penicillin and RGI- Relative Growth Index
Procedure
streptomycin in the medium which helps to suppress the growth of many bacteria.
1. Allow the agar surface to dry before incubating.
Formula* 2. Inoculate and streak the specimen as soon as possible after collection. If the
Ingredients in grams per liter specimen to be cultured is on a swab, roll the swab over a small area of the
Mycological peptone 2.50 agar surface.
Dextrose 5.00
Disodium hydrogen phosphate 5.00
3. Streak for isolation with a sterile loop.
Sodium sulphite 5.00 4. Incubate plate in an inverted position.
Bismuth sulphite indicator 3.00 5. Once incubated, the medium should be protected from light and incubated
Agar 15.00
aerobically at 25-300C with increase humidity for 48 hrs.
Final pH (at 250C) 7.6 ±0.2
* Formula adjusted to suit performance parameters Interpretation of Results
Directions Refer to appropriate reference and procedures for results.
1. Suspend the 35.5 gms of powder in 1000 ml distilled water. Storage

2. Mix thoroughly. Store at 22-300C and prepared medium at 2-80C.

Shelf Life
3. Boil with frequent agitation to dissolve the powder completely. DO NOT
Use before expiry date as mentioned on the label.
AUTOCLAVE.

Carbohydrate Consumption Broth AM50220

Use purple than recommended by FDA(2.1) and ISO(46.2) committee.
Carbohydrate Consumption Broth is recommended for cultivation and Differentiation is based on fermentation of glucose, dulcitol, raffinose, rhamnose
differentiation of Listeria species. and salicin.
Summary Principle
Carbohydrate Consumption Broth is used for cultivation and differentiation of Peptone and beef extract provide carbon and nitrogen compounds including
Listeria species (1.1). It has slightly different concentration of Bromo cresol essential amino acids, vitamins and trace elements for bacterial metabolism.

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Bromo cresol purple is the pH indicator which turns yellow in acidic condition. Prepared Appearance
Formula* Purple coloured, clear solution without any precipitate.
Ingredients in grams per liter Cultural Response
Proteose peptone 10.00 Cultural characteristics after18-48 hours at 35-370C.
Sodium chloride 5.00 Organisms (ATCC) Growth Without With
Beef extract 1.00 carbohydrate 1% Dextrose
Bromocresol purple 0.10 Acid Gas Acid Gas
Final pH (at 250C) 6.8 ±0.2 Listeria monocytogenes Luxuriant — ---- + ---
* Formula adjusted to suit performance parameters (19118)
Directions Escherichia coli Luxuriant — ---- + +
(25922)
1. Suspend the16.1 gms of powder in 990 ml distilled water.
Staphylococcus aureus Luxuriant — ---- + ---
2. Heat if necessary to dissolve the medium completely. (25923)
3. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes. Interpretation of Results

4. Aseptically add 10 ml separately sterilized carbohydrate solution to give a Refer to appropriate reference and procedures for results.
final concentration of 0.5%. Storage

5. Mix well and pour into sterile test tubes. Store at 22-300C and prepared medium at 2-80C.
Quality Control Shelf Life
Dehydrated Appearance Use before expiry date as mentioned on the label.
Beige coloured, homogeneous, free flowing powder.

Cetrimide Agar (Harmonized Media) AMH5022

Use phosphorous to be released from bacterial cells other than Pseudomonas
Cetrimide Agar is used as a selective medium for the isolation of Pseudomonas aeruginosa. The magnesium chloride and potassium sulphate in the medium
aeruginosa from clinical and non-clinical specimens. stimulates the production of pyocyanin. Pancreatic digest of gelatin provides
Summary nitrogenous compounds.
Cetrimide Agar is based on the formulation described by King et al., and is widely Formula*
recommended for use in the examination of cosmetics, pharmaceuticals and Ingredients in grams per liter
clinical specimens for the presence of P.aeruginosa, as well as for evaluating the Pancreatic digest of gelatin 20.0
Magnesium chloride 1.4
efficacy of disinfectants against this organism. Strains of P.aeruginosa are
Dipotassium sulphate 10.3
identified from specimens by the production of pyocyanin, a blue, water-soluble,
Cetrimide 0.3
nonfluorescent, phenazine pigment in addition to their colonial morphology and Agar 13.6
the characteristic grapelike odour of aminoacetophenone. P.aeruginosa is the Final pH (at 250C) 7.2 ±0.2
only species of Pseudomonas or gram-negative rod known to excrete pyocyanin. * Formula adjusted to suit performance parameters
Cetrimide Agar Base is therefore, a valuable culture medium in the identification Directions
of this organism. It is also included in the Bacteriological Analytical Manual for 1. Suspend 45.6gms of the powder in 1000 ml distilled water containing 10
cosmetics testing and recommended by the USP, BP and IP in Microbial Limit ml glycerol.
Tests . 2. Mix thoroughly.
Principle
3. Heat with frequent agitation and boil to dissolve the powder completely.
Cetrimide (Cetyltrimethylammonium bromide) is a quaternary ammonium
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
compound, cationic detergent, which is inhibitory to a wide variety of bacteria
5. If desired, add rehydrated contents of 1 vial of Nalidixic Selective
including Pseudomonas species other than P.aeruginosa. It causes nitrogen and
Supplement (AS020) aseptically to sterile molten medium.

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Quality Control 2. Most non-Pseudomonas species are inhibited, and some species of
Dehydrated Appearance Pseudomonas may also be inhibited.
Light yellow coloured, homogeneous, free flowing powder.
3. The type of peptone used in the base may affect pigment production.
Prepared Appearance
Light amber coloured, opalescent gel, with slight precipitate. 4. No single medium can be depended upon to exhibit all pigment producing
Cultural Response P.aeruginosa strains.
Cultural characteristics after 24-48 hours at 35-370C. 5. Occasionally some enterics will exhibit a slight yellowing of the medium;
Organisms (ATCC) Growth RGI however, this colouration is easily distinguished from fluorescein production
Escherichia coli (25922) Inhibited 0% since this yellowing does not fluoresce.
Pseudomonas aeruginosa (27853) Luxuriant More than 70%
6. Some non-fermenters and some aerobic spore formers may exhibit a water-
Pseudomonas maltophila (13637) Inhibited 0%
soluble tan to brown pigmentation on this medium. Serratia strains may
Staphylococcus aureus (25923) Inhibited 0%
For growth RGI should be more than 70%
exhibit a pink pigmentation.
For Inhibition RGI should be 0% 7. If swarming colonies of Proteus species are a problem in food samples then
RGI- Relative Growth Index the incubation temperature can be lowered to 20°C for a period of 3-5 days.
Procedure 8. Molten agar should not be kept longer than 4 hours. Medium should not be
1. Use standard procedures to obtain isolated colonies from specimens. stored and remelted.
2. Incubate at 35-370C for 24-48 hours. Storage
Precautions / Limitations Store at 22-300C and prepared medium at 2-80C.
1. Certain strains of P.aeruginosa may not produce pyocyanin while other Shelf Life
species of Pseudomonas do not produce pyocyanin but fluoresce under UV Use before expiry date as mentioned on the label.
light.

Cetrimide Agar Base AM1022/AM5022

Use including Pseudomonas species other than P.aeruginosa. It causes nitrogen and
Cetrimide Agar Base is used as a selective medium for the isolation of phosphorous to be released from bacterial cells other than Pseudomonas
Pseudomonas aeruginosa from clinical and non-clinical specimens. aeruginosa. The magnesium chloride and potassium sulphate in the medium
Summary stimulates the production of pyocyanin. Pancreatic digest of gelatin provides
Cetrimide Agar Base is based on the formulation described by King et al (55) and nitrogenous compounds.
is widely recommended for use in the examination of cosmetics, pharmaceuticals Formula*
and clinical specimens for the presence of P.aeruginosa, as well as for evaluating Ingredients in grams per liter
the efficacy of disinfectants against this organism. Strains of P.aeruginosa are Pancreatic Digest of Gelatin 20.0
Potassium Sulphate 10.0
identified from specimens by the production of pyocyanin, a blue, water-soluble,
Magnesium Chloride 1.4
non-fluorescent, phenazine pigment in addition to their colonial morphology and
Cetrimide 0.3
the characteristic grapelike odour of aminoacetophenone. P.aeruginosa is the Agar 15.0
only species of Pseudomonas or gram-negative rod known to excrete pyocyanin. Final pH (at 250C) 7.2 ± 0.2
Cetrimide Agar Base is therefore, a valuable culture medium in the identification * Formula adjusted to suit performance parameters
of this organism. It is also included in the Bacteriological Analytical Manual for Directions
cosmetics testing (113) and recommended by the USP and IP in Microbial Limit 1. Suspend 46.7 gms of the powder in 1000 ml distilled water containing 10
Tests (114, 46). ml glycerol.
Principle 2. Mix thoroughly.
Cetrimide (Cetyltrimethylammonium bromide) is a quaternary ammonium 3. Heat with frequent agitation and boil to dissolve the powder completely.
compound, cationic detergent, which is inhibitory to a wide variety of bacteria
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.

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5. If desired, add rehydrated contents of 1 vial of Nalidixic Selective species of Pseudomonas do not produce pyocyanin but fluoresce under UV
Supplement (AS020) aseptically to sterile molten medium. light.
Quality Control 2. Most non-Pseudomonas species are inhibited, and some species of
Dehydrated Appearance Pseudomonas may also be inhibited.
Light yellow coloured, homogeneous, free flowing powder.
2. The type of peptone used in the base may affect pigment production.
Prepared Appearance
Light amber coloured, opalescent gel, with slight precipitate. 3. No single medium can be depended upon to exhibit all pigment producing
Cultural Response P.aeruginosa strains.
Cultural characteristics after 24-48 hours at 35-370C. 4. Occasionally some enterics will exhibit a slight yellowing of the medium;
Organisms (ATCC) Growth RGI however, this colouration is easily distinguished from fluorescein production
Escherichia coli (25922) Inhibited More than 70% since this yellowing does not fluoresce.
Pseudomonas aeruginosa (27853) Luxuriant More than 70%
5. Some non-fermenters and some aerobic spore formers may exhibit a water-
Pseudomonas maltophila (13637) Inhibited 0%
soluble tan to brown pigmentation on this medium. Serratia strains may
Staphylococcus aureus (25923) Inhibited 0%
Procedure
exhibit a pink pigmentation.
1. Use standard procedures to obtain isolated colonies from specimens. 6. If swarming colonies of Proteus species are a problem in food samples then
the incubation temperature can be lowered to 20°C for a period of 3-5 days.
2. Incubate at 35-370C for 24-48 hours.
Interpretation of Results 7. Molten agar should not be kept longer than 4 hours. Medium should not be
1. Colonies that are surrounded by a blue-green pigment and fluoresce under stored and remelted.
ultraviolet light (wavelength 254 nm) may be presumptively identified as Storage

Pseudomonas aeruginosa. Store at 22-300C and prepared medium at 2-80C.

Precautions / Limitations Shelf Life
1. Certain strains of P.aeruginosa may not produce pyocyanin while other Use before expiry date as mentioned on the label.

Cetrimide Agar IP AM10221/AM50221

Cetrimide Agar USP AM10222/AM50222
Cetrimide Agar EP AM10223/AM50223
Cetrimide Agar BP AM10224/AM50224
Use cosmetics testing and recommended by the USP, BP and IP in Microbial Limit
Cetrimide Agar Base is used as a selective medium for the isolation of Tests .
Pseudomonas aeruginosa from clinical and non-clinical specimens. Principle
Summary Cetrimide (Cetyltrimethylammonium bromide) is a quaternary ammonium
Cetrimide Agar Base is based on the formulation described by King et al., and is compound, cationic detergent, which is inhibitory to a wide variety of bacteria
widely recommended for use in the examination of cosmetics, pharmaceuticals including Pseudomonas species other than P.aeruginosa. It causes nitrogen and
and clinical specimens for the presence of P.aeruginosa, as well as for evaluating phosphorous to be released from bacterial cells other than Pseudomonas
the efficacy of disinfectants against this organism. Strains of P.aeruginosa are aeruginosa. The magnesium chloride and potassium sulphate in the medium
identified from specimens by the production of pyocyanin, a blue, water-soluble, stimulates the production of pyocyanin. Pancreatic digest of gelatin provides
nonfluorescent, phenazine pigment in addition to their colonial morphology and nitrogenous compounds.
the characteristic grapelike odour of aminoacetophenone. P.aeruginosa is the Formula*
only species of Pseudomonas or gram-negative rod known to excrete pyocyanin. Ingredients in grams per liter
Cetrimide Agar Base is therefore, a valuable culture medium in the identification Pancreatic digest of gelatin 20.0
of this organism. It is also included in the Bacteriological Analytical Manual for Magnesium chloride 1.4

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Dipotassium sulphate 10.0 2. Incubate at 35-370C for 24-48 hours.
Cetrimide 0.3 Precautions / Limitations
Agar 13.6
1. Certain strains of P.aeruginosa may not produce pyocyanin while other
Final pH (at 250C) 7.2 ±0.2
species of Pseudomonas do not produce pyocyanin but fluoresce under UV
* Formula adjusted to suit performance parameters
Directions
light.
1. Suspend 45.3 gms of the powder in 1000 ml distilled water containing 10 2. Most non-Pseudomonas species are inhibited, and some species of
ml glycerol. Pseudomonas may also be inhibited.
2. Mix thoroughly. 3. The type of peptone used in the base may affect pigment production.
3. Heat with frequent agitation and boil to dissolve the powder completely. 4. No single medium can be depended upon to exhibit all pigment producing
0 P.aeruginosa strains.
4. Sterilize by autoclaving at 121 C (15 lbs pressure) for 15 minutes.
5. Occasionally some enterics will exhibit a slight yellowing of the medium;
5. If desired, add rehydrated contents of 1 vial of Nalidixic Selective
however, this colouration is easily distinguished from fluorescein production
Supplement (AS020) aseptically to sterile molten medium.
since this yellowing does not fluoresce.
Quality Control
Dehydrated Appearance 6. Some non-fermenters and some aerobic spore formers may exhibit a water-
Light yellow coloured, homogeneous, free flowing powder. soluble tan to brown pigmentation on this medium. Serratia strains may
Prepared Appearance exhibit a pink pigmentation.
Light amber coloured, opalescent gel, with slight precipitate.
7. If swarming colonies of Proteus species are a problem in food samples then
Cultural Response
the incubation temperature can be lowered to 20°C for a period of 3-5 days.
Cultural characteristics after 24-48 hours at 35-370C.
Organisms (ATCC) Growth RGI 8. Molten agar should not be kept longer than 4 hours. Medium should not be
Escherichia coli (25922) Inhibited 0% stored and remelted.
Pseudomonas aeruginosa (27853) Luxuriant More than 70% Storage
Pseudomonas maltophila (13637) Inhibited 0% Store at 22-300C and prepared medium at 2-80C.
Staphylococcus aureus (25923) Inhibited 0%
Shelf Life
For growth RGI should be more than 70%
Use before expiry date as mentioned on the label.
RGI- Relative Growth Index
Procedure
1. Use standard procedures to obtain isolated colonies from specimens.

Cetrimide Broth AM1023/AM5023

Use Pseudomonas aeruginosa. Sodium chloride maintains osmotic balance.
Cetrimide Broth is used for cultivation of Pseudomonas aeruginosa. Formula*
Summary Ingredients in grams per liter
Cetrimide Broth is the modification of the formula described by King, Ward and Beef Extract 10.0
Peptone 10.0
Raney (55). It is a selective medium for the cultivation of Pseudomonas
Cetrimide 0.3
aeruginosa due to the presence of cetrimide.
Sodium Chloride 5.0
Principle
Final pH (at 250C) 7.2 ± 0.2
Cetrimide (Cetyltrimethylammonium bromide) is a quaternary ammonium * Formula adjusted to suit performance parameters
compound, cationic detergent, which is inhibitory to a wide variety of bacteria Directions
including Pseudomonas species other than P.aeruginosa. It causes release of 1. Suspend 25.3 gms of the powder in 1000 ml distilled water.
phosphorous and nitrogen from bacterial cells other than Pseudomonas 2. Boil to dissolve the powder completely.
aeruginosa. Peptone and beef extract provide necessary nutrients for
3. Dispense in tubes as per requirements.

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4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. Organisms (ATCC) Growth
Quality Control Escherichia coli (25922) Inhibited
Dehydrated Appearance Pseudomonas aeruginosa (27853) Luxuriant
Yellow coloured, homogeneous, free flowing powder. Staphylococcus aureus (25923) Inhibited
Prepared Appearance Storage
Yellow coloured, clear to very slightly opalescent solution. Store at 22-300C and prepared medium at 2-80C.
Cultural Response Shelf Life
Cultural characteristics after 24-48 hours at 350C. Use before expiry date as mentioned on the label.

Chapman Stone Agar AM50231

Use Agar 15.0
Chapman Stone Agar is recommended for the selective isolation of Staphylococci Final pH (at 250C) 7.0 ±0.2
* Formula adjusted to suit performance parameters
causing food poisoning.
Directions
Summary
Chapman (14.1) developed Staphylococcus Agar No. 110 and subsequently 1. Suspend 20.25gms of the powder in 100 ml distilled water.
modified it by reducing sodium chloride concentration and incorporating 2. Boil to dissolve the powder completely.
ammonium sulphate in the formulation (16.1). 3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 10 minutes.
Principle Quality Control
Casein enzymic hydrolysate, yeast extract provide nitrogen, carbon, sulphur, Dehydrated Appearance
Light yellow coloured, coarse, free flowing powder.
vitamin B and trace elements. Sodium chloride acts as a selective agent which
Prepared Appearance
inhibits most of the bacterial species. Mannitol is the fermentable carbohydrate
Light amber coloured, opalescent gel forms in petri plates.
and its fermentation can be detected by adding a few drops of bromo cresol purple
Cultural Response
resulting in production of yellow colour. Gelatin hydrolysis id observed as clear Cultural characteristics after 18-48 hours at 300C.
zones around colonies. Due to the presence of ammonium sulphate in the medium Organisms (ATCC) Growth Gelatinase Mannitol RGI
itself there is no need to flood the plate with ammonium sulphate solution for production fermentation
detection of gelatin liquefaction by the isolates which is knows as stone's method Staphylococcus Luxuriant + + More than
(103.1). After incubation of 48 hours at 350C, cream to golden yellow colonies aureus (25923) 70%
surrounded by clear zone are presumptively identified as Staphylococcus aureus. Staphylococcus Luxuriant + – More than
epidermidis(12228) 70%
White or non-pigmented colonies, with or without a clear zone, are presumptively
Escherichia coli Inhibited – – 0%
identified as Staphylococcus epidermidis.
(25922)
Formula*
* Key: Gelatinase + = clearing or halo.
Ingredients in grams per liter
Procedure
Casein enzyme hydrolysate 10.00
Yeast extract 2.50 1. Use standard procedures to obtain isolated colonies from
Gelatin 30.00 Storage
D- mannitol 10.00 Store at 22-300C and prepared medium at 2-80C.
Sodium chloride 55.00 Shelf Life
Ammonium sulphate 75.00 Use before expiry date as mentioned on the label.
Dipotassium Phosphate 5.0

Chloramphenicol Yeast Glucose Agar AM1024/AM5024

Use enumeration of yeasts and moulds in milk and milk products.
Chloramphenicol Yeast Glucose Agar is a selective medium used for isolation and

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Summary Saccharomyces cerevisiae (9763) Good to luxuriant More than 70%
The antibiotic method for enumerating yeasts and moulds in dairy products has Staphylococcus aureus (25923) Inhibited More than 0%
For growth RGI should be more than 70%
become the method of choice, thereby replacing the traditional acidified method.
For Inhibition RGI should be 0%
The use of antibiotics for suppressing bacteria results in better recovery of injured
RGI- Relative Growth Index
fungal cells, which are sensitive to an acid environment and in less interference Procedure
from precipitated food particles during the counting. Chloramphenicol Yeast
1. Prepare initial sample dilutions using 10 gms or 10 ml of sample in 90 ml of
Glucose Agar is a nutrient medium that inhibits the growth of organisms other
diluent, as listed below:
than yeasts and moulds due to the presence of chloramphenicol. Chloramphenicol
SAMPLE DILUENT PREPARATION
Yeast Glucose Agar is recommended by APHA for the examination of dairy products Milk, Liquid milk ¼-strength Ringer's Mix.
(39). The ISO committee recommends this medium for the enumeration of yeasts product solution
and moulds. Dried milk, Whey ¼-strength Ringer's Shake at 470C.
Principle powder, Buttermilk solution
powder, Lactose
Yeast extract provides nitrogen and vitamin B complex. Dextrose is the Casein 2% Dipotassium Shake at 470C.
carbohydrate source. Chloramphenicol, a thermostable antibiotic, suppresses phosphate solution
accompanying bacterial flora, which improves the shelf life of the prepared Cheese 2% Sodium citrate Shake at 470C.
medium; therefore the prepared medium can be used over a period of at least 4 solution
months. Butter, Edible ice ¼-strength Ringer's Shake at 470C.
solution
Formula*
Custard, Dessert ¼-strength Ringer's Shake.
Ingredients in grams per liter
Fermented milk, solution
Dextrose 20.0 Yogurt
Yeast Extract 5.0
2. Add 10 ml from the initial dilution prepared above to 90 ml of ¼-strength
Chloramphenicol 0.1
Agar 14.9
Ringer's solution. One milliliter (1ml) of this dilution corresponds to 0.01
Final pH (at 250C) 6.6 ± 0.2 gm/ml of sample.
* Formula adjusted to suit performance parameters 3. Prepare further dilutions by adding 10 ml of the above 0.01 gm/ml dilution
Directions to 90 ml of diluent.
1. Suspend 40 gms of the powder in 1000 ml distilled water. 4. Pipette 1ml of each dilution into two petri plates.
2. Heat to boiling to dissolve the powder completely. 5. Pour 10 ml of sterile molten agar (cooled to 450C) into each sterile petri
0
3. Sterilize by autoclaving at 121 C (15 lbs pressure) for 15 minutes. plate. Mix thoroughly.
Warning: Chloramphenicol is a potent carcinogen and must be handled 6. Incubate at 250C for 4 days.
with care so as to avoid inhalation or contact with skin.
Interpretation of Results
Quality Control
Dehydrated Appearance 1. Select plates containing 30-300 colonies and count the colonies.
Light yellow coloured, homogeneous, free flowing powder. 2. Distinguish yeasts from moulds by colony morphology.
Prepared Appearance
3. Express results as yeasts and moulds “per gram” or “per milliliter”.
Yellow coloured clear to slightly opalescent gel.
Storage
Cultural Response
Cultural characteristics after 2-5 days at 22-250C. Store at 22-300C and prepared medium at 2-80C.
Organisms (ATCC) Growth RGI Shelf Life
Aspergillus niger (16404) Good to luxuriant More than 70% Use before expiry date as mentioned on the label.
Candida albicans (10231) Good to luxuriant More than 70%
Escherichia coli (25922) Inhibited More than 0%

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Christensen Citrate Agar AM1025/AM5025
Use Directions
Christensen Citrate Agar is used for the differentiation of enteric pathogens and 1. Suspend 24.81 gms of the powder in 1000 ml distilled water and mix well.
coliforms on the basis of citrate utilization. 2. Boil with frequent agitation to dissolve the powder completely.
Summary
3. Dispense into test tubes.
Christensen Citrate Agar is a modification of Christensen Iron Agar (18).
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Christensen reported that all members of genera Escherichia, Enterobacter,
Citrobacter, Salmonella and Alkalescens-Dispar group were capable of utilizing 5. Cool the tubes in a slanted position.
Quality Control
citrate as a source of energy while on the other hand Shigella failed to do so.
Dehydrated Appearance
Christensen Citrate Agar is recommended by APHA for the examination of foods
Light pink coloured, homogeneous, free flowing powder.
(20). Prepared Appearance
Principle Orange red coloured, very slightly opalescent gel.
Yeast extract provide nitrogen and vitamin B complex. L-cysteine hydrochloride is Cultural Response
a reducing agent. Dextrose is the carbohydrate source. Sodium citrate is the Cultural characteristic after 24-48 hours at 370C
energy source for citrate utilizing organisms. Sodium chloride maintains the Organisms (ATCC) Growth Colour of the
slant
osmotic balance. Monopotassium phosphate acts as the buffer. Organisms that
Enterobacter aerogenes (13048) Luxuriant Cerise
metabolize citrate as the sole source of carbon cleave citrate to oxaloacetate and
Escherichia coli (25922) Luxuriant No change
acetate via the citritase enzyme. Another enzyme, oxaloacetate decarboxylase, Klebsiella pneumoniae (13883) Luxuriant Orange to pink
then converts oxaloacetate to pyruvate and CO2. This CO2 combines with sodium Salmonella serotype Enteritidis (13076) Luxuriant Cerise
and water to form an alkaline sodium carbonate compound. As a result the pH of For growth RGI should be more than 70%
the medium rises and the indicator, phenol red changes from orange red to cerise. RGI- Relative Growth Index
Presence of cerise colour indicates a positive finding for citrate utilization. Interpretation of Results
Formula* 1. Presence of cerise colour indicates a positive finding for citrate utilization.
Ingredients in grams per liter Precautions / Limitations
Sodium Citrate 3.0 1. Care should be taken while inoculating, as, too heavy an inoculum may give
Yeast Extract 0.5
a false positive result.
Monopotassium Phosphate 1.0
Storage
L-Cysteine Hydrochloride 0.1
Phenol Red 0.012 Store at 22-300C and prepared medium at 2-80C.
Dextrose 0.2 Shelf Life
Sodium Chloride 5.0 Use before expiry date as mentioned on the label.
Agar 15.0
Final pH (at 250C) 6.9 ± 0.2
* Formula adjusted to suit performance parameters

Chromogenic Coliform Agar AM10251/AM50251

Use ingestion of raw or uncooked beef, from person to person and recreational water
Chromogenic Coliform Agar is recommended for the simultaneous detection of sources. Thus, the presence of E.coli in foods is an indicator of direct and indirect
Escherichia coli and total coliforms in water and food samples. fecal contamination and possible presence of enteric pathogens in water,
Summary shellfish, dairy products and other foods (74, 75, 90).
Escherichia coli is a bacterium whose natural habitat is the enteric tract of Hence, Chromogenic Coliform Agar is recommended for simultaneous detection of
humans and warm-blooded animals. Transmission most often occurs through Escherichia coli and total coliforms in water and food samples. The chromogenic

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mixture in this medium consists of chromogenic substrates, which release Cultural Response
insoluble coloured compounds when hydrolysed by a specific enzyme, which Cultural characteristics after 24-48 hours at 35-370 C.
Organisms Colony Salmon X-Glucuro- Indole
permits differentiation of pathogenic microorganisms.
(ATCC) Colour GAL nide
Principle
Escherichia coli Dark blue/violet + + +
Peptone special and sodium pyruvate provide essential growth nutrients. (25922)
Dipotassium hydrogen phosphate and Potassium dihydrogen phosphate act as Enterobacter cloacae Salmon to red + - -
buffers. Sodium lauryl sulphate inhibits the growth of gram-positive organisms. (13047)
The chromogenic mixture in this medium contains two substrates namely Salmon- Citrobacter freundii Salmon to red + - -
GAL and X-glucuronide. (8090)
Klebsiella pneumoniae Light pink + - -
The characteristic enzyme for coliforms, ß-D-galactosidase cleaves the Salmon-
(13883)
GAL substrate and causes a salmon to red colour of the coliform colonies. S. serotype Enteridis Colourless - - -
The substrate X-glucuronide is used for the identification of ß-D-glucuronidase, (13076)
which is characteristic for E. coli. E. coli cleaves both Salmon-GAL and X- Shigella flexneri Colourless - - -
glucuronide, so that positive colonies take on a dark-blue to violet colour. These (12022)
are easily distinguished from other coliform colonies, which have a salmon to red Enterococcus faecalis Inhibited - - -
(29212)
colour. As part of an additional confirmation of E. coli, the inclusion of tryptophan
For growth RGI should be more than 70%
improves the indole reaction, thereby increasing detection reliability when it is
For Inhibition RGI should be 0%
used in combination with the Salmon-GAL and X-glucuronide reaction. RGI- Relative Growth Index
Formula* Procedure
Ingredients in grams per liter
1. Inoculate the medium by the pour plate method or by spreading the sample
Peptone special 3.0
Sodium chloride 5.0
material on the surface of the plates. In addition the membrane-filter-
Sodium pyruvate 1.0 technique can also be used.
Tryptophan 1.0 2. Incubate for 24 hours at 35 - 37°C.
Sodium lauryl sulphate 0.1 Interpretation of Results
Dipotassium hydrogen phosphate 3.0
1. E. coli: dark-blue to violet colonies (Salmon-GAL and X-glucuronide
Potassium dihydrogen phosphate 1.7
reaction).
Chromogenic mixture 0.2
Agar 12.0 2. Total coliforms: salmon to red colonies (Salmon-GAL reaction) and dark-blue
Final pH (at 250 C) 6.8 ± 0.2 to violet colonies (E. coli ).
* Formula adjusted to suit performance parameters 3. Other gram-negative microorganisms: colourless colonies, except for some
Directions
organisms, which possess ß-D-glucuronidase activity. These colonies appear
1. Suspend 27 grams of the powder in 1000 ml distilled water. light blue to turquoise.
2. Boil with frequent agitation to dissolve the powder completely. 4. In order to confirm E. coli, coat the dark-blue to violet colonies with a drop of
3. When a high number of gram-positive accompanying bacteria are expected, Kovac's indole reagent. If the reagent turns to a cherry-red colour after some
add 5 mg/L Novobiocin. seconds, a positive indole formation confirms the presence of E. coli.
4. Sterilize by autoclaving at 1210 C (15 lbs pressure) for 15 minutes. Storage
Quality Control Store at 22-300C and prepared medium at 2-80C.
Dehydrated Appearance Shelf Life
Beige coloured, homogeneous, free flowing powder. Use before expiry date as mentioned on the label.
Prepared Appearance
Colourless, clear to very slightly opalescent gel.

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Chromogenic E. coli Agar AM10252/AM50252
Use Directions
Chromogenic E.coli Agar is recommended for the detection and enumeration of E. 1. Suspend 36.58 grams of the powder in 1000 ml distilled water.
coli in foods without further confirmation on membrane filter or by indole reagent. 2. Sterilize by autoclaving at 1210 C (15 lbs pressure) for 15 minutes.
Summary
3. Cool to 500 C and pour into sterile petriplates.
Escherichia coli is a common Gram-negative microorganism, which may be Quality Control
present in food and water samples. The detection of E.coli and its differentiation Dehydrated Appearance
from other coliforms is important in medical and environmental analyses Light yellow coloured, homogeneous, free flowing powder.
(75,90). Chromogneic E.coli Agar is a differential agar for presumptive Prepared Appearance
identification of E.coli from other coliforms by presence of an enzyme Light yellow coloured, clear to slightly opalescent gel.
glucoronidase produced by E.coli strains. The chromogenic agent in this medium Cultural Response

consists of chromogenic substrates, which release insoluble coloured compounds Cultural characteristics after 18-24 hours at 440 C.
Organisms (ATCC) Growth Colony Colour RGI
when hydrolysed by a specific enzyme, which permits differentiation of
Escherichia coli (25922) Luxuriant Blue More than 70%
pathogenic microorganisms.
S. serotype Enteridis (13076) Luxuriant Colourless More than 70%
Principle
Staphylococcus aureus (25923) Inhibited -- 0%
Tryptone and Peptone special provide essential growth nutrients. Bile salts For growth RGI should be more than 70%
mixture inhibits the growth of gram-positive organisms. Sodium chloride For Inhibition RGI should be 0%
maintains the osmotic equilibrium. Disodium hydrogen phosphate and Sodium RGI- Relative Growth Index
dihydrogen phosphate act as buffers. The chromogenic agent X-glucuronide is Procedure

used for identification of E. coli. E.coli cleaves X-glucuronide, giving a 1. Dry the surface of the medium in the prepared plates.
colouration to the colonies, which are easily distinguished from other coliform 2. Prepare the food sample by diluting 1 in 5 or 1 in 10 (as appropriate) with
colonies. 0.1% (w/v) sterile Peptone Water and homogenize in a stomacher or
Formula* laboratory blender.
Ingredients in grams per liter
3. Pipette 0.5 ml or 1.0 ml (as appropriate) of the homogenate on to the
Tryptone 14.0
petriplate and spread over the surface with a sterile glass spreader.
Peptone, special 5.0
Sodium chloride 2.4 4. Incubate plates for 18 - 24 hours at 35 - 370 C.
Bile Salts mixture 1.5 Interpretation of Results
X-Glucuronide 0.075 1. Clear differentiation of blue coloured E.coli colonies observed.
Disodium hydrogen phosphate 1.0 Precautions / Limitations
Sodium dihydrogen phosphate 0.6
1. Avoid contact with eyes, wear appropriate mask while handling the product.
Agar 12.0
0 Storage
Final pH (at 25 C) 7.2 ± 0.2
* Formula adjusted to suit performance parameters Store at 22-300C and prepared medium at 2-80C.
Shelf Life
Use before expiry date as mentioned on the label.

Chromogenic Enterococci Broth AM10253/AM50253

Use serves as an indicator for faecal contamination. It is more specific than the
Chromogenic Enterococci Broth is recommended for identification and presence of coliforms, which may originate from non-faecal sources (74).
differentiation of Enterococci from water samples. Chromogenic Enterococci Broth is recommended for identification and
Summary differentiation of Enterococci from other coliforms. The chromogenic mixture in
The presence of Enterococci, which account for most of the faecal streptococci, this medium consists of chromogenic substrates, which release insoluble coloured

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compounds when hydrolysed by a specific enzyme, which permits differentiation Quality Control

of pathogenic microorganisms. Dehydrated Appearance

Light yellow coloured, homogeneous, free flowing powder.
Principle
Prepared Appearance
Peptone special provides essential nutrients and nitrogenous compounds to the
Light yellow coloured, clear solution.
medium. Sodium azide inhibits the growth of gram-negative organisms. Sodium Cultural Response
chloride maintains the osmotic equilibrium. Disodium hydrogen phosphate acts Cultural characteristics after 24-48 hours at 35-370 C.
as a buffer. The chromogenic substrate is cleaved by the enzyme -D-glucosidase Organisms (ATCC) Growth Colour of the Broth
produced by Enterococci resulting in colour change of the medium from light Enterococcus faecalis (29212) Luxuriant Blue
yellow to blue green. Escherichia coli (25922) None to poor Light yellow

Formula* Staphylococcus aureus (25923) None to poor Light yellow

Ingredients in grams per liter Pseudomonas aeruginosa (27853) None to poor Light yellow

Peptone, special 10.0 Procedure

Sodium chloride 5.0 1. Inoculate the medium by the pour-plate method or by spreading the sample
Sodium Azide 0.3 material on the surface of the medium. In addition, the membrane-filter
Chromogenic Mixture 0.04 technique may also be used.
Disodium hydrogen phosphate 1.0
2. Incubate at 24 ± 4 hours at 35-37°C. If there is no color change or any
Final pH (at 250 C) 7.5 ± 0.2
visible growth after 24±4 hours, continue the incubation up to 44±4
* Formula adjusted to suit performance parameters
hours.
Directions
Warning: Sodium azide has a tendency to form explosive metal azides
1. Suspend 16.59 grams of the powder in 1000 ml distilled water. with plumbing materials and it is advisable to flush off the disposables
with water.
2. Boil with frequent agitation to dissolve the powder completely.
Storage
3. Sterilize by autoclaving at 1210 C (15 lbs pressure) for 15 minutes. Store at 22-300C and prepared medium at 2-80C.
4. Dispense as desired. Shelf Life
Use before expiry date as mentioned on the label.

Chromogenic Improved Salmonella Agar AM10255/AM50255

Use Principle
Chromogenic Improved Salmonella Agar for identification and differentiation of Specially selected peptone supply the nutrients. Gram-positive organisms are
Salmonella from water sample. generally inhibited as a result of the selective medium base used.
Summary Formula
Salmonella Medium was developed by A. Rambach. Salmonella is ubiquitous in Ingredients Gms/Liter
Peptone special 8.0
animal populations and is generally isolated from the intestines of animals and
Yeast extract 2.0
humans. It is one of the most prevalent organisms associated with food borne
Sodium deoxycholate 1.0
illness, which is often linked to animal origin. Illness caused by Salmonella have Chromogenic substance 3.25
been associated with poultry, chocolate, dairy and vegetable products. Agar 12.0
Salmonella medium is intended for the identification and differentiation of Final pH (at 250C) 7.3 ±0.2
Salmonella species based on the formation of an insoluble pigment. The Directions
addition of chromogenic substrate in the medium facilitrates detection of 1. Suspend 26.25 gms of the powder in 1000 ml-distiled water and mix
Salmonella species from other normal flora. thoroughly.

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2. Boil with frequent agitation to dissolve the powder completely. Salmonella Serotype Luxuriant Light pink More than 70%
Typhimurium (14028)
3. DO NOT AUTOCLAVE.
S. Serotype Enteritidis (13076) Luxuriant Pink More than 70%
4. Cool to 500C and pour into sterile petriplates. Proteus vulgaris (13315) Good Light brown More than 70%
Quality Control Staphylococcus aureus (25923) Inhibited – 0%
Dehydrated Appearance Bacillus subtillus (6633) Inhibited – 0%
Red coloured, coarse, free flowing powder. For growth RGI should be more than 70%
Prepared Appearance: For Inhibition RGI should be 0%
Reddish pink coloured slightly opalescent gel forms in periplates. RGI- Relative Growth Index
Cultural Response Storage
Cultural characteristics after 18-24 hours at 35-370C. Store at 22-300C and prepared medium at 2-80C.
Organisms (ATCC) Growth Colour RGI Shelf Life
of colony Use before expiry date as mentioned on the label.
Escherichia coli (25922) Luxuriant Blue More than 70%

Chromogenic UTI Agar AM10254/AM50254

Use Directions
Chromogenic UTI Agar is a differential medium recommended for the presumptive 1. Suspend 32.45 grams of the powder in 1000 ml distilled water.
identification of microorganisms mainly causing urinary tract infections. 2. Boil with frequent agitation to dissolve the powder completely.
Summary
3. Sterilize by autoclaving at 1210 C (15 lbs pressure) for 15 minutes.
Escherichia coli, Enterococci, Klebsiella, and Proteus are frequently encountered
4. Cool to 500 C and pour into sterile petriplates.
microorganisms in urinary tract infections. For many years, blood, CLED and
Quality Control
MacConkey Agars have been used for the detection of urinary pathogens as well Dehydrated Appearance
as for differentiation of a few of them (123). However, chromogenic media have Light yellow coloured, homogeneous, free flowing powder.
proved more specific for direct differentiation of microorganisms. Chromogenic Prepared Appearance
UTI Agar has been formulated on these lines. It facilitates and expedites the Light amber coloured, clear to slightly opalescent gel.
identification of some gram-negative and gram-positive bacteria based on Cultural Response
reactions of the chromogenic substrates with specific enymes produced by these Cultural characteristics after 24 hours at 35-370 C.
pathogens. The chromogenic mixture in this medium consists of chromogenic Organisms (ATCC) Growth Colony Colour RGI
Escherichia coli Luxuriant Pink-red More than 70%
substrates, which release insoluble coloured compounds when hydrolysed by a
(25922)
specific enzyme, which permits differentiation of pathogenic microorganisms.
Enterococcus faecalis Luxuriant Blue, small More than 70%
Principle
(29212)
Peptone special provides essential growth nutrients. The chromogenic mixture Staphylococcus aureus Luxuriant Golden yellow More than 70%
consists of artificial substrates, which release insoluble coloured compounds when (25923)
hydrolysed by a specific enzyme, permitting differentiation of Enterococci, E. coli Pseudomonas aeruginosa Luxuriant Colourless More than 70%
and coliforms. (27853)
Formula* Proteus mirabilis Luxuriant Light brown More than 70%
Ingredients in grams per liter (10975)
Peptone, special 15.0 Klebsiella pneumoniae Luxuriant Blue to purple, More than 70%
Chromogenic mixture 2.45 (13883) mucoid
Agar 15.0 For growth RGI should be more than 70%
Final pH (at 250 C) 6.8 ± 0.2 RGI- Relative Growth Index
* Formula adjusted to suit performance parameters

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Procedure substrates must be further differentiated with appropriate biochemical or
1. Inoculate the urine specimen on to the medium. serological tests.
2. Incubate plates aerobically at 35-370 C for not less than 20-24 hours. 4. In rare cases, Listeria species might be present in urine. Hence, it is necessary
3. Observe for growth. to perform a gram stain of all microorganisms isolated. Isolates of
Precautions / Limitations Aeromonas hydrophila may produce rose colonies and can be differentiated
1. Do not incubate the plates in an atmosphere supplemented with carbon from E. coli by performing the oxidase test.
dioxide. 5. This medium will not support growth of fastidious microorganisms such as
2. Avoid exposure to light during incubation as light may destroy the Neisseria species.
chromogens. However, once the colony colour develops, exposure to light is Storage

permissible. Store at 22-300C and prepared medium at 2-80C.

Shelf Life
3. Colonies that show their natural colour and do not react with chromogenic
Use before expiry date as mentioned on the label.

C.L.E.D. Agar with Andrade Indicator AM1026/AM5026

Use 6.8 pale grey
C.L.E.D. Agar with Andrade indicator is used for the isolation, enumeration and 6.6 pinkish grey
presumptive identification of microorganisms from urine, giving good colonial
6.4 bright red with whitish tinge
differentiation.
6.0 bright red
Summary
Formula*
Sandys (101) observed that restricting the electrolytes on a solid medium might Ingredients in grams per liter
prevent the swarming of Proteus. Previous chemical methods used to inhibit Lactose 10.0
swarming of Proteus included the addition of chloral hydrate, alcohol, sodium Tryptone 4.0
azide, surface-active agents, boric acid and sulphonamides to the culture Peptone 4.0
medium. This electrolyte-deficient medium was modified for use in urine culture Beef Extract 3.0
by substituting lactose and sucrose instead of mannitol and increasing the L-Cystine 0.128
concentrations of bromothymol blue indicator and agar. The medium was further Andrade Indicator 0.10
Bromothymol Blue 0.02
modified by the incorporation of cystine in order to enhance the growth of cystine-
Agar 15.0
dependent “dwarf colony ” coliforms and by the deletion of sucrose. This new
Final pH (at 250C) 7.5 ± 0.2
medium, Cystine-Lactose-Electrolyte-Deficient (C.L.E.D.) Agar is ideal for dip- * Formula adjusted to suit performance parameters
inoculation techniques and for urinary bacteriology in general. Directions
Principle
1. Suspend 36.25 gms of the powder in 1000 ml distilled water.
C.L.E.D. Agar with Andrade indicator is similar to C.L.E.D. Agar with Bromothymol
2. Mix thoroughly.
blue except in this medium Andrade indicator (Acid Fuchsin in 1N Sodium
Hydroxide) is incorporated. The essential nutrients are supplied by peptone, 3. Boil with frequent agitation to dissolve the powder completely.
tryptone and beef extract. Lactose is the carbohydrate source. L-cystine permits 4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
the growth of "dwarf colony" coliforms. Addition of Andrade indicator enhances Quality Control
the appearance of colony and aids in the identification of microorganisms. At Dehydrated Appearance
Yellow coloured, homogeneous, free flowing powder.
different pH values, the colour of the medium varies from the standard medium.
Prepared Appearance
pH Colour of the medium Greenish blue coloured, clear to slightly opalescent gel.
7.4 deep blue Cultural Response
Cultural characteristics after 18-24 hours at 35-370C.
7.0 bluish grey

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Organisms (ATCC) Growth Colour of colony RGI Interpretation of Results
Enterobacter Luxuriant Greyish green, More than 70% 1. Count the number of colonies on the plate or dipstick. Multiply by the dilution
aerogenes (13048) mucoid
factor to convert the count to CFU per ml of the sample.
Enterococcus faecalis Luxuriant Orange, yellow More than 70%
(29212) or green 2. Contaminant bacteria usually appear in low numbers and vary in colony
Escherichia coli Luxuriant Bright pink with More than 70% morphology.
(25922) halo 3. Urinary pathogens will usually yield high counts having uniform colonial
Proteus mirabilis Luxuriant Blue-green More than 70% morphology and should not be sub cultured directly to routine media for
(25933)
identification and susceptibility testing.
Staphylococcus Luxuriant Golden yellow More than 70%
Precautions / Limitations
aureus (25923)
Streptococcus Luxuriant Greyish green More than 70% 1. The medium should not be incubated for more than 24 hours since, if lactose
pyogenes (19615) fermenters predominate, the whole medium may turn pink, masking the
For growth RGI should be more than 70% presence of non-lactose fermenters.
RGI- Relative Growth Index 2. Factors that may cause urine counts from infected patients to be low include:
Procedure
apid rate of urine flow, prior initiation of antimicrobial therapy, a urine pH of
1. Inoculate the medium as soon as possible after the specimen is received in less than 5 and a specific gravity of less than 1.003.
the laboratory.
3. Shigella species may not grow on this medium.
2. It is recommended that quantitative methods be used for culturing urine Storage
specimens. Store at 22-300C and prepared medium at 2-80C.
0
3. Incubate at 35-37 C for 24 hours. Shelf Life
Use before expiry date as mentioned on the label.

C.L.E.D. Agar with Bromothymol Blue AM1027/AM5027

Use
lactose fermenters from non-lactose fermenters. Organisms that ferment lactose
C.L.E.D. Agar with Bromothymol Blue is used for the isolation, enumeration and
will lower the pH and change the colour of the medium from green to yellow.
presumptive identification of microorganisms from urine.
Electrolyte sources are reduced in order to restrict the swarming of Proteus
Summary
species.
Sandys (101) observed that restricting the electrolytes on a solid medium might
Formula*
prevent the swarming of Proteus. Previous chemical methods used to inhibit
Ingredients in grams per liter
swarming of Proteus included the addition of chloral hydrate, alcohol, sodium
Tryptone 4.0
azide, surface-active agents, boric acid and sulphonamides to the culture
Peptone 4.0
medium. This electrolyte medium was modified for use in urine culture by
Lactose 10.0
substituting lactose and sucrose instead of mannitol and increasing the
Beef Extract 3.0
concentrations of bromothymol blue indicator and agar. The medium was further
L-Cystine 0.128
modified by the incorporation of cystine in order to enhance the growth of cystine-
Bromothymol Blue 0.02
dependent “dwarf colony ” coliforms and by the deletion of sucrose. This new
Agar 15.0
medium, Cystine-Lactose-Electrolyte-Deficient (C.L.E.D.) Agar is ideal for dip- 0
Final pH (at 25 C) 7.3 ± 0.2
inoculation techniques and for urinary bacteriology in general.
* Formula adjusted to suit performance parameters
Principle
Directions
The essential growth nutrients are supplied by peptone, tryptone and beef extract.
1. Suspend 36.15 gms of the powder in 1000 ml distilled water.
Lactose is the carbohydrate source. L-cystine permits the growth of “dwarf
2. Mix thoroughly.
colony” coliforms. Bromothymol blue is used as the pH indicator to differentiate

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3. Boil with frequent agitation to dissolve the powder completely. Interpretation of Results
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. 1. Count the number of colonies on the plate or dipstick. Multiply by dilution
Quality Control factor to convert the count to CFU per ml of the sample.
Dehydrated Appearance 2. Contaminant bacteria usually appear in low numbers, which vary in colony
Yellow coloured, homogenous, free flowing powder. morphology. Urinary pathogens will usually yield high counts having
Prepared Appearance uniform colonial morphology and should not be subcultured directly to
Green coloured, very slightly opalescent gel. routine media for identification and susceptibility testing.
Cultural Response 3. Typical colony morphology on C.L.E.D. Agar is as follows:
Cultural characteristics after 18-24 hours at 35- 370C. E.coli Yellow colonies, opaque, center slightly deeper
Organisms (ATCC) Growth Colour of colony RGI yellow.
Enterococcus faecalis Luxuriant Slight yellowish More than 70% Klebsiella Yellow to whitish-blue colonies, extremely mucoid.
(29212) or greenish P.aeruginosa Green colonies with typical matted surface and
Escherichia coli Luxuriant Yellow opaque, center More than 70% rough periphery.
(25922) slightly deep yellow Enterococci Small yellow colonies, about 0.5 mm in diameter.
Klebsiella pneumoniae Luxuriant Yellow to whitish blue More than 70% S.aureus Deep yellow colonies, uniform in colour.
(13883) Coagulase negative Pale yellow colonies, more opaque than E.faecalis.
Proteus vulgaris Luxuriant Blue More than 70% staphylococci
(13315) Precautions / Limitations
Staphylococcus Luxuriant Golden yellow More than 70% 1. The medium should not be incubated for more than 24 hours since, if lactose
aureus (25923) fermenters predominate, the whole medium will turn pink, thus masking the
For growth RGI should be more than 70% presence of non-lactose fermenters.
RGI- Relative Growth Index 2. Factors that may cause urine counts from infected patients to be low include:
Procedure rapid rate of urine flow, prior initiation of antimicrobial therapy, a urine pH
1. Inoculate the medium as soon as possible after the specimen is received in of less than 5 and a specific gravity of less than 1.003.
the laboratory. 3. Shigella species may not grow on this medium.?
2. It is recommended that quantitative methods be used for culturing urine Storage
specimens. Store at 22-300C and prepared medium at 2-80C.
3. Incubate at 35-370C for 18-24 hours. Shelf Life
Use before expiry date as mentioned on the label.

Coagulase Mannitol Agar Base AM1028/AM5028

Use selective medium for isolating and differentiating staphylococcal species.
Coagulase Mannitol Agar Base with added plasma is used for isolation and Several years later, Zebovit et al (124) and Marwin (79) introduced tellurite-
differentiation of staphylococci from clinical specimens or for classifying pure glycine media designed to selectively isolate coagulase positive staphylococcal
cultures. species. The present formulation is based on Esber and Faulconer (26)
Summary formulation.
Coagulase-positive and coagulase-negative Staphylococcus species have major Principle
medical significance. Coagulase producing staphylococci (S.aureus ) may be Coagulase production is dependent on the presence of mannitol and a protein
differentiated and presumptively identified on this medium based on production factor in the brain heart infusion and blood serum (plasma). During utilization of
of coagulase and mannitol utilization. Chapman (16) introduced the first mannitol, the pH of the medium drops, causing the bromocresol purple indicator

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to change from purple to yellow, producing yellow zones around these colonies. Cultural Response
An opaque area of coagulated plasma forms around the colonies of organisms Cultural characteristics after 18-48 hours at 35-370C.
Organisms Growth Mannitol Coagulase RGI
that also produce coagulase. In contrast some coagulase-negative species that do
(ATCC) Fermentation production
not utilize mannitol, such as Staphylococcus epidermidis, do not change the
Staphylococcus Luxuriant + (yellow) + More than
colour of the medium and it remains clear. Other coagulase-negative species may aureus (25923) (opaque zone) 70%
utilize mannitol and produce a yellow zone around the colony, but an opaque Staphylococcus Luxuriant - (purple) - More than
zone will not be produced. epidermidis (12228) 70%
Formula* For growth RGI should be more than 70%
Ingredients in grams per liter RGI- Relative Growth Index
Tryptone 10.5 Procedure
Mannitol 10.0 1. Inoculate and incubate at 35-370C, and examine for growth after 18-24
Brain Heart Infusion 5.0 hours.
Sodium Chloride 3.5
2. Avoid prolonged incubation because it may cause the opaque zones
Soya Peptone 3.5
surrounding coagulase-positive organisms to become clear.
Bromocresol Purple 0.02
Agar 14.5 Interpretation of Results
0
Final pH (at 25 C) 7.4 ± 0.2 1. Coagulase-positive organisms will produce opaque zones; coagulase-
* Formula adjusted to suit performance parameters negative organisms will produce no opacity.
Directions
2. Organisms that utilize mannitol produce yellow zones. S.aureus may be
1. Suspend 47.02 gms of the powder in 1000 ml distilled water. presumptively identified as those colonies with opaque, yellow zones around
2. Mix thoroughly. them.
3. Boil with frequent agitation to dissolve the powder completely. Precautions
0 0
4. Sterilize by autoclaving at 118 C -121 C (12-15 lbs pressure) for 15 1. Some old or mutant strains of S.aureus may be weak coagulase producers
minutes. or exhibit negative coagulase reaction and should be subcultured and retested
5. Cool to 45-500C. if in doubt.

6. Add 7-15% v/v sterile, pre-tested, rabbit plasma to the basal medium. 2. E.coli also uses mannitol and may be weakly coagulase-positive. Colonial
morphology and a gram stain should readily allow for differentiation from
7. Mix well and pour into sterile petri plates.
S.aureus.
Quality Control
Storage
Dehydrated Appearance
Light grey coloured, homogeneous, free flowing powder. Store at 22-300C and prepared medium at 2-80C.
Prepared Appearance Shelf Life
Purple coloured, slightly opalescent gel. Use before expiry date as mentioned on the label.

Columbia Blood Agar Base AM1029/AM5029

Use purpose medium. It is also used for the selective cultivation of Brucella and
Columbia Blood Agar Base is used as an efficient base for the preparation of Blood Campylobacter species by the addition of the respective selective supplement.
Agar, Chocolate Agar and for various selective and identification media in Columbia Blood Agar Base is recommended by APHA for the examination of foods
isolating and cultivating fastidious microorganisms. (20).
Summary Principle
Columbia Blood Agar Base was developed by Ellner et al (24) and is used for Peptone special provides essential nutrients. Corn starch serves as the energy
isolating, cultivating and determining the haemolytic reactions of fastidious source and also neutralizes toxic metabolites. Columbia Blood Agar Base is used
pathogenic microorganisms. Without enrichment, it can be used as a general- as a base for media containing blood and for selective media formulations in

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which different combinations of antimicrobial agents are added as additives. It Organisms (ATCC) Growth with Haemolysis RGI
also promotes typical colonial morphology; better pigment production and more 5% Blood
Staphylococcus aureus Luxuriant Beta or More than 70%
sharply defined hemolytic reaction. Sheep blood permits the detection of
(25923) gamma
hemolysis and also provides heme (X factor), which is required for the growth of
Staphylococcus Luxuriant Gamma More than 70%
many bacteria. However, it is devoid of V factor (Nicotinamide Adenine epidermidis (12228)
Dinucleotide) and hence Haemophilus influenzae, which needs both X and V Streptococcus Luxuriant Alpha More than 70%
factors, will not grow on this medium. pneumoniae (6303)
Formula* Streptococcus Luxuriant Beta More than 70%
Ingredients in grams per liter pyogenes (19615)
Peptone Special 23.0 Neisseria meningitidis Luxuriant None More than 70%
Sodium Chloride 5.0 (13090)
Corn Starch 1.0 For growth RGI should be more than 70%
Agar 15.0 RGI- Relative Growth Index
Final pH (at 250C) 7.3 ± 0.2 Procedure
* Formula adjusted to suit performance parameters 1. Use standard procedures to obtain isolated colonies from specimens.
Directions
2. Incubate plates at 35-370C for 18-48 hours.
1. Suspend 44 gms of the powder in 1000 ml distilled water and mix well.
3. Since many pathogens require carbon dioxide on primary isolation, plates
2. Boil with frequent agitation to dissolve the powder completely.
may be incubated in an atmosphere containing approximately 3-10% CO2.
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. Interpretation of Results
4. Cool to 45-500C before the addition of heat sensitive compounds. 1. After incubation most plates will show an area of confluent growth.
·For Blood Agar: Add 5% sterile defibrinated sheep blood to sterile cool 2. Because the streaking procedure is in effect, a “dilution” technique,
base. diminishing numbers of microorganisms are deposited on the streaked areas.
·For Chocolate Agar: Add 10% sterile defibrinated sheep blood to sterile Consequently, one or more of these areas should exhibit isolated colonies of
cool base. Heat to 800C for 10 minutes with constant agitation. The the organisms contained in the specimen.
medium may be made selective by the addition of different 3. Further, growth of each organism may be semi-quantitatively scored on the
antimicrobials to sterile base. basis of growth in each of the streaked areas.
·For Brucella species: Add rehydrated contents of 1 vial of Brucella Precautions / Limitations
Selective Supplement, Modified (AS006) to 500 ml of sterile molten 1. Brucella cultures are highly infective and must be handled under properly
base containing 5-10% v/v inactivated Horse Serum (AS015) and 1% protected conditions.
w/v sterile dextrose. 2. Campylobacter species are best grown at 420C (except C.fetus subspecies fetus
·For Cocci: Add rehydrated contents of 1 vial of Staph-Strepto ) in a microaerophilic atmosphere.
Supplement (AS025) or Strepto Supplement (AS026) to 500 ml of 3. Staph/Strepto supplemented plates should be incubated aerobically at 350C
sterile molten base along with 25ml sterile defibrinated horse blood. for 18 hours. Incubation in carbon dioxide-enriched air will cause inhibition of
Quality Control staphylococcal growth.
Dehydrated Appearance
4. Strepto supplemented plates should be incubated aerobically or
Light yellow coloured, homogeneous, free flowing powder.
Prepared Appearance
anaerobically at 350C for 18 hours.
Basal Medium - Light amber coloured, clear to slightly opalescent gel. 5. Since the nutritional requirements of organisms vary, some strains may be
With addition of 5% defibrinated blood - Cherry red coloured, opaque gel. encountered that fail to grow or grow poorly on this medium.
Cultural Response
6. Columbia Blood Agar Base has a relatively high carbohydrate content and
Cultural characteristics after 18-48 hours at 35-370C.
therefore beta-hemolytic streptococci may produce a greenish hemolytic
reaction that may be mistaken for alpha-hemolysis

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7. Hemolytic reactions of some strains of group D streptococci are affected by Storage
differences in animal blood. Such strains are beta-hemolytic on horse, Store at 22-300C and prepared medium at 2-80C.
human and rabbit Blood Agar and alpha-hemolytic on sheep Blood Agar. Shelf Life
8. Prepared plates of supplemented media should be used within 18 hours of Use before expiry date as mentioned on the label.
preparation for maximum selectivity.

Columbia Agar (Harmonized) AMH5029

Columbia Agar Base EP AM50291
Columbia Agar Base BP AM50292
Columbia Agar Base USP (Agar Medium Q) AM50293
Use Light yellow coloured, homogeneous, free flowing powder.
Columbia Agar Base is used for detection of Clostridium perfringens from Prepared Appearance
Basal Medium- Light amber coloured, clear to slightly opalescent gel.
pharmaceutical products.
Cultural Response
Summary
Cultural characteristics after 18-24 hours at 35ºC.
Columbia Blood Agar Base was developed by Ellner et al., and used for isolating, Organisms (ATCC) Growth RGI
cultivating and determining the haemolytic reactions of fastidious pathogenic Staphylococcus aureus (25923) Luxuriant More than 70%
microorganisms. Without enrichment, it can be used as a general-purpose Staphylococcus epidermidis (12228) Luxuriant More than 70%
medium. Streptococcus pneumoniae (6303) Luxuriant More than 70%
Principle Streptococcus pyogenes (19615) Luxuriant More than 70%
Neisseria meningitidis (13090) Luxuriant More than 70%
Pancreatic digest of casein, meat peptic digest, heart pancreatic digest and yeast
Clostridium sporogenes (11437) Luxuriant More than 70%
extract provide essential nutrients. Maize starch serves as the energy source and
For growth RGI should be more than 70%
also neutralizes toxic metabolites. Sodium chloride maintains the osmotic RGI- Relative Growth Index
pressure. Procedure
Formula*
1. Use standard procedures to obtain isolated colonies from specimens.
Ingredients in grams per liter
Pancreatic digest of casein 10.0 2. Incubate plates at -37ºC for 18-48 hours.
Meat peptic digest 5.0 3. Since many pathogens require carbon dioxide on primary isolation, plates
Heart pancreatic digest 3.0 may be incubated in an atmosphere containing approximately 3-10% CO2.
Yeast extract 5.0
Interpretation of Results
Maize starch 1.0
Sodium chloride 5.0
1. After incubation most plates will show an area of confluent growth.
Agar 15.0 2. Because the streaking procedure is in effect, a “dilution”technique,
Final pH (at 250C) 7.3± 0.2 diminishing numbers are deposited on the streaked areas. Consequently,
Formula adjusted to suit performance parameters one or more of these areas should be exhibit-isolated colonies of the
Directions organisms contained in the specimen.
1. Suspend 44 gms of the powder in 1000ml distilled water and mix 3. Further growth of each organism may be semi quantitatively scored on the
thoroughly. basis of growth in each of the streaked areas.
2. Boil with frequent agitation to dissolve the powder completely. Storage
3. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes. Store at 22-300C and prepared medium at 2-80C.
Shelf Life
4. Cool to 45-50°C before the addition of heat sensitive compounds.
Quality Control
Use before expiry date as mentioned on the label.
Dehydrated Appearance

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Cooked Meat Medium AM1030/AM5030
Use Cultural Response
Cooked Meat Medium is used for the cultivation and maintenance of aerobes and Cultural characteristics after 40-48 hours at 350C.
anaerobes, especially Clostridium species. Organisms (ATCC) Growth RGI
Clostridium sporogenes (11437) Luxuriant More than 70%
Summary
Enterococcus faecalis (29215) Luxuriant More than 70%
Cooked Meat Medium is a modification of Robertson's (93) original formula and it Streptococcus pneumoniae (6303) Luxuriant More than 70%
is also called Chopped Meat Medium (69). Cooked Meat Medium is used for the Clostridium perfringenes (12924) Luxuriant More than 70%
cultivation and maintenance of clostridia and for determining the proteolytic For growth RGI should be more than 70%
activity of anaerobes. It also supports the growth of many spore forming and non- RGI- Relative Growth Index
spore forming strict anaerobes. This medium is recommended by FDA's Procedure
Bacteriological Analytical Manual for enumeration and identification of 1. Preferably use freshly reconstituted, sterile medium, which is inoculated as
Clostridium perfringens from foods and cosmetics (113) and by APHA for the soon as it has cooled to approximately 350C.
examination of foods (20). 2. Inoculate at the bottom of the tube in the meat particles.
Principle
3. For aerobic organisms, incubate up to 7 days at 350C with loosened caps,
Proteose peptone provides carbon and nitrogen. Sodium chloride maintains the examine daily for turbidity, gas or changes in the meat particles.
osmotic balance. The muscle protein in the heart tissue is a source of amino acids
4. For anaerobic organisms, incubate up to 21 days at 350C, make films and
and other nutrients, it also provides reducing substances, particularly
subculture at regular intervals.
glutathione, which allows the growth of strict anaerobes. The sulphydryl groups,
5. For maintenance of stock culture, hold at room temperature after initial
which exert the reducing effect, are more available in denatured protein; hence
meat particles are cooked for use in this medium. Growth is indicated by turbidity incubation at 350C. Subculture every 4-6 months.
Interpretation of Results
or bubble formation by some organisms. Blackening and disintegration of meat
particles indicate proteolysis. 1. Saccharolytic organisms usually produce acid and gas.
Formula* 2. Proteolytic organisms generally cause blackening and dissolution of the
Ingredients in grams per liter meat particles.
Beef Heart, Infusion from 454.0 Precautions / Limitations
Proteose Peptone 20.0
1. Tubes not used on the day of preparation should be placed in a boiling water
Sodium Chloride 5.0
Dextrose 2.0
bath or steamed for about 15 minutes to remove dissolved oxygen, cooled
Final pH (at 250C) 7.2 ± 0.2 without agitation, then inoculated.
* Formula adjusted to suit performance parameters 2. Meat particles in the medium may cause turbidity, which could be
Directions interpreted as positive growth.
1. Suspend 12.5 gms of the powder in 100 ml distilled water. 3. Meat particles blacken only in the presence of alkali, which is a result of the
2. Mix thoroughly. ammonia produced by proteolytic organisms.
3. Allow to stand for 15 minutes until all the particles are thoroughly wetted. 4. The reaction observed in the medium is useful for characterization, not
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. speciation, of the organism.
Quality control Storage
Dehydrated Appearance Store below 300C and prepared medium at 2-80C.
Brown coloured granules.
Shelf Life
Prepared Appearance
Medium amber coloured, clear to slightly opalescent supernatant over
Use before expiry date as mentioned on the label.
insoluble granules.

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Cooked Meat Medium BIS AM103011/AM503011
Use Cultural Response
A medium for cultivation and maintenance of aerobes, especially Clostridium Cultural characteristics after 40-48 hours at 350C.
species in compliance with BIS specification IS:5887(Part 2) 1976. Organisms (ATCC) Growth
Clostridium botulinum (25763) Luxuriant
Summary
Clostridium perfringens (12924) Luxuriant
Cooked Meat Medium was originally developed by Robertson for the cultivation of Clostridium sporogenes (11437) Luxuriant
certain anaerobes isolated from wounds. The present formulation is Streptococcus pneumoniae (6303) Luxuriant
recommended by BIS for the detection and enumeration of bacteria responsible Enterococcus faecalis (29212) Luxuriant
for food poisoning especially Clostridium welchii (11.1). This medium with For growth RGI should be more than 70%
addition of further 10% Sodium chloride is used as a salt medium for isolation of RGI- Relative Growth Index
Staphylococci (11.2). It is used for cultivation and maintenance of Clostridia and Procedure

for determining proteolytic activity of anaerobes (77.1). FDA has recommended a 1. Preferably use freshly reconstituted, sterile medium, which is inoculated as
slight modification of this medium for enumeration and identification of soon as it has cooled to approximately 350C.
Clostridium perfringens from foods. 2. Inoculate at the bottom of the tube in the meat particles.
Principle
3. For aerobic organisms, incubate up to 7 days at 350C with loosened caps,
Cooked Meat Medium contains beef heart, the muscle protein which provides examine daily for turbidity, gas or changes in the meat particles.
amino acids and other nutrients. It also contains glutathione a reducing
4. For anaerobic organisms, incubate up to 21 days at 350C, make films and
substance which permits the growth of obligate anaerobes. The sulphydryl groups
subculture at regular intervals.
which impart reducing effect are more available in denatured protein and hence
5. For maintenance of stock culture, hold at room temperature after initial
the cooked meat is added in the medium. The growth in this medium is indicated
incubation at 350C. Subculture every 4-6 months.
by the turbidity or bubble formation by some organisms. Blackening and
Interpretation of Results
disintegration of the meat particles indicate proteolysis. For best results, medium
should be used on the day it is prepared, otherwise it should be boiled or steamed 1. Saccharolytic organisms usually produce acid and gas.
for a few minutes and allowed to cool without agitation and then inoculated. 2. Proteolytic organisms generally cause blackening and dissolution of the
Formula* meat particles.
Ingredients in grams per liter Precautions / Limitations
Beef Heart, Infusion from(500gm) 107.90 1. Tubes not used on the day of preparation should be placed in a boiling water
Peptic digest of animal tissue 5.0 bath or steamed for about 15 minutes to remove dissolved oxygen, cooled
Sodium Chloride 2.5
without agitation, then inoculated.
Final pH (at 250C) 7.8 ±0.2
* Formula adjusted to suit performance parameters 2. Meat particles in the medium may cause turbidity, which could be
Directions interpreted as positive growth.
1. Suspend 115.40 gms of the powder in 1000 ml distilled water. 3. Meat particles blacken only in the presence of alkali, which is a result of the
2. Mix thoroughly. ammonia produced by proteolytic organisms.
3. Allow to stand for 15 minutes until all the particles are thoroughly wetted. 4. The reaction observed in the medium is useful for characterization, not
specialization, of the organism.
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Storage
Quality control
Dehydrated Appearance Store at 22-300C and prepared medium at 2-80C.
Brown coloured granules. Shelf Life
Prepared Appearance Use before expiry date as mentioned on the label.
Medium amber coloured, clear to slightly opalescent supernatant over
insoluble granules.

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Corn Meal Agar AM10301/AM50301
Use Candida albicans Luxuriant + More than 70%
Corn Meal Agar is a general-purpose medium for the cultivation of fungi. (10231)
Candida kefyr Good _ More than 70%
Summary
(8553)
To facilitate a more rapid differentiation of strains of Candida albicans from other Saccharomyces Luxuriant _ More than 70%
yeasts and from other species of Candida, there arose a need for culture uvarum (9080)
conditions, which would result in the rapid formation of mycelia, chlamydospores Saccharomyces Luxuriant _ More than 70%
or both. Pollack and Benham reported the usefulness of Corn Meal Agar for cerevisiae (9763)
studying the morphology of Candida (89). In 1960, Walker and Huppert For growth RGI should be more than 70%
modified the basic formulation of Corn Meal Agar by adding Polysorbate 80, RGI- Relative Growth Index
which stimulated rapid and abundant chlamydospore formation (117). This Procedure

modified formulation is recommended for the production and visualization of 1. To prepare plated media, place the medium in a boiling water bath until the
chlamydospores. Besides, the addition of dextrose enhances fungal growth and medium becomes liquefied.
pigment production. 2. Pour the molten medium into a sterile petriplate and allow to solidify.
Principle 3. With the help of an inoculating needle, streak the medium with growth from
Corn Meal Agar is a relatively simple medium, consisting of an infusion of corn 0
a pure culture and incubate at 25 ± 2 C.
meal and agar. This infusion product contains sufficient nutrients to support the 4. Examine at regular intervals for up to 28 days for growth and pigmentation.
growth of fungal species. The polysorbate 80 is a mixture of oleic esters which
5. If Dextrose is added to the medium, it is necessary to incubate for up to 4
when added to the basal medium, stimulates the production of chlamydospores.
weeks to allow sufficient time for pigmentation to develop.
Dextrose when added to Corn Meal Agar provides an energy source that enhances
fungal growth and chromogenesis. 6. If Polysorbate 80 is added to the medium, the production of chlamydospores
Formula* should be tested using the Dalmau Plate Method.
Ingredients in grams per liter Interpretation of Results
Corn meal, infusion from 50.0 1. Observe cultures for growth and morphology.
Agar 15.0 2. If Polysorbate 80 is added to a medium, most strains of C. albicans and C.
Final pH (at 250 C) 6.0 ± 0.2
stellatoidea form chlamydospores within 24-48 hours.
* Formula adjusted to suit performance parameters
Directions 3. If Dextrose is added to the medium, observe for chromogenesis
1. Suspend 17 grams of the powder in 1000 ml distilled water. If desired, add macroscopically.
Precautions / Limitations
1% Polysorbate 80 or 1% Dextrose and mix thoroughly.
1. Some Candida species lose their ability of chlamydospores formation by
2. Heat with frequent agitation to dissolve the powder completely.
repeated subculturing.
3. Sterilize by autoclaving at 1210 C (15 lbs pressure) for 15 minutes.
2. When streaking, with a sterile inoculating needle, lightly touch the yeast
Quality Control
Dehydrated Appearance
colony and make two separate streaks. Do not dig into the agar. Flame a
Light yellow coloured, coarse, free flowing powder. cover slip and after it cools, place it over the central area of the stab marks to
Prepared Appearance provide slightly reduced oxygen tension.
Light amber coloured, opalescent gel. 3. Glucose supplemented Corn Meal Agar should not be used for
Cultural Response chlamydospores production by Candida species.
Cultural characteristics after 4 days when incubated at 25 ± 20 C when Storage
incubated for 4 days.
Organisms (ATCC) Growth Chlamydospores RGI Store at 22-300C and prepared medium at 2-80C.
Asperigillus niger Luxurinat _ More than 70% Shelf Life
(16404) Use before expiry date as mentioned on the label.

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Czapek Dox Agar AM10302/AM50302
Use 2. Mix thoroughly.
Czapek Dox Agar is used for general cultivation of fungi, yeasts and soil bacteria. 3. Boil with frequent agitation to dissolve the powder completely. Do not
Summary overheat.
Czapek Dox agar has been developed according to the formula of Thom and 4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Church. This medium has a defined chemical composition. Czapek Dox Agar is a
Quality Control
semi synthetic medium containing sodium nitrate as the sole source of nitrogen.
Dehydrated Appearance
Principle
Creamish white, homogeneous, free flowing powder.
Sucrose serves, as a sole source of carbon and sodium nitrate is the source of Prepared Appearance
nitrogen. Dipotassium phosphate is the buffering agent. Magnesium sulphate, Light yellow coloured, clear to slightly opalescent solution or gel with slight
Potassium chloride and Ferrous sulphate are source of essential cations. medium precipitate forms in petriplates.
Cultural Response
is also a highly satisfactory substrate for chlamydospore production by Candida
Cultural characteristics after 48-72 hours at 30ºC.
albicans. The pH is slightly alkaline. Czapek Dox Agar supports abundant growth
Organisms (ATCC) Growth RGI
of almost all saprophytic Aspergilli with characteristic mycelia and conidia Aspergillus niger (16404) Luxuriant More than 70%
formation. Saccharomyces cerevisiae (9763) Luxuriant More than 70%
Formula* Candida albicans (10231) Luxuriant More than 70%
Ingredients in grams per liter For growth RGI should be more than 70%
Sucrose 30.0 RGI- Relative Growth Index
Sodium nitrate 2.0 Procedure
Dipotassium phosphate 1.0
1. Prepare the medium. After plating inoculate the medium.
Magnesium sulphate 0.50
Potassium chloride 0.50 2. Incubate the medium at 30ºC.
Ferrous sulphate 0.01 Interpretation of Results
Agar 15.0 Refer to appropriate references and procedures for result.
Final pH (at 250C) 7.3 ± 0.2 Storage
* Formula adjusted to suit performance parameters
Store at 22-300C and prepared medium at 2-80C.
Directions
Shelf Life
1. Suspend the 49gms of powder in 1000 ml distilled water. Use before expiry date as mentioned on the label.

Decarboxylase Agar Base AM503023

Use nutrients for the bacterial growth. Dextrose is the fermentable carbohydrate.
Decarboxylase Agar Base is used to differentiate bacteria on the basis of their Bromo cresol purple is the pH indicator which change colour from purple to yellow
ability to decarboxylate the amino acid added to the medium. in acidic condition. Decarboxylase activity is stimulated by acidic pH and hence
Summary the amino acids are decarboxylated or degraded to form corresponding amine.
Decarboxylase agar base is formulated as described by Moeller to differentiate Production of these amines increase the pH of the medium changing the colour of
bacteria on the basis of their ability to decarboxylate the amino acids. the indicator and inturn the medium from yellow to purple violet.
Principle Each isolate must be inoculated into a tube of the basal medium without amino
The medium is useful for the identification of the Enterobacteriaceae and other acid. If this tube becomes alkaline then the best is invalid. Exposure of the
gram-negative bacilli. Production of ornithine decarboxylase is especially useful medium to air may cause alkalinization so the inoculated tubes if covered with a
for differentiating Enterobacter and Klebsiella species as the former produces layer of sterile mineral oil will give best result.
this enzyme and are motile while later are nonmotile and do not synthesize this Formula*
enzyme. Peptic digest of animal tissue and yeast extract supply nitrogenous Peptic digest of animal tissue 5.0

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Yeast extract 3.0 Organisms (ATCC) Lysine Arginine Ornithine
Dextrose 1.00 Citrobacter freundii (8090) - + +
Bromo Cresol Purple 0.02 Enterobacter aerogenes (13048) + - +
Agar 15.0 Escherichia coli (25922) + + +
Final pH (at 250C)6.5+0.2 Klebsiella pneumoniae (13883) + - -
* Formula adjusted to suit performance parameters Proteus mirabilis (25933) - - +
Directions Salmonella serotype - (+)or+
1. Suspend 24.02 gms in 1000 ml distilled water. Paratyphi A
Salmonella serotype Typhi (6539) + (+) or - -
2. Boil to dissolve the medium completely. Shigella flexneri (12022) - - or (+) -
3. Add 5 gms of desired L-Amino acid (L-Lysine, L-Arginine, L-Ornithine) in Shigella sonnei (25931) - + +
hydrochloride form per liter of the medium. Shigella dysenteriae (13313) - - or (+) -
Serratia marcescens (8100) + - +
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Key: - = negative reaction, yellow colour
5. Dispense into sterile test tubes and cool in a slanted position. + = positive reaction, purple colour
6. When L-Orinithine hydrochloride is used, readjustment of pH is necessary. (+) = delayed positive reaction
Quality Control Storage
Dehydrated Appearance Store at 22-300C and prepared medium at 2-80C.
Creamish yellow, homogeneous, free flowing powder. Shelf Life
Prepared Appearance Use before expiry date as mentioned on the label.
Purple coloured, clear gel forms in tubes as slants.
Cultural Response
Cultural characteristics after upto 4 days at 35-37ºC.

Deoxycholate Agar AM10303/AM50303

Use the solidifying agent. Sodium chloride provides sodium ions for the membrane
A medium for direct differential count of coliforms in dairy products and for transport and maintains osmotic equilibrium of the medium. Bacteria that
isolation of enteric pathogens from rectal swabs, faeces and other pathological ferment lactose produce acid and form red colonies. Bacteria that do not ferment
specimens. lactose form colourless colonies. The majority of normal intestine bacteria
Summary ferment lactose except Salmonella and Shigella.
Deoxycholate Agar is a modification of Leifson’s original formula. It demonstrates Formula*
improved recovery of intestinal pathogens from specimens containing normal Ingredients in grams per liter
intestinal flora by using sodium deoxycholate and sodium citrate in specified Peptic digest of animal tissue 10.0
Lactose 10.0
amounts as inhibitors of gram-positive bacteria. It is used for isolation of enteric
Sodium chloride 5.00
pathogens from rectal swabs, faeces or other specimens for the routine
Dipotassium phosphate 2.00
examination of clinical samples, it is recommended that MacConkey Agar (AM Sodium deoxycholate 1.00
1059/5059), Bismuth Sulphite Agar (AM 1013/5013) etc. should be used in Sodium citrate 1.00
parallel with this media. Ferric citrate 1.00
It is also used for enumeration of coliforms in dairy products & water. Neutral red 0.03
Principle Agar 15.00
Final pH (at 250C) 7.3 ± 0.2
Peptic Digest of Animal Tissue supply nutrients, nitrogen compounds and amino
* Formula adjusted to suit performance parameters
acids. Lactose is the fermentable carbohydrate. Sodium citrate and sodium Directions
deoxycholate inhibit gram-positive bacteria. Neutral red is a pH indicator. Agar is
1. Suspend the 45.03 gms of powder in 1000 ml distilled water.

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2. Mix thoroughly. Escherichia coli Good Pink with bile More than 70%
(25922) precipitate.
3. Boil with frequent agitation to dissolve the powder completely. Avoid
Staphylococcus Inhibited – 0%
excessive heating, as it is detrimental to the medium. DO NOT AUTOCLAVE aureus (25923)
OR REHEAT. For growth RGI should be more than 70%
4. Pour into adequate containers homogenizing the medium well enough to For Inhibition RGI should be 0%
distribute the calcium carbonate. RGI- Relative Growth Index
Quality Control Procedure
Dehydrated Appearance 1. Inoculate directly on the surface of the medium.
Light pink coloured, homogeneous, free flowing powder. 2. Incubate plates at 35-370C for 18-24 hours. Plates can be incubated for an
Prepared Appearance
additional 24 hours if no lactose fermenters observed.
Reddish orange coloured, Clear to very slightly opalescent gel .
Interpretation of Results
Cultural Response
Cultural characteristics after 18-24 hours at 35-370C. 1. Non lactose fermenters produce transparent, Colourless to pink or tan
Organisms (ATCC) Growth Colour of RGI coloured colonies with or without bile precipitation.
colonies 2. Lactose fermenters produce red colonies with or without bile precipitation.
S. serotype Good Colourless More than 70%
Storage
Typhimurium (14028)
S. serotype Enteritidis Good Colourless More than 70%
Store at 22-300C and prepared medium at 2-80C.
(13076) Shelf Life
Use before expiry date as mentioned on the label.

Deoxycholate Citrate Agar AM1031/AM5031

Use is used. Proteose peptone provides carbon, nitrogen, vitamins and minerals.
Deoxycholate Citrate Agar is a selective medium used for the isolation of enteric Lactose is the fermentable carbohydrate. Sodium citrate and sodium
pathogens particularly Salmonella and Shigella species. deoxycholate inhibit gram-positive bacteria, coliforms and Proteus species. Ferric
Summary ammonium citrate aids in the detection of H2S-producing bacteria. Neutral red is
Deoxycholate Citrate Agar is a modification of Deoxycholate Agar formulated by a pH indicator.
Leifson (67), which demonstrates improved recovery of intestinal pathogens from Bacteria that ferment lactose produce acid and form red colonies. Bacteria that do
specimens containing normal intestinal flora by using citrates and sodium not ferment lactose form colourless colonies. Bacteria producing H2S will have
deoxycholate in specified amounts as inhibitors of gram-positive bacteria. In black centers. The majority of normal intestinal bacteria ferment lactose and do
comparison, Deoxycholate Citrate Agar has increased concentrations of sodium not produce H2S (red colonies without black centers). Salmonella and Shigella
citrate and sodium deoxycholate for reliably isolating many Salmonella and species do not ferment lactose but Salmonella may produce H2S (colourless
Shigella species while inhibiting coliforms and many Proteus species. This
colonies with or without black centers). Lactose fermenting colonies may have a
medium is used for the isolation and maximum recovery of intestinal pathogens
zone of precipitation around them caused by the precipitation of deoxycholate in
belonging to Salmonella and Shigella groups from foods. The selectivity of this
the presence of acid.
medium permits the use of fairly heavy inocula without danger of overgrowth of
Formula*
Shigella and Salmonella by other micro flora. Deoxycholate Citrate Agar is Ingredients in grams per liter
recommended by APHA for the examination of foods (20) and in the IP for use in Sodium Citrate 20.0
Microbial Limit test (46). Proteose Peptone 10.0
Principle Heart Infusion Solids 10.0
Heart infusion is a source of carbon and nitrogen and is preferred because the Lactose 10.0
Sodium Deoxycholate 5.0
inhibition of coliforms produced is greater than when an extract or simple peptone

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Ferric Ammonium Citrate 2.0 Procedure
Neutral Red 0.02 1. Inoculate directly onto the surface of the medium.
Agar 13.5
2. Incubate plates at 35-370C for 18-24 hours. Plates can be incubated for an
Final pH (at 250C) 7.5 ± 0.2
* Formula adjusted to suit performance parameters additional 24 hours if no lactose fermenters are observed.
Directions Interpretation of Results

1. Suspend 70.52 gms of the powder in 1000 ml distilled water. 1. Non-lactose fermenters produce transparent, colourless to light pink or tan
coloured colonies with or without black centers.
2. Mix thoroughly.
2. Lactose fermenters produce red colonies with or without bile precipitation.
3. Boil with frequent agitation to dissolve the powder completely. Avoid
Precautions / Limitations
excessive heating, as it is detrimental to the medium.
1. Deoxycholate Citrate Agar is heat sensitive. Avoid excessive or prolonged
4. DO NOT AUTOCLAVE.
heating during reconstitution. Do not autoclave or remelt.
Quality Control
Dehydrated Appearance 2. The medium is best used freshly prepared.
Light pink coloured, homogeneous, free flowing powder. 3. Stock cultures of Shigella species may predominantly be in the R-phase
Prepared Appearance when subcultured away from Deoxycholate Citrate Agar. Such cultures are
Reddish orange coloured, clear to very slightly opalescent gel.
difficult to use for control purposes without first heavily streaking the culture
Cultural Response
on Deoxycholate Citrate Agar plates and picking off the few S-phase colonies
Cultural characteristics after 18-24 hours at 35-370C.
i.e. the macro-colonies on the agar surface, for further subculture.
Organisms (ATCC) Growth Colour of colony H2S RGI
Enterococcus Inhibited - - More than 70% 4. When performing biochemical tests on colonies picked from the surface of
faecalis (29212) DCA plates, purity of subcultures should be carried out because the colony may
Escherichia coli Poor Pink with - 0% be contaminated with E.coli present as micro colonies.
(25922) bile precipitate
Salmonella serotype Luxuriant Colourless + More than 70%
5. Coliform strains may be encountered that will grow on this medium, making it
Enteritidis (13076) difficult to detect pathogens.
Salmonella serotype Luxuriant Colourless + More than 70% 6. Heavy inocula should be distributed over the entire surface of the medium to
Typhimurium (14028)
prevent complete masking of pathogens by coliform organisms.
Shigella flexneri (12022) Good Colourless - More than 70%
Storage
For growth RGI should be more than 70%
For Inhibition RGI should be 0% Store at 22-300C and prepared medium at 2-80C.
RGI- Relative Growth Index Shelf Life
Use before expiry date as mentioned on the label.

Deoxycholate Citrate Agar IP AM10311/AM50311

Use Shigella species while inhibiting coliforms and many proteus species. This
Deoxycholate Citrate Agar is a selective medium used for the isolation of enteric medium is used for the isolation maximum recovery of intestinal pathogens
pathogens particularly Salmonella and Shigella species in compliance with IP. belonging to Salmonella and Shigella groups from foods. The selectivity of this
Summary medium permits the use of fairly heavy inocula without danger of overgrowth of
Deoxycholate Citarte Agar is a modification of Deoxycholate agar formulated by Shigella and Salmonella by other micro flora. Deoxycholate Citrate Agar is
Leifson’s (67), which demonstrates improved recovery of intestinal pathogens recommended by APHA for the examination of foods and in the IP for use in
from specimens containing normal intestinal flora by using sodium deoxycholate Microbial Limit Test.
and sodium citrate in specified amounts as inhibitors of gram-positive bacteria. In Principle
comparison, deoxycholate Citrate Agar has incresed concentrations of sodium Peptone supply nutrients, nitrogen compounds and amino acids. Lactose is the
citrate and sodium deoxycholate for reliably isolating many Salmonella and fermentable carbohydrate. Sodium citrate and sodium deoxycholate inhibit

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gram-positive bacteria, coliforms and Proteus species. Ferric ammonium citrate Prepared Appearance
aid in the detection of H2S producing bacteria. Neutral red is a pH indicator. Reddish orange coloured, Clear to very slightly opalescent gel .
Cultural Response
Bacteria that ferment lactose produce acid and form red colonies. Bacteria that do Cultural characteristics after 18-24 hours at 35-370C.
not ferment lactose form colourless colonies. Bacteria producing H2S will have Organisms (ATCC) Growth Colour of H2S RGI
black centers. The majority of normal intestine bacteria ferment lactose and do colonies
not produce H2S (red colonies without black centers). Lactose fermenting colonies Enerococcus Inhibited – – 0%
may have a zone of precipitation around them caused by the precipitation of faecalis (29212)
Escherichia coli Poor Pink with bile 0%
deoxycholate in the presence of acid.
(25922) precipitate –
Formula*
S. serotype Luxuriant Colourless + More than 70%
Ingredients in grams per liter
Enteritidis (13076)
Trisodium citrate 8.5
S. serotype Luxuriant Colourless + More than 70%
Beef extract 5.0
Typhimurium (14028)
Peptone 5.0
Shigella flexneri Good Colourless – More than 70%
Lactose 10.0
(12022)
Sodium deoxycholate 5.00
For growth RGI should be more than 70%
Ferric citrate 1.00
For Inhibition RGI should be 0%
Neutral red 0.02
RGI- Relative Growth Index
Agar 13.5
Procedure
Final pH (at 250C) 7.5 ± 0.2
* Formula adjusted to suit performance parameters 1. Inoculate directly on the surface of the medium.
Directions 2. Incubate plates at 35-370C for 18-24 hours. Plates can be incubated for an
1. Suspend the 69.52 gms of powder in 1000 ml distilled water. additional 24 hours if no lactose fermenters observed.
2. Mix thoroughly. Interpretation of Results

3. Boil with frequent agitation to dissolve the powder completely. Avoid 1. Non lactose fermenters produce transparent, Colourless to pink or tan
excessive heating, as it is detrimental to the medium. DO NOT AUTOCLAVE coloured colonies with or without bile precipitation.
OR REHEAT. 2. Lactose fermenters produce red colonies with or without bile precipitation
Quality Control Storage
Dehydrated Appearance Store at 22-300C and prepared medium at 2-80C.
Light pink coloured, homogeneous, free flowing powder. Shelf Life
Use before expiry date as mentioned on the label.

Deoxycholate Citrate Agar EP AM10312/AM50312

Deoxycholate Citrate Agar BP AM50313
Use comparison, deoxycholate Citrate Agar has increased concentrations of sodium
Deoxycholate Citrate Agar is a selective medium used for the isolation of enteric citrate and sodium deoxycholate for reliably isolating many Salmonella and
pathogens particularly Salmonella and Shigella s pecies. Shigella species while inhibiting coliforms and many Proteus species. This
Summary medium is used for the isolation maximum recovery of intestinal pathogens
Deoxycholate Citarte Agar is a modification of Deoxycholate Agar formulated by belonging to Salmonell a and Shigella groups from foods. The selectivity of this
Leifson’s (67), which demonstrates improved recovery of intestinal pathogens medium permits the use of fairly heavy inocula without danger of overgrowth of
from specimens containing normal intestinal flora by using sodium deoxycholate Shigella and Salmonella by other micro flora. Deoxycholate Citrate Agar is
and sodium citrate in specified amounts as inhibitors of gram-positive bacteria. In recommended by APHA for the examination of foods and in the BP for use in
Microbial Limit Test.

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Principle Quality Control
Heart Infusion is a source of carbon and nitrogen and is preferred because the Dehydrated Appearance
Light pink coloured, homogeneous, free flowing powder.
inhibition of coliforms produced is greater than when an extract or simple peptone
Prepared Appearance
is used. Proteose peptone provides carbon, nitrogen vitamins and minerals.
Reddish orange coloured, Clear to very slightly opalescent gel .
Lactose is the fermentable carbohydrate. Sodium citrate and sodium Cultural Response
deoxycholate inhibit gram-positive bacteria, coliforms and Proteus species. Ferric Cultural characteristics after 18-24 hours at 35-370C.
ammonium citrate aid in the detection of H2S producing bacteria. Neutral red is a Organisms (ATCC) Growth Colour of H2S RGI
pH indicator. colonies
Bacteria that ferment lactose produce acid and form red colonies. Bacteria that do Enerococcus faecalis Inhibited – – 0%
not ferment lactose form colourless colonies. Bacteria producing H2S will have (29212)
Escherichia coli Poor Pink with – 0%
black centers. The majority of normal intestine bacteria ferment lactose and do
(25922) bile precipitate
not produce H2S (red colonies without black centers). Lactose fermenting colonies S. serotype Luxuriant Colourless + More than 70%
may have a zone of precipitation around them caused by the precipitation of Enteritidis (13076)
deoxycholate in the presence of acid. S. serotype Luxuriant Colourless + More than 70%
Formula* Typhimurium (14028)
Ingredients in grams per liter Shigella flexneri Good Colourless – More than 70%
Sodium citrate 20.0 (12022)
Beef extract 10.0 For growth RGI should be more than 70%
Meat peptone 10.0 For Inhibition RGI should be 0%
Lactose monohydrate 10.0 RGI- Relative Growth Index
Sodium deoxycholate 5.00 Procedure
Iron (lll) citrate 1.00 1. Inoculate directly on the surface of the medium.
Neutral red 0.02
2. Incubate plates at 35-370C for 18-24 hours. Plates can be incubated for an
Agar 13.50
Final pH (at 250C) 7.3 ± 0.2
additional 24 hours if no lactose fermenters observed.
* Formula adjusted to suit performance parameters Interpretation of Results
Directions 1. Non lactose fermenters produce transparent, Colourless to pink or tan
1. Suspend the 69.52 gms of powder in 1000 ml distilled water. coloured colonies with or without bile precipitation.
2. Mix thoroughly. 2. Lactose fermenters produce red colonies with or without bile precipitation.
Storage
3. Boil with frequent agitation to dissolve the powder completely. Avoid
excessive heating, as it is detrimental to the medium. DO NOT AUTOCLAVE Store at 22-300C and prepared medium at 2-80C.
OR REHEAT. Shelf Life
Use before expiry date as mentioned on the label.

Dextrose Agar AM1032/AM5032

Dextrose Broth AM1033/AM5033
Use many fastidious bacteria, including Haemophilus and Neisseria. This medium
Dextrose Agar with or without blood is used for cultivation of a wide variety of has a high concentration of dextrose, which makes it suitable for the production of
microorganisms. Dextrose Broth is used for the cultivation of fastidious early, abundant organism growth and shortens the lag period of older cultures.
microorganisms and for detecting gas production from enteric bacilli. Dextrose Agar facilitates anaerobic growth and aids in dispersion of reducing
Summary substances and CO2 formed in the environment. The low agar concentration
Dextrose Agar with 5% defibrinated blood is recommended for the isolation of provides suitable conditions for both aerobic growth in the clear upper zones and

1
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for microaerophilic growth in the lower, flocculent agar zones. 5. OPTIONAL: To prepare Blood Agar add 5% v/v sterile defibrinated sheep
Dextrose Broth is a highly nutritious broth for the isolation of fastidious blood to sterile Dextrose Agar cooled to 45-500C. Mix well and dispense as
microorganisms and for specimens containing a low inoculum. This medium is desired.
used for antibiotic sensitivity testing using tube dilution method as it is found to Quality Control
be superior when compared to Soyabean Peptone Medium, particularly for Dehydrated Appearance
Light yellow coloured, homogeneous, free flowing powder.
sensitivity testing of Neomycin and Chlortetracycline. Dextrose Agar and Dextrose
Prepared Appearance
Broth are both specified in the Compendium of Methods for the microbiological
Dextrose Agar - Light yellow coloured, clear to slightly opalescent gel.
examination of foods (20). Dextrose Broth - Light yellow coloured clear solution.
Principle Cultural Response
Dextrose serves as the carbon source, the high concentration of which in Dextrose Cultural characteristics after 18-24 hours at 35-370C.
Agar is a distinguishing characteristic of this medium from other formulations Organisms (ATCC) Growth on Dextrose Growth with RGI
used as Blood Agar bases. Beef extract and tryptose provide nitrogen, sulphur, Agar and in blood on
Dextrose Broth Dextrose
carbon, amino acids, minerals and vitamins. Sodium chloride maintains the
Agar
osmotic balance. Bordetella pertussis Good to luxuriant Luxuriant More than 70%
Formula* (8467)
Ingredients in grams per liter Dextrose Agar Dextrose Broth Clostridium Fair to good Luxuriant More than 70%
Tryptose 10.0 10.0 perfringenes (12919)
Dextrose 10.0 5.0 Neisseria meningitidis Good to luxuriant Luxuriant More than 70%
Sodium Chloride 5.0 5.0 (13090)
Beef Extract 3.0 3.0 Escherichia coli Good to luxuriant Luxuriant More than 70%
Agar 15.0 - (25922)
Final pH (at 250C) 7.3 ± 0.2 7.2 ± 0.2 Streptococcus Good to luxuriant Luxuriant More than 70%
* Formula adjusted to suit performance parameters pyogenes (19615)
Directions For growth RGI should be more than 70%
RGI- Relative Growth Index
1. Suspend the powder in 1000 ml distilled water.
Precautions / Limitations
Dextrose Agar - 43 gms
1. Dextrose Agar is not suitable for observation of haemolysis when
Dextrose Broth - 23 gms supplemented with 5% sheep, rabbit or horse blood, because of the high
2. Mix thoroughly. concentration of dextrose.
3. Boil with frequent agitation to dissolve the powder completely. Storage
0
4. Sterilize by autoclaving at 121 C (15 lbs pressure) for 15 minutes. Store at 22-300C and prepared medium at 2-80C.
Shelf Life
Use before expiry date as mentioned on the label.

Dextrose Peptone
Microxpress Agar
Dehydrated AM1034/AM5034
Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits

Dextrose Peptone Broth AM1035/AM5035

Use microorganisms that are fastidious, or present in small numbers. Dextrose
Dextrose Peptone Agar and Dextrose Peptone Broth are general-purpose media Peptone Agar is an excellent basal medium for the preparation of Glucose Blood
used for the cultivation of a wide variety of microorganisms. Agar, which supports good growth of anaerobic microorganisms. Dextrose
Summary Peptone Agar is also used for the enumeration of thermophilic bacteria
Dextrose Peptone Media are formulated as suggested by Williams (120). responsible for flat sour spoilage of canned foods and both are recommended for
Dextrose Peptone Agar and Dextrose Peptone Broth are used for the cultivation of routine cultivation purpose by AOAC.

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Principle Quality Control
Dextrose serves as the carbon and energy source. Peptone provides amino acids, Dehydrated Appearance
Light yellow coloured, homogeneous, free flowing powder.
peptides etc. Sodium chloride maintains the osmotic balance.
Prepared Appearance
Formula*
Dextrose Peptone Agar - Light yellow coloured, clear to slightly opalescent
Ingredients in grams Dextrose Peptone Dextrose Peptone
gel.
per liter Agar Broth
Dextrose Peptone Broth - Light yellow coloured, clear to slightly opalescent
Dextrose 10.0 10.0 solution.
Peptone 20.0 20.0 Cultural Response
Sodium Chloride 5.0 5.0 Cultural characteristics after 18-48 hours at 35-370C.
Agar 15.0 - Organisms (ATCC) Growth RGI
Final pH (at 250C) 7.2 ± 0.2 Escherichia coli (25922) Luxuriant More than 70%
* Formula adjusted to suit performance parameters Pseudomonas aeruginosa (27853) Luxuriant More than 70%
Directions Staphylococcus aureus (25923) Luxuriant More than 70%
1. Suspend the powder in 1000 ml distilled water. Streptococcus pyogenes (19615) Luxuriant More than 70%
Dextrose Peptone Agar - 50 gms For growth RGI should be more than 70%
RGI- Relative Growth Index
Dextrose Peptone Broth - 35 gms Storage
2. Mix thoroughly. Store at 22-300C and prepared medium at 2-80C.
3. Boil with frequent agitation to dissolve the powder completely. Shelf Life
0 Use before expiry date as mentioned on the label.
4. Sterilize by autoclaving at 121 C (15 lbs pressure) for 15 minutes.

Dextrose Tryptone Agar AM1036/AM5036

Dextrose Tryptone Broth AM1037/AM5037
Use enumerating and enriching mesophilic and thermophilic organisms respectively
Dextrose Tryptone Agar is used for the cultivation and enumeration of mesophilic in cereals and cereal products, dehydrated fruits and vegetables and spices.
and thermophilic aerobic microorganisms associated with food spoilage while Dextrose Tryptone Agar and Dextrose Tryptone Broth are recommended by APHA
Dextrose Tryptone Broth is used for enrichment of mesophilic and thermophilic for the examination of foods (20).
microorganisms associated with food spoilage. Principle
Summary Dextrose serves as the carbohydrate source. Tryptone provides carbon and
Williams (120) formulated Dextrose Tryptone Agar, a suitable medium for the nitrogen for growth. Bromocresol purple is the pH indicator.
cultivation and enumeration of thermophilic bacteria. Dextrose Tryptone Broth is Formula*
used for enriching “flat sour” organisms from food products while Dextrose Ingredients in grams Dextrose Tryptone Dextrose Tryptone
Tryptone Agar is used for isolating “flat sour” organisms from food products. per liter Agar Broth
“Flat sour” spoilage of canned foods is caused by Bacillus coagulans (Bacillus Tryptone 10.0 10.0
Dextrose 5.0 5.0
thermodurans ). Bacterial growth results in a 0.3 - 0.5 drop in pH, while the ends
Agar 15.0 -
of the can remain flat. B.coagulans is a soil microorganism that can be found in
Bromocresol Purple 0.04 0.04
canned tomato products and dairy products. Conditions favourable for Final pH (at 250C) 6.7 ± 0.2 6.7 ± 0.2
multiplication of the bacterium can result in spoilage of the food product. * Formula adjusted to suit performance parameters
Dextrose Tryptone Agar and Dextrose Tryptone Broth are also used for isolating Directions
and enriching other food spoilage bacteria respectively, mesophilic aerobic spore 1. Suspend the powder in 1000 ml distilled water and mix well.
formers in the genera Bacillus and Sporolactobacillus and thermophilic flat sour Dextrose Tryptone Agar - 30 gms
spore formers such as B.stearothermophilus. This media are used for
Dextrose Tryptone Broth - 15 gms

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2. Boil with frequent agitation to dissolve the powder completely. RGI- Relative Growth Index
0 Interpretation of Results
3. Sterilize by autoclaving at 121 C (15 lbs pressure) for 15 minutes.
Quality Control
Dextrose Tryptone Agar
Dehydrated Appearance 1. Acid producing organisms form yellow coloured colonies surrounded by a
Yellow coloured, homogeneous, free flowing powder. yellow zone.
Prepared Appearance
Dextrose Tryptone Broth
Dextrose Tryptone Agar - Purple coloured, slightly opalescent gel.
Dextrose Tryptone Broth - Purple coloured, slightly opalescent solution. 1. A change in the colour of the medium from purple to yellow indicates
Cultural Response dextrose fermentation.
Cultural characteristics after 48 hours at 550C. Precautions / Limitations
Organisms (ATCC) Growth on Dextrose Colour of colony RGI 1. Dextrose Tryptone Agar must be incubated at 550C under humid conditions
Tryptone Agar & in on Tryptone
Dextrose Agar
e.g. wrapped dishes or in a high humid environment.
Tryptone Broth Storage
Bacillus Good to luxuriant Yellow More than 70% Store at 22-300C and prepared medium at 2-80C.
stearothermophilus (7953)
Shelf Life
Bacillus coagulans (8038) Good to luxuriant Yellow More than 70%
For growth RGI should be more than 70%
Use before expiry date as mentioned on the label.

Dey-Engley Neutralizing Agar (D/E Agar Disinfectant Testing) AM50371

Use Yeast extract 2.50
Dey Engley Neutralizing Agar is recommended for disinfectant testing where Dextrose 10.0
Sodium thiosulphate 6.0
neutralization of the antiseptics and disinfectant is important for determining its
Sodium thioglycollate 1.0
bactericidal activity.
Sodium bisulphate 2.50
Summary
Lecithin 7.0
Dey and Engley described a procedure of neutralizer evaluation and also Polysorbate 80 5.0
formulated a medium, known as Dey-Engley Neutralizing medium (25.1). This Bromo cresol purple 0.02
medium neutralizes a broad spectrum of antiseptics and disinfectants including Agar 15.0
quaternary ammonium compounds, phenolics, iodine and chlorine preparations, Final pH (at 250C) 7.6± 0.2
mercurials, formaldehyde and glutaraldehyde. Sodium thioglycollate, sodium Directions

thiosulfate, sodium bisulfite, soya lecithin and polysorbate 80 act as neutralizing 1. Suspend the 54 gms of powder in 1000 ml of distilled water.
components. 2. Mix thoroughly.
Principle 3. Boil with frequent agitation to dissolve the powder completely. DO NOT
Casein enzymic hydrolysate serves as a rich source of nitrogen and amino acid. OVERHEAT.
Yeast extract provides a source of trace elements and vitamins. Dextrose is a 4. Sterilize by autoclaving at 15lbs pressure (121ºC) for 15 minutes.
source of energy. Five neutralizers are incorporated into the medium to inactivate Quality Control
different types of biocides. Sodium thiosulfate neutralizes iodine and chlorine; Dehydrated Appearance
sodium thioglycollate neutralizes mercurials; sodium bisulphate neutralizes Bluish grey coloured, homogeneous, free flowing powder.
aldehydes; lecithin neutralizes quaternary ammonium compounds; and Prepared Appearance
Polysorbate 80 neutralizes substituted phenolics. Bromocresol purple acts as an Purple coloured opalescent gel forms in petriplates.
indicator, which indicates the utilization of dextrose. Cultural Response
Formula* Cultural characteristics after 40-48 hours at 35-370C.
Ingredients in grams per liter Organisms (ATCC) Growth RGI
Casein enzymic hydrolysate 5.0 Bacillus subtilis (6633) Luxuriant More than 70%

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Escherichia coli (25922) Luxuriant More than 70% 2. Incubate exposed plates at 35-37ºC for 48 hours.
Pseudomonas aeruginosa (27853) Luxuriant More than 70% Interpretation of Results
Salmonella serotype Typhimurium Luxuriant More than 70%
Growth is indicated by a colour change from purple to yellow, and visible colonies
(14028)
Staphylococcus aureus (25923) Luxuriant More than 70%
are found on incubated plate.
For growth RGI should be more than 70% Storage
RGI- Relative Growth Index Store at 2- 80C and prepared medium at 2-80C.
Procedure Shelf Life
1. Selected surface are sampled by firmly pressing the agar medium against Use before expiry date as mentioned on the label.
the test area. Slightly curved surfaces may be sampled with a rolling motion.
Areas (walls, floors, etc.) to be assayed may be divided into sections or grids
and samples taken from specific points within the grid.

Dey-Engley Neutralizing Broth (D/E Broth Disinfectant Testing) AM50372

Use Lecithin 7.0
Dey Engley Neutralizing Broth is recommended for disinfectant testing where Polysorbate 80 5.0
Bromo cresol purple 0.02
neutralization of the antiseptics and disinfectant is important for determining its
Final pH (at 250C) 7.6± 0.2
bactericidal activity.
* Formula adjusted to suit performance parameters
Summary
Directions
Dey and Engley described a procedure of neutralizer evaluation and also
1. Suspend the 39 gms of powder in 1000 ml distilled water.
formulated a medium, known as Dey-Engley Neutralizing medium (25.1). This
2. Mix thoroughly.
medium neutralizes a broad spectrum of antiseptics and disinfectants including
quaternary ammonium compounds, phenolics, iodine and chlorine preparations, 3. Boil with frequent agitation to dissolve the powder completely. DO NOT
mercurials, formaldehyde and glutaraldehyde. Sodium thioglycollate, sodium OVERHEAT.
thiosulfate, sodium bisulfite, soya lecithin and polysorbate 80 act as neutralizing 4. Dispense in tubes or adequate containers and sterilize by autoclaving at 15
components. lbs pressure (121ºC) for 15 minutes.
Principle Quality Control
Casein enzymic hydrolysate serves as a rich source of nitrogen and amino acid. Dehydrated Appearance
Bluish grey coloured, homogeneous, free flowing powder.
Yeast extract provides a source of trace elements and vitamins. Dextrose is a
Prepared Appearance
source of energy. Five neutralizers are incorporated into the medium to inactivate
Purple coloured opalescent solution forms in tubes.
different types of biocides. Sodium thiosulfate neutralizes iodine and chlorine; Cultural Response
sodium thioglycollate neutralizes mercurials; sodium bisulphate neutralizes Cultural characteristics after 40-48 hours at 35-370C.
aldehydes; lecithin neutralizes quaternary ammonium compounds; and Organisms (ATCC) Growth
Polysorbate 80 neutralizes substituted phenolics. Bromocresol purple acts as an Bacillus subtilis (6633) Luxuriant
indicator, which indicates the utilization of dextrose. Escherichia coli (25922) Luxuriant
Formula* Pseudomonas aeruginosa (27853) Luxuriant
Ingredients in grams per liter Salmonella serotype Typhimurium (14028) Luxuriant
Casein enzymic hydrolysate 5.0 Staphylococcus aureus (25923) Luxuriant
Yeast extract 2.50 Procedure
Dextrose 10.0 1. Add 1 ml of disinfectant solution to one tube of Dey-Engley Neutralizing
Sodium thiosulphate 6.0 Broth.
Sodium thioglycollate 1.0
2. Add desired amount of culture.
Sodium bisulphate 2.50
3. Incubate tubes at 35ºC for 40-48 hours.

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4. Do further testing to determine bacteriostatic or bactericidal activity of the Growth on the plates from negative broth tubes indicates a bacteriostatic
solution by inoculating samples from broth onto Dey-Engley Neutralizing substance.
Agar (AM 50371). No growth the plates from negative broth tubes indicates a bactericidal
Interpretation of Results substance. All positive broth tubes should be positive on the plates.
Growth is indicated by a colour change from purple to yellow, or pellicle Storage

formation. Store at 2- 80C and prepared medium at 2-80C.

Shelf Life
Use before expiry date as mentioned on the label.

Dichloron 18% Glycerol (DG18) Agar AM50373

Use * Formula adjusted to suit performance parameters
Dichloran Glycerol Medium Base with Chloramphenicol is recommended for Directions

selective isolation of xerophilic moulds from food samples. 1. Suspend 15.8 grams in 500 ml distilled water.
Summary 2. Heat to boiling to dissolve the medium completely.
Hocking and Pitt (45.5) formulated Medium Dichloran Glycerol and is 3. Add 110 grams of glycerol.
recommended for isolation and enumeration of xerophilic moulds from dried and 4. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
semidried foods. The glycerol at 18% (w/w) lowers the water activity from 0.999 Quality Control
to 0.95 (6.2) without causing any problem. This restrictive characteristic makes Dehydrated Appearance
the medium especially suitable for foods. Cream to yellow homogeneous free flowing powder
Principle Prepared Appearance
Peptic digest of animal tissue provides nitrogen, vitamins and minerals. Dextrose Medium amber coloured, clear to slightly opalescent gel forms in Petri plates
Cultural Response
is a carbohydrate source. Phosphate buffers the medium. Magnesium sulfate
Cultural characteristics after an incubation at 25°C for upto 6 days.
provides divalent cations and sulfate. Dichloran is an antifungal agent, added to
Organisms (ATCC) Growth RGI
the medium to reduce colony diameters of spreading fungi. Chloramphenicol is Bacillus subtilis (6633) Inhibited 0%
included to inhibit the growth of bacteria present in environmental and food Candida albicans (10231) Good-luxuriant More than 70%
samples. Escherichia coli (25922) Inhibited 0%
Formula* Mucor racemosus (42647) Good-luxuriant More than 70%
Ingredients in grams per liter Saccharomyces cerevisiae (9763) Good-luxuriant More than 70%
Peptic digest of animal tissue 5.00 For growth RGI should be more than 70%
Dextrose 10.00 For Inhibition RGI should be 0%
Monopotassium phosphate 1.00 RGI- Relative Growth Index
Magnesium sulphate 0.50 (9763)
Dichloran 0.002 Storage
Chloramphenicol 0.10
Store at 22-300C and prepared medium at 2-80C.
Agar 15.00
Shelf Life
Final pH ( at 25°C) 5.6±0.2
Use before expiry date as mentioned on the label.

Dichloran Rose Bengal chloramphenicol (DRBC) Agar AM50374

Use Summary
Dichloran Rose Bengal chloramphenicol (DRBC) Agar used as a selective medium Dichloran Rose Bengal chloramphenicol (DRBC) Agar is based on the Dichloran
for the enumeration of yeast and moulds in food industries Rose Bengal Chlortetracycline agar formula described by King, Hocking and Pitt

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(55.4). DRBC Agar conforms with APHA guidelines for the mycological Directions
examination of foods, containing chloramphenicol rather than chlortetracycline 1. Suspend the 31.6 gms of powder in 1000 ml distilled water.
as originally proposed (6.2.1). DRBC Agar is a selective medium that supports 2. Mix thoroughly.
good growth of yeasts and molds. 3. Heat with frequent agitation to dissolve the powder completely.
Principle
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Peptone provides nitrogen, vitamins and minerals. Dextrose is a carbohydrate
5. Cool at 500C and pour into plates.
source. Phosphate is a buffering agent. Magnesium sulfate is a source of divalent
Quality Control
cations and sulfate. The antifungal agent, dichloran, is added to the medium to
Dehydrated Appearance
reduce colony diameters of spreading fungi. Rose bengal suppresses the growth Pink colour, homogeneous, free flowing powder.
of bacteria and restricts the size and height of colonies of the more rapidly Prepared Appearance
growing molds. Chloramphenicol is included in this medium to inhibit the growth Bright pink coloured, slightly opalescent.
of bacteria present in environmental and food samples. Agar is the solidifying Cultural Response
agents. Cultural characteristics up to 5 days at 25+20C.
Formula* Organisms(ATCC) Growth RGI
Ingredients in grams per liter Aspergillus niger (16404) Luxuriant More than 70%
Bacteriological peptone 5.0 Candida albicans (10231) Luxuriant More than 70%
Glucose 10.0 Escherichia coli (25922) Inhibited 0%
KH2PO4 1.0 For growth RGI should be more than 70%
MgSO4.7H2O 0.5 For Inhibition RGI should be 0%
Rose Bengal (5% aqueous soln. w/v) 0.05 RGI- Relative Growth Index
Dichloran (0.2% in ethanol w/v) 0.002 Storage
Chloramphenicol 0.1 Store at 22-300C and prepared medium at 2-80C.
Agar 15.0 Shelf Life
Final pH (at 250C)5.6 ± 0.2 Use before expiry date as mentioned on the label.
* Formula adjusted to suit performance parameters

Differential Reinforced Clostridial Broth AM1038/AM5038

Use Formula*
Differential Reinforced Clostridial Broth is used for the cultivation of clostridia Ingredients in grams per liter
Peptone 10.0
from water.
Yeast Extract 1.5
Summary
Sodium Acetate 5.0
Differential Reinforced Clostridial Broth is based on the formulation described by Beef Extract 10.0
Barnes and Ingram (5) and Gibbs and Freame (34). It is used for cultivation of Glucose 1.0
sulphite reducing clostridia from food and enumeration in water by multiple tube L-Cysteine Hydrochloride 0.5
method. Differentiation is based on the ability to reduce sulphite to sulphide to Starch 1.0
form black coloured iron sulphide. Final pH (at 250C) 7.2 ± 0.2
Principle * Formula adjusted to suit performance parameters
Directions
Peptone, beef extract, yeast extract, starch and L-cysteine hydrochloride provide
nutrients and co-factors. Dextrose serves as the energy source. Partial selectivity of 1. Suspend 29 gms of the powder in 1000 ml distilled water.
the medium is achieved by the addition of sodium acetate. L-cysteine 2. Boil with frequent agitation to dissolve the powder completely.
hydrochloride also acts as a reducing agent. 3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.

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4. Just before use add 0.5 ml filter sterilized solution prepared by mixing equal Organisms (ATCC) Growth H2S production
volumes of 4% w/v solution of sodium sulphite and 7% w/v ferric citrate to Clostridium perfringens (13124) Good to luxuriant +
Clostridium sporogenes (11437) Good to luxuriant +
25 ml of single strength medium or 0.4 ml and 2 ml to 10ml and 50 ml of
Key:
double strength medium respectively.
+ = Blackening of the medium
5. Mix well.
Interpretation of Results
Quality Control
Dehydrated Appearance 1. Blackening of the medium presumptively indicates the presence of
Yellow coloured, homogeneous, free flowing powder. clostridia.
Prepared Appearance Storage
Light yellow coloured, clear solution without any precipitate. Store at 22-300C and prepared medium at 2-80C.
Cultural Response Shelf Life
Cultural characteristics within 1 week at 300C. Use before expiry date as mentioned on the label.

Differential Reinforced Clostridial Broth Base ISO AM503811

Use 3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Differential Reinforced Clostridial Broth is used for the cultivation of clostridia 4. Just before use add 0.5 ml filter sterilized solution prepared by mixing equal
from water, in compliance with ISO specification ISO 6461-1: 1986. volumes of 4% w/v solution of sodium sulphite an 7% w/v ferric citrate to 25
Summary ml of single strength medium or 0.4 ml and 2 ml to 10ml and 50 ml of double
Differential Reinforced Clostridial Broth is based on the formulation described by strength medium respectively.
Barnes and Ingram and Gibbs and Freame. It is used for cultivation of sulphite 5. Mix well.
reducing clostridia from food and enumeration in water by multiple tube method. Quality Control
Differentiation is based on the ability to reduce sulphite to sulphide to form black Dehydrated Appearance
coloured iron sulphide. Yellow coloured, homogeneous, free flowing powder.
Principle Prepared Appearance
Tryptose, meat extract, yeast extract, starch and L-cysteine hydrochloride provide Light yellow coloured, clear solution without any precipitate.
nutrients and co-factors. Dextrose serves as the energy source. Partial selectivity of Cultural Response
Cultural characteristics within 1 week at 30ºC.
the medium is achieved by the addition of sodium acetate. L-cysteine
Organisms (ATCC) Growth H2S
hydrochloride also acts as a reducing agent.
production
Formula*
Clostridium Good to luxuriant +
Ingredients in grams per liter
perfringens (13124)
Tryptose 10.0
Clostridium Good to luxuriant +
Yeast extract 1.5
sporogenes (11437)
Sodium acetate 5.0
Key: + = Blackening of the medium
Meat extract 10.0
Interpretation of results
Glucose 1.0
L-Cysteine hydrochloride 0.5 1. Blackening of the medium presumptively indicates the presence of
Starch 1.0 clostridia.
Final pH (at 25ºC) 7.2 ± 0.2 Storage
* Formula adjusted to suit performance parameters Store below 300C and prepared medium at 2-80C.
Directions Shelf Life
1. Suspend 29 gms of the powder in 1000 ml distilled water. Use before expiry date as mentioned on the label.
2. Boil with frequent agitation to dissolve the powder completely.

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DNase Test Agar Base AM10381
Use 4. Cool to 450 C and pour into sterile petriplates.
DNase Test Agar Base is a differential medium recommended for the detection of Quality Control
deoxyribonuclease activity to aid in the identification of bacteria and fungi Dehydrated Appearance
isolated from clinical specimens especially pathogenic staphylococci. Light yellow coloured, homogeneous, free flowing powder.
Summary Prepared Appearance
Light yellow coloured, clear to slightly opalescent gel.
Weckman and Catlin in their study observed that DNase activity might prove to be
Cultural Response
a taxonomic characteristic useful in the detection of strains of the micrococcus- Cultural characteristics after 18-24 hous at 35-370 C.
staphylococcus group (118). Subsequently, DiSalvo reported a correlation Organisms (ATCC) Growth RGI
between coagulase production and DNase activity. This test proves useful in Staphylococcus aureus (25923) Luxuriant More than 70%
differentiating Serratia from Enterobacter, Staphylococcus aureus from Staphyloccus epidermidis (12228) Luxuriant More than 70%
coagulase-negative staphylococci and Moraxella catarrhalis from Neisseria Streptococcus pyogenes (19615) Luxuriant More than 70%
species (125). Serratia marcescens (8100) Luxuriant More than 70%
Principle For growth RGI should be more than 70%
RGI- Relative Growth Index
Tryptone and Papaic Digest of Soyabean Meal provide amino acids and other
Procedure
complex nitrogenous substances required to support bacterial growth. Sodium
1. Inoculate each plate / tube with the sample for testing by drawing a single
chloride maintains the osmotic equilibrium. DNA is the substrate for DNase
streak line.
activity. DNase is an extracellular enzyme that breaks the DNA down into subunits
composed of nucleotides. 2. Incubate at 35 ± 20 C for 24-48 hours. Plates should be incubated in an
inverted position and tubes with caps loosened.
The depolymerization of the DNA may be detected by flooding the surface of the
medium with 1 N HCl and observing for clear zones around the medium 3. On completion of the incubation period, flood the plates / tubes with 1 N HCl
surrounding growth. In the absence of DNase activity, the reagent reacts with the reagent to precipitate the DNA in the medium.
intact nucleic acid, resulting in the formation of a cloudy precipitate. 4. Observe for reaction.
Formula* Interpretation of Results
Ingredients in grams per liter 1. A clear area surrouding growth on the medium after the addition of 1 N HCl
Tryptone 15.0 indicates a positive reaction i.e.; DNase activity.
Papaic Digest of Soyabean Meal 5.0
Deoxyribonucleic Acid (DNA) 2.0
2. No clearing and a cloudy precipitate around colonies throughout the
Sodium Chloride 5.0 medium indicate a negative reaction. This occurs due to the formation of
Agar 15.0 precipitated salts in the medium.
Final pH (at 250 C) 7.3 ± 0.2 3. Gram positive, catalase positive cocci that produce DNase can be
* Formula adjusted to suit performance parameters provisionally classified as S. aureus and confirmed by tube coagulase or
Directions
thermostable DNase tests.
1. Suspend 42 grams of the powder in 1000 ml distilled water. Storage
2. Heat with frequent agitation to dissolve the powder completely. Store at 22-300C and prepared medium at 2-80C.
0 0
3. Sterilize by autoclaving at 12 to 15 lbs pressure (118 C to 121 C) for 15 Shelf Life

minutes. Use before expiry date as mentioned on the label.

Double Sugar Agar, Russell AM50382

Use gas formation.
Double Sugar Agar, Russel is used for the differentiation of gram-negative enteric Summary
bacilli on the basis of their ability to ferment dextrose and lactose with or without In 1911, Russell described a new double sugar tube medium for the isolation of

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typhoid bacilli from urine and feces (97). Six years later, Kligler developed a Directions
simple lead acetate medium for the differentiation of the typhoid-paratyphoid 1. Suspend 44 grams of the powder in 1000 ml distilled water. Mix thoroughly.
group. Subsequently, Kligler evaluated culture media used in the isolation and 2. Boil with frequent agitation to dissolve the powder completely.
differentiation of typhoid, dysentery and allied bacilli and endorsed Russell's 3. Dispense in tubes or as desired.
medium. Bailey and Lacey substituted phenol red for the Andrade indicator
4. Sterilize by autoclaving at 1180C (12-15 lbs pressure) for 15 minutes.
previously used as a pH indicator.
Princple 5. Allow the tubes to solidify in slanting position to form generous butt.
Quality Control
This medium is based upon the original formula of Russel except the litmus is new
Dehydrated Appearance
substituted by phenol red and used for differentiating gram-negative enteric
Pink coloured, homogeneous, free flowing powder.
bacilli especially the colon-typhoid-salmonellae dysentery groups based on the Prepared Appearance
fermentation of dextrose and lactose. After the incubation the acid production in Red coloured, clear to very slightly opalescent gel forms in tubes as slants..
aerobic condition (on the slant) and under anaerobic condition (in the butt) can Cultural Response
be detected by the change in colour of the indicator. Phenol red is pH indicator in Cultural characteristics after 18-40 hours at 35-37ºC.
the medium. Gaseous fermentation is indicated by the splitting of the agar or by Organisms (ATCC) Growth Slant Butt Gas RGI
the bubble formation in the butt. Organism like Salmonella typhi capable of Escherichia coli Luxuriant A A + More than 70%
(25922)
fermenting dextrose but not lactose, will show an initial acid slant in short
Proteus vulgaris Luxuriant K A + More than 70%
incubation period. As the dextrose is consumed the reaction under aerobic
(13315)
condition reverts and becomes alkaline. Under anaerobic condition in the butt, Pseudomonas
the same organisms fail to revert the reaction and remain acidic. aeruginosa (27853) Luxuriant K K --- More than 70%
Formula* Salmonella serotype
Ingredients in grams per liter Typhimurium (14028) Luxuriant K A + More than 70%
Peptic digest of animal tissue 2.50 Shigella dysenteriae Luxuriant K A --- More than 70%
Casein enzymic hydrolysate 7.50 (13313)
Beef extract 3.00 Key:
Lactose 10.0 A = acidic reaction, yellowing of the medium
Dextrose 1.00 K= alkaline reaction, red colour of the medium
Sodium chloride 5.00 For growth RGI should be more than 70%
Phenol red 0.025 RGI- Relative Growth Index
Agar 15.00 Storage
Final pH (at 25ºC) 7.3 ± 0.2
Store at 22-300C and prepared medium at 2-80C.
* Formula adjusted to suit performance parameters
Shelf Life
Use before expiry date as mentioned on the label.

E. C. Broth AM1039/AM5039
E. C. Broth ISO AM503911
Use for the recovery of E.coli from frozen foods and nut meats. This medium is
EC Broth is used for detection of coliform bacteria at 350C and Escherichia coli at recommended for use in Most Probable Number (MPN) procedure for examination
an elevated temperature of 44.5 or 45.50C. of dairy products (39) water and wastewater (36) and foods (20). The procedure
Summary employing EC Broth provides information regarding the source of the coliform
EC Broth was developed by Hajna and Perry (42) and is used for the examination group (faecal or non-faecal) when used as a confirmatory test. It is included in the
of water, milk, shellfish and other material for evidence of faecal pollution. Bacteriological Analytical Manual for food testing (114).
Tennant et al (110) reported the use of this medium for the estimation of E.coli Principle
densities in seawater and shellfish. Fishbein and Surkiewicz (32) used EC Broth Tryptone provides nutrients for growth while lactose is the fermentable

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carbohydrate. Bile salts mixture inhibits gram-positive organisms especially Prepared Appearance
bacilli and faecal streptococci. The medium contains a strong potassium Yellow coloured, clear solution without any precipitate.
Cultural Response
phosphate buffering system to control the pH during fermentation of lactose.
Cultural characteristics after 24 hours at 44.50C ± 0.20C.
Sodium chloride maintains osmotic balance.
Organisms (ATCC) Growth Gas
Formula*
Bacillus subtilis (6633) Inhibited -
Ingredients in grams per liter
Enterobacter aerogenes (13048) Inhibited -
Tryptone 20.0
Enterococcus faecalis (29212) Inhibited -
Lactose 5.0
Escherichia coli (25922) Luxuriant +
Sodium Chloride 5.0
Klebsiella pneumoniae (13883) Luxuriant +
Monopotassium Phosphate 1.5
Pseudomonas aeruginosa (27853) Fair to good -
Bile Salts Mixture 1.5
Interpretation of Results
Dipotassium Phosphate 4.0
Final pH (at 250C) 6.9 ± 0.2
1. Lactose fermenting organisms produce gas, which is detected by the
* Formula adjusted to suit performance parameters appearance of bubbles in the inverted Durham's tube within 24 hours, which
Directions is a presumptive evidence of the presence of coliform bacteria.
1. Suspend 37 gms of the powder in 1000ml distilled water and mix well. 2. The development of turbidity and gas production within 48 hours at 350C or at
2. Boil with frequent agitation to dissolve the powder completely. 45.50C indicates the presence of coliforms.
Precautions / Limitations
3. Dispense into tubes containing inverted Durham's tubes.
1. This medium should not be used for the direct isolation of coliforms since
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
prior enrichment in a presumptive test medium for optimal recovery of faecal
5. Adjust the concentration of the medium as per the sample size. coliforms is required.
Quality Control
Storage
Dehydrated Appearance
Yellow coloured, homogeneous, free flowing powder.
Store at 22-300C and prepared medium at 2-80C.
Shelf Life
Use before expiry date as mentioned on the label.

E. E. Broth, Mossel AM103913/AM503913

Use fermenting organisms.
E E Broth, Mossel is used for selective enrichment and detection of Principle
Enterobacteriaceae in bacteriological examination of food and feedstuffs. Peptic Digest of Animal Tissue supply nutrients, nitrogen compounds and amino
Summary acids. Ox bile supports the growth of enteric bacteria and inhibits other bacteria,
Mossel, Visser and Cornelissen developed a culture media for the selective which do not normally live in the intestine. Brilliant-green specifically inhibits the
enrichment of Enterobacteriaceae that enable the Enterobacteriaceae to Gram-positive accompanying flora.Sodium chloride provides sodium ions for the
multiply freely and inhibit accompanying other organisms (81.1). Media membrane transport and maintains osmotic equilibrium of the medium.
contains dextrose to facilitate growth of most Enterobacteriaceae including Disodium phosphate and Mono-potassium phosphate are buffering agents.
Salmonella and other non-lactose fermenting organisms. Formula*
EE Broth should be used as an enrichment broth in conjunction with Violet Red Ingredients in grams per liter
Peptic Digest of Animal Tissue 10.0
Bile Glucose Agar (AM51073). When specific organisms, rather than
Dextrose 5.0
Enterobacteriaceae in general, are required subcultures must be made onto
Ox-bile Purified 20.0
lactose differential media e.g. Deoxycholate Citrate Agar (AM1031/5031), Disodium phosphate 6.45
Brilliant Green Agar, Modified (AM1018/5018), or MacConkey Agar Mono-potassium phosphate 2.0
(AM1059/5059) for the detection of non-lactose fermenting or delayed lactose Brilliant Green 0.0135

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Final pH (at 250C) 7.2 ± 0.2 Escherichia coli (25922) Luxuriant +
* Formula adjusted to suit performance parameters Enterobacter aerogenes (13048) Luxuriant +
Directions Proteus mirabilis (25933) Luxuriant +
1. Suspend the 43.50 gms of powder in 1000 ml distilled water. Staphylococcus aureus (25923) Inhibited -
* Key: + = Positive, yellow colouration
2. Mix thoroughly.
– = Negative, no colour change green
3. Heat with frequent agitation to dissolve the powder completely. Do not boil. Procedure
DO NOT AUTOCLAVE OR REHEAT. 1. Inoculate the E E Broth, Mossel with food or other test specimen.
4. Pour into adequate containers. 2. Mix well the inoculated medium.
Quality Control
Dehydrated Appearance
3. Incubate at 35-370C for 20-24 hours.
Greenish yellow coloured, homogeneous, free flowing powder. Interpretation of Results
Prepared Appearance Acid production causes the color of EE Broth Mossel to become yellow. A negative
Emerald green coloured, clear solution without any precipitate.
reaction results in no colour change and the medium remains green.
Cultural Response
Storage
Cultural characteristics after 18-24 hours at 35-370C.
Organisms (ATCC) Growth Acid Store at 22-300C and prepared medium at 2-80C.
Shigella boydii (12030) Luxuriant - Shelf Life
S.serotype Enteritidis (13076) Luxuriant ± Use before expiry date as mentioned on the label.

ECD Agar AM503912

Use Directions
ECD Agar is used for selective isolation of coliforms, specially Escherichia coli in 1. Suspend the 53 gms of powder in 1000 ml distilled water.
water and food by membrane filter technique. 2. Mix thoroughly.
Summary
3. Heat with frequent agitation to dissolve the powder completely.
The medium complies with the German-DIN-Norm 10110 for the examination of
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
meat, with the regulation according to §35 LMBG (06.00/36) for the
examination of food and with ISO standard 6391 (1996) for the enumeration of 5. Pour into sterile petri plates.
Quality Control
E.coli in meat and meat products.
Dehydrated Appearance
Principle
Bright beige coloured, homogeneous, free flowing powder.
Casein peptone and yeast extract provides essential growth nutrients. Bile salts Prepared Appearance
inhibit gram-positive bacteria specially bacilli and faecal streptococci. Potassium Yellowing brown coloured, clear gel.
phosphates control the pH. Cultural Response
Formula* Cultural characteristics up to18-24 hour at 440C.
Ingredients in grams per liter Organisms (ATCC) Growth Indole RGI
Casein peptone (tryptic) 20.00 formation
Yeast extract 5.00 Escherichia coli (8739) Luxuriant + More than 70%
Bile salt 1.50 Escherichia coli (25922) Luxuriant + More than 70%
Sodium chloride 5.00 Enterobacter aerogenes Good - More than 70%
Disodium hydrogen phosphate 5.00 (13048)
Potassium dihydrogen phosphate 1.50 Klebsiella pneumoniae Good More than 70%
Agar 15.00 (13883)
Final pH (at 250C)7.0 ±0.2 Pseudomonas aeruginosa Good More than 70%
* Formula adjusted to suit performance parameters (27853)

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Proteus mirabilis Good More than 70% For Inhibition RGI should be 0%
(14153) RGI- Relative Growth Index
Citrobacter freundii Good More than 70% Storage
(8090) Store at 22-300C and prepared medium at 2-80C.
Clostridium None-poor 0%
Shelf Life
perfringens (10543)
Use before expiry date as mentioned on the label.
For growth RGI should be more than 70%

ECD MUG Agar AM5039121

Use Final pH ( at 25°C) 7.0±0.2
ECD MUG Agar is recommended for demonstrating the presence of Escherichia coli * Formula adjusted to suit performance parameters
Directions
by fluorescence in UV and positive indole test while inhibiting accompanying
intestinal flora. 1. Suspend 53.0 grams in 1000 ml distilled water.
Summary 2. Heat to boiling to dissolve the medium completely.
Feng and Hartman (30.1) developed a rapid assay for E. coli by incorporating 4- 3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to
methylumbilliferyl-ß-gluconide (MUG) in to Lauryl Trytose Broth. E.C Medium 45-50oC.
with MUG is prepared according to the formula specified by the U.S 4. Mix well and pour into sterile Petri plates.
Environmental Protection Agency and Standard methods for water and food Quality Control
testing (17.1). Dehydrated Appearance
Principle Cream to yellow homogeneous free flowing powder
Casein enzymic hydrolysate provides the nitrogen, vitamins and amino acids in EC Prepared Appearance
medium with MUG. Lactose is the carbon source in this medium. Bile salts mixture Light amber coloured, clear to slightly opalescent gel forms in Petri plates
Cultural Response
is the selective agent against gram-positive bacteria, particularly bacilli and
Cultural characteristics after 18-24 hours at 35-370C.
fecal streptococci. Dipotassium phosphate and mono potassium phosphate are Organisms Growth RGI Indole production Fuorescence
buffering agents. Sodium chloride maintains the osmotic balance of the medium. (under 366nm)
Enterobacter Good-luxuriant More than 70% Negative reaction Negative
E.coli produces the enzyme glucoronidase that hydrolysis MUG to yield a aerogenes
fluorogenic product that is detectable under long wave (366 nm) UV light. (13048)
Escherichia coli Good-luxuriant More than 70% Positive reaction, Red positive zone
Tryptophan serves as the substrate for indole reaction. (25922) around the colony
Formula* Staphylococcus Inhibited 0%
aureus (25923)
Ingredients in grams per liter
For growth RGI should be more than 70%
Casein peptone 20.00
For Inhibition RGI should be 0%
Lactose 5.00
RGI- Relative Growth Index
Sodium chloride 5.00
Storage
Bile salts mixture 1.50
Dipotassium hydrogen phosphate 4.00 Store at 22-300C and prepared medium at 2-80C.
Potassium hydrogen phosphate 1.50 Shelf Life
Tryptophan 1.00 Use before expiry date as mentioned on the label.
4-Methylumbelliferyl ß-D-Glucuronide (MUG) 0.07
Agar 15.00

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Enterobacteria Enrichment, Mossel (Harmonized) AMH503913
EE Broth, Mossel EP AM503914
EE Broth, Mossel BP AM503915
EE Broth, Mossel IP AM5039151
EE Broth, Mossel USP AM5039152
Use Directions
E E Broth, Mossel is used for selective enrichment and detection of 1. Suspend the 45.015 gms of powder in 1000 ml distilled water.
Enterobacteriaceae in bacteriological examination of food and feedstuffs. 2. Mix thoroughly.
Summary
3. Heat in free flowing steam or boiling water for 30 minutes. Avoid
Mossel, Visser and Cornelissen developed a culture media for the selective overheating of the medium. DO NOT AUTOCLAVE OR REHEAT.
enrichment of Enterobacteriaceae that enable the Enterobacteriaceae to
4. Pour into adequate containers.
multiply freely and inhibit accompanying other organisms (81.1). Media
Quality Control
contains dextrose to facilitate growth of most Enterobacteriaceae including Dehydrated Appearance
Salmonella and other non-lactose fermenting organisms. EE Broth should be Greenish yellow coloured, homogeneous, free flowing powder.
used as an enrichment broth in conjunction with Violet Red Bile Glucose Agar Prepared Appearance
(AM51073). When specific organisms, rather than Enterobacteriaceae in Emerald green coloured, clear solution without any precipitate.
general, are required subcultures must be made onto lactose differential media Cultural Response
e.g. Deoxycholate Citrate Agar (AM1031/5031), Brilliant Green Agar, Modified Cultural characteristics after 18-24 hours at 35-370C.
(AM1018/5018), or MacConkey Agar (AM1059/5059) for the detection of non- Organisms (ATCC) Growth Acid
Shigella boydii (12030) Luxuriant -
lactose fermenting or delayed lactose fermenting organisms.
S.serotype Enteritidis (13076) Luxuriant ±
Principle
Escherichia coli (25922) Luxuriant +
Pancreatic digest of gelatin provides nutrients, nitrogen compounds and amino Enterobacter aerogenes (13048) Luxuriant +
acids. Ox bile supports the growth of enteric bacteria and inhibits other bacteria, Proteus mirabilis (25933) Luxuriant +
which do not normally live in the intestine. Brilliant-green specifically inhibits the Staphylococcus aureus (25923) Inhibited -
Gram-positive accompanying flora. Disodium phosphate and Mono-potassium Key: + = positive, yellow colouration.
phosphate are buffering agents. - = negative, no colour change, green.
Formula* Procedure
Ingredients in grams per liter AM5039151 AM503914, 1. Inoculate the E E Broth, Mossel with food or other test specimen.
AM503915, 2. Mix well the inoculated medium.
AM5039152 &
AMH503913
3. Incubate at 35-370C for 20-24 hours.
Pancreatic digest of gelatin 10.0 10.0 Interpretation of Results
Glucose monohydrate - 5.0 Acid production causes the color of EE Broth Mossel to become yellow. A negative
Dextrose monohydrate 5.0 -
reaction results in no color change and the medium remains green.
Dehydrated Ox-bile 20.0 20.0
Storage
Disodium hydrogen phosphate, dihydrate 8.0 8.0
Potassium dihydrogen phosphate 2.0 2.0 Store at 22-300C and prepared medium at 2-80C.
Brilliant Green 0.015 0.015 Shelf Life
0
Final pH (at 25 C) 7.2 ± 0.2 Use before expiry date as mentioned on the label.
* Formula adjusted to suit performance parameters

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E. E. Broth Mossel with Oxgall AM503916
Use 2. Mix thoroughly.
E E Broth, Mossel with Oxgall is used for selective enrichment and detection of 3. Heat at 1000C for 30 minutes and cool immediately. Do not boil. DO NOT
Enterobacteriaceae in bacteriological examination of foods. AUTOCLAVE OR REHEAT.
Summary
4. Pour into adequate containers.
Mossel, Visser and Cornelissen developed a culture media for the selective Quality Control
enrichment of Enterobacteriaceae that enable the Enterobacteriaceae to multiply Dehydrated Appearance
freely and inhibit accompanying other organisms. Media contains dextrose to Greenish yellow coloured, homogeneous, free flowing powder.
facilitate growth of most Enterobacteriaceae including Salmonella and other Prepared Appearance
non-lactose fermenting organisms. EE Broth should be used as an enrichment Emerald green coloured, clear solution without any precipitate.
broth in conjunction with Violet Red Bile Glucose Agar (AM51073). When specific Cultural Response
organisms, rather than Enterobacteriaceae in general, are required subcultures Cultural characteristics after 18-24 hours at 35-370C.
Organisms (ATCC) Growth Acid
must be made onto lactose differential media e.g. Deoxycholate Citrate Agar
Shigella boydii (12030) Luxuriant -
(AM1031/5031), Brilliant Green Agar, Modified (AM1018/5018), or
S.serotype Enteritidis (13076) Luxuriant ±
MacConkey Agar (AM1059/5059) for the detection of non-lactose fermenting or Escherichia coli (25922) Luxuriant +
delayed lactose fermenting organisms. Enterobacter aerogenes (13048) Luxuriant +
Principle Proteus mirabilis (25933) Luxuriant +
Pancreatic digest of gelatin provides nutrients, nitrogen compounds and amino Staphylococcus aureus (25923) Inhibited -
acids. Brilliant-green or oxgall specifically inhibits the Gram-positive Key: + = positive, yellow colouration.
- = negative, no colour change, green.
accompanying flora. Disodium phosphate and Mono-potassium phosphate are
Procedure
buffering agents.
Formula* 1. Inoculate the E E Broth, Mossel with Oxgall, with food or other test specimen.
Ingredients in grams per liter 2. Mix well the inoculated medium.
Pancreatic digest of casein 12.0
3. Incubate at 35-370C for 20-24 hours.
Proteose peptone No. 3 8.0
Interpretation of Results
Dextrose 5.0
Oxgall 10.0 Acid production causes the color of EE Broth Mossel with Oxgall to become yellow.
Disodium phosphate 8.0 A negative reaction results in no color change and the medium remains green.
Monopotassium phosphate 2.0 Storage
Brilliant Green 0.0135 Store at 22-300C and prepared medium at 2-80C.
0
Final pH (at 25 C) 7.2 ±0.2 Shelf Life
* Formula adjusted to suit performance parameters Use before expiry date as mentioned on the label.
Directions
1. Suspend the 45.01 gms of powder in 1000 ml distilled water.

Eijkman Lactose Broth AM503917

Use from the faeces of warm blooded and cold blooded animals. This method had
Eijkman Lactose Broth is used for the detection and differentiation of Escherichia limitation due to the inability to obtain growth after subculturing from positive
coli from other coliform organisms on the basis of their ability to grow and tubes incubated at 460C, as acidity and high temperature results in death of the
liberate gas from Lactose. culture within 24-48 hours. Perry and Hajna (87.1) modified Eijkman original
Summary method by decreasing carbohydrate content and adding a phosphate buffer
Eijkman (23.2) described a method for separating the strains of Escherichia coli enabling to subculture Escherichia coli after incubation at 460C for 96 hours or

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longer where pH was 5.6 unlike 4.5 of Eijkman medium. 3. Dispense into tubes with inverted Durham's fermentation tubes.
Principle 4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Tryptose provides nitrogen, carbon and other growth factors. Perry modified Quality Control
Eijkman medium using lactose for isolation of Escherichia coli. This medium can Dehydrated Appearance
also be used for water filtration control work. Dipotassium phosphate and Mono- Light yellow coloured, homogeneous, free flowing powder.
potassium phosphate are buffering agents. Prepared Appearance
Formula* Light yellow coloured, clear solution without any precipitate.
Ingredients in grams per liter Cultural Response
Tryptose 15.00 Cultural characteristics after 24-48 hours at 45.5-460C.
Lactose 3.00 Organisms (ATCC) Growth Gas
Dipotassium phosphate 4.00 Escherichia coli (25992) Luxuriant +
Monopotassium phosphate 1.50 Enterobacter aerogenes Poor -
Sodium chloride 5.00 (13048)
Final pH (at 250C) 6.8 ±0.2 Storage
* Formula adjusted to suit performance parameters Store at 22-300C and prepared medium at 2-80C.
Directions Shelf Life
1. Suspend the 28.5gms of powder in 1000 ml distilled water. Use before expiry date as mentioned on the label.
2. For examination of 10 ml portion of water samples, use 57 grams per 1000
ml distilled water.

EMB Agar AM10391/AM50391

EMB Broth AM10401/AM50401
Use Lactose 5.0 5.0
Eosin Methylene Blue (EMB) Agar and Eosin Methylene Blue (EMB) Broth are Sucrose 5.0 5.0
Eosin Y 0.4 0.4
slightly selective and differential media recommended for the isolation,
Methylene Blue 0.065 0.065
cultivation and differentiation of gram-negative enteric bacilli from clinical and
Agar 13.5 -
non-clinical specimens. Final pH (at 250 C) 7.2 ± 0.2
Summary * Formula adjusted to suit performance parameters
Eosin Methylene Blue (EMB) media were originally developed by Holt-Harris and Directions
Teague. Eosin Y and Methylene Blue are the two dyes incorporated in these 1. Suspend the powder in 1000 ml distilled water.
media. This formulation gives a sharp and distinct differentiation between the
EMB Agar - 36 grams
colonies of lactose fermenting and non-lactose fermenting microorganisms.
EMB Broth - 22.5 grams
Principle
The media contain Eosin Y and Methylene Blue dyes that inhibit gram-positive 2. Mix thoroughly until the suspension is uniform.
bacteria to a limited degree. In addition, these dyes also serve as differential 3. Heat with frequent agitation to dissolve the powder completely. AVOID
indicators in response to lactose/sucrose fermentation by the microorganisms. OVERHEATING.
Sucrose is added to the media as an alternative carbohydrate source for typical 4. Sterilize by autoclaving at 1210 C (15 lbs pressure) for 20 minutes.
lactose fermenting, gram-negative bacilli, which may not ferment lactose or may 5. Cool to 500 C and shake the medium to oxidize the methylene blue and to
do so slowly. suspend the flocculent precipitate.
Formula*
6. Pour into sterile petriplates.
Ingredients in grams per liter EMB Agar EMB Broth
Quality Control
Tryptone 10.0 10.0
Dehydrated Appearance
Dipotassium Phosphate 2.0 2.0
Light purple coloured, homogeneous, free flowing powder.

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Prepared Appearance 4. Examine plates for colonial morphology.
Reddish-purple coloured, opalescent gel or solution having greenish cast
forms in petriplates / tubes. 5. Plates can be incubated for an additional 24 hours if no growth is observed.
Cultural Response Interpretation of Results
Cultural response after 18 - 24 hours at 350 C. 1. Coliforms produce blue-black colonies due to the taking up of an eosin-
Organisms (ATCC) Growth Colour of RGI methylene blue dye complex by the bacterial cells when the pH drops.
Colony
2. Salmonella and Shigella colonies appear colourless or have a transparent
Escherichia coli Luxuriant Purple with black More than 70%
amber colour.
(25922) center with green
metallic sheen 3. Escherichia coli colonies may show a characteristic green metallic sheen due
Proteus mirabilis Luxuriant Colourless More than 70% to rapid fermentation of lactose.
(25933) 4. Some gram-positive bacteria, such as fecal streptococci, staphylococci and
Salmonella serotype Luxuriant Colourless More than 70%
yeasts, usually form pinpoint colonies.
Typhimurium (14028)
Precautions / Limitations
Enterobacter aerogenes Good Pink, without More than 70%
(13048) sheen 1. Store the prepared medium away from light to avoid photo oxidation.
Klebsiella pneumoniae Good Pink, mucoid More than 70% 2. If EMB Agar is inoculated on the same day, as it is prepared, it may be used
(13883) without autoclave sterilization.
Staphylococcus aureus Inhibited ---- 0%
3. A number of non-pathogenic, lactose non-fermenting gram-negative
(25923)
For growth RGI should be more than 70%
bacteria will grow on this medium and must be distinguished from the
For Inhibition RGI should be 0% pathogenic bacterial strains by additional biochemical tests.
RGI- Relative Growth Index 4. Serial inoculation may be required to assure adequate isolation of mixed flora
Procedure samples.
1. Obtain isolated colonies from specimens using standard procedures. Storage
2. Allow plates to attain room temperature. Ensure that the agar surface is dry Store at 22-300C and prepared medium at 2-80C.
before inoculation. Shelf Life
0
3. Incubate plates, protected from light, at 35 ± 2 C for 18-24 hours. Use before expiry date as mentioned on the label.

EMB Agar, Levine AM1040/AM5040

EMB Agar, Levine IP AM104011/AM504011
(Levine Eosin-Methylene Blue Agar Medium)
EMB Agar, Levine USP AM104012/AM504012
EMB Agar, Levine BIS AM104013/AM504013
Use Levine EMB Agar differs from the other formulation in not containing sucrose. The
EMB Agar, Levine is a slightly selective and differential medium used for the original medium could not discriminate between which carbohydrate (lactose or
isolation, enumeration and differentiation of members of Enterobacteriaceae. sucrose) was being fermented. Also, Y.enterocolitica, which ferments sucrose but
Summary not lactose, produced same colonies as that of lactose fermenters. Levine
Holt-Harris and Teague (45) developed a culture medium for the differentiation modified the formula by omitting sucrose and doubling the level of lactose. This
of enteric microorganisms through the use of eosin and methylene blue dyes. medium was mainly developed to improve upon the differentiating properties of
Levine (70) brought about a modification of their formulation, which he claimed Endo Agar.
gave a better differentiation between Escherichia and Enterobacter species. This EMB Agar, Levine is mainly used for the differentiation of Escherichia coli and

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Enterobacter aerogenes and also for the rapid identification of Candida albicans. essential part of the medium.
It is recommended for use in microbial examination of dairy products (39), foods Quality Control
(20) and water (36) by APHA and for use in the performance of microbial limit test Dehydrated Appearance
by USP and IP (114, 46). It is also included in the Bacteriological Analytical Light purple coloured, homogeneous free flowing powder.
Prepared Appearance
Manual for food testing (113).
Reddish purple coloured, slightly opalescent gel with greenish cast and finely
Weld proposed the use of EMB Agar, Levine with added chlortetracycline dispersed precipitate.
hydrochloride, for the rapid identification of Candida albicans in clinical Cultural Response
specimens. A positive identification of C.albican s can be made after 24-48 hours Cultural characteristics after 24-48 hours at 35-370C.
incubation at 35-370C in 10% carbon dioxide atmosphere, from specimens such Organisms (ATCC) Growth Colour of RGI
as faeces, oral and vaginal secretions and nail or skin scrapings. Menolasino et al colony
Candida albicans* Good to Colourless More than 70%
used this medium for identification of coagulase positive staphylococci, which
(10231) luxuriant
grew, as characteristic colourless pinpoint colonies.
Enterobacter aerogenes Good Pink to red More than 70%
Principle
(13048)
Peptone provides essential nutrients while lactose is the fermentable Enterococcus faecalis Inhibited - 0%
carbohydrate. Dipotassium phosphate is the buffer. The Eosin Y and methylene (29212)
blue dye in this medium inhibit gram-positive organisms to a limited degree Escherichia coli Luxuriant Blue black with More than 70%
making the medium only slightly selective besides; it also helps in differentiating (25922) metallic sheen
between lactose fermenters and non-lactose fermenters. Lactose fermenters Pseudomonas Luxuriant Colourless More than 70%
aeruginosa (27853)
produce blue-black colonies due to taking up of the dye when the pH drops.
Salmonella serotype Luxuriant Colourless More than 70%
Lactose non-fermenters like Salmonella and Shigella probably raise the pH of
Typhimurium (14028)
the surrounding medium by oxidative deamination of the protein, solubilising the Staphylococcus aureus None to Colourless 0%
methylene blue-eosin complex and appear as colourless or transparent colonies. (25923) poor
Some gram-positive bacteria like faecal streptococci, staphylococci and yeasts For growth RGI should be more than 70%
grow on this medium to form pinpoint colonies. For Inhibition RGI should be 0%
Formula* RGI- Relative Growth Index
Ingredients in grams per liter Key * = incubation in 10 % CO2
Peptone 10.0 Procedure
Lactose 10.0 1. Allow the agar surface to dry before inoculating.
Dipotassium Phosphate 2.0
2. Inoculate and streak the specimen as soon as possible after collection.
Eosin Y 0.4
Methylene Blue 0.065 3. If the specimen to be cultured is on a swab, roll the swab over a small area of
Agar 15.0 the agar surface.
Final pH (at 250C) 7.1 ± 0.2 4. Use standard procedures like the streak plate method to obtain isolated
* Formula adjusted to suit performance parameters
colonies.
Directions
5. A non-selective medium should also be streaked simultaneously to increase
1. Suspend 37.5 gms of the powder in 1000 ml distilled water.
the chances of recovery when the population of gram-negative organisms is
2. Mix thoroughly. low and to detect other organisms present in the medium.
3. Boil with frequent agitation to dissolve the powder completely. 6. Incubate plates aerobically protected from light at 35-370C for 24 hours.
0
4. Sterilize by autoclaving at 121 C (15 lbs pressure) for 15 minutes. 7. If negative, reincubate for an additional 24 hours.
5. AVOID OVERHEATING. Interpretation of Results
6. Cool to 45-500C and shake the medium in order to restore the blue colour Typical colonial morphology on EMB Agar, Levine
(i.e. oxidize the methylene blue) and to suspend the precipitate, which is an Escherichia coli------------------- Large, blue-black with green metallic sheen

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Enterobacter/ Klebsiella---------- Large, mucoid, blue-black 2. Some strains of Salmonella and Shigella will not grow in the presence of
Proteus--------------------------- Large, colourless eosin Y and methylene blue.
Salmonella----------------------- Large, colourless 3. Some gram-positive bacteria such as staphylococci, enterococci and yeasts
may grow on this medium.
Shigella-------------------------- Large, colourless
Pseudomonas-------------------- Irregular, colourless 4. Non-pathogenic, non-lactose fermenting organisms may also grow on this
medium.
Gram-positive bacteria------------ No growth to slight growth
5. Serial inoculation may be required to assure adequate isolation of mixed flora
Candida albicans------------------ After 24-48 hours, at 350C in 10% CO2,
samples.
colonies are spidery or feathery
Storage
Precautions / Limitations
Store at 22-300C and prepared medium at 2-80C.
1. STORE THE MEDIUM AWAY FROM LIGHT TO AVOID PHOTO OXIDATION.
Shelf Life
Use before expiry date as mentioned on the label.

Endo Agar AM1041/AM5041

Use Peptone 10.0
Endo Agar is a differential and slightly selective culture medium for the detection Sodium Sulphite 2.5
Dipotassium Phosphate 3.5
of coliforms and other enteric microorganisms.
Basic Fuchsin 0.5
Summary
Agar 15.0
Endo (25) developed a culture medium for the differentiation of lactose Final pH (at 250C) 7.5 ± 0.2
fermenters from non-lactose fermenters in which no bile salts were used. * Formula adjusted to suit performance parameters
Inhibition of gram-positive organisms was achieved by the combination of Directions
sodium sulphite and basic fuchsin. Endo's Fuchsin Sulphite Infusion Agar was the 1. Suspend 41.5 gms of the powder in 1000 ml distilled water.
original name for this medium, which is known today as Endo Agar. This medium
2. Mix thoroughly.
is used for the microbial examination of potable water and wastewater (36) and
3. Boil with frequent agitation to dissolve the powder completely.
food (20); however, the current compendia of standard methods recommend
alternative media formulations for the examination of these materials. 4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Principle 5. Cool to 45-500C.
Peptone provides nitrogen and carbon while lactose is the fermentable 6. Re-suspend precipitate by gently mixing before use.
carbohydrate. Dipotassium phosphate is the buffer. Sodium sulphite and basic Warning:
fuchsin inhibit gram-positive organisms to a limited degree. This medium is Basic fuchsin is a potential carcinogen. Avoid inhalation of the powder
and contact with skin.
classified as only slightly selective because other media contain more potent
Quality Control
inhibitors of gram-positive organisms. Lactose fermenting coliforms produce
Dehydrated Appearance
aldehyde and acid. The aldehyde in turn liberates the fuchsin from the fuchsin-
Light purple coloured, homogeneous, free flowing powder.
sulphite complex, giving rise to red colouration of the colonies and similar Prepared Appearance
colouration of the medium. Non-lactose fermenters form faint to colourless Orange pink coloured, clear to slightly opalescent gel with fine precipitate.
colonies against the pink background of the medium. With E.coli, this reaction is Cultural Response
very pronounced as the fuchsin crystallizes, exhibiting a permanent greenish Cultural characteristics after 18-24 hours at 35-370C.
metallic luster to the colonies. Organisms (ATCC) Growth Colour of colony RGI
Formula* Enterobacter Luxuriant Pink, mucoid More than 70%
Ingredients in grams per liter aerogenes (13048)
Lactose 10.0 Enterococcus None to Pink, small 0%

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faecalis (29212) poor 3. A non-selective medium should also be streaked to increase the chances of
Escherichia coli Luxuriant Pink to rose red More than 70% recovery when the population of gram-negative organisms is suspected to be
(25922) with metallic sheen
Klebsiella Luxuriant Pink mucoid More than 70%
low and to provide an indication of other organisms present in the specimen.
pneumoniae (13883) 4. If negative, reincubate an additional 24 hours.
Proteus vulgaris Luxuriant Colourless to More than 70% Interpretation of Results
(13315) pale pink
Typical colony morphology
Pseudomonas Luxuriant Colourless, More than 70%
aeruginosa (27853) irregular Escherichia coli------------------------- Pink to rose red, green metallic sheen
Salmonella serotype Luxuriant Colourless to More than 70% Enterobacter/ Klebsiella--------------- Large, mucoid, pink
Typhimurium (14028) pale pink
Proteus--------------------------------- Colourless to pale pink
Shigella sonnei Luxuriant Colourless to More than 70%
(25931) pale pink Salmonella----------------------------- Colourless to pale pink
Staphylococcus Inhibited - 0% Shigella--------------------------------- Colourless to pale pink
aureus (25923)
Pseudomonas-------------------------- Irregular, colourless
For growth RGI should be more than 70%
For Inhibition RGI should be 0% Gram-positive bacteria----------------- No growth to slight growth
RGI- Relative Growth Index Precautions / Limitations
Procedure 1. Store the medium away from light to avoid photo oxidation.
1. Use standard procedures like the streak plate method to obtain isolated Storage
colonies. Store at 22-300C and prepared medium at 2-80C.
2. Incubate plates protected from light, at 35-370C for 18-24 hours. Shelf Life
Use before expiry date as mentioned on the label.

Emerson Agar AM504101

Use Yeast extract 1.0
Emerson Agar is used for isolation and cultivation of Actinomycetaceae , Peptic digest of animal tissue 4.0
Dextrose 10.0
Streptomycetaceae , fungi and moulds.
Sodium chloride 2.5
Summary
Agar 20.0
Emerson Agar was originally formulated by Emerson et al (24.3) and is used for Final pH ( at 25°C) 7.0±0.2
the cultivation of moulds and bacterial species resembling moulds (45.6). This * Formula adjusted to suit performance parameters
medium was further modified by Gottlieb et al (34.2) and is used for screening Directions
potent antibiotic-producing organisms (102.2). In their study, they stored 1. Dissolve 41.5 gms of medium in 1000 ml distilled water.
Streptomyces in soil for long time and transferred them as needed, to slants of 2. Heat to boiling to dissolve the medium completely.
Emerson Agar. The slant cultures were incubated for 3-7 days. The spores were
3. Add 0.05 grams / litre cycloheximide, or 0.005 gm/l captan if desired..
gently scraped from the cultures surface to form a spore inoculum.
Principle 4. Sterilize by autoclaving at 15lbs pressure (121ºC) for 15 minutes. Mix well
Yeast extract provides a source of trace elements, vitamins and amino acids. For and pour into sterile Petri plates.
Quality Control
the selective isolation of Streptomyces species, cycloheximide is incorporated in
Dehydrated Appearance
the medium, which limits the growth of moulds. This medium is also used for
Cream to yellow homogeneous, free flowing powder.
routine cultivation and maintenance of pure cultures. Prepared Appearance
Formula* Light amber coloured clear to slightly opalescent gel forms in Petri plates
Ingredients in grams per liter Cultural Response
Beef extract 4.0 Cultural characteristics after 48-72 hours at 30ºC.

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Organisms (ATCC) Growth RGI Storage
Aspergillus niger (16404) Luxuriant More than 70% Store at 22-300C and prepared medium at 2-80C.
Saccharomyces cerevisiae (9763) Luxuriant More than 70% Shelf Life
Streptomyces albus subsp albus (3004) Luxuriant More than 70%
Use before expiry date as mentioned on the label.
Streptomyces lavendulae (8664) Luxuriant More than 70%
Streptomyces achromogenes (12767) Luxuriant More than 70%
(13048)
For growth RGI should be more than 70%
RGI- Relative Growth Index

Eugonic Agar AM50410111

Use Final pH ( at 25°C) 7.0±0.2
Eugonic Agar is recommended for the cultivation of fastidious microorganisms * Formula adjusted to suit performance parameters
Directions
like Haemophillus, Neisseria, Posteurella, Brucella and Lactobacillus species.
Summary 1. Dissolve 44.4 gms in 1000 ml distilled water.
Eugonic Agar was developed by Pelczar and Vera (87.2) for cultivation of 2. Heat to dissolve the medium completely.
fastidious organisms like Brucella . These media can also be used to grow 3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
Mycobacteria and various pathogenic fungi including Nocardia, Histoplasma 4. Cool to 45°C and add 5 -10% v/v sterile defibrinated blood if desired. The
and Blastomyces , Eugonic Agar was developed to obtain eugonic (luxuriant) blood may be chocolated by heating, if chocolate agar plates are required.
growth of fastidious microorganisms like Brucella that are otherwise difficult to Quality Control
cultivate (77.1). The unnourished medium supports rapid growth of lactobacilli Dehydrated Appearance
associated with cured meat products, dairy products and other foods. APHA Cream to Yellow coloured, homogeneous, free flowing powder.
recommends Eugonic agar, which is also used in germinating anaerobic spores Prepared Appearance
Principle Yellow coloured clear to slightly opalescent gel forms in petri plates
Cultural Response
Casein enzymic hydrolysate and papaic digest of soyabean meal provide the
Cultural characteristics after 48 hours at 35-37ºC for bacteria and 20-25ºC for
nitrogen, vitamins and amino acids, which supports the growth of fastidious fungi with added 5-10%
microbial species. The high concentration of dextrose is the energy source for rapid Organisms (ATCC) Growth RGI
growth of bacteria. L-Cystine and sodium sulphite are added to stimulate growth. Bacillus pumilus ATCC (14884) Good More than 70%
Sodium chloride maintains the osmotic balance of the media. The high Candida albicans ATCC (26790) Good More than 70%
carbohydrate content along with high sulfur (cystine) content improves growth Lactobacillus fermentum ATCC (9338) Good More than 70%
with chromogenicity. Neisseria meningitidis ATCC (13090) Good More than 70%
Streptococcus pneumoniae ATCC (6303) Luxuriant More than 70%
Formula*
Streptococcus pyogenes ATCC (19615) Luxuriant More than 70%
Ingredients in grams per liter
Brucella abortus (4315) Good More than 70%
Casein enzymic hydrolysate 15.0
For growth RGI should be more than 70%
Papaic digest of soyabean meal 5.0
RGI- Relative Growth Index
Dextrose 5.0
Storage
Sodium chloride 4.0
Sodium sulphite 0.2 Store at 22-300C and prepared medium at 2-80C.
L-Cystine 0.2 Shelf Life
Agar 15.0 Use before expiry date as mentioned on the label.

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Fluid Casein Digest Soya Lecithin Medium (Twin Pack) IP AM10411/AM50411
Fluid Casein Digest Soya Lecithin Medium (Twin Pack) USP AM10412/AM50412
Use 3. Add 40 ml of Part B, mix well.
Fluid Casein Digest Soya Lecithin Medium (Twin Pack) is used a medium for 4. Dispense in tubes or adequate containers and sterilize by autoclaving at
detection of microbes on 15lbs pressure (121ºC) for 15 minutes.
sanitized surfaces in compliance with IP and USP. Quality Control
Summary Dehydrated Appearance
Fluid Casein Digest Soya Lecithin Medium is recommended for sanitary Part A: Yellow coloured, homogeneous, free flowing powder.
Part B: Colourless, clear, viscous liquid.
examination of surfaces in compliance with IP and USP . NASA also recommends
Prepared Appearance
this medium for the microbiological sampling of environmental surfaces sanitized
Yellow coloured clear solution without any precipitate.
with quaternary ammonium compounds(84.2). Cultural Response
Principle Cultural characteristics after 18-24 hours at 35ºC.
Casein enzymic hydrolysate provides the essential nutrients for the growth of Organisms (ATCC) Growth
bacteria. Soya lecithin neutralizes the quaternary ammonium compounds Bacillus subtilis (6633) Good-luxuriant
whereas polysorbate 20 neutralizes phenolic disinfectants, hexachlorophene and *Candida albicans (10231) Good-luxuriant
formalin. Escherichia coli (25922) Good-luxuriant
Staphylococcus aureus (25923) Good-luxuriant
Formula*
Key: * Incubate at 300C for 24-48 hours.
Ingredients in grams per liter
Procedure
Part A:
Casein enzymic hydrolysate 20.0 Refer to appropriate references for specific procedures.
Soy lecithin 5.0 Interpretation of Results
Part B: Refer to appropriate references and procedures for results.
Polysorbate 20 40.0 ml Storage
Final pH (at 250C) 7.3 ± 0.2
Store at 22-300C and prepared medium at 2-80C.
*Formula adjusted to suit performance parameters
Shelf Life
Directions
Use before expiry date as mentioned on the label.
1. Dissolve 25 gms of Part A powder in 960 ml distilled water.
2. Heat gently to dissolve the medium completely. Do not boil.

Fluid Lactose Medium AM1042/AM5042

Fluid Lactose Medium IP AM10421/AM50421
Fluid Lactose Medium USP AM104211/AM504211
Use Principle
Fluid Lactose Medium is used for the detection of coliforms and the study of Beef extract and pancreatic digest of gelatin provide the essential nutrients while
lactose fermentation by common bacteria. lactose is the fermentable carbohydrate. Growth with gas formation is a
Summary presumptive test for coliforms. Multiple strength Fluid Lactose Broth can be used
Fluid Lactose Medium is used for testing water, dairy products and foods. It is also for larger inocula. The final concentration of the ingredients is maintained at a
used in the performance of microbial limit test for Escherichia coli and constant level. i.e. 13 grams per litre.
Salmonella and in the Completed Test for coliforms in dairy products (113, 46). Formula*
Ingredients in grams per liter
Pancreatic Digest of Gelatin 5.0

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Lactose 5.0 concentration of the medium. Regardless of the sample size, Fluid Lactose
Beef Extract 3.0
Medium must have a concentration of 13 grams per litre. For example, if
Final pH (at 250C) 6.9 ± 0.2
10 ml sample is to be added to 10 ml of Lactose Broth, the broth must be
* Formula adjusted to suit performance parameters
double strength.
Directions
2. Allow the medium to warm to room temperature before inoculation.
1. Suspend 13 gms of the powder in 1000 ml distilled water.
3. Inoculate tubes of Fluid Lactose Medium with the dilutions of the sample.
2. Mix thoroughly.
4. Incubate aerobically for 24 hours at 35-370C.
3. Boil with frequent agitation to dissolve the powder completely.
5. Examine for turbidity and gas formation.
4. Distribute into tubes containing inverted Durham's tube.
6. Reincubate the tubes for additional 24 hours (48 hours) if the results are
5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. negative at 24 hours.
6. For large inocula, prepare multiple strength Fluid Lactose Broth. Interpretation of Results
Quality Control
1. Turbidity in the medium accompanied by formation of gas in any amount in
Dehydrated Appearance
the Durham's tubes within 48 hours is a positive presumptive test for the
Light yellow coloured, homogeneous, free flowing powder.
Prepared Appearance
presence of coliforms.
Light amber coloured, clear solution without any precipitate. Precautions / Limitations
Cultural Response 1. Durham's tube must be free from air bubbles before inoculation.
Cultural characteristics after 18-48 hours at 35-370C. 2. Avoid overheating multiple strength broth as inhibitory products may be
Organisms Growth Gas formed.
Enterobacter aerogenes (13048) Good to luxuriant +
Storage
Enterococcus faecalis (29212) Good to luxuriant -
Escherichia coli (25922) Good to luxuriant + Store at 22-300C and prepared medium at 2-80C.
Pseudomonas aeruginosa (27853) Good to luxuriant - Shelf Life
Procedure Use before expiry date as mentioned on the label.
1. The amount of sample added to Fluid Lactose Medium is dependent on the

Fluid Lactose Medium with Lecithin And Tween 80* AM50422

Use 80 neutralizes formalin, phenolic disinfectants, hexachlorophene etc.
Fluid Lactose Medium with Lecithin And Tween 80 is used for detection of coliform Formula*
and lactose fermentation by common bacteria. Ingredients in grams per liter
Summary Pancreatic digest of gelatin 5.0
Lactose 5.0
Fluid Lactose Medium is formulated in accordance with the recommendation of
Beef extract 3.0
APHA and used for testing water, dairy products(78.1) and foods (115.1).
Polysorbate 80 (Tween 80) 5.0
Lactose Broth is used in the completed test for coliforms in dairy products. It is also Lecithin 0.70
used in the performance of microbial limit test for Salmonella species and Final pH (at 250C) 6.9± 0.2
Escherichia coli (111.1). *Formula adjusted to suit performance parameters
Principle Directions
Beef extract and pancreatic digest of gelatin provide essential nutrients for 1. suspend 18.7 gms of the powder in 1000 ml distilled water. Mix thoroughly.
bacterial metabolism. Lactose is the source of fermentable carbohydrate. Growth 2. Boil with frequent agitation to dissolve the powder completely.
with gas formation is a presumptive test for coliforms. Tween 80 and lecithin act
3. Distribute into tubes with inverted Durham's tubes.
as neutralizers to inactivate the residual disinfectants where the samples are
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
collected. Lecithin inactivates quaternary ammonium compounds whereas tween

132 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
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Quality Control Enterococcus faecalis (29212) Good-luxuriant -
Dehydrated Appearance Procedure
Light yellow coloured, homogenous, free flowing powder. Refer to appropriate references for specific procedures.
Prepared Appearance
Interpretation of Results
Light amber coloured, clear solution without any precipitate.
Cultural Response
Refer to appropriate references and procedures for result
Cultural characteristics after 18-48 hours at 35-37ºC. Storage
Organisms (ATCC) Growth Gas Store at 22-300C and prepared medium at 2-80C.
Enterobacter aerogenes (13048) Good-luxuriant + Shelf Life
Escherichia coli (25922) Good-luxuriant + Use before expiry date as mentioned on the label.
Pseudomonas aeruginosa (27853) Good-luxuriant -

Fluid Sabouraud Medium AM1043/AM5043

Use Quality Control
Fluid Sabouraud Medium is a liquid medium used for sterility testing of moulds Dehydrated Appearance
Light yellow coloured, homogeneous, free flowing powder.
and lower bacteria in pharmaceutical preparations.
Prepared Appearance
Summary
Light amber coloured, clear solution without any precipitate.
Fluid Sabouraud Medium is a modification of the formulation of Sabouraud Cultural Response
(100). It is a mycological sterility test medium conforming to the medium Cultural characteristics at 25-300C for 48-72 hours or up to 10 days if
described in the USP (114) and FDA's Bacteriological Analytical Manual (113) for necessary.
the determination of fungistatic activity of pharmaceutical and cosmetic products Organisms (ATCC) Growth
in order to avoid false sterility tests. It is also used for the cultivation of yeasts, Aspergillus niger (16404) Luxuriant
Candida albicans (10231) Luxuriant
moulds and aciduric microorganisms. In clinical microbiology, this medium has
* Escherichia coli (25922) Luxuriant
shown to increase the isolation rate of Candida albicans in blood culture. The acid
*Lactobacillus casei (9595) Luxuriant
reaction of the final medium is inhibitory to a large number of bacteria and makes Saccharomyces cerevisiae (9763) Luxuriant
the medium particularly well suited for cultivating fungi and aciduric Key:
microorganisms. * = incubated at 350C
Principle Procedure
Peptone and Tryptone provide nitrogen and carbon. Dextrose is the carbohydrate 1. Incubate the positive control and the product to be tested at 25-300C for 10
source. days.
Formula* Interpretation of Results
Ingredients in grams per liter
1. If growth in both the tubes is comparable, then the product is non fungistatic.
Dextrose 20.0
Peptone 5.0 2. If the product is fungistatic, add a suitable sterile inactivating agent, or use a
Tryptone 5.0 larger ratio of medium to product in order to determine the ratio of product to
Final pH (at 250C) 5.7 ± 0.2 medium in which growth of the test organism is not affected.
* Formula adjusted to suit performance parameters Precautions / Limitations
Directions 1. Some fungi may be inhibited by the acidic pH of the medium.
1. Suspend 30 gms of the powder in 1000 ml distilled water. 2. Transfer of growth from broth to plated medium may be required in order to
2. Mix thoroughly. obtain pure cultures of fungi.
3. Boil with frequent agitation to dissolve the powder completely. Storage
0
4. Sterilize by autoclaving at 121 C (15 lbs pressure) for 15 minutes. Store at 22-300C and prepared medium at 2-80C.
Shelf Life
Use before expiry date as mentioned on the label.

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Fluid Selenite Cystine Medium (Twin Pack) AM1044/AM5044
Fluid Selenite Cystine Medium IP (Twin Pack) AM10441/AM50441
Fluid Selenite Cystine Medium USP (Twin Pack) AM10442/AM50442
Fluid Selenite Cystine Medium ISO (Twin Pack) AM50443
Use 3. Sterilize in a boiling water bath or free flowing steam for 10 minutes.
Fluid Selenite Cystine Medium is used as a selective enrichment medium for the 4. Discard if large amount of selenite is reduced which is indicated by a red
isolation of salmonellae from faeces, foods, pharmaceutical articles, water and precipitate at the bottom of the tube.
other materials of sanitary importance. Warning: Sodium Hydrogen Selenite (Sodium biselenite) is very toxic,
Summary corrosive agent and causes teratogenicity and should be handled with
care. Upon contact with skin, wash with plenty of water.
Klett (56) first demonstrated the selective inhibitory effects of selenite and Guth
Quality Control
(38) used it to isolate Salmonella typhi. Leifson found that selenite inhibited Dehydrated Appearance
faecal streptococci and coliforms during the first 12 hours of incubation, allowing Cream coloured, homogeneous, free flowing powder.
Salmonella to replicate. Fluid Selenite Cystine Medium is a modification of Prepared Appearance
Leifson's formula (65) with added cystine. This medium is included in the USP Light yellow coloured, clear to slightly opalescent solution.
(114) and IP (46) for use in the performance of microbial limit test for Salmonella Cultural Response
species and is recommended by the FDA's Bacteriological Analytical Manual Cultural characteristics after 18-24 hours at 35-370C when subcultured on
MacConkey Agar (AM1059/AM5059).
(113), AOAC International and APHA for detecting Salmonella in foods (20),
Organisms (ATCC) Recovery Colour
particularly in egg products, milk (39) and water (36). of colony
Principle Escherichia coli (25922) Little to none Pink with bile
Tryptone provides nitrogen and other amino acids. Lactose is the carbohydrate precipitate
source and also maintains the pH in the medium as selenite is reduced by Salmonella serotype Good to excellent Colourless
bacterial growth and alkali is produced. An increase in pH lessens the toxicity of Choleraesuis (12011)
Salmonella serotype Typhi (6539) Good to excellent Colourless
the selenite and results in the overgrowth of other bacteria. The acid produced by
Procedure
bacteria due to lactose fermentation helps to maintain a neutral pH. Sodium
1. For faeces, food sample or other materials, suspend 1-2 gms of the
phosphate buffers the medium to maintain the pH and also lessens the toxicity of
specimen in the broth (approximately 10-15% by volume) and emulsify if
selenite. Sodium selenite inhibits gram-positive bacteria and suppresses the
necessary.
growth of most gram-negative bacteria other than Salmonella. L-cystine
2. Solid material is added to the normal strength broth.
improves the recovery of Salmonella and also acts as a reducing agent.
Formula* 3. Liquid samples are mixed with double strength medium in the ratio of 1:1.
Ingredients in grams per liter 4. Incubate for 12-24 hours at 35-37oC.
DisodiumPhosphate 10.0 5. Sub-culture after 12-18 hours of incubation.
Tryptone 5.0
Interpretation of Results
Sodium Hydrogen Selenite 4.0
Lactose 4.0
1. After incubation, there must be an increase in the number of pathogens that
L-Cystine 0.01 the medium is designed to select for and enrich.
Final pH (at 250C) 7.0 ± 0.2 2. Subculture onto any combination of greater and lesser inhibitory, selective
* Formula adjusted to suit performance parameters and differential media for Enterobacteriaceae. e.g. MacConkey Agar, XLD
Directions Agar, etc to isolate pathogens for identification.
1. Suspend 23 gms of the powder in 1000 ml distilled water and mix well. Precautions / Limitations
2. Warm or just boil to dissolve the medium completely and dispense in tubes 1. Discard the prepared medium if large amounts of reduced selenite can be
as required. AVOID OVERHEATING. DO NOT AUTOCLAVE. seen as a red precipitate at the bottom of the tube.

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2. Do not incubate for longer than 24 hours because the inhibitory effect of chances of isolating pathogens, particularly when they may be present in
selenite is reduced after 6-12 hours incubation and coliforms may overgrow small numbers.
the pathogens. Storage

3. Take subcultures from the upper third of the broth column , which should be Store at 22-300C and prepared medium at 2-80C.
at least 5 cm in depth. Shelf Life

4. Enrichment broths should not be used as the sole isolation medium. Use in Use before expiry date as mentioned on the label.
conjunction with selective and non-selective plating media to increase the

Fluid Tetrathionate Medium USP AM504431

Use brilliant green solution.
An enrichment broth for isolation of Salmonellae from specimens suspected to be 4. Mix well and dispense in 10 ml quantities.
contaminated with Salmonellae in compliance with USP.
(This complete medium should be used on the same day of preparation. Do
Summary
not heat after the addition of Iodine solution. Use the medium immediately
Tetrathionate broth base was originally described by Muller (81.2) and found that after addition of Iodine).
the medium selectively inhibit coliforms and permit unrestricted growth of enteric Quality Control
pathogens. The medium is now formulated according to USP which specify this Dehydrated Appearance
medium as enrichment medium for Salmonella species Cream coloured, homogeneous, free flowing powder.
Principle Prepared Appearance
Fluid Tetrathionate Medium USP as enrichment medium for Salmonella species. Complete medium with added brilliant green and iodine solution forms light
green coloured, opalescent solution with heavy white precipitate.
Bile salts inhibit gram-Positive microorganisms. The selectivity depends on the
Cultural Response
ability of thiosulphate and tetrathionate in combination to suppress commensal Cultural characteristics after 18-24 hours at 37ºC when subcultured on
coliform organisms. Calcium carbonate neutralizes the acidic tetrathionate MacConkey Agar after enrichment in Tetrathionate medium.
decomposition products. For further confirmation, streak the enriched cultures Organisms (ATCC) Recovery Colony
after incubation, on the plates of Brilliant Green Agar, MacConkey agar, Bismuth Escherichia coli (25922) Little or on increase white to pink
Sulphite Agar. in number with bile
precipitate
Formula *
S. serotype Choleraesuis (12011) Good to excellent Colourless
Pancreatic digest of casein 2.5g
S. serotype Typhi (6539) Good to excellent Colourless
Peptic digest of animal tissue 2.5g
S. serotype Typhimurium (14028) Good to excellent Colourless
Bile salts 1.0g
Procedure
Calcium carbonate 10.0g
Sodium thiosulphate 30.0g Refer to appropriate references for specific procedures.
*Formula adjusted, standardized to suit performance parameters. Interpretation of Results
Directions Refer to appropriate references and procedures for results.
1. Suspend 46 grams in 1000 ml of distilled water. Storage

2. Heat to boiling to dissolve the medium completely. DO NOT AUTOCLAVE. Store at 22-300C and prepared medium at 2-80C.
Shelf Life
3. Cool below 450C and add 20 ml Iodine solution (Iodine 6 grams and
Use before expiry date as mentioned on the label.
Potassium Iodide-5 grams in 20 ml distilled water) and 10 ml of 0.1%

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Fluid Thioglycollate Medium AM1045/AM5045
Fluid Thioglycollate Medium IP AM10451/AM50451
Fluid Thioglycollate Medium USP AM10452/AM50452
Fluid Thioglycollate Medium EP AM10453/AM50453
Fluid Thioglycollate Medium BP AM10454/AM50454
Use Yeast extract 5.00 5.00 5.00 5.00 5.00
Fluid Thioglycollate Medium is used for sterility testing of biologicals and for Glucose monohydrate – – – 5.50 5.50
Dextrose 5.50 – – – –
cultivation of aerobes, anaerobes and microaerophiles.
Dextrose monohydrate – 5.50 5.50 – –
Summary
Sodium chloride 2.50 2.50 2.50 2.50 2.50
Falk, Bucca and Simmons (29) showed the advantage of using small quantities L-Cystine 0.50 0.50 0.50 0.50 0.50
of agar in detecting contaminants during sterility testing of biologicals. Brewer Sodium thioglycollate 0.50 0.50 0.50 0.50 0.50
(9) demonstrated that in a liquid medium containing 0.05% agar, anaerobes Resazurin Sodium 0.001 0.001 0.001 0.001 0.001
grew equally well in the presence or absence of sodium thioglycollate and Agar 0.75 0.75 0.75 0.75 0.75
therefore formulated Fluid Thioglycollate Medium for rapid cultivation of aerobes Final pH (at 25ºC) 7.1 ± 0.2
* Formula adjusted to suit performance parameters
as well as anaerobes by adding a reducing agent and a small amount of agar.
Directions
Fluid Thioglycollate Medium is recommended by APHA (20) and the AOAC
1. Suspend 29.75 gms of the powder in 1000 ml distilled water.
International for the examination of food, and for determining the phenol
coefficient and sporicidal effect of disinfectants . This medium is also specified for 2. Mix thoroughly.
sterility checks on banked blood. It is recommended in the USP (114) and IP (46) 3. Boil with frequent agitation to dissolve the powder completely.
for use in sterility testing of articles supposed to be sterile and is also included in 4. Dispense as desired into containers.
the Bacteriological Analytical Manual for food testing (113). 5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Principle 6. Tighten lids of the containers immediately (while still warm) to reduce
Tryptone, yeast extract and L-cystine provide sources of nitrogen, carbon and oxidation.
other growth factors while dextrose is the carbohydrate source. Sodium chloride 7. Cool to 250C and store in a cool dark place preferably below 250C.
provides essential ions and maintains the osmotic balance. Sodium thioglycollate
Note: If more than the upper one third of the medium is pink prior to use,
is a reducing agent, which prevents the accumulation of peroxides that is lethal to
reheat once (1000C) in a water bath to drive off absorbed oxygen (till pink
bacterial growth and neutralizes the antibacterial effect of mercurial
colour disappears).
preservatives. L-cystine is also a reducing agent, since it contains sulphydryl
Quality Control
groups that inactivate heavy metal compounds, which exert a bacteriostatic effect Dehydrated Appearance
in the materials under examination, and also maintains a low redox potential, Yellow coloured, homogeneous, free flowing powder.
thereby maintaining anaerobiosis. Resazurin is the oxidation-reduction indicator; Prepared Appearance
increased oxidation raises the Eh, causing resazurin to change colour to red. The Light straw coloured, clear to very slightly opalescent solution with upper 10%
small amount of added agar assists in maintaining a low redox potential by or less medium turning pink on standing.
stabilizing the medium, thereby maintaining anaerobiosis in the lower depths of Cultural Response
Cultural characteristics after 48-72 hours at 350C.
the medium.
Organisms (ATCC) Growth
Formula*
*Bacillus subtilis (6633) Luxuriant
Ingredients in grams/liter FTM FTM FTM FTM FTM
*Bacteroides vulgatus (8482) Luxuriant
IP USP EP BP
*Candida albicans (10231) Luxuriant
Tryptone 15.00 – – – –
*Clostridium sporogenes (11437) Luxuriant
Pancreatic digest of casein - 15.00 15.00 15.00 15.00

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*Micrococcus luteus (9341) Luxuriant 3. A slight turbidity or haziness may be present due to the small amount of agar
Streptococcus pyogenes (19615) Luxuriant present in the medium. When the medium has been boiled, generally it
Staphyloccocus aureus (6538) Luxuriant
appears clear.
Key:
* These cultures may be incubated at 25-300C for 2-7 days. 4. Anaerobes can be overgrown by more rapidly growing facultative organisms.
Interpretation of Results Gram stain and examine broth if plating medium reveals no growth.
1. After incubation, growth is indicated by the presence of turbidity compared to 5. Some anaerobes may be inhibited by metabolic products or acids produced
an un-inoculated control. from more rapidly growing facultative anaerobes.
2. Strict aerobes tend to grow in a thin layer at the surface of the broth; obligate 6. Do not rely on broth cultures exclusively for isolation of anaerobes.
anaerobes will grow only in the portion of the broth that is below the upper 7. Do not reheat the medium more than once as it may give rise to toxicity.
oxidized layer. 8. If more than one third of the medium is oxidized (pink), it must be discarded.
Precautions / Limitations
9. Store the prepared medium at room temperature away from light.
1. Some dextrose fermenting organisms, which are able to reduce the pH of the Storage
medium to a critical level, may not survive in this medium. Early subculture
Store at 22-300C and prepared medium at 2-80C.
is required to isolate these organisms.
Shelf Life
2. In test samples, the proper surface to volume ratio of the medium must be Use before expiry date as mentioned on the label.
maintained to avoid oxidation of the medium, which is unsuitable for
microaerophilic and anaerobic growth.

Fraser Broth Base AM50455

Fraser Broth Base ISO AM50456
Use Formula*
Fraser Broth Base is used for the isolation, cultivation and enrichment of Listeria Ingredients in grams per liter
Sodium chloride 20.0
monocytogenes from food and environmental samples.
Disodium phosphate 2H2O 12.0
Summary
Meat peptone 5.0
Listeria monocytogenes is a Gram-positive, non-sporeforming, aerobic to Tryptone 5.0
facultatively anaerobic, rod shaped bacterium, which exhibits pathogenicity Yeast extract 5.0
towards humans and other animals. Although not generally recognized as a food- Beef extract 5.0
borne pathogen, three recent outbreaks of listeriosis may indicate that this Lithium chloride 3.0
organism is becoming more prevalent as an agent of food-borne disease. Mono potassium phosphate 1.30
Esculin 1.00
Fraser Broth Base and Fraser Supplements are based on the formulation of Fraser
Acriflavin 0.025
and Sperber. Fraser supplements results in a higher detection rate of Listeria
Nalidixic acid 0.020
monocytogenes. Final pH (at 25ºC) 7.2 ± 0.2
Principle Formula adjusted to suit performance parameters
Casein enzyme hydrolysate, pancreatic digest of casein, beef extract and yeast Directions
extract serves as a source of carbon, nitrogen, vitamins and minerals. Disodium 1. Suspend the 57.35 gms of powder in 1000 ml distilled water.
phosphate and mono potassium phosphate are buffering agents. Addition of 2. Mix thoroughly.
ferric ammonium citrate in the medium helps to differentiate the esculin
3. Boil with frequent agitation to dissolve the powder completely. Do not
hydrolysis, resulting in the blackening of the medium by Listeria species. Lithium
overheat.
chloride and high salt concentration makes the medium selective for Listeria
species. 4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.

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5. Cool the medium to -50°C. Procedure

6. Add 2 vial of rehydrated Fraser Enrichment Supplement (AS0114). 1. For fecal and biological specimens, the sample is homogenized in 0.1%
Peptone water (AM1079/5079) and transfer 0.1 ml of the incubated broth to
7. After addition, the medium must be gently but thoroughly mixed to ensure
Fraser broth base
that the antibiotics are uniformly distributed throughout the medium.
Warning: Lithium chloride is harmful. Avoid bodily contact and 2. Incubate the medium at 35ºC for 26±2 hours and examine the result.
inhalation of vapours. On contact with skin, wash with plenty of 3. After 24-48 hours, streak the Fraser Broth culture to Listeria Oxford Medium
water immediately. Base (AM 105512 / AM 505512).
Quality Control
4. Incubate the plates at 35ºC for 24-48 hours.
Dehydrated Appearance
Interpretation of Results
Tan coloured, homogeneous, free flowing powder.
Prepared Appearance Examine the agar plates for suspected colonies. For further identification and
Amber coloured, clear to slightly opalescent with a fine precipitate. confirmation of Listeria species, consult appropriate references.
Cultural Response Precautions / Limitations
Cultural characteristics after 18-24 hours at 35ºC. 1. Since Listeria spp. other than L. monocytogenes can grow on these media,
Organisms (ATCC) Growth Esculin
biochemical and serological testing should be done to identify L.
hydrolysis
monocytogenes.
Enterococcus faecalis (29212) Inhibited -
Escherichia coli (25922) Inhibited - 2. Poor growth and a weak esculin reaction may be seen after 40 hours incubation
Listeria monocytogenes (19117) Good + for some enterococci.
Listeria monocytogenes (19115) Good + Storage
Staphylococcus aureus (25923) Inhibited - Store at 22-300C and prepared medium at 2-80C.
Key : + = Black zones around colonies Shelf Life
Use before expiry date as mentioned on the label.

Fraser Secondary Enrichment Broth Base AM50457

Use Principle
Fraser Secondary Enrichment Broth Base with added supplement is Proteose peptone, casein enzyme hydrolysate, beef extract and yeast extract
recommended for isolation, cultivation and enrichment of Listeria monocytogenes serves as a source of carbon, nitrogen, vitamins and minerals. Lithium chloride
from food and environmental samples. inhibits the growth of Enterococci. All Listeria species hydrolyze esculin to
Summary esculatin which with ferric ions results in forming dark-brown to black complex.
Listeria monocytogenes is a Gram-positive, non-sporeforming, aerobic to Ferric ammonium citrate enhances the growth of Listeria monocytogenes. Fraser
facultatively anaerobic, rod shaped bacterium, which exhibits pathogenicity Secondary Enrichment Broth is inoculated with primary Enrichment Broth. All
towards humans and other animals. Although not generally recognized as a food- Fraser Broth Enrichment cultures should be subcultured on plating medium for
borne pathogen, three recent outbreaks of listeriosis may indicate that this confirmation of presence or absence of Listeria species.
organism is becoming more prevalent as an agent of food-borne disease. Formula*
Ingredients in grams per liter
Fraser secondary enrichment broth base is based on the original formulation
Proteose peptone 5.0
described by Catherine W. Donnelly and Gregory J. Baigent (19.1), and its later
Casein enzyme hydrolysate 5.0
modifications of United States Department of Agriculture Food Safety Inspection Yeast extract 5.0
Service (USDA-FSIS) UVM Secondary Enrichment Broth. Fraser Secondary Beef extract 5.0
Enrichment Broth Base and Fraser supplements are based on the formulation of Sodium chloride 20.0
Fraser and Sperber. (32.2) This modification and two-step selective enrichment Lithium chloride 3.0
method developed (USDA-FSIS method) results in a higher detection rate of Disodium phosphate 12.0
Listeria monocytogene. Mono potassium phosphate 1.35
Esculin 1.00

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Ferric ammonium citrate 0.50 Staphylococcus aureus (25923) Inhibited -
Final pH (at 25ºC) 7.2 ± 0.2 Key : + = Black zones around colonies
Formula adjusted to suit performance parameters * = Subcultured on Listeriaidentification Agar Base (PALCAM) ((AM1055/
Directions 5055)
Procedure
1. Suspend the 57.85 gms of powder in 990 ml distilled water.
1. For fecal and biological specimens, the sample is homogenized in 0.1%
2. Heat to boiling to dissolve the medium completely.
Peptone water (AM1079/5079) and transfer 0.1 ml of the incubated broth to
3. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Fraser broth base
4. Cool the medium to -50°C.
2. Incubate the medium at 35ºC for 26±2 hours and examine the result.
6. Add 1 vial of rehydrated Fraser Selective Supplement (AS0112) to prepare
3. After 24-48 hours, streak the Fraser Broth culture to Listeria Oxford Medium
selective medium for Listeria.
Base (AM 105512/AM 505512).
7. After addition, the medium must be gently but thoroughly mixed to ensure
4. Incubate the plates at 35ºC for 24-48 hours.
that the antibiotics are uniformly distributed throughout the medium. Interpretation of Results
Warning: Lithium chloride is harmful. Avoid bodily contact and
inhalation of vapours. On contact with skin, wash with plenty of 1. Examine the agar plates for suspected colonies. For further identification and
water immediately. confirmation of Listeria species, consult appropriate references.
Quality Control Precautions / Limitations
Dehydrated Appearance 1. Since Listeria spp. other than L. monocytogenes can grow on these media,
Yellow coloured, homogeneous, free flowing powder.
biochemical and serological testing should be done to identify
Prepared Appearance
Yellow coloured, clear to slightly precipitate. With addition of supplement, L. monocytogenes.
fluorescent yellow coloured solution forms with slight precipitate. 2. Poor growth and a weak esculin reaction may be seen after 40 hours incubation
Cultural Response for some Enterococci.
Cultural characteristics after 24-48 hours at 35ºC.
Storage
Organisms (ATCC) Growth Esculin
hydrolysis*
Store at 22-300C and prepared medium at 2-80C.
Enterococcus faecalis (29212) Inhibited - Shelf Life
Escherichia coli (25922) Inhibited - Use before expiry date as mentioned on the label.
Listeria monocytogenes (19117) Luxuriant +

G.C. Agar Base AM1046/AM5046

Use prevents change in the pH due to amine production that can effect the survival of
G.C. Agar Base with various additives is used to isolate and cultivate gonococci the organisms. Sodium chloride maintains the osmotic balance.
and other fastidious organisms. Chocolate Agar is prepared from G.C. Agar Base with the addition of 2%
Summary Haemoglobin. Haemoglobin provides hemin, which enhances growth of
G.C. Agar Base with added blood or haemoglobin and other supplements is Neisseria. The medium can also be made selective with the addition of selective
recommended for selective isolation and cultivation of fastidious organisms like supplements.
Neisseria gonorrhoeae. Johnston (49) developed a medium that could produce Formula*
colonies of N.gonorrhoeae in 24 hours rather than 48 hours. The accelerated Ingredients in grams per liter
growth rates were primarily due to the decreased agar content. Carpenter and Peptone Special 15.0
Morton (13) later on improved the medium with the addition of haemoglobin. Sodium Chloride 5.0
Principle Dipotassium Phosphate 4.0
Monopotassium Phosphate 1.0
Peptone special provides nitrogen, vitamins and amino acids. Corn starch absorbs
Corn Starch 1.0
and neutralizes the toxic metabolites and phosphates buffers the medium and Agar 10.0

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Final pH (at 250C) 7.2 ± 0.2 3. Specimens should be streaked on the surface of the plates so as to get some
* Formula adjusted to suit performance parameters areas heavily seeded and other areas lightly seeded.
Directions
4. Incubate at 370C in an atmosphere of 5-10% CO2 and 70% humidity.
1. Suspend 18 gms of the medium in 235 ml distilled water.
Precautions / Limitations
2. Boil with frequent agitation to dissolve the powder completely.
1. Avoid cotton wool for specimen collection.
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
2. Any suspected Neisseria containing specimen should be inoculated onto a
4. Cool to 45-500C and aseptically add separately prepared Haemoglobin primary isolation medium immediately on collection. If this is not possible, then
(250 ml sterile 2% solution) (AS014) and 1 vial of G.C. Supplement N.gonorrhoea swabs are better held at 400C for not more then 3 hours.
(AS012).
3. It is seen that the usual transport media are not totally reliable for
5. Mix well and pour into sterile petri plates. N.gonorrhoea. Inoculation of the sample onto the surface of the medium
Quality Control slants is preferable.
Dehydrated Appearance
Yellow coloured, homogeneous, free flowing powder.
4. Humidity is essential for the successful isolation of gonococci. If the plates look
Prepared Appearance dry, moisten the surface with a few drops of sterile broth and allow it to soak into
Basal Medium - Light yellow coloured, clear to slightly opalescent gel. the agar before inoculation. Do not flood the plate with broth. Place damp gauze
With addition of haemoglobin - Chocolate brown coloured opaque gel. or paper towels in the CO2 chamber before incubation.
Cultural Response
5. Agar varies widely in their toxicity for N.gonorrhoea and may be a major
Cultural characteristics after 40-48 hours at 35-370C on Chocolate Agar
prepared from GC Agar Base incubated in 5-10% CO2 and 70% humidity.
factor in preventing the growth of gonococci on solid media.
Organisms (ATCC) Growth RGI 6. Enrichments including haemoglobin and coenzymes must preferably be
Haemophilus influenzae (19418) Luxuriant More than 70% added to obtain a good growth of Neisseria species.
*Neisseria meningitidis (13090) Luxuriant More than 70% 7. Improper specimen collection, environment, temperature, CO2 level, moisture
Streptococcus pneumoniae (6303) Luxuriant More than 70%
and pH can adversely effect the growth and viability of the organisms.
Streptococcus pyogenes (19615) Luxuriant More than 70%
For growth RGI should be more than 70% 8. Inactivation or deterioration of antibiotics in the selective medium may allow
RGI- Relative Growth Index the growth of contaminants.
Key: 9. The medium has sufficient buffering capacity to offset the very low pH of the
*with antibiotic supplement small amounts of nutritive enrichments added. However, the pH of some
Procedure
media may have to be adjusted with 1% NaOH after the addition of
1. Infectious material should be submitted directly to the laboratory protected enrichments.
from excessive heat and cold. Storage
2. If there is to be delay in processing, the specimen should be inoculated onto Store at 22-300C and prepared medium at 2-80C.
an appropriate transport medium. Shelf Life
Use before expiry date as mentioned on the label.

Glucose Agar AM50461

Use Principle
Glucose agar is used for determining the fermentation reaction of presumptive Tryptone provides carbon and nitrogen compounds glucose is the sole source of
Enterobacteriaceae. fermentable carbohydrate while sodium chloride maintains the osmotic balance.
Summary Formula*
Glucose agar is used for the differentiation of Enterobacteriaceae in urine, water Ingredients in grams per liter
Tryptone 10.0
and food. It differentiates species on the basis of glucose fermentation. Glucose
Glucose 10.0
agar is used for deep stab and shake cultures of anaerobes (77.2).
Sodium chloride 5.0

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Yeast extract 1.5 Organisms (ATCC) Growth Colour of RGI
Bromocresol purple 0.015 colony/Medium
Agar 15.0 Enterobacter aerogenes Luxuriant Yellow More than 70%
Final pH (at 250C) 7.0 ± 0.2 (13048)
* Formula adjusted to suit performance parameters Escherichia coli Luxuriant Yellow More than 70%
Directions (25922)
1. Suspend the 41.52 gms of powder in 1000 ml distilled water. Pseudomonas aeruginosa Luxuriant Colourless More than 70%
(27853) colony with no
2. Mix thoroughly. change in medium.
3. Heat gently with frequent agitation to dissolve the powder completely. For growth RGI should be more than 70%
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes. RGI- Relative Growth Index
Quality Control Procedure
Dehydrated Appearance Refer to appropriate references for specific procedures.
Light yellow coloured, homogeneous, free flowing powder. Interpretation of Results
Prepared Appearance Refer to appropriate references and procedures for results.
Purple coloured clear to slightly opalescent gel forms in petri plate. Storage
Cultural Response
Cultural characteristics after 18-24 hours at 37°C.
Store at 22-300C and prepared medium at 2-80C.
Shelf Life
Use before expiry date as mentioned on the label.

Gelatin Broth AM504611

Use Directions
Gelatin Broth is used for isolation and enumeration of Bacillus cereus. 1. Suspend the 45.0 gms of powder in 1000 ml distilled water.
Summary 2. Mix thoroughly.
Gelatin is a protein of uniform molecular constitution derived chiefly by the 3. Warm slightly with frequent agitation to dissolve the powder completely.
hydrolysis of collagen. Collagens are a class of albuminiod found abundantly in
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
bones, skin, tendon, cartilage and similar animal tissues.
Dehydrated Appearance
Koch introduced gelatin into bacteriology when he invented the gelatin tube Light yellow coloured, homogeneous, free flowing powder.
method in 1875 and the plate method in 1881. Prepared Appearance:
Principle Dark yellow coloured clear solution.
The nitrogen, carbon, vitamins, and amino acids are provided by Yeast Extract for Cultural Response
Cultural characteristics after 18-24 hours at 37°C.
general growth requirements in Gelatin Broth. Gelatin is the substrate for
Organisms (ATCC) Growth
determining if microorganisms elaborate the proteolytic enzyme to hydrolyze
Bacillus cereus (10876) Luxuriant
(liquefy) gelatin. Bacillus subtilis (6633) Luxuriant
Formula* Procedure
Ingredients in grams per liter
Refer to appropriate references for specific procedures.
Gelatin 30.0
Interpretation of Results
Peptone 5.0
Yeast extract 5.0 Refer to appropriate references and procedures for results.
Potassium dihydrogenphosphate 5.0 Storage
0
Final pH (at 25 C) 7.0 ± 0.2 Store at 22-300C and prepared medium at 2-80C.
* Formula adjusted to suit performance parameters Shelf Life
Use before expiry date as mentioned on the label.

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Glucose Broth AM1047/AM5047

Use 2. Mix thoroughly.
Glucose Broth is used in glucose fermentation studies where a pH indicator is not 3. Boil with frequent agitation to dissolve the powder completely.
desired.
4. Dispense in tubes containing inverted Durham's tubes.
Summary
5. Sterilize by autoclaving at 1180C (12 lbs pressure) for 15 minutes.
Waisbren, Carr and Dunnett used Glucose Broth for the study of glucose
Quality Control
fermentation and for testing the sensitivity of microorganisms to antibiotics by
Dehydrated Appearance
the tube dilution method. This medium being highly nutritious, hastens the early Light yellow coloured, homogeneous, free flowing powder.
recovery of injured cells. Glucose Broth is included in the Bacteriological Prepared Appearance
Analytical Manual for food testing (113). Light yellow coloured, clear solution without any precipitate.
Principle Cultural Response
Tryptone provides carbon and nitrogen compounds, glucose is the sole source of Cultural characteristics after 18-24 hours at 370C.
fermentable carbohydrate while sodium chloride maintains the osmotic balance. Organisms (ATCC) Growth Gas
Escherichia coli (25922) Luxuriant +
Formula*
Salmonella serotype Typhi (6539) Luxuriant -
Ingredients in grams per liter
For growth RGI should be more than 70%
Glucose 5.0
RGI- Relative Growth Index
Tryptone 10.0
Storage
Sodium Chloride 5.0
Final pH (at 250C) 7.3 ± 0.2 Store at 22-300C and prepared medium at 2-80C.
* Formula adjusted to suit performance parameters Shelf Life
Directions Use before expiry date as mentioned on the label.
1. Suspend 20 gms of the powder in 1000 ml distilled water.

Glucose Salt Teepol Broth (Twin Pack) AM10471/AM50471

Use provides sodium ions for the membrane transport and maintains osmotic
Glucose Salt Teepol Broth is used for selective enrichment and enumeration of equilibrium of the medium.
Vibrio parahaemolyticus from marine isolates in compliance with BIS Formula*
specification IS: 5887. Ingredients in grams per liter
Summary Part A: Peptic digest of animal tissue 10.0
Beef extract 3.0
Glucose Salt Teepol Broth is recommended by APHA for isolation of Vibrio
Sodium chloride 30.0
parahaemolyticus from marine isolates (32.1). It is a selective enrichment
Glucose 5.0
medium for Vibrio parahaemolyticus that enables faster growth of the organism Methyl violet 0.002
(40.1). Medium contains teepol, inhibits the migration of halophilic organisms Part B: Teepol 4.0 ml
and the growth of the Gram-positive organism while the organism utilizes glucose Final pH (at 250C)8.8 ± 0.2
of the medium. It is also used to enumerate the bacteria by MPN technique. *Formula adjusted to suit performance parameters
Glucose Salt Teepol Broth should be used as an enrichment broth in conjunction Directions
with TCBS Agar (AM1095/5095). 1. Dissolve 48gms of Part A in 1000ml distilled water and add 4ml of Part B.
Principle 2. Mix thoroughly.
Peptic Digest of Animal Tissue supply nutrients, nitrogen compounds and amino 3. Heat gently to dissolve the medium completely. Do not boil.
acids. Beef extract also serves as an essential source of nitrogen. High percentage
4. Dispense in tubes or adequate containers and sterilize by autoclaving at
of chloride enhances the growth of halophilic Vibrio parahaemolyticus. It also
15lbs pressure (121ºC) for 15 minutes.

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Quality Control 2. Mix well the inoculated medium.
Dehydrated Appearance
3. Incubate at 35-370C for 18-24 hours.
Part A: Light yellow coloured, homogeneous, free flowing powder.
Part B: Light yellow coloured viscous liquid. 4. Transfer 0.1 ml of incubated broth to TCBS agar. Incubate at 35-370C for 18-
Prepared Appearance 24 hours.
Purple colour clear solution with very slight precipitate. Interpretation of Results
Cultural Response Examine agar plates for suspect colonies. For further identification and
Cultural characteristics after 18-24 hours at 35ºC.
confirmation of V. parahaemolyticus, consult appropriate references.
Organisms (ATCC) Growth
Storage
Vibrio parahaemolyticus (17802) Good-luxuriant
Vibrio alginolyticus (17749) Good-luxuriant Store at 22-300C and prepared medium at 2-80C.
Procedure Shelf Life

1. Inoculate the Glucose Salt Teepol Broth with marine or other food specimens. Use before expiry date as mentioned on the label.

Glucose Yeast Extract Agar AM1048/AM5048

Use Agar 15.0
Glucose Yeast Extract Agar is used for enumeration of lactobacilli in * Formula adjusted to suit performance parameters
Directions
pharmaceutical preparations.
Summary 1. Suspend 28.4 gms of the powder in 1000 ml distilled water.
Glucose Yeast Extract Agar was formulated by Evans and Niven (27) and Rogosa 2. Mix thoroughly.
et al (95). 3. Boil with frequent agitation to dissolve the powder completely.
Principle 4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Glucose is the carbohydrate source. Peptone and yeast extract provides essential Quality Control
nutrients while salts like sulphates and phosphates support the growth of Dehydrated Appearance
lactobacilli. The metallic salts are sources of ions essential for the replication of Beige coloured, homogeneous, free flowing powder.
lactic acid bacteria. Prepared Appearance
Formula* Yellow coloured, clear to slightly opalescent gel.
Ingredients in grams per liter Cultural Response
Yeast Extract 5.0 Cultural characteristics after 24-48 hours at 350C.
Peptone 5.0 Organisms (ATCC) Growth RGI
Glucose 2.0 Lactobacillus casei (9595) Good to luxuriant More than 70%
Dipotassium Phosphate 0.5 Lactobacillus bulgaricus Good to luxuriant More than 70%
Monopotassium Phosphate 0.5 (11842)
Magnesium Sulphate 0.3 For growth RGI should be more than 70%
Manganese Sulphate 0.01 RGI- Relative Growth Index
Sodium Chloride 0.01 Storage
Cobalt Sulphate 0.0016
Store at 22-300C and prepared medium at 2-80C.
Copper Sulphate 0.0016
Shelf Life
Zinc Sulphate 0.0016
Use before expiry date as mentioned on the label.

Heart Infusion Agar AM10481/AM50481

Heart Infusion Broth AM10482/AM50482
Use range of microorganisms from a variety of clinical and non-clinical specimens.
Heart Infusion Agar is a general-purpose medium used in the cultivation of a wide Heart Infusion Broth is used for cultivating fastidious microorganisms.

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Summary
3. Sterilize by autoclaving at 1210 C (15 lbs pressure) for 15 minutes.
It has long been known that the growth of bacteria on culture media is facilitated
4. To prepare blood agar, aseptically add 5% sterile defibrinated blood to the
by the addition of certain supplements, soluble in water, thermostable at 1000C
medium at 45-500 C.
and readily adsorbed from solution by filtration through paper or cotton. Huntoon
demonstrated that pathogenic microorganisms could be grown on an infusion 5. Mix well and pour into sterile petriplates or tubes.
Quality Control
agar without supplements. Heart infusion media are specified for the isolation of
Dehydrated Appearance
Vibrio cholerae and Vibrio species.
Light yellow coloured, homogeneous, free flowing powder.
Heart Infusion Agar is one such medium, which due to its nutritious composition is Prepared Appearance
used as a basal medium for primary isolation of pathogenic organisms such as Light yellow coloured basal medium, cherry red coloured opaque gel.
meningococci and pneumococci. Cultural Response
Heart Infusion Broth is a non-selective general-purpose medium used for the Cultural response after 18 - 48 hours at 350 C.
Organisms Growth Growth Growth Haemolysis RGI
isolation of nutritionally fastidious microorganisms. Huntoon using fresh Beef (ATCC) on HI Agar on HI Agar on HI Broth With 5%
Heart and Peptone prepared a “hormone” broth to retain growth-promoting sheep blood More than70%
Escherichia Good to Luxuriant Luxuriant Beta More than70%
substances. However, the formulation contains tryptose, which is better suited to
coli (25922) Luxuriant
the nutritional requirements of pathogenic bacteria than peptone. Neisseria Good to Luxuriant Luxuriant None More than 70%
Principle meningitidis Luxuriant
Beef Heart Infusion and Tryptose provide nitrogenous compondounds, sulphur, (13090)
Streptococcus Good to Good to Luxuriant Alpha More than 70%
vitamins, amino acids and trace ingredients. Sodium Chloride maintains the
pneumoniae Luxuriant Luxuriant
osmotic equilibrium. Agar is the solidifying agent. Heart Infusion broth can be (6303)
modified by adding dextrose, 5% sheep blood or other ingredients to determine Streptococcus Good to Good to Luxuriant Beta More than 70%
pyogenes Luxuriant Luxuriant
hemolytic reactions and many other purposes. (19615)
Formula* Procedure
Ingredients in grams per liter Agar Broth
1. Obtain isolated colonies from specimens using standard procedures.
Beef Heart, Infusion from 500.0 500.0
Tryptose 10.0 10.0 2. Since many pathogens require carbon dioxide on primary isolation, incubate
Sodium Chloride 5.0 5.0 plates in an atmosphere containing approximately 3-10% CO2 at 35±20 C
Agar 15.0 -- for 18-48 hours.
Final pH (at 250 C) 7.4 ± 0.2
3. Examine plates for colonial morphology.
* Formula adjusted to suit performance parameters
Interpretation of Results
Directions
1. Refer to U.S.P. and other appropriate references for interpretation of results.
1. Suspend the powder in 1000 ml distilled water.
Storage
Heart Infusion Agar - 40 grams
Store at 22-300C and prepared medium at 2-80C.
Heart Infusion Broth - 25 grams Shelf Life
2. Heat with frequent agitation and boil for 1 minute to dissolve the powder Use before expiry date as mentioned on the label.
completely.

Hektoen Enteric Agar AM104821/AM504821

Use selective isolation of Shigella and other pathogenic species from clinical
Hektoen Enteric Agar is used for isolation and differentiation of gram-negative specimens(55.1). They found in their comparative study that H E medium was
enteric pathogens, particularly Shigella species from clinical and nonclinical more superior than SS agar for recovery of Salmonella and Shigella species
specimens. (55.2). The present formulation of H E agar has less amount of bile salt and
Summary deoxycholate is absent. High level of peptones and sugars reduce the inhibitory
King and Metzger developed H E Agar at the Hektoen institute in Chicago for effect of bile salt and enable it to be moderately selective for Salmonella and

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Shigella species. H E agar is suitable for isolation of Salmonella and Shigella S. Choleraesuis (12011) Luxuriant Greenish blue More than 70%
species from food, clinical, dairy and other specimens. colonies with black
center
Principle
Shigella flexneri (12022 ) Luxuriant Green to blue-green More than 70%
Peptone and Yeast extract serve as a source of nitrogen. Bile salts act as a selective Enterobacter fair to Salmon orange to More than 70%
agent by inhibiting gram-positive and other than enteric organisms. Sodium aerogenes (13048) Good yellowish orange
chloride provides sodium ions for the membrane transport and maintains osmotic Enterococcus faecalis Inhibited - 0%
equilibrium of the medium. Salicin, Sucrose and Lactose provide differentiation (29212)
of gram-negative enteric pathogens. Bromothymol blue and Acid fuchsin are Escherichia coli (25922) fair Orange (may have 0%
acid-base indicator. The additions of ferric ammonium sulphate and sodium bile precipitate)
thiosulphate enable the detection of H2S production. Agar is the solidifying agent. For growth RGI should be more than 70%
For Inhibition RGI should be 0%
Formula*
RGI- Relative Growth Index
Ingredients in grams per liter
Procedure
Proteose peptone 12.00
Yeast extract 3.00 1. Use standard procedures like the streak plate method to obtain isolated
Lactose 12.00 colonies.
Sucrose 12.00 2. If the specimen to be cultured is on a swab, roll the swab on a small area of
Salicin 2.00 the agar surface and streak for isolation with a sterile loop.
Bile salts 9.00
Sodium chloride 5.00 3. Incubate plates aerobically, protected from light, at 35-370C for 18-24
Sodium thiosulfate 5.00 hours.
Ferric ammonium citrate 1.50 4. If negative, incubate for an additional 24 hours.
Bromthymol blue 0.065
5. Examine colony morphology.
Acid fuchsin 0.10
Agar 15.00 6. Note: A non-selective medium should also be streaked to increase the
0
Final pH (at 25 C) 7.5 ±0.2 chances of recovery when the population of gram-negative organisms is low
* Formula adjusted to suit performance parameters and to provide an indication of other organisms present in the specimen.
Directions Interpretation of Results
1. Suspend 76.67 gms of the powder in 1000 ml distilled water Typical colonial morphology on H E Agar
2. Mix thoroughly. E. coli ...................................... Large, yellow to salmon color; some strains
may be inhibited
3. Boil with frequent agitation to dissolve the powder completely. Salmonella............................... Blue-green to blue; most strains with black
4. AVOID OVERHEATING. DO NOT AUTOCLAVE. center
Enterobacter/Klebsiella ........... Large, yellow to salmon color
5. Cool the medium to approximately 45-500C, pour in to sterile petriplates.
Shigella .................................... Green and moist, raised
Quality Control
Proteus .................................... Variable, blue-green to blue or salmon, most
Dehydrated Appearance strains with black center
Light greenish coloured, may have a slight green cast, homogeneous, free Pseudomonas ......................... Irregular, green to brown
flowing powder.
Gram-positive bacteria ........... No growth to slight growth
Prepared Appearance
Limitations
Green coloured, clear to slightly opalescent gel.
Cultural Response
1. Colonies of proteus may resemble Salmonella or Shigella.
Cultural characteristics after 18-24 hours at 35-370C. 2. It is preferable that biochemical and / or serological tests be performed on
Organisms(ATCC) Growth Colour of colony RGI colonies from pure culture for complete identification.
S. serotype Enteritidis Luxuriant Greenish blue More than 70% Storage
(13076) with black center
Store at 22-300C and prepared medium at 2-80C.
S. serotype Luxuriant Greenish blue to More than 70%
Shelf Life
Typhimurium (14028) green with black
Use before expiry date as mentioned on the label.
center

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Hottinger Broth AM104822
Use 3. Dispense in tubes or adequate containers and sterilize by autoclaving at
Hottinger Broth is used for cultivation of less fastidious microorganisms and 15lbs pressure (121ºC) for 15 minutes.
determination of indole production. Quality Control
Summary Dehydrated Appearance
Hottinger Broth is recommended for the determination of indole production. Yellow coloured, homogeneous, free flowing powder.
Prepared Appearance
Indole is a component of amino acid tryptophan. Some bacteria have the ability
Light amber coloured clear solution without any precipitate.
to breakdown tryptophan for nutritional needs using the enzyme tryptophanase
Cultural Response
and indole is produced as end product. Cultural characteristics observed after 18-48 hours at 35-370C.
Principle Organisms (ATCC) Growth Indole
Fish peptone and yeast extract provides the essential nutrients. Tryptophan Escherichia coli (25922) Good +
present in the medium is utilized by some of the bacteria and produced indole. Pseudomonas aeruginosa (27853) Good -
Formula* Staphylococcus aureus (25923) Good -
Ingredients in grams per liter Streptococcus pyogenes (19615) Good -
Fish peptone 20.00 For growth RGI should be more than 70%
Tryptophan 1.00 RGI- Relative Growth Index
Yeast extract 2.00 Procedure
Final pH (at 250C) 7.4 ± 0.2 Refer to appropriate references for specific procedures.
* Formula adjusted to suit performance parameters Interpretation of Results
Directions
Refer to appropriate references and procedures for results.
1. Suspend 23 gms in 1000 ml distilled water. Soak for 5 minutes. Storage
2. Warm slightly with frequent agitation to dissolve the powder completely. DO Store at 22-300C and prepared medium at 2-80C.
NOT OVERHEAT. Shelf Life
Use before expiry date as mentioned on the label.

Hoyle Medium Base AM104823/AM504823

Use Formula*
Hoyle Medium Base is used for the selective isolation and differentiation of Ingredients in grams per liter
Peptic digest of animal tissue 10.0
Corynebacterium diphtheriae.
Beef extract 10.0
Summary
Sodium chloride 5.0
Hoyle Medium is the modification (45.2) of Neill’s medium for the cultural Agar 15.0
isolation and differentiation of Corynebacterium diphtheriaetypes. Hoyle Final pH (at 250C) 7.8 ± 0.2
medium does not exert the inhibitory effect manifested by Neill’s on some mitis * Formula adjusted to suit performance parameters
types, but supports very rapid growth with all types of Corynebacterium Directions
diphtheriae, so that diagnosis is possible after 18 hours incubation. 1. Suspend the 40 gms of powder in 1000 ml distilled water.
Principle
2. Mix thoroughly.
Peptic digest of animal tissue and beef extract supply nutrients, nitrogen
3. Boil with frequent agitation to dissolve the powder completely. DO NOT
compounds and amino acids. Potassium tellurite is a selective agent which
OVERHEAT.
inhibits most of the normal flora of the upper respiratory tract except
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Corynebacterium.chloride provides sodium ions for the membrane transport and
maintains osmotic equilibrium of the medium. Agar is the solidifying agent. 5. Cool to 45-500C and aseptically add 50 ml of laked blood and 10 ml of

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3.5% Potassium Tellurite Solution (AS023). Corynebacterium diphtheriae Good to Grey colonies with More than 70%
type intermedius (14779) Luxuriant Darker centers.
6. Mix thoroughly, but gently and pour into sterile petri plates. Enterococcus faecalis (29212) Good to black minute More than 70%
Quality Control Luxuriant colonies
Dehydrated Appearance Escherichia coli (25922) Inhibited - 0%
Light yellow coloured, homogeneous, free flowing powder. Note: It should be noted that not all corynebacteriaproduce the typical colonies
Prepared Appearance described above - so in all cases it is advisable to use Hoyle medium in
Basal medium yields amber coloured gel. conjunction with the non-selective media such as Loeffler Medium Base
With addition of laked blood and tellurite, brownish red coloured, opalescent
(AM1056/5056) and Blood Agar Base (AM 1014/5014).
gel.
Storage
Cultural Response
0
Cultural characteristics after 18-24 hours at 35-37 C. Store at 22-300C and prepared medium at 2-80C.
Organisms (ATCC) Growth Colony RGI Shelf Life
Characteristics Use before expiry date as mentioned on the label.
Corynebacterium diphtheriae Good to Grey colonies with More than 70%
type intermedius (11913) Luxuriant Darker centers.

Inactivator Broth (Twin Pack) AM104824/AM504824

Use Soya lecithin 3.0
This media is recommended for the detection and isolation of microbial Histidine 1.0
Cysteine 1.0
contamination present on clean surfaces in environmentally controlled areas and
Part B
accidentally contaminated raw material samples of pharmaceutical
Tween 80 30 ml.
formulations. Final pH (at 250C) 7.3 ±0.2
Summary * Formula adjusted to suit performance parameters
This media may be employed to establish and monitor cleaning techniques in Directions
'clean' rooms. This media is also used for detection and isolation of 1. Suspend 35gms of Powder (Twin Pack A) in 970 ml distilled water.
microorganisms present in raw material samples of pharmaceutical formulations
2. Add 30 ml of Tween 80 (Twin Pack B)
which may have accidentally contaminated by various disinfectants.
3. Heat with frequent agitation to dissolve the powder completely
Principle
Casein and Soya Peptone are sources of carbon and nitrogen. Dextrose is a source 4. Dispense the media in tubes.
of fermentable Carbohydrate. Sodium Chloride maintains the osmotic 5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
equilibrium while mono and dipotassium phosphates are the buffers. Lecithin Quality Control
and Tween 80 inactivate many residual disinfectants. Lecithin neutralizes Dehydrated Appearance
Part A-Yellow coloured, homogeneous, free flowing powder.
quaternary Ammonium compounds and Tween 80 neutralizes Phenols,
Part B- Yellowish viscous liquid.
hexachlorophene & formalin. Cysteine acts as detoxicant, it neutralizes toxic
Prepared Appearance
chemicals. Yellow coloured slight opalescent solution.
Formula* Cultural Response
Part A Cultural characteristics observed after an incubation of 24-48 hrs at 35-370C
Ingredients Gms/lit for bacteria and 48-72 hrs at 25-300C for fungi.
Pancreatic digest of casein 17.0 Organisms(ATCC) Growth
Soya peptone 3.0 Staphylococcus aureus (25923) Good
Sodium chloride 5.0 Bacillus subtillis (6633) Good
Dipotassium hydrogen phosphate 1.25 Streptococcus pyogenes (19615) Good
Potassium dihydrogen phosphate 1.25 Bacteroides vulgatus (8482) Good
Dextrose 2.5 Candida albicans (10231) Good

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Pseudomonas aeruginosa (9027) Good Interpretation of Results
Aspergillus niger (16404) Good Growth in the broth is indicated by the presence of turbidity compared to an
Procedure uninoculated control.
1) If specimen is being cultured from a swab (rolled on clean surface), dip the Storage
swab directly in the medium. Store at 22-300C and prepared medium at 2-80C.
2) Incubate tubes at 370C + 20C for 48 hrs and 250C-300C for 72 hrs. Shelf Life
3) When incubation has been completed check for growth. Use before expiry date as mentioned on the label.

Iron Sulphite Agar AM10483/AM50483

Use Quality Control
Iron Sulphite Agar is recommended for the detection of thermophilic anaerobic Dehydrated Appearance
Yellow coloured, homogeneous, free flowing powder.
organisms causing sulphide spoilage in foods.
Prepared Appearance
Summary
Yellow coloured, slightly opalescent gel forms in petriplates / tubes.
Iron Sulphite Agar is a modification of Cameron Sulphite Agar developed by the Cultural Response
National Canners Association of America. The medium was modified and Cultural response after 24 - 78 hours at 55 - 560 C under anerobic conditions.
improved by reducing the concentration of sodium sulphite. But Beerens showed Organisms (ATCC) Growth Colour of RGI
that some 0.1% sulphite concentration was inhibitory to some strains of colonies
Clostridium sporogenes. This observation was later confirmed by Mossel et. al. Clostridium sporogenes Luxuriant Black More than 70%
who showed that a concentration of 0.05% sulphite was not inhibitory to the (19404)
Clostridium botulinum Luxuriant Black More than 70%
organisms.
(25763)
Principle
Desulfotomaculum Luxuriant Black More than 70%
Mossel et al. (1956) and Mossel (1959), reported an iron-sulfite agar medium nigrificans (19858)
containing 0.05 % sodium sulfite which yielded quantitative recovery of pure Escherichia coli (25922) Good No More than 70%
cultures of several species of clostridia in Miller-Prickett tubes. Iron Sulphite Agar blackening
is particularly suitable for the detection of thermophilic anaerobic organisms Procedure

causing sulphide spoilage in foods. For detection of such organisms, two methods Deep-Shake Culture Method
can be used: (a) Deep-Shake Culture Method and (b) Attenborough and Scarr 1. Dispense the medium in 10 ml amounts in tubes.
Method. 2. Inoculate the sample on the medium when it is about 500C.
Formula* 3. Allow to set and incubate at 550C for 24-48 hours.
Ingredients in grams per liter Attenborough and Scarr Method
Tryptone 10.0
1. Filter diluted samples of sugar or any other food particles though membrane
Sodium Sulphite 0.5
filters.
Iron (III) Citrate 0.5
Agar 15.0 2. Roll up the filters and place in tubes containing melted Iron Sulphite Agar
Final pH (at 250 C) 7.1 ± 0.2 sufficient enough to cover them at 500C.
* Formula adjusted to suit performance parameters 3. Allow the medium to set and incubate at 55-560C for 24-48 hours.
Directions Interpretation of Results
1. Suspend 26 grams in 1000 ml distilled water. Deep-Shake Culture Method
2. Heat with frequent agitation and boil for 1 minute to dissolve the powder 1. Desulfotomaculum nigrificans, a typical thermophilic species, produces
completely. distinct black spherical colonies in the depth of the medium.
3. Sterilize by autoclaving at 1210 C (15 lbs pressure) for 15 minutes. Attenborough and Scarr Method
1. After incubation, count the number of black colonies formed.

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Precautions / Limitations Storage
1. A number of thermophilic anaerobic organisms like Proteus and Salmonella Store at 22-300C and prepared medium at 2-80C.
grow on this medium and must be confirmed by additional biochemical tests. Shelf Life
2. The membrane filter technique is quicker, of comparable accuracy and Use before expiry date as mentioned on the label.
permits the examination of larger samples.

Iron Sulphite Agar ISO AM504831

Use Quality Control
Iron Sulphite Agar is recommended for the detection of thermophilic anaerobic Dehydrated Appearance
Yellow coloured, homogeneous, free flowing powder.
organisms causing sulphide spoilage in foods in compliance with ISO.
Prepared Appearance
Summary
Yellow coloured, slightly opalescent gel forms in petriplates.
Iron Sulphite Agar is a modification of Cameron Sulphite Agar developed by the Cultural Response
National Canners Association of America. The medium was modified and Cultural response after 24 - 78 hours at 55 - 560C under anerobic conditions.
improved by reducing the concentration of sodium sulphite. But Beerens showed Organisms (ATCC) Growth Colour of RGI
that some 0.1% sulphite concentration was inhibitory to some strains of colonies
Clostridium sporogenes. This observation was later confirmed by Mossel et al., Clostridium sporogenes Luxuriant Black More than 70%
who showed that a concentration of 0.05% sulphite was not inhibitory to the (19404)
Clostridium botulinum Luxuriant Black More than 70%
organisms.
(25763)
Principle
Desulfotomaculum Luxuriant Bblack More than 70%
Mossel et al., (1956) and Mossel (1959), reported an iron-sulfite agar medium nigrificans (19858)
containing 0.05 % sodium sulfite which yielded quantitative recovery of pure Escherichia coli Good No blackening More than 70%
cultures of several species of clostridia in Miller- Prickett tubes. Iron Sulphite Agar (25922)
is particularly suitable for the detection of thermophilic anaerobic organisms For growth RGI should be more than 70%
causing sulphide spoilage in foods. For detection of such organisms, two methods RGI- Relative Growth Index
can be used: (a) Deep-Shake Culture Method and (b) Attenborough and Scarr Procedure

Method. Deep-Shake Culture Method

Formula* 1. Dispense the medium in 10 ml amounts in tubes.
Ingredients in grams per liter
2. Inoculate the sample on the medium when it is about 500C.
Enzymatic digest of casein 15.0
Pancreatic digest of soya 5.0 3. Allow to set and incubate at 550C for 24-48 hours.
Yeast extract 5.0 Attenborough and Scarr Method
Disodium disulfite 1.0 1. Filter diluted samples of sugar or any other food particles though membrane
Iron (lll) ammonium citrate 1.0 filters.
Agar 12.0
2. Roll up the filters and place in tubes containing melted Iron Sulphite Agar
Final pH (at 250C) 7.2 ±0.2
* Formula adjusted to suit performance parameters
sufficient enough to cover them at 500C.
Directions 3. Allow the medium to set and incubate at 55-560C for 24-48 hours.
1. Suspend 39 grams in 1000 ml distilled water. Interpretation of Results
2. Heat with frequent agitation and boil for 1 minute to dissolve the powder Deep-Shake Culture Method
completely. 1. Desulfotomaculum nigrificans, a typical thermophilic species, produces
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. distinct black spherical colonies in the depth of the medium.

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Attenborough and Scarr Method 2. The membrane filter technique is quicker, of comparable accuracy and
1. After incubation, count the number of black colonies formed. permits the examination of larger samples.
Precautions / Limitations Storage
1. A number of thermophilic anaerobic organisms like Proteus and Salmonella Store at 22-300C and prepared medium at 2-80C.
grow on this medium and must be confirmed by additional biochemical tests. Shelf Life
Use before expiry date as mentioned on the label.

IUT Medium Base AM10484/AM50484

Use Directions
IUT Medium Base with added glycerol and egg yolk emulsion is used for 1. Suspend 7.3gms of Powder in 600 ml distilled water containing 12 ml
cultivation of Mycobacterium tuberculosis. glycerol.
Summary 2. If necessary, heat to dissolve the medium completely
IUT Medium is recommended by the International Union Against Tuberculosis for 3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
the diagnosis of Mycobacterial infections (46.3). It differs from Lowenstein-
4. Cool to 500C and aseptically add 1 liter of sterile whole egg emulsion.
Jensen medium since it does not contain potato flour/starch. It is reported to be
Prepared under aseptic conditions.
giving higher proportion of positives. This medium supports rapid and luxuriant
growth of primary cultures. 5. Mix well and dispense in screw capped containers. Sterilize by inspissation
Principle at 850C for 1 hour.
Quality Control
Malachite green inhibits the growth of other bacteria and also acts as a pH
Dehydrated Appearance
indicator. L-Asparagine serves as a source of nutrients. Inorganic salts provide Peacock blue coloured, homogeneous, free flowing powder.
ions for the metabolism of Mycobacteria. Prepared Appearance
Formula* Pale blue coloured opaque smooth slants.
Ingredients Gms/600 ml Cultural Response
L-Asparagine 3.60 Cultural characteristics observed after 2-4 weeks at 350C.
Monpotassium phosphate 2.46 Organisms (ATCC) Growth
Mgnesium sulphate 0.24 Mycobacterium tuberculosis H37RV (25618) Luxuriant
Magnesium citrate 0.60 Mycobacterium smegmatis (14468) Luxuriant
Malachite green 0.40 Storage
Final pH (at 250C) 7.0 ±0.2
Store at 22-300C and prepared medium at 2-80C.
* Formula adjusted to suit performance parameters
Shelf Life
Use before expiry date as mentioned on the label.

Jensen’s Broth AM50485

Use Principle
Jensen’s Broth is recommended for detection and cultivation of nitrogen fixing Sucrose serves as energy source. Different salts support the bacterial growth.
bacteria. Sodium chloride maintains the osmotic of the medium. Agar is the solidifying
Summary agent.
Nitrogen fixing bacteria are capable of taking gaseous nitrogen and combining it Formula*
with hydrogen to make ammonia. The plant for growth can use fixed nitrogen. Ingredients in grams per liter
Sucrose 20.0
Thus nitrogen-fixing bacteria increases the soil productivity. To isolate the
Calcium carbonate 2.0
nitrogen fixing bacteria nitrogen free Jensen’s medium has been formulated and
Dipotassium phosphate 1.0
used (115.2). Sodium chloride 0.50

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Ferrous sulphate 0.10 Cultural Response
Sodium molybdate 0.005 Cultural characteristics after 7 days at 30°C.
Magnesium Sulphite 0.50 Organisms(ATCC) Growth
Final pH (at 250C) 7.5± 0.2 Rhizobium leguminosarum (10004) Luxuriant
* Formula adjusted to suit performance parameters Rhizobium meliloti (9930) Luxuriant
Directions For growth RGI should be more than 70%
1. Suspend 24.1 gms of the powder in 1000ml distilled water. RGI- Relative Growth Index
Procedure
2. Mix thoroughly.
Refer to appropriate references for specific procedures for the cultivation of
3. Boil with frequent agitation to dissolve the powder completely. Do not
phosphate solubilizing soil microorganisms.
overheat.
Interpretation of Results
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes. Refer to appropriate references and procedures for results.
Quality Control
Storage
Dehydrated Appearance
Cream to yellow coloured, homogeneous, free flowing powder.
Store at 22-300C and prepared medium at 2-80C.
Prepared Appearance Shelf Life
Cream coloured, clear to slightly opalescent with a slightly precipitated gel Use before expiry date as mentioned on the label.
forms in petri plates.

Jensen’s Medium AM10486/AM50486

Use Directions
Jensen’s Medium is recommended for detection and cultivation of nitrogen fixing 1. Suspend 39 gms of the powder in 1000ml distilled water.
bacteria. 2. Mix thoroughly.
Summary 3. Boil with frequent agitation to dissolve the powder completely. Do not
Nitrogen fixing bacteria are capable of taking gaseous nitrogen and combining it overheat.
with hydrogen to make ammonia. The plant for growth can use fixed nitrogen. 4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Thus nitrogen-fixing bacteria increases the soil productivity. To isolate the Quality Control
nitrogen fixing bacteria nitrogen free Jensen’s medium has been formulated and Dehydrated Appearance
used (115.2). Cream to yellow coloured, homogeneous, free flowing powder.
Prepared Appearance
Principle
Cream coloured, clear to slightly opalescent with a slightly precipitated gel
Sucrose serves as energy source. Different salts support the bacterial growth. forms in petri plates.
Sodium chloride maintains the osmotic of the medium. Agar is the solidifying Cultural Response
agent. Cultural characteristics after 7 days at 30°C.
Formula* Organisms (ATCC) Growth
Ingredients in grams per liter Rhizobium leguminosarum (10004) Luxuriant
Sucrose 20.0 Rhizobium meliloti (9930) Luxuriant
Calcium carbonate 2.0 Procedure
Dipotassium phosphate 1.0 Refer to appropriate references for specific procedures for the cultivation of
Sodium chloride 0.50 phosphate solubilizing soil microorganisms.
Ferrous sulphate 0.10 Interpretation of Results
Sodium molybdate 0.005 Refer to appropriate references and procedures for results.
Agar 15.0 Storage
Final pH (at 250C) 6.8 ± 0.2
Store at 22-300C and prepared medium at 2-80C.
* Formula adjusted to suit performance parameters
Shelf Life
Use before expiry date as mentioned on the label.

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Karmali Campylobacter Agar Base AM1049/AM5049
Use 3. Boil with frequent agitation to dissolve the powder completely.
Karmali Campylobacter Agar Base is a blood free medium for selective isolation of 4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
thermotolerant Campylobacter species from clinical and non-clinical specimens. 5. Cool to 40-500C and aseptically add 1 vial of Campylobacter Selective
Summary
Supplement with Hemin, Karmali Modified (AS007).
Karmali et al (50) formulated Karmali Campylobacter Agar Base, a highly
6. Mix well before pouring into sterile petri plates.
nutritious basal medium, which after the addition of antimicrobial agents, is used Quality Control
for the selective isolation of Campylobacter species from fecal specimens, food Dehydrated Appearance
and environmental specimens. The addition of antimicrobial agents suppresses Greyish black coloured free flowing, homogeneous powder.
the growth of normal enteric flora. Prepared Appearance
Principle Black coloured opalescent gel.
Campylobacter species, which are microaerophilic, are inhibited by the normal Cultural Response
Cultural characteristics after 42-48 hours at 420C.
atmospheric oxygen levels as microaerophilic bacteria are more sensitive to toxic
Organisms (ATCC) Growth RGI
forms of oxygen (super oxide, peroxide) that occur in aerobic culture media.
Campylobacter jejuni (29428) Good to luxuriant More than 70%
Special peptone and corn starch provide essential vitamins. The selective Escherichia coli (25922) None to poor 0%
supplement contains hemin, which provides essential nutrients, sodium pyruvate, For growth RGI should be more than 70%
which enhances the aero tolerance of microaerophilic bacteria by quenching the For Inhibition RGI should be 0%
toxic forms of oxygen, vancomycin, which suppresses gram-positive organisms, RGI- Relative Growth Index
amphotericin B, which inhibits yeasts, and cefoperazone, which inhibits gram- Procedure
negative organisms other than Campylobacter. 1. Use standard procedures to obtain isolated colonies from specimens.
Formula* 2. If immediate inoculation of Karmali Campylobacter Agar Base cannot be
Ingredients in grams per liter
performed, the use of a suitable holding medium is recommended.
Peptone Special 23.0
Sodium Chloride 5.0 3. Incubate inoculated plates at 420C for 42-48 hours.
Charcoal Activated 4.0 Precautions / Limitations
Corn Starch 1.0 1. Since C.jejuni is thermophilic, it is important to incubate the plates at 420C;
Agar 12.0 otherwise, growth will be delayed. The higher temperature also improves
pH (at 250C) 7.4 ± 0.2 selectivity by inhibiting the normal flora.
* Formula adjusted to suit performance parameters
Storage
Directions
Store at 22-300C and prepared medium at 2-80C.
1. Suspend 22.5 gms of the powder in 490 ml distilled water.
Shelf Life
2. Mix thoroughly. Use before expiry date as mentioned on the label.

King’s Medium A Base AM50491

Use Principle
King’s Medium A Base is used for isolation, cultivation and pigment production of Proteose peptone supplies the essential carbon and nitrogen source to the medium.
Pseudomonas species. Metallic salts are the source of ions, which support the growth of the bacteria. Agar
Summary is the solidifying agent.
King’s Medium A Base is recommended for Pseudomonas species. King first Formula*
formulated this medium (33.1). Ingredients in grams per liter
Proteose peptone 20.0

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Potassium sulphate 10.0 Cultural Response
Magnesium chloride, 1.64 Cultural characteristics after 18-24 hours at 35-370C.
Anhydrous Organisms (ATCC) Growth RGI
Agar 15.0 Pseudomonas aeruginosa (17934) Luxuriant More than 70%
Final pH (at 250C) 7.3 ± 0.2 Pseudomonas aeruginosa (27853) Luxuriant More than 70%
* Formula adjusted to suit performance parameters For growth RGI should be more than 70%
Directions RGI- Relative Growth Index
1. Suspend 46.64 gms of powder in 1000ml distilled water. Procedure

2. Add 10 ml of glycerrol and mix thoroughly. Refer to appropriate references for specific procedures
Interpretation of Results
3. Boil with frequent agitation to dissolve the powder completely.
Refer to appropriate references and procedures for results.
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Storage
Quality Control
Dehydrated Appearance
Store at 22-300C and prepared medium at 2-80C.
Light yellow coloured, homogeneous, free flowing powder. Shelf Life
Prepared Appearance Use before expiry date as mentioned on the label.
Light yellow coloured clear gel forms in petri plates.

King’s Medium B Base AM50492

Use 2. Add 15 ml of glycerol and mix thoroughly.
King’s Medium B Base is used for isolation, cultivation and pigment production of 3. Boil with frequent agitation to dissolve the powder completely.
Pseudomonas species.
4. Sterilize by autoclaving at 1210C (15lbs pressure) for 15 minutes.
Summary
Quality Control
King’s Medium B Base is recommended for Pseudomonas species. King first Dehydrated Appearance
formulated this medium (33.1). Light yellow coloured, homogeneous free flowing powder.
Principle Prepared Appearance
Proteose peptone provides the essential carbon and nitrogen. Dipotassium Light yellow coloured, clear to slightly opalescent gel forms in petri plates.
hydrogen phosphate acts as a buffering agent. Metallic ion supports the growth of Cultural Response
Cultural characteristics after 18-24 hours at 35-370C.
bacteria.
Organisms (ATCC) Growth
Formula*
Pseudomonas aeruginosa (27853) Luxuriant
Ingredients in grams per liter
Procedure
Proteose peptone No. 3 20.0
Magnesium Sulphate 7H2O 1.5 Refer to appropriate references for specific procedures
Dipotassium hydrogen phosphate 1.5 Interpretation of Results
Agar 20.0 Refer to appropriate references and procedures for results.
Final pH (at 250C) 7.2 ± 0.2 Storage
* Formula adjusted to suit performance parameters
Store at 22-300C and prepared medium at 2-80C.
Directions
Shelf Life
1. Suspend 43 gms of powder in 1000ml of distilled water. Use before expiry date as mentioned on the label.

Kirschner Medium Base, Modified AM50493

Use Summary

Kirschner Medium Base with added glycerol and enrichment is used for cultivation Kirschner Medium was first developed by Kirchner based on the formulation of
of Mycobacterium tuberculosis. Long's Medium (4.1) and further modified with addition of glycerol and

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enrichments for the cultivation of Mycobacterium tuberculosis. It is agents and 3. Dispense in 9 ml aliquotes.
some time in differential culture of Mycobacterium tuberculosis from unhealthy 4. Sterilize by autoclaving at 1150C (10lbs pressure) for 15 minutes.
materials. 5. Just before use, aseptically add 1 ml of Horse serum and 100 IU Penicillin per
Principle
9 ml medium.
Krichner medium contains two phosphates, a sulphate and citrate which buffers Quality Control
the medium well. Hence direct inoculum can be adopted without any Dehydrated Appearance
neutralization. L-asparagine in the medium supports the growth of White coloured, homogeneous free flowing powder.
Mycobacterium tuberculosis as it is a good nutrient for the organism. Horse serum Prepared Appearance
also promotes the growth of the organism. Penicillin inhibits the growth of Colourless solution having slight white precipitate at the bottom..
Cultural Response
contaminating bacteria.
Cultural characteristics after 2-4 weeks at 35-370C.
Formula *
Organisms (ATCC) Growth
Ingredients in grams per liter
Mycobacterium tuberculosis H37 RV (25618) Luxuriant
Disodium phosphate 3.00
Mycobacterium smegmatis (14468) Luxuriant
Monopotassium phosphate 4.00
Mycobacterium fortuitum (6841) Luxuriant
Magnesium sulphate 0.60
Procedure
Sodium citrate 2.50
L-Asparagine 5.00 Refer to appropriate references for specific procedures
Final pH (at 250C) 7.4 ± 0.2 Interpretation of Results
* Formula adjusted to suit performance parameters Refer to appropriate references and procedures for results.
Directions Storage
1. Suspend 15.1 gms of powder in 700 ml of distilled water. Add 200 ml Store at 22-300C and prepared medium at 2-80C.
glycerol. Shelf Life
2. Heat to boiling to dissolve the powder completely. Use before expiry date as mentioned on the label.

Kligler Iron Agar AM1050/AM5050

Use of B group vitamins while sodium chloride maintains the osmotic balance.
Kligler Iron Agar is a differential medium used for the differentiation of members Lactose and dextrose enable the differentiation of enteric bacilli due to change in
of Enterobacteriaceae on the basis of their ability to ferment dextrose and lactose colour of the phenol red pH indicator in response to the acid produced during the
and to produce hydrogen sulphide. fermentation of these sugars. Ferrous sulphate and sodium thiosulphate enable
Summary the detection of hydrogen sulphide production. Fermentation of dextrose is
Russel (97) described a double sugar tubed medium for the isolation of typhoid indicated by yellow butt and that of lactose by yellow slant.
bacilli from urine and faeces. Some years later, Kligler (57) developed a simple Non-lactose fermenters (Salmonella and Shigella ) initially produce acid (yellow
lead acetate medium for the differentiation of typhoid-paratyphoid group. slant) as a result of dextrose fermentation. The concentration of dextrose being
Subsequently, he evaluated culture media used in the isolation of typhoid, very small is rapidly exhausted. Once the dextrose is depleted in the aerobic
dysentery and allied bacilli. Bailey and Lacey substituted phenol red for the environment of the slant, the reaction reverts to alkaline (red slant) due to
Andrade indicator previously used in the formulation, enabling the differentiation oxidation of the acids. The reversion does not occur in the anaerobic environment
of gram-negative bacilli based on their ability to ferment dextrose, lactose and in the butt where an acidic environment is maintained. Lactose fermenting
hydrogen sulphide production. Kligler Iron Agar is recommended by APHA for the organisms produce yellow slants and butts. Organisms incapable of fermenting
examination of foods (20). either of the carbohydrates produce red slants and butts. H2S production results in
Principle the blackening of the medium, either throughout the butt or in a ring formation
Tryptone and peptone provide essential growth nutrients. Yeast extract is a source near the top of the butt. Gas production is demonstrated by the presence of
bubbles or cracks in the medium.

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Formula* 5. Incubate tubes with loosened caps for 18-24 hours at 35-370C in an aerobic
Ingredients in grams per liter atmosphere.
Peptone 15.0
Interpretation of Results
Yeast Extract 3.0
Beef Extract 3.0 1. An alkaline slant-acid butt (red / yellow) indicates fermentation of dextrose
Proteose Peptone 5.0 only.
Sodium Chloride 5.0 2. An acid slant-acid butt (yellow / yellow) indicates fermentation of both
Lactose 10.0
dextrose and lactose.
Dextrose 1.0
Sodium Thiosulphate 0.3 3. An alkaline slant-alkaline butt (red / red) indicates that neither dextrose nor
Ferrous Sulphate 0.2 lactose was fermented.
Phenol Red 0.024 4. Cracks, splits or bubbles in the medium indicate gas production.
Agar 15.0
Final pH (at 250C) 7.4 ± 0.2 5. A black precipitate in the butt indicates H2S production.
* Formula adjusted to suit performance parameters Typical reactions produced by members of the Enterobacteriaceae are
Directions Slant Butt Gas H2S
1. Suspend 57.5 gms of the powder in 1000 ml distilled water and mix well. Citrobacter Alkaline Acid + ±
2. Boil with frequent agitation to dissolve the powder completely. Edwardsiella Alkaline Acid + +
3. Mix well and distribute into tubes. E. coli Acid Acid + -
Enterobacter Acid (may revert to alkaline Acid + -
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
even though lactose fermented-
5. Allow to set as slopes with 1 inch butts. E. aerogenes)
6. Best reactions are obtained on freshly prepared medium. Morganella Alkaline Acid ± -
Quality Control Proteus Alkaline or acid Acid + +
Dehydrated Appearance
Providencia Alkaline Acid ± -
Light pink coloured, homogeneous, free flowing powder.
Salmonella Alkaline Acid + +
Prepared Appearance
Shigella Alkaline Acid - -
Red coloured, clear to slightly opalescent gel.
Precautions / Limitations
Cultural Response
Cultural characteristics after 18-48 hours at 370C. 1. It is essential that Kligler Iron Agar be examined and reported after 18-24
Organisms (ATCC) Growth Slant Butt Gas H2S hours. Early or late readings may give false results.
Escherichia coli (25922) Luxuriant Acid Acid + - 2. Kligler Iron Agar will grow both oxidative and fermentative organisms and
Enterobacter aerogenes (13048) Luxuriant Acid Acid + - care should be taken to distinguish between the two groups.
Klebsiella pneumoniae (13883) Luxuriant Acid Acid + -
Pseudomanas aeruginosa (9027) Luxuriant Alkaline Alkaline - -
3. It is essential that air is freely available for growth on the slant and
Salmonella serotype Enteritidis Luxuriant Alkaline Acid + + therefore do not use screw cap bottles or tubes for testing.
(13076) 4. Several colonies from each primary plate should be studied separately, since
Salmonella serotype Typhimurium Luxuriant Alkaline Acid - + mixed infections may occur.
(6539)
5. It is important to stab the butt of the medium but the integrity of the medium
Shigella flexneri (12022) Luxuriant Alkaline Acid - -
Procedure
must be maintained while stabbing. Use a straight wire for inoculation to
avoid splitting the agar.
1. Obtain a pure culture of the organism to be tested.
6. An organism that produces H2S may mask acid production in the butt of the
2. Touch the center of an isolated colony with an inoculating needle.
medium. However, H2S production requires an acid environment, thus the
3. Stab the center of the medium into the deep of the butt to within 3-5 mm of
butt portion should be considered acidic if H2S is produced.
the bottom.
7. Certain species of organisms may give delayed reactions or completely fail to
4. Withdraw the inoculating needle, and streak the surface of the slant.

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ferment the carbohydrate. However, in most cases, if the organism fails to Storage
ferment dextrose within 48 hours and growth is definitely present, the Store at 22-300C and prepared medium at 2-80C.
organism, most likely does not belong to the family Enterobacteriaceae. Shelf Life
Use before expiry date as mentioned on the label.

Kligler Iron Agar ISO AM50501

Use Directions
Kligler Iron Agar ISO is a differential medium recommended for identification of 1. Suspend the 57.7 gms of powder in 1000 ml distilled water.
Pseudomonas species and members of Enterobacteriaceae on the basis of their 2. Mix thoroughly.
ability to ferment dextrose and lactose and to produce hydrogen sulphide in 3. Boil to dissolve the powder completely.
compliance with ISO specification ISO/DIS/13720:1995.
4. Dispense in tubes and sterilize by autoclaving at 121°C (15 lbs pressure) for
Summary
15 minutes.
Kligler Iron agar ISO is the modification of Kligler medium. ISO committee has
recommenced for identification of Pseudomonas species. 5. Cool in slanted position for use as slants.
Principle 6. Best reactions are obtained on freshly prepared media.
Casein and meat peptones provide essential growth nutrients. Yeast extract is a Quality Control
Dehydrated Appearance
good source of B vitamins. Lactose and dextrose are the fermentable sugars which
Light pink coloured, homogeneous, free flowing powder.
enable the differentiation of species due to the colour changes of phenol red – the
Prepared Appearance
pH indicator. Sodium thiosulphate accelerate H2S production which is evidenced Red coloured, clear to slightly opalescent gel form as slants in tubes.
by a black colour either throughout the butt or in a ring formation near the top of Cultural Response
butt. Gas production is detected as individual bubbles or splitting or displacement Cultural characteristics after 24-48 hours at 370C.
of agar. Fermentation of dextrose is indicated by yellow butt and that of lactose by Organisms (ATCC) Growth Slant Butt Gas H2S
yellow slant and H2S production is indicated by blacking in the butt. Escherichia coli (25922) Luxuriant A A + –
Formula* Enterobacter aerogenes (13047) Luxuriant A A + –
Ingredients in grams per liter Protus vulgaris (13315) Luxuriant K A – +
Beefextract 3.00 S. serotype Typhi (6539) Luxuriant K A – +
Yest extract 3.00 S. serotype Enteridis (13076) Luxuriant K A + +
Casein enzymic hydrolysate 20.00 Klebsiella pneumoniae (13883) Luxuriant A A + –
Sodium chloride 5.00 Shigella flexneri (12022) Luxuriant K A – –
Lactose 10.00 Pseudomonas aeruginosa (27853) Luxuriant K K – –
Glucose anhydrous 1.00 Key: A = acid production (yellow)
Ferrous ammonium sulphate, 6H2O 0.50 K= alkaline reaction
+= positive or blackening
Sodium thiosulphate, pentahydrate 0.50
Storage
Phenol red 0.025
Agar 15.00 Store at 22-300C and prepared medium at 2-80C.
0
Final pH (at 25 C) 7.4 ±0.2 Shelf Life
* Formula adjusted to suit performance parameters Use before expiry date as mentioned on the label.

Lactobacillus MRS Agar AM1051/AM5051

Use Summary
Lactobacillus MRS Agar is used for the isolation, enumeration and cultivation of Lactobacillus MRS Agar is based on the formulation of deMann, Rogosa and
all Lactobacillus species. Sharpe (19) with slight modification. It supports luxuriant growth of lactobacilli
from oral cavity, faeces, foods (20) and dairy products (39).

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Principle Prepared Appearance
Proteose peptone and beef extract provide carbon and nitrogen compounds. Yeast Medium amber coloured, clear to slightly opalescent gel.
Cultural Response
extract provides vitamin B complex while dextrose is the fermentable
Cultural characteristics at 18-24 hours or longer at 350C.
carbohydrate. Polysorbate 80, sodium acetate, magnesium sulphate and
Organisms (ATCC) Growth RGI
manganese sulphate provide growth factors. Sodium acetate and ammonium
Lactobacillus leichmanni (7830) Luxuriant More than 70%
citrate inhibits streptococci, moulds and many other microorganisms. Lactobacillus plantarum (8014) Luxuriant More than 70%
Dipotassium phosphate is the buffer. Lactobacillus fermentum (9338) Luxuriant More than 70%
Formula* Procedure
Ingredients in grams per liter
1. For quantitative test follow the pour plate method using 1 ml volumes of
Dextrose 20.0
diluted test sample.
Beef Extract 10.0
Proteose Peptone 10.0 2. Alternatively, the streak plate method can be used for recovery of organisms.
Yeast Extract 5.0 3. Incubate the plates at 350C for 3 days, or at 300C for 5 days in an aerobic
Sodium Acetate 5.0 atmosphere supplemented with carbon dioxide.
Ammonium Citrate 2.0
Interpretation of results
Dipotassium Phosphate 2.0
Polysorbate 80 1.0
1. Lactobacilli appear as large, white colonies in or on the surface of the
Magnesium Sulphate 0.1 medium.
Manganese Sulphate 0.05 2. Count the number of colonies and express as colony forming units (CFU) per
Agar 12.0 gram or ml of sample, taking into account the applicable dilution factor.
Final pH (at 250C) 6.5 ± 0.2 Precautions / Limitations
* Formula adjusted to suit performance parameters
1. Organisms other than lactobacilli may grow in these media and therefore
Directions
isolates must be confirmed as lactobacilli by appropriate biochemical
1. Suspend 67.15 gms of the powder in 1000 ml distilled water.
testing.
2. Mix thoroughly. Storage
3. Boil with frequent agitation to dissolve the powder completely. Store at 22-300C and prepared medium at 2-80C.
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. Shelf Life
Quality Control Use before expiry date as mentioned on the label.
Dehydrated Appearance
Yellow coloured, homogeneous, free flowing powder.

Lactobacillus MRS Agar ISO AM50511

Use citrate inhibit streptococci, moulds and many other microorganisms.
Lactobacillus MRS Agar is used for isolation and enumeration of lactic acid Formula*
bacteria from meat and meat products in compliance with ISO. Dextrose 20.0
Summary Beef extract 10.0
Proteose peptone 10.0
Lactobacillus MRS media are based on the formulation of deMan, Rogosa and
Yeast extract 5.0
sharpe with slight modification. It support luxuriant growth of all Lactobacilli
Sodium acetate 5.0
from oral cavity, dairy products, foods faeces and other sources. Ammonium citrate 2.0
Principles Dipotassium phosphate 2.0
Proteose peptone and beef extract supply nitrogenous and carbonaceous Polysorbate 80 1.0
compounds. Yeast extract provides vitamin B complex and dextrose is the Magnesium sulphate 0.1
fermentable carbohydrate and energy source. Polysorbate 80 supplies fatty acids Manganese sulphate 0.05
Agar 12.0
required for the metabolism of Lactobacilli. Sodium acetate and ammonium

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Final pH (at 250C) 6.5 ± 0.2 Organisms (ATCC) Growth RGI
* Formula adjusted to suit performance parameters Lactobacillus fermentum (9338) Luxuriant More than 70%
Directions Lactobacillus leichmanii (7830) Luxuriant More than 70%
1. Suspend 67.15 gms of powder in 1000 ml of distilled water. Lactobacillus plantarum (8014) Luxuriant More than 70%
For growth RGI should be more than 70%
2. Mix thoroughly. RGI- Relative Growth Index
3. Boil with frequent agitation to dissolve the powder completely. Procedure
4. Sterilize by autoclaving at 1210C (15lbs pressure) for 15 minutes. Refer to appropriate references for specific procedures
Quality Control Interpretation of Results
Dehydrated Appearance Refer to appropriate references and procedures for results.
Yellow coloured, homogeneous, free flowing powder. Storage
Prepared Appearance Store at 2- 80C and prepared medium at 2-80C.
Medium to amber coloured, clear to slightly opalescent gel.
Shelf Life
Cultural Response
Use before expiry date as mentioned on the label.
Cultural characteristics after 18-24 hours or longer at 350C.

Lactobacillus MRS Broth AM10512/AM50512

Use *Formula adjusted to suit performance parameters
Lactobacillus MRS Broth is used for isolation and enumeration and cultivation of Directions

all Lactobacillus species. 1. Suspend 55.15 gms of the powder in 1000 ml distilled water.
Summary 2. Mix thoroughly.
Lactobacillus MRS Broth is based on the formulation of deMann, Rogosa and 3. Boil with frequent agitation to dissolve the powder completely.
Sharpe with slight modification. It supports luxuriant growth of lactobacilli from 4. Sterilize by autoclaving at 121ºC (15 lbs pressure) for 15 minutes.
oral cavity, faeces, foods and dairy products. Quality Control
Principle Dehydrated Appearance
Proteose peptone and beef extract provide carbon and nitrogen compounds. Yeast Yellow coloured, homogeneous, free flowing powder.
extract provides vitamin B complex while dextrose is the fermentable Prepared Appearance
carbohydrate. Polysorbate 80, sodium acetate, magnesium sulphate and Medium amber coloured, clear to slightly opalescent solution forms in tubes.
manganese sulphate provide growth factors. Sodium acetate and ammonium Cultural Response
Cultural characteristics at 18-24 hours or longer at 35ºC.
citrate inhibits streptococci, moulds and many other microorganisms.
Organisms (ATCC) Growth
Dipotassium phosphate is the buffer.
Lactobacillus leichmanni (7830) Luxuriant
Formula*
Lactobacillus plantarum (8014) Luxuriant
Ingredients in grams per liter
Lactobacillus fermentum (9338) Luxuriant
Dextrose 20.0
Procedure
Beef extract 10.0
Proteose peptone 10.0
1. For quantitative test follow the pour plate method using 1 ml volumes of
Yeast extract 5.0 diluted test sample.
Sodium acetate 5.0 2. Alternatively, the streak plate method can be used for recovery of organisms.
Ammonium citrate 2.0
3. Incubate the plates at 35ºC for 3 days, or at 30ºC for 5 days in an aerobic
Dipotassium phosphate 2.0
atmosphere supplemented with carbon dioxide.
Polysorbate 80 1.0
Interpretation of results
Magnesium sulphate 0.1
Manganese sulphate 0.05 1. Lactobacilli appear as large, white colonies in or on the surface of the
Final pH (at 25ºC) 6.5 ± 0.2 medium.

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2. Count the number of colonies and express as colony forming units (CFU) per Storage
gram or ml of sample, taking into account the applicable dilution factor. Store at 2- 80C and prepared medium at 2-80C.
Precautions / Limitations Shelf Life
Organisms other than lactobacilli may grow in these media and therefore isolates Use before expiry date as mentioned on the label.
must be confirmed as lactobacilli by appropriate biochemical testing.

Lactobacillus Selection agar AM50513

Use Directions
Lactobacillus Selection Agar Base is recommended for isolation and enumeration 1. Suspend 84.7 grams in 1000 ml distilled water containing 1.32 ml glacial
of Lactobacilli from foods. acetic acid.
Summary 2. Heat with frequent stirring.
Lactobacillus Selection Agar is used for isolation and enumeration of Lactobacilli. 3. Boil for 1-2 minutes to dissolve the medium completely. DO NOT
Rogosa et al (95, 95.1) developed LBS Agar as a selective medium for isolation AUTOCLAVE.
and enumeration of Lactobacilli from oral, faecal specimens (24.2), food
4. If storage is necessary, autoclave at 12 lbs pressure (118°C) for 15
(103.4) and dairy products (91.2). Lactobacillus Selection Medium was
minutes. Mix well and pour into sterile Petri plates.
demonstrated to be more suitable for growth of lactobacilli than Tomato Juice
Quality Control
Medium traditionally used to isolate lactobacilli. Lactobacilli Selection Media Dehydrated Appearance
can be further enriched by addition of tomato juice (100.1). Cream to yellow homogeneous free flowing powder
Principle Prepared Appearance
Casein enzymic hydrolysate, yeast extract and dextrose are the nitrogen and Yellow coloured slightly opalescent gel forms in Petri plates
carbon sources. Polysorbate 80 provides fatty acids required for the metabolism of Cultural Response
Lactobacilli. Selectivity of the medium is obtained due to the presence of Cultural characteristics after 48 hours at 35-370C.
Organisms (ATCC) Growth Growth RGI
ammonium citrate and sodium acetate. Addition of acetic acid lowers the pH
Recovery
which is inhibitory to many microorganisms but favours the growth of Lactobacilli.
Enterococcus faecalis (29212) Inhibited 0% 0%
Formula*
Lactobacillus acidophilus (4356) Luxuriant >=50% More than 70%
Ingredients in grams per liter
Lactobacillus casei (9595) Luxuriant >=50% More than 70%
Casein enzymic hydrolysate 10.00
Lactobacillus plantarum (8014) Luxuriant >=50% More than 70%
Yeast extract 5.00
Proteus vulgaris (13315) Inhibited 0% 0%
Glucose 20.00
Staphylococcus aureus (25923) Inhibited 0% 0%
Sodium acetate 25.00
Escherichia coli (25922) Inhibited 0% 0%
Monopotassium hydrogen phosphate 6.00
For growth RGI should be more than 70%
Ammonium citrate 2.00
For Inhibition RGI should be 0%
Polysorbate 80 1.00
RGI- Relative Growth Index
Magnesium sulphate 0.575
Storage
Manganese sulphate 0.12
Ferrous sulphate 0.034 Store at 22-300C and prepared medium at 2-80C.
Agar 15.00 Shelf Life
Final pH ( at 25°C) 5.5±0.2 Use before expiry date as mentioned on the label.
* Formula adjusted to suit performance parameters

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Lactose Broth AM1052/AM5052
Lactose Broth IP AM10521/AM50521
Lactose Broth BIS AM10524/AM50524
Use Prepared Appearance
Lactose Broth is used for the detection of coliforms, as a pre-enrichment broth for Light to medium amber coloured, clear solution without any precipitate.
Cultural Response
salmonellae and in the study of lactose fermentation in general.
Cultural characteristics after 18-48 hours at 350C.
Summary
Organisms (ATCC) Growth Gas
Lactose Broth is recommended by APHA in the performance and confirmation of Enterobacter aerogenes (13048) Luxuriant +
the presumptive test for coliform bacteria in milk (39). This medium is Enterococcus faecalis (29212) Luxuriant -
recommended in the Compendium of Methods for the Microbiological Escherichia coli (25922) Luxuriant +
Examination of foods for pre-enrichment when Salmonella is suspected in foods Pseudomonas aeruginosa (10145) Luxuriant -
(20) and is included in the USP (114) and IP (46) for use in the performance of Salmonella serotype Typhi (6539) Luxuriant -
Microbial Limit Test for salmonellae and E. coli. It is also included in the Procedure
Bacteriological Analytical Manual for food testing (113). 1. The amount of sample added to Lactose Broth is dependent on the
Principle concentration of the medium. Regardless of the sample size, Lactose Broth
Peptone and beef extract provide nitrogen and carbon compounds while lactose is must have a concentration of 13 grams per liter. For example, if 10 ml
the fermentable carbohydrate. In processed foods and in debilitated conditions samples are to be added to 10 ml of Lactose Broth, the broth must be double
Salmonella may be present in low numbers. The medium provides for repair of strength.
cell damage, dilutes toxic substances that may be present and favours the growth 2. Inoculate tubes of Lactose Broth with the dilutions of the sample.
of Salmonella. Growth with the production of gas is a presumptive test for 3. Incubate aerobically for 24 hours at 350C.
coliforms.
4. Examine for turbidity and gas formation.
Formula*
Ingredients in grams per liter 5. Reincubate the tubes for a further 24 hours (48 hours) if the results are
Pancreatic digest of gelatin 5.0 negative at 24 hours.
Beef extract 3.0 Interpretation of Results
Lactose 5.0 1. A positive test for coliforms is the production of turbidity in the medium and
Peptone 5.0 gas in the Durham's tube within 48 hours.
Beef Extract 3.0 Precautions / Limitations
Final pH (at 250C) 6.9 ± 0.2
1. The results should be confirmed by additional standard testing.
* Formula adjusted to suit performance parameters
Directions 2. The Durham's tubes must be free from air bubbles before inoculation.
1. Suspend 13 gms of the powder in 1000 ml distilled water. 3. Avoid overheating double strength broth as inhibitory products may be
2. Mix thoroughly. formed.
3. Heat if necessary to dissolve the powder completely. 4. When used for the pre-enrichment of samples for the recovery of Salmonella,
the growth from the medium should be subcultured to an appropriate
4. Dispense in test tubes containing inverted Durham's tubes, in 10 ml
medium for the identification of Salmonella species.
amounts for testing samples of 1 ml or less. For testing larger samples (10
Storage
ml), prepare double strength broth (26 gms in 1000 ml) and distribute in
10 ml amounts. Store at 22-300C and prepared medium at 2-80C.
Shelf Life
5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. Use before expiry date as mentioned on the label.
Quality Control
Dehydrated Appearance
Yellow coloured, homogeneous, free flowing powder.

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Lactose Broth EP AM10522/AM50522
Lactose Broth BP AM10523/AM50523
Use Prepared Appearance
Lactose Broth is used for the detection of coliforms, in water, food and dairy Light to medium amber coloured, clear solution without any precipitate.
Cultural Response
products.
Cultural characteristics after 18-48 hours at 350C.
Summary
Organisms (ATCC) Growth Gas
Lactose Broth is recommended by APHA in the performance and confirmation of Enterobacter aerogenes (13048) Luxuriant +
the presumptive test for coliform bacteria in milk . This medium is recommended Enterococcus faecalis (29212) Luxuriant -
in the Compendium of Methods for the Microbiological Examination of foods for Escherichia coli (25922) Luxuriant +
pre-enrichment when Salmonella is suspected in foods and is included in the Pseudomonas aeruginosa (10145) Luxuriant -
USP and IP for use in the performance of Microbial Limit Test for salmonellae and Salmonella serotype Typhi (6539) Luxuriant -
E. coli. It is also included in the Bacteriological Analytical Manual for food testing. 1. The amount of sample added to Lactose Broth is dependent on the
Principle concentration of the medium. Regardless of the sample size, Lactose Broth
Pancreatic digest of gelatin and beef extract provide nitrogen and carbon must have a concentration of 13 grams per liter. For example, if 10 ml
compounds while lactose is the fermentable carbohydrate. In processed foods and samples are to be added to 10 ml of Lactose Broth, the broth must be double
in debilitated conditions Salmonella may be present in low numbers. The strength.
medium provides for repair of cell damage, dilutes toxic substances that may be 2. Inoculate tubes of Lactose Broth with the dilutions of the sample.
present and favours the growth of Salmonella. Growth with the production of gas 3. Incubate aerobically for 24 hours at 350C.
is a presumptive test for coliforms.
4. Examine for turbidity and gas formation.
Formula*
Ingredients in grams per liter 5. Reincubate the tubes for a further 24 hours (48 hours) if the results are
Lactose monohydrate 5.0 negative at 24 hours.
Pancreatic digest of gelatin 5.0 Interpretation of Results
Beef Extract 3.0 1. A positive test for coliforms is the production of turbidity in the medium and
Final pH (at 250C) 6.9 ±0.2 gas in the Durham's tube within 48 hours.
* Formula adjusted to suit performance parameters
Precautions / Limitations
Directions
1. The results should be confirmed by additional standard testing.
1. Suspend 13 gms of the powder in 1000 ml distilled water.
2. The Durham's tubes must be free from air bubbles before inoculation.
2. Mix thoroughly.
3. Avoid overheating double strength broth as inhibitory products may be
3. Heat if necessary to dissolve the powder completely.
formed.
4. Dispense in test tubes containing inverted Durham's tubes, in 10 ml
4. When used for the pre-enrichment of samples for the recovery of Salmonella,
amounts for testing samples of 1 ml or less. For testing larger samples (10
the growth from the medium should be subcultured to an appropriate
ml), prepare double strength broth (26 gms in 1000 ml) and distribute in 10
medium for the identification of Salmonella species.
ml amounts.
Storage
5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. Store at 22-300C and prepared medium at 2-80C.
Quality Control
Shelf Life
Dehydrated Appearance
Use before expiry date as mentioned on the label.
Yellow coloured, homogeneous, free flowing powder.

Lactose Monohydrate Sterile (g

irradiated) AM505241/AM505241-5K
Use filling injectable.
Lactose Monohydrate Sterile (g
irradiated) used for the media fill runs in dry

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Summary used for Media Fill Run. g
irradiation also assumes that sterile products is free
Routine sampling for sterility testing is not sensitive enough to detect any low from Mycoplasma.
level contamination in sterile pharmaceutical formulations. Sample numbers are Principle
too small and only gross contamination is likely to be detected. Pharmaceutical During Media Fill Run for validation of dry injectable g irradiated Lactose is
manufactures therefore need other means of guaranteeing the quality of their dispersed into individual vial/ampules. After completion of filling process
product. This is why process stimulations (Media Fill Run) supported by individual vial is reconstituted with sterile distilled water (As per label claim of
environmental monitoring is must in pharmaceutical industry. injection). Reconstituted vials an incubated at 35-370C and monitored till 14
The FDA guidelines have recommended using SCDM for liquid injectable and days.
Lactose for dry injectable. Regular dehydrated culture media or Lactose is usually Quality Control
supplied in non sterile form which carries high bioburden and should not be Dehydrated Appearance
directly taken into a controlled area therefore irradiated sterile SCDM/Lactose is Light yellow coloured, homogeneous, free flowing powder.
Prepared Appearance

Lactose Sulphite Broth Base AM50525

Lactose Sulphite Broth Base (Medium R) EP AM50526
Lactose Sulphite Broth Base (Medium R) BP AM50527
Use Yeast extract 2.5
Lactose sulphite Medium is recommended for detection and enumeration of Sodium chloride 2.5
Lactose monohydrate 10.0
Closterium perfringens in pharmaceutical products.
Cysteine hydrochloride 0.3
Summary
Final pH (at 250C) 7.1 ±0.2
Lactose sulfite broth base is a selective medium used to detect and enumerate * Formula adjusted to suit performance parameters
Clostridium perfringens based on lactose fermentation and production of Directions
hydrogen sulfide. 1. Suspend 20.3 gms of the powder in 1000 ml distilled water.
The Eruopean Pharmacopoeia recommends to prepare samples using 1:100 and 2. Mix thoroughly.
1:1000 dilutions with Buffered Peptone Water (AM10211). Mix sample with
3. Heat if necessary to dissolve the powder completely.
medium with minimum shaking and incubate at 45.5 - 46.5°C for 24 – 48
hours. Colonies producing hydrogen sulfide are characterized by blackening due 4. Dispense 8 ml in test tubes containing inverted Durham's tubes, 16 mm x
to the reaction of Sodium bisulfite and the Ferric ammonium citrate salt. The 160 mm of size.
containers showing a blackening and abundant formation of gas in the Durham 5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
tube (at least 1/10 of the volume) indicate the presence of C. perfringens. Quality Control
Dehydrated Appearance
The principle of the medium relies on the ability of Clostridium perfringens to
Yellow coloured, homogeneous, free flowing powder.
ferment lactose whlie producing gas (observable in Durham bell jar) and to Prepared Appearance
reduce sulphite to sulphide at 46 °C (black iron sulphide precipitate in base of the Light to medium amber coloured, clear solution without any precipitate.
tube. Cultural Response
Principle Cultural characteristics after 24-48 hours at 45-460C.
The nutrient base provides optimal conditions for the development of Clostridia. Organisms (ATCC) Growth Gas Blackening
Casein peptone and Yeast extract provide the basic nutrients of the medium: Closterium perfringens (12924) luxuriant + +
Closterium paraperfringens (27639) inhibited - -
nitrogen, vitamins, minerals and amino acids. Lactose is a complex carbohydrate
Storage
energy source. Cysteine hydrochloride is the reducing agent.
Formula* Store at 22-300C and prepared medium at 2-80C.
Shelf Life
Ingredients in grams per liter
Pancreatic digest of casein 5.0 Use before expiry date as mentioned on the label.

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Lauryl Tryptose Broth (Lauryl Sulphate Broth) AM1053/AM5053
Use 5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Lauryl Tryptose Broth is used for the detection of coliform organisms in materials Quality Control
of sanitary importance. Dehydrated Appearance
Summary Light yellow coloured, homogeneous, free flowing powder.
Prepared Appearance
Lauryl Tryptose Broth is based on the formulation of Mallmann and Darby (78).
Light yellow coloured, clear solution without any precipitate.
This medium is recommended by APHA for the presumptive detection of coliforms
Cultural Response
in water, effluent or sewage by Most Probable Number (MPN) technique (36) Cultural response after 18-24 hours at 370C.
and for detection of coliforms in foods (20) and milk (39). Mallmann and Darby Organisms (ATCC) Growth
Gas Indole
0
found that Lauryl Tryptose Broth gave higher colon index than confirmatory Production (44 C)
standard methods media and that gas production served not only as a Enterobacter aerogenes (13048) Luxuriant + -
presumptive test but also confirmatory of the presence of coliforms for routine Enterococcus faecalis (29212) Inhibited - -
Escherichia coli (25922) Luxuriant + +
testing of water. Lauryl Tryptose Broth is listed in the Official Methods of Analysis
Salmonella serotype Typhimurium (14028) Luxuriant - -
of AOAC International and in the Bacteriological Analytical Manual for food
Staphylococcus aureus (25923) Inhibited - -
testing (113). This medium is designed to promote rich growth and copious gas
Procedure
production from small inocula of coliform organisms, while inhibiting the aerobic
1. Use single strength medium for inocula of 1 ml or less. For inocula of 10 ml
spore formers, which may give a false reaction. This medium gave a higher colon
or more, double strength or proportionate medium must be used.
index than most of the media and the gas production served not only as a
2. After inoculation, incubate the tubes at 370C for 24-48 hours. For every tube
presumptive test but also a confirmatory test for the presence of coliforms in
showing fermentation (primary fermentation), inoculate two tubes of Lauryl
routine testing of water. The advantage in using this medium is that in addition to
Tryptose Broth from the tube showing primary fermentation and incubate at
giving the fermentation reaction typical of MacConkey Broth, it can also be
directly used for the detection of indole. 370C and 440C respectively.
Interpretation of results
Principle
1. If fermentation occurs in the tube incubated at 440C after 8-24 hours,
Tryptose provides the essential growth factors, sulphur and trace elements while
perform indole test by adding Kovac's reagent.
lactose is the carbohydrate source. Mono and dipotassium phosphates are the
buffers while sodium chloride maintains the osmotic balance. Sodium lauryl 2. A positive indole test in the broth tube showing gas production at 440C
sulphate selectively inhibits organisms other than coliforms. indicates the presence of E. coli.
Formula* 3. If no fermentation occurs in the tube incubated at 370C after 24 hours, the
Ingredients in grams per liter primary fermentation is assumed to be due to organisms other than
Tryptose 20.0 coliforms.
Sodium Chloride 5.0 Precautions / Limitations
Lactose 5.0
1. If stored at 2-80C, the broth becomes cloudy or forms a precipitate. This
Monopotassium Phosphate 2.75
Dipotassium Phosphate 2.75
should clear at room temperature.
Sodium Lauryl Sulphate 0.1 2. Gas formation is the criterion of growth and not turbidity.
Final pH (at 250C) 6.8 ± 0.2 3. Prior to inoculation of the medium, it may be required to invert the tube to
* Formula adjusted to suit performance parameters release the bubbles that may have formed in the Durham's tubes.
Directions
4. Since the nutritional requirements of the organisms vary, some strains may
1. Suspend 35.6 gms of the powder in 1000 ml distilled water. fail to grow or grow poorly on this medium.
2. Mix thoroughly. Storage
3. Warm slightly to completely dissolve the powder. Store at 22-300C and prepared medium at 2-80C.
4. Dispense into tubes containing inverted Durham's tubes. Shelf Life
Use before expiry date as mentioned on the label.

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Lauryl Tryptose Mannitol Broth with Tryptophan AM505311
Use L-Tryptophan 0.20
Lauryl Tryptose Mannitol Broth with Tryptophan is a single tube medium used for Final pH (at 250C) 6.8 ±0.2
* Formula adjusted to suit performance parameters
confirmation of Escherichia coli in drinking water.
Directions
Summary
Lauryl Tryptose Mannitol Broth with Tryptophan has been recommended as a 1. Suspend 35.8 gms of the powder in 1000 ml distilled water.
single tube confirmatory test in report 71 (19.2). It is also recommended by ISO 2. Mix thoroughly.
Committee (46.4) and is a suitable medium for the requirements of the EC 3. Warm gently to dissolve the medium completely.
directive (49.1) related to the quality of drinking water. 4. Dispense in fermentation test tubes containing inverted Durham's tubes.
Principle
5. Sterilize by autoclaving at 1150C (10 lbs pressure) for 10 minutes.
This medium may be used parallel to Lauryl Tryptose Broth (AM1053) to detect Quality Control
non-lactose fermenting strains of Escherichia coli. Escherichia coli is confirmed by Dehydrated Appearance
gas and indole production when incubated at 440C for 24 hours. If the indole test Yellow coloured, homogeneous, free flowing powder.
is negative even if in a single tube medium, repeat the test in Tryptone water Prepared Appearance
(AM1104). Each tube showing acid and gas in the multiple tube test is Yellow coloured, clear solution without any precipitate.
subcultured to a tube of Lauryl Tryptose Mannitol Broth with Tryptophan and Cultural Response
Cultural characteristics after 24 hours at 440C.
incubated at 440C.
Organisms (ATCC) Growth Indole Gas
Formula*
Escherichia coli (25922) Luxuriant + +
Ingredients in grams per liter
Enterobacter aerogenes (13048) Luxuriant - -
Tryptose 20.00
Staphylococcus aureus (25923) Luxuriant - -
Mannitol 5.00
Storage
Sodium chloride 5.00
Dipotassium phosphate 2.75 Store below 300C and prepared medium at 2-80C.
Monopotassium phosphate 2.75 Shelf Life
Sodium lauryl sulphate 0.10 Use before expiry date as mentioned on the label.

Lecithin Agar AM10531/AM50531

Use many residual disinfectants. Polysorbate 80 neutralizes phenols,
Lecithin Agar is used for the detection of bacterial contamination of surfaces in hexachlorophene and formalin.
unprotected and protected areas. Formula*
Summary Ingredients in grams per liter
Monitoring the microbial flora of environmental surfaces, walls, ceilings, and Tryptone 15.0
L-Histidine 1.0
equipment is an important stage in achieving good manufacturing practices in
Lecithin 0.7
factories handling foods, cosmetics or pharmaceuticals. To maintain good
Papaic Digest of Soyabean Meal 5.0
hygiene standards in hotels and restaurant kitchens, microbiological Polysorbate 80 5.0
contamination must also be monitored. Lecithin Agar is one such medium used for Sodium Chloride 5.0
the detection of surface contaminants. Sodium Thiosulphate 1.0
Principle Agar 20.5
Tryptone and Papaic Digest of Soyabean Meal are sources of carbon and nitrogen. Final pH (at 250 C) 7.2 ± 0.2
Sodium Thiosulphate neutralizes iodine and chlorine. Lecithin neutralizes * Formula adjusted to suit performance parameters
Directions
quaternary ammonium compounds. Sodium Chloride maintains the osmotic
equilibrium. Agar is a solidifying agent. Lecithin and Polysorbate 80 inactivate 1. Suspend 53.2 grams of powder in 1000 ml distilled water.

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2. Heat with frequent agitation to dissolve the powder completely. For growth RGI should be more than 70%
RGI- Relative Growth Index
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Procedure
Quality Control
Dehydrated Appearance
1. Cool the medium to about 450C, mix well and pour in petriplates or RODAC
Yellowish brown coloured, homogeneous free flowing powder. (Replicate Organism Detection and Counting) plates.
Prepared Appearance 2. Inoculate onto the medium by spreading method.
Yellowish brown coloured, slightly opalescent gel. 3. Using RODAC plates for checking the cleanliness and disinfection efficiency of
Cultural Response surfaces, press the plates with even pressure onto the surface.
0
Cultural characteristics after 24-48 hours at 35±2 C aerobically.
4. Avoid rubbing to prevent damage of the agar bed. Clean the surface to
Organisms Growth Pigment Colony Colour RGI
(ATCC)
remove any remains of the agar.
Staphylococcus Good to -- Yellow to white More than 70% Storage
aureus (25923) Luxuriant Store at 22-300C and prepared medium at 2-80C.
Pseudomonas Good to + Green to blue More than 70% Shelf Life
aeruginosa Luxuriant Use before expiry date as mentioned on the label.
(10145)

Lee’s Agar AM50532

Use Lactose 5.0
Lee Agar is used as differential enumeration of yoghurt starter bacteria i.e Sucrose 5.0
Calcium carbonate 3.0
lactobacillus bulgaris & streptococcus thermophilus.
Dipotassium phosphate 0.5
Summary
Bromo cresol purple 0.0
Lee's Agar is formulated as per APHA (115.1) for differential enumeration of Agar 18.0
yoghurt starter bacteria, homofermentative Lactobacillus bulgaricus and Final pH (at 250C) 7.0 ±0.2
heterofermentative Streptococcus thermophilus. Yoghurt is made by the * Formula adjusted to suit performance parameters
controlled fermentation of milk held at 430C using a starter culture of Directions
Streptococcus thermophilus and Lactobacillus bulgaricus. Streptococci grow 1. Suspend 51.52 gms of the powder in 1000 ml distilled water.
forst and produce a creamy buttery aroma from diacetyl and similar metabolites. 2. Mix thoroughly.
Redox potential is also lowered by streptococci which enables Lactobacilli to grow
3. Warm gently to dissolve the medium completely.
thereby growth stimulatory products for streptococci are synthesized by
Lactobacilli. Hence the typical sharp acetaldehyde flavour of mature yoghurt is 4. Dispense and sterilize by autoclaving at 1210C, 15 lbs pressure for 20
formed (18.2). minutes.
Principle 5. While dispensing, mix carefully to suspend calcium carbonate evenly.
Casein enzymic hydrolysate and yeast extarct provide the essential nitrogenous 6. Pour into sterile petri plates to obtain 4-5 mm thick gel.
nutrients to the yoghurt (lactic) starter bacteria. Lactose and sucrose are the Quality Control
fermentable carbohydrates. Calcium carbonate is added to medium along with Dehydrated Appearance
Light grey coloured, homogeneous, free flowing powder.
the dipotassium phosphate to buffer the medium and avoid the drastic drop in pH
Prepared Appearance
due to lactic acid formation. Bromo cresol Purple is the pH indicator which turns
Purple coloured, opaque gel forms in petri plates.
yellow in acidic condition and imparts the yellow colour of the colony. It is
Cultural Response
recommended to dry the media plates for 18-24 hours prior to use. Cultural characteristics after 48 hours at 370C in a CO2 incubator.
Formula*
Organisms (ATCC) Growth Colour of RGI
Ingredients in grams per liter
colony
Casein enzymic hydrolysate 10.0
Lactobacillus bulgaricus Luxuriant White More than 70%
Yeast extract 10.0
(11842)

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Streptococcus thermophilus Luxuriant Yellow More than 70% Storage
(14486 Store at 22-300C and prepared medium at 2-80C.
For growth RGI should be more than 70% Shelf Life
RGI- Relative Growth Index)
Use before expiry date as mentioned on the label.

Lee’s Multi-differential Agar AM50533

Lee's Multi-differential Agar is used for the cultivation and identification of all overheating.
brewing bacteria in the brewing industry. 4. Stir the medium while dispensing to prevent settling of calcium carbonate.
Principle Quality Control
Lee's Multidifferential Agar is used for cultivation and identification of brewing Dehydrated Appearance
bacteria. The medium contains Tomato juice broth which provides nutrients and Greenish yellow coloured, homogeneous, free flowing powder.
acid environment for the growth of acidophilic bacteria. Peptonized milk provides Prepared Appearance
Light blue coloured, opaque gel forms in petri plates.
lactose as an energy source. The low pH of the medium inhibits bacteria other
Cultural Response
than acidophilic bacteria. Polysorbate 80 serves as a source of fatty acids. Bromo
Cultural characteristics observed after an incubation of 48-72 hours at 30°C.
cresol green acts as a pH indicator. Acid producing bacteria produce a clear yellow Organisms (ATCC) Growth RGI
halo around the colonies. Other bacteria produce colonies in colours ranging from A. calcoaceticus (19606) None-poor 0%
colourless to yellow green and blue depending on species and strain. Further tests L. acidophilus (4356) Luxuriant with clear More than 70%
should be carried out for their identification. yellow halo
Formula* Lact. Fermentum (9338) Luxuriant with clear More than 70%
Ingredients in grams per liter yellow halo
Tomato juice broth 41.00 Lact. leichmannii (4797) Luxuriant with clear More than 70%
Peptonized milk 20.00 yellow halo
Calcium pantothenate 2.00 Lact. Plantarum (8014) Luxuriant with clear More than 70%
Citric acid 1.10 yellow halo
Calcium carbonate 5.00 P. vulgaris (13315) Inhibited 0%
Polysorbate 80 0.50 Storage and Shelflife :
Bromo cresol green 0.022 For growth RGI should be more than 70%
Cycloheximide 0.007 For Inhibition RGI should be 0%
Agar 15.00 RGI- Relative Growth Index
Final pH (at 25°C) 5.5 ± 0.2 Store below 8°C and the prepared medium at 2-8°C Use before expiry date on the
** Formula adjusted, standardized to suit performance parameters label.
Directions
Storage
1. Suspend 85 grams in 1000 ml distilled water. Store below 300C and prepared medium at 2-80C.
2. Heat gently to dissolve the medium completely. Shelf Life
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 10 minutes. Avoid Use before expiry date as mentioned on the label.

Legionella Agar Base AM1054/AM5054

Use non-clinical specimens. Feely et al (30) developed Legionella Agar as a
Legionella Agar Base with added supplements is used for cultivation of Legionella modification of F-G Agar by replacing the starch with activated charcoal and
species. casein hydrolysate with yeast extract. Pasculle (84) reported that the agar could
Summary be improved by buffering the medium with ACES (N- 2-acetamido-2-amino
Legionella Agar Base is used for isolation of Legionella species from clinical and ethane sulphonic acid) buffer. Edelstein (22) further increased the sensitivity of
the medium by adding a -Ketoglutarate.

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Principle Quality Control
Yeast extract provides nutrients for bacterial growth. a
-Ketoglutarate meets the Dehydrated Appearance
Grey coloured, homogeneous, free flowing powder.
specific nutritional requirements of Legionella species. The activated charcoal
Prepared Appearance
decomposes hydrogen peroxide, a toxic metabolic product, and may also collect
Black coloured, opaque gel.
CO2 and modify surface tension. ACES buffer helps in maintaining proper pH of Cultural Response
the medium for the optimal growth of Legionella species. Antibiotics in the Cultural characteristics after 48-72 hours at 350C.
Selective Supplement inhibit the growth of various contaminating bacteria and Organisms (ATCC) Growth Colour of RGI
fungi. the colony
Formula* Legionella pneumophila Good to luxuriant Light blue More than 70%
Ingredients in grams per liter (33153) to grey white
Yeast Extract 10.0 For growth RGI should be more than 70%
ACES Buffer 6.0 RGI- Relative Growth Index
Potassium Hydroxide 1.5 Procedure
Charcoal Activated 1.5 1. Use standard procedures like the streak plate method to obtain isolated
a-Ketoglutarate 1.0 colonies.
Agar 17.0
2. Incubate the plates at 350C for a minimum of 3 days.
Final pH (at 250C) 6.9 ± 0.2
Interpretation of results
* Formula adjusted to suit performance parameters
Directions 1. Growth is usually visible after 3 days, but may take up to 2 weeks to appear.
1. Suspend 18.5 gms of the powder in 500 ml distilled water and mix well. 2. L.pneumophila produce small to large, smooth, colourless to pale blue-grey,
2. DO NOT HEAT PRIOR TO STERILIZATION. slightly mucoid colonies that fluoresce yellow-green when exposed to long-
wave UV light.
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Precautions / Limitations
4. Cool to 45-500C and aseptically add rehydrated contents of 1 vial each of
1. Legionella species are highly pathogenic if inhaled. Handle liquid cultures in
Legionella Growth Supplement (AS016) and Legionella Selective
a protective cabinet and avoid creating aerosols.
Supplement (AS017).
2. Decontaminate working surfaces with 5% hypochlorite solution and
5. Mix well and stir the medium while dispensing into sterile petri plates to
autoclave all materials before discarding.
prevent charcoal particles from settling down. Storage
Warning: Potassium Hydroxide is corrosive. On contact with skin, wash
immediately with plenty of water. Store at 22-300C and prepared medium at 2-80C.
Shelf Life
Use before expiry date as mentioned on the label.

Letheen Agar AM50541

Use polysorbate 80.
Letheen Agar is recommended to inactivate quaternary ammonium compounds american Society for Testing and Materials (ASTM) specifies Letheen Agar in the
and other preservatives when determining the number of bacteria present in Standard TestMethod for Preservatives in Water-Containing (1.2)
cosmetics. Principle
Summary
Pancreatic digest of protein and beef extract provide the carbon and nitrogen
Letheen Agar is a highly nutritive medium with neutralizing agents for quaternary source. Dextrose is the source of energy. Lecithin and polysorbate 80 are added to
ammonium compounds. So this medium is very useful for sanitary and neutralize surface disinfectants.
disinfectant testing. It neutralizes the disinfectants especially quaternary Formula*
ammonium compounds and other water containing cosmetics. Letheen Agar is a Ingredients in grams per liter
modification of Tryptone Glucose Extract agar with the addition of lecithin and Part A

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Casein enzymic hydrolysate 5.0 Quality Control
Beef extract 3.0 Dehydrated Appearance
Dextrose 1.0 Yellow coloured, homogeneous, free flowing powder.
Lecithin 1.0 Prepared Appearance
Agar 15.0 Light yellow coloured, clear to slightly opalescent gel.
Part B Cultural Response
Polysorbate 80 7.0 Cultural characteristics after 24-48 hours at 35-370C.
Organisms (ATCC) Growth
0
Final pH (at 25 C) 7.0 ±0.2
* Formula adjusted to suit performance parameters Escherichia coli (25922) Good to luxuriant
Directions Staphylococcus aureus (6538) Good to luxuriant
1. Suspend 32 gms of the powder in 1000ml of distilled water. Procedure

2. Mix thoroughly. Refer to appropriate references for specific procedures.

Interpretation of Results
3. Boil with frequent agitation to dissolve the powder completely. DO NOT
OVERHEAT. Refer to appropriate references and procedures for results.
Storage
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Store at 22-300C and prepared medium at 2-80C.
Shelf Life
Use before expiry date as mentioned on the label.

Letheen Agar, Modified AM505411

Use Polysorbate 80 7.0
Letheen Agar, modified is recommended to inactivate quaternary ammonium Dextrose 1.0
Agar 15.0
compounds and other preservatives when determining the number of bacteria
Yeast extract 2.0
present in cosmetics.
Sodium chloride 5.0
Summary
Sodium bisulphite 0.1
Letheen Agar, Modified is based on Letheen Agar as described as in the U. S. Food Final pH (at 250C) 7.2 ± 0.2
and Drug Administration (FDA) Bacteriological Analytical Manual (43.1). It is a * Formula adjusted to suit performance parameters
highly nutritive medium with neutralizing agents for quaternary ammonium Directions
compounds. So this medium is very useful for sanitary and disinfectant testing. It 1. Suspend 54 gms powder in 1000ml-distilled water.
neutralizes the disinfectants especially quaternary ammonium compounds and 2. Mix thoroughly.
other water containing cosmetics.
3. Boil with frequent agitation to dissolve the powder completely. DO NOT
Principle
OVERHEAT.
Peptic digest of animal tissue, casein enzymic hydrolysate, yeast extract and beef
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
extract provide the carbon and nitrogen source. Increased amount of peptone in
Quality Control
the modified Letheen Agar serves as a better medium for microorganisms.
Dehydrated Appearance
Dextrose is the source of energy. Sodium chloride maintains the osmotic balance. Yellow coloured, homogeneous free flowing powder..
Lecithin, polysorbate 80 and sodium bisulfite are added to neutralize surface Prepared Appearance
disinfectants. Yellow coloured, slightly opalescent gel forms in petri plates.
Formula* Cultural Response
Ingredients in grams per liter Cultural characteristics after 24-48 hours at 35-370C.
Peptic digest of animal tissue 10.0 Organisms (ATCC) Growth RGI
Casein enzymic hydrolysate 10.0 Escherichia coli ( 25922) Luxuriant More than 70%
Beef extract 3.0 Staphylococcus aureus ( 25923) Luxuriant More than 70%
Lecithin 1.0 For growth RGI should be more than 70%

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RGI- Relative Growth Index Storage
Procedure Store at 22-300C and prepared medium at 2-80C.
Refer to appropriate references for specific procedures. Shelf Life
Interpretation of Results Use before expiry date as mentioned on the label.
Refer to appropriate references and procedures for results.

Letheen Broth, Modified (Twin Pack) AM50543

Use * Formula adjusted to suit performance parameters
Letheen Broth, Modified is used for determination of bactericidal activity of Directions

quaternary ammonium compounds using Escherichia coli and Staphylococcus 1. Suspend 37.8 gms of the powder in 995 ml-distilled water.
aureus. 2. Add 5 gms of polysorbate 80 (Twin Pack B)
Summary 3. Mix thoroughly.
Letheen Broth, Modified is based on Letheen Broth as described as in the U. S. 4. Boil with frequent agitation to dissolve the powder completely. DO NOT
Food and Drug Administration (FDA) Bacteriological Analytical Manual. It is a OVERHEAT.
highly nutritive medium with neutralizing agents for quaternary ammonium
5. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
compounds. So this medium is very useful for sanitary and disinfectant testing. It
Quality Control
neutralizes the disinfectants especially quaternary ammonium compounds and
Dehydrated Appearance
other water containing cosmetics. Part A: Yellow coloured, homogeneous free flowing powder.
Principle Part B: Yellow colour clear viscous liquid.
Peptic digest of animal tissue and beef extract provide the carbon and nitrogen Prepared Appearance
source. Sodium chloride maintains the osmotic balance. Lecithin, polysorbate 80 Yellow coloured, clear solution without any precipitate.
and sodium bisulfite are added to neutralize surface disinfectants. Cultural Response
Formula* Cultural characteristics after 24-48 hours at 35-370C.
Ingredients in grams per liter Organisms (ATCC) Growth
Part A Escherichia coli ( 25922) Good to luxuriant
Peptic digest of animal tissue 20.0 Staphylococcus aureus ( 6538) Good to luxuriant
Beef extract 5.0 Procedure
Lecithin 0.7 Refer to appropriate references for specific procedures.
Sodium chloride 5.0 Interpretation of Results
Sodium bisulfite 0.1 Refer to appropriate references and procedures for results.
Yeast extract 2.0
Storage
Casein enzymic hydrolysate 5.0
Part B Store at 22-300C and prepared medium at 2-80C
Polysorbate 80 5.0 Shelf Life
Final pH (at 250C) 7.2 ± 0.2 Use before expiry date as mentioned on the label.

Letheen Broth (Twin Pack) AM50542

Use quaternary ammonium compounds in disinfectant testing. Letheen Broth is
Letheen Broth is used for determining the phenol coefficient of cationic surface- recommended in the Official Methods of Analysis of AOAC International for use
active materials. with disinfectants containing cationic surface active materials (45.2). American
Summary Society for Testing and Materials (ASTM) specifies Letheen Agar and Letheen
Letheen Broth was developed as a subculture medium for the neutralization of Broth in the Standard Test Method for Preservatives in Water-Containing (1.2).

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Principle
5. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Peptic digest of animal tissue, and beef extract provide the carbon and nitrogen Quality Control
source. Sodium chloride maintains the osmotic balance. Lecithin and polysorbate Dehydrated Appearance
80 are added to neutralize surface disinfectants. Yellow coloured, homogeneous, free flowing powder.
Formula* Prepared Appearance
Ingredients in grams per liter Light yellow coloured, clear solution.
Part A Cultural Response
Peptic digest of animal tissue 10.0 Cultural characteristics after 24-48 hours at 35-370C.
Beef extract 5.0 Organisms (ATCC) Growth
Lecithin 0.7 Escherichia coli (25922) Good to luxuriant
Sodium chloride 5.0 Staphylococcus aureus (6538) Good to luxuriant
Procedure
Part B
Polysorbate 80 5.0 Refer to appropriate references for specific procedures.
Final pH (at 250C) 7.0 ±0.2 Interpretation of Results
* Formula adjusted to suit performance parameters Refer to appropriate references and procedures for results.
Directions Storage
1. Suspend 20.7 gms (Twin pack) of the powder in 995 ml-distilled water. Store at 22-300C and prepared medium at 2-80C.
2. Add 5 gms of polysorbate 80 (Twin Pack B) Shelf Life
3. Mix thoroughly. Use before expiry date as mentioned on the label.
4. Boil with frequent agitation to dissolve the powder completely. DO NOT
OVERHEAT.

Listeria Enrichment Broth AM50544

Use Dipotassium phosphate 2.5
Listeria Enrichment Broth is used for enrichment of Listeria from food. Yeast extract 6.0
Cycloheximide 0.05
Summary
Acriflavine Hcl 0.0015
Food borne listeriosis was first reported in 1985, since then, microbiological and Nalidixic acid 0.04
epidemiological evidence from both sporadic and epidemic cases of listeriosis has Final pH (at 250C) 7.3 ± 0.2
shown that the principle route of transmission is via the consumption of foodstuffs * Formula adjusted to suit performance parameters
contaminated with Listeria monocytogenes. Directions
Principle 1. Suspend 36.1 gms powder in 1000 ml distilled water.
Pancreatic digest of casein and yeast extract provide nitrogen, vitamins and 2. Heat with frquent agitation to dissolve the medium completely.
minerals. Dextrose is a carbohydrate source, sodium chloride maintains the
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
osmotic balance of the medium. Phosphate provides buffering capacity. Nalidixic
Quality Control
acid inhibits growth of gram-negative organisms. Acriflavine Hcl suppresses the Dehydrated Appearance
growth of gram-positive bacteria. Cycloheximide is incorporated to inhibit Light beige coloured, homogeneous, free flowing powder.
saprophytic fungi. Prepared Appearance
Formula* Light yellowish - amber coloured, clear to slightly opalescent solution.
Ingredients in grams per liter Cultural Response
Pancreatic digest of casein 17.0 Cultural characteristics after 18-48 hours at 30 + 20C.
Soytone 3.0 Prepared Appearance
Dextrose 2.5 Light yellow coloured, clear solution.
Sodium chloride 5.0 Cultural Response
Cultural characteristics after 24-48 hours at 35-370C.

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Organisms (ATCC) Growth procedures for isolating Listeria.
Enterococcus faecalis (29212) Inhibition Storage
Escherichia coli (25922) Inhibition
Store at 22-300C and prepared medium at 2-80C.
Listeria monocytogenes (19114) Luxuriant
Shelf Life
Saccharomyces cerevisiae (9080) Inhibition
Procedure Use before expiry date as mentioned on the label.
For food samples, use Listeria Enrichment Broth in recommended laboratory

Listeria Identification Agar Base (PALCAM) AM1055/AM5055

Use Formula*
Listeria Identification Agar Base, PALCAM with added supplement is used for Ingredients in grams per liter
Peptone 23.0
selective isolation and identification of Listeria species.
Lithium Chloride 15.0
Summary
Mannitol 10.0
Van Netten et al (115) formulated Listeria Identification Agar Base, which is also Sodium Chloride 5.0
called Polymixin Acriflavin lithium chloride Ceftazidime Esculin Mannitol Starch 1.0
(PALCAM) and is used for the isolation of Listeria monocytogens from foods. Phenol Red 0.08
PALCAM is a highly selective medium due to the presence of Lithium chloride, and Esculin 0.80
selective supplement containing Ceftazidime, Polymixin B and Acriflavin Ammonium Ferric Citrate 0.50
hydrochloride. It is a differential diagnostic medium utilizing two indicator Dextrose 0.50
Agar 13.0
systems as esculin-ferric citrate and mannitol-phenol red.
Final pH (at 250C) 7.0 ± 0.2
Listeria monocytogenes hydrolyze esculin, which is evidenced by blackening of * Formula adjusted to suit performance parameters
the medium. This blackening by esculin-hydrolyzing bacteria results from the Directions
formation of 6,7 dihydroxycoumarin, which reacts with ferric ions that are present 1. Suspend 69 gms of the powder in 1000 ml distilled water.
in the medium as ammonium ferric citrate. It does not ferment mannitol but the
2. Mix thoroughly.
contaminants such as enterococci and staphylococci ferment mannitol and is
indicated by the change of colour from red to yellow. Under strict microaerophilic 3. Boil with frequent agitation to dissolve the powder completely.
conditions strict aerobes such as Bacillus and Pseudomonas species are 4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
inhibited. 5. Cool to 45-500C and aseptically add rehydrated contents of 2 vials of Listeria
The addition of egg yolk (2.5%v/v) to Listeria Identification Agar Base, PALCAM Selective Supplement (PALCAM) (AS018).
has been reported to aid repair of damaged cells. A medium containing blood 6. Mix well and pour into sterile petri plates.
when overlaid on Listeria Identification Agar Base, PALCAM enables to Warning: Lithium chloride is harmful, bodily contact and inhalation of
vapours must be avoided. On contact with skin, wash with plenty of
differentiate and enumerate haemolytic Listeria species. Listeria Identification
water immediately.
Agar Base is recommended by APHA for the examination of milk (39) and foods Quality Control
(20). Dehydrated Appearance
Principle Light pink coloured, homogeneous, free flowing powder.
Peptone provides nitrogen, vitamins and other nutrients. Dextrose is the Prepared Appearance
carbohydrate source while sodium chloride maintains the osmotic balance. High Red coloured, clear to slightly opalescent gel.
amounts of lithium chloride and added selective supplement inhibit Cultural Response
Cultural characteristics after 48 hours at 350C, in microaerophilic environment.
accompanying micro flora and allow the growth of only Listeria species. The
Organisms (ATCC) Growth Colony RGI
combination of mannitol and phenol red helps in the detection of mannitol
characteristics
fermentation while esculin and ammonium ferric citrate helps in the detection of Listeria monocytogenes Luxuriant Grey-green with More than 70%
esculin hydrolysis. (19112) black center and
a black halo.

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Staphylococcus aureus None to Yellow colonies 0% Interpretation of Results
(25923) poor with yellow halo. 1. Typical Listeria species form colonies that are approximately 2 mm in
Enterococcus faecalis None to Grey colonies 0%
diameter, green-grey in colour with a black sunken center and a black halo
(29212) poor with a brown
against a red background.
green halo.
Precautions / Limitations
For growth RGI should be more than 70%
For Inhibition RGI should be 0% 1. Acriflavin Hydrochloride in the Listeria Selective Supplement should be
RGI- Relative Growth Index protected from light because photo oxidation causes it to become inhibitory
Procedure to Listeria growth.
1. The technique for the isolation of Listeria monocytogenes depends on the 2. Occasionally Enterococcus or Staphylococcus strains develop on Listeria
material under test. The sample may be first inoculated into an enrichment Identification Agar Base, PALCAM to form grey colonies with a brown-green
broth to allow multiplication before isolation and identification. halo or yellow colonies with a yellow halo.
2. Inoculate a loopful of the selective enrichment broth onto the Listeria Storage
Identification Agar Base, PALCAM plates. Store at 22-300C and prepared medium at 2-80C.
3. Incubate at 370C for 48 hours under microaerophilic conditions. Shelf Life
Use before expiry date as mentioned on the label.

Listeria Identification Broth Base (PALCAM) AM105511/AM505511

Use amounts of lithium chloride and added selective supplement (73.1) inhibit
Listeria Identification Broth Base (PALCAM) with added supplement accompanying micro flora and allow the growth of only Listeria species. The
recommended for selective enrichment and identification of Listeria species combination of mannitol and phenol red helps in the detection of mannitol
Summary fermentation while esculin and ammonium ferric citrate helps in the detection of
Listeria Identification Broth also known as Polymyxin Acriflavin Lithium-chloride esculin hydrolysis.
Ceftazidime Aesculin Manitol (PALCAM) Broth is prepared as described by van Formula*
Netten et al., for selective enrichment of Listeria species. Ingredients Gms/Liter

Listeria monocytogenes hydrolyze esculin, which is evidenced by blackening of Peptic digest of animal tissue 23.00
the medium. This blackening by esculin-hydrolyzing bacteria results from the Yeast Extract 5.00
formation of 6,7 dihydroxycoumarin, which reacts with ferric ions that are present Lithium chloride 10.00
in the medium as ammonium ferric citrate. It does not ferment mannitol but the Esculin 0.80
contaminants such as enterococci and staphylococci ferment mannitol and is
Ammonium ferric citrate 0.50
indicated by the change of colour from red to yellow. Under strict microaerophilic
conditions strict aerobes such as Bacillus and Pseudomonas species are D-Mannitol 5.00
inhibited. The addition of egg yolk (2.5%v/v) to Listeria Identification Agar Soya Lecithin 1.00
Base, PALCAM has been reported to aid repair of damaged cells. A medium Polysorbate 80 2.00
containing blood when overlaid on Listeria Identification Agar Base, PALCAM Phenol red 0.08
enables to differentiate and enumerate haemolytic Listeria species. Listeria 0
Final pH (at 25 C) 7.4 ±0.2
Identification Agar Base is recommended by APHA for the examination of milk
and foods. * Formula adjusted to suit performance parameters
Directions
Principle
Peptone provides nitrogen, vitamins and other nutrients. Dextrose is the 1. Suspend 23.7 gms powder in 500 ml distilled water. Heat if necessary to
carbohydrate source while sodium chloride maintains the osmotic balance. High dissolve the medium completely.
2. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.

172 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
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3. Cool to 45-500C and aseptically add sterile reconstituted contents of vial of Procedure
Listeria Selective Supplement (AS018). 1. The technique for the isolation of Listeria monocytogenes depends on the
4. Mix well before dispensing. material under test. The sample may be first inoculated into an enrichment
Quality Control broth to allow multiplication before isolation and identification.
Dehydrated Appearance 2. Inoculate a loopful of the selective enrichment broth onto the Listeria
Light pink coloured, homogeneous, free flowing powder. Identification Agar Base, PALCAM plates.
Prepared Appearance
3. Incubate at 370C for 48 hours under microaerophilic conditions.
Red coloured, clear solution without any precipitate.
Interpretation of Results
Cultural Response
Cultural characteristics after 48 hours at 300C. 1. Typical Listeria species form colonies that are approximately 2 mm in
Organisms (ATCC) Growth Colour diameter, green-grey in colour with a black sunken center and a black halo
Listeria monocytogenes (19118) Good Black against a red background.
Staphylococcus aureus (25923) Inhibited – Storage
Enterococcus faecalis (29212) Inhibited –
Store at 22-300C and prepared medium at 2-80C.
Micrococcus luteus (10240) Inhibited –
Shelf Life
For growth RGI should be more than 70%
Use before expiry date as mentioned on the label.
For Inhibition RGI should be 0%
RGI- Relative Growth Index

Listeria Oxford Medium Base AM105512/AM505512

Use Esculin 1.0
Listeria Oxford Medium Base is used for isolation of Listeria species from Ammonium ferric citrate 0.50
Agar 10.0
pathological specimens.
Final pH (at 250C) 7.0± 0.2
Summary
Formula adjusted to suit performance parameters
In 1926 Murray et al., first described that Listeria monocytogenes is a Directions
widespread problem in public health and the food industries. Identification of
1. Suspend the 55.50 gms of powder in 1000 ml distilled water.
Listeria is based on successful isolation of the organism. According to Curtis et al.,
2. Mix thoroughly.
description Listeria Oxford Medium Base is formulated for isolation of Listeria
monocytogenes (18.1). Various techniques are followed to isolate the organism 3. Boil with frequent agitation to dissolve the powder completely. Do not
from different specimen. For all specimens selective and cold enrichment is overheat.
recommended (33.2). Antimicrobial supplements are used for selective isolation 4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
of Listeria species. 5. Cool the medium to -50°C.
Principles
6. Add 1 vial of rehydrated Oxford Listeria Supplement (AS0201) to prepare
Peptone serves as a source of nitrogen and amino acids. Lithium chloride inhibits Oxford’s medium or add 1 vial of rehydrated Listeria Moxalactam
Gram negative bacteria specially enterococci. Addition of various antimicrobial Supplement (AS0171) to prepare selective medium for Listeria.
agents makes the base more selective. Sodium chloride maintains the osmotic
7. After addition, the medium must be gently but thoroughly mixed to ensure
balance. Listeria monocytogenes hydrolyzes esculin to esculatin and dextrose.
that the antibiotics are uniformly distributed throughout the medium.
Esculatin reacts with ferric ions and produces black zones around the colonies. Warning: Lithium chloride is harmful. Avoid bodily contact and
Formula* inhalation of vapours. On contact with skin, wash with plenty of water
Ingredients in grams per liter immediately.
Peptone, special 23.0 Quality Control
Lithium chloride 15.0 Dehydrated Appearance
Sodium chloride 5.0 Dark yellow coloured, homogeneous, free flowing powder.
Corn starch 1.0 Prepared Appearance
Dark amber coloured, clear gel with blue cast, forms in petri plates.

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Cultural Response Interpretation of Results
Cultural characteristics after 24-48 hours at 35ºC. Black zone is found around esculin positive colonies. For further identification do
Organisms (ATCC) Growth* Esculin hydrolysis
biochemical testing. For additional information, refer to appropriate references.
Listeria monocytogenes (19117) Luxuriant +
Precautions / Limitations
Listeria monocytogenes (19111) Luxuriant +
Listeria monocytogenes (19112) Luxuriant + Since Listeria species other than L. monocytogenes can grow on these media,
Staphylococcus aureus (25923) Good - biochemical and serological testing should be done to identify L. monocytogenes.
Enterococcus faecalis (29212) Inhibited - Use freshly prepared antimicrobial agent solutions or aliquot portions and store at
Enterococcus hirae (10541) Inhibited - - 20ºC or below. Poor growth and a weak esculin reaction may be seen after 40
Bacillus subtilis (6633) Inhibited - hours incubation for some enterococci.
Key : + = Black zones around colonies Storage
Procedure
Store at 22-300C and prepared medium at 2-80C.
For faecal and biological specimens, the sample is homogenized in 0.1% Peptone Shelf Life
water (AM1079/5079) and 0.1ml amount is directly plated on Listeria Selective Use before expiry date as mentioned on the label.
Medium. Incubate the plates at 35ºC for 24-48 hours and examine the plates.

Liver Infusion Agar AM10551/AM50551

Liver Infusion Broth AM10552/AM50552
Use * Formula adjusted to suit performance parameters
Liver Infusion Agar is used for the cultivation of Brucella and other pathogenic Directions

bacteria. 1. Suspend the powder in 1000 ml distilled water.

Liver Infusion Broth is used for the cultivation of highly fastidious Liver Infusion Agar - 55 grams
microorganisms, particularly Brucella species and anaerobes. Liver Infusion Broth - 35 grams
Summary 2. Boil with frequent agitation to dissolve the medium completely.
Brucellosis is a zoonotic disease and is usually transmitted by milk, milk products, 3. Sterilize by autoclaving at 1210 C (15 lbs pressure) for 15 minutes.
meat and direct contact with infected animals. Most strains of Brucella grow on Quality Control
chocolate agar or blood agar. However, the nutritive factors of Liver Infusion Dehydrated Appearance
media permit luxuriant growth of Brucella and other fastidious pathogens. Dark beige to light tan, homogeneous, free flowing powder.
Prepared Appearance
For isolating Brucella strains from contaminated milk, crystal violet can be added
Medium to dark amber, slightly opalescent gel.
to Liver Infusion Agar to suppress the growth of gram-positive microorganisms
Cultural Response
and 5% heated horse or rabbit serum to enhance Brucella growth. 0
Cultural response at 35 ± 2 C after 18 - 48 hours up to 72 hours.
Liver Infusion Broth supports the growth of anaerobic microorganisms, especially Organisms (ATCC) Growth RGI
Clostridium species. Brucella abortus (4315) Good More than 70%
Principle Brucella melitensis (4309) Good More than 70%
Brucella suis (4314) Good More than 70%
Proteose Peptone and Beef Liver Infusion provide sources of nitrogen, amino
Clostridium sporogenes (11437) Good More than 70%
acids, vitamins and carbon. Sodium Chloride maintains the osmotic equilibrium.
Streptococcus pneumoniae (6305) Good More than 70%
Agar is the solidifying agent. For growth RGI should be more than 70%
Formula* For Inhibition RGI should be 0%
Ingredients in grams per liter Agar Broth RGI- Relative Growth Index
Beef Liver, Infusion from 500.0 500.0
Storage
Proteose Peptone 10.0 10.0
Sodium Chloride 5.0 5.0 Store at 22-300C and prepared medium at 2-80C.
Agar 20.0 -- Shelf Life
Final pH (at 250 C) 6.9 ± 0.2 Use before expiry date as mentioned on the label.

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Loeffler Medium Base AM1056/AM5056
Use With added serum and after coagulation - Opalescent slants.
Loeffler Medium Base with added horse serum is used for the cultivation of Cultural Response

Corynebacterium diphtheriae from clinical specimens and in pure cultures. Cultural characteristics after 3-4 days at 350C.
Organisms(ATCC) Growth
Summary
Corynebacterium diphtheriae (19113) Fair to good
Loeffler (72) devised Loeffler Medium Base, which was further modified by Pery Pseudomonas aeruginosa (10145) Good (green colonies)
and Petran (88) and Buck (11). This medium is used for the promotion of growth Procedure
and morphological characterization of members of the genus Corynebacterium; 1. Inoculate the tubed medium as soon as possible after specimen collection,
formation of metachromatic granules is enhanced within the cells of these using direct inoculation of the specimen by swabs or by means of an
organisms. This medium is also useful for demonstrating colonial pigmentation, inoculating loop.
ascospore production and proteolytic activity of microorganisms.
2. Incubate the tubes with caps loosened for 18-24 hours and up to 45 days at
Principle
35-370C in an aerobic atmosphere.
Bovine serum and peptone provides amino acids and other nitrogenous
Interpretation of Results
substances necessary to support growth of corynebacteria. Dextrose is the
1. Examine cultures and smears with Loefflers methylene blue after incubation.
carbohydrate source.
Formula* 2. Observe for typical cellular morphology of Corynebacterium species and for
Ingredients in grams per liter the presence of metachromatic granules, which take up the methylene blue
Beef Extract 2.5 dye.
Peptone, Special 2.5 3. Colonies that are catalase positive and exhibit typical morphology are
Dextrose 2.5
subcultured on to blood agar plates to provide growth for identification
Sodium Chloride 1.25
procedures.
Final pH (at 250C) 7.3 ± 0.2
* Formula adjusted to suit performance parameters 4. Observe for pigmentation of specific organisms; e.g. Pseudomonas
Directions aeruginosa (green) and Staphylococcus aureus (yellow to gold).
1. Suspend 8.8 gms of the powder in 250 ml distilled water. 5. Proteolytic activity is observed by destruction of the integrity of the
2. Mix thoroughly. coagulated medium.
3. Boil with frequent agitation to dissolve the powder completely. Precautions / Limitations
0
4. Sterilize by autoclaving at 115 C (10 lbs pressure) for 20 minutes. 1. Though the production of metachromatic granules on this medium is
characteristic of Corynebacterium genus, other organisms such as
5. Cool to 50-550C, aseptically add 750 ml of sterile horse serum (AS 015).
Propionibacterium, some Actinomyces and pleomorphic streptococcal
6. Mix well and aseptically dispense into sterile tubes.
strains display stained granules resembling those of the corynebacteria.
7. Inspissate the medium at 80-850C for 2 hours in free flowing steam for at
2. Loeffler Medium Base must be used in parallel with a tellurite-containing
least 3 consecutive days.
medium for the selective isolation of pathogens, particularly C.diphtheria.
Quality Control
Storage
Dehydrated Appearance
Yellow coloured, homogeneous, free flowing powder. Store at 22-300C and prepared medium at 2-80C.
Prepared Appearance Shelf Life
Basal Medium - Light amber coloured clear solution. Use before expiry date as mentioned on the label.

Lowenstein Jensen Medium Base AM1057/AM5057

Use Summary
Lowenstein Jensen Medium Base supplemented with eggs is used for the Lowenstein Jensen Medium is an inspissated, egg based medium developed from
cultivation and isolation of mycobacteria, especially M.tuberculosis. Jensen's modification (48) of Lowensteins (73) formula. Gruft (37) modified this
medium by adding two antimicrobials penicillin and nalidixic acid for selective

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isolation of mycobacteria. He also found that addition of ribonucleic acid (RNA) Cultural Response
increased the percentage of tubercle bacilli recovered from clinical specimens Cultural characteristics after 2-4 weeks at 350C with 5-10% CO2.

compared to recovery on the standard Lowenstein Jensen Medium. Organism (ATCC) L-J Medium L-J Medium
Base with Gruft
Principle
supplement
Lowenstein Jensen Medium Base is a relatively simple formulation that requires Mycobacterium tuberculosis Luxuriant growth, Good to
supplementation in order to support the growth of mycobacteria. Glycerol and egg (H37Rv strain) granular luxuriant
mixture is added prior to the inspissation process. They provide fatty acids and rough, warty, dry,
protein required for the metabolism of mycobacteria. Glycerol also serves as a friable colonies
carbon source and is favourable to the human type tubercle bacilli while being Mycobacterium smegmatis (14468) Luxuriant growth, Good to
unfavourable to the bovine type. The coagulation of egg albumin during wrinkled, creamy luxuriant
sterilization provides a solid medium for inoculation purposes. Malachite green white colonies
Mycobacterium kansasii (12478) Luxuriant growth, Good to
serves as a pH indicator and inhibits majority of contaminants surviving
photochromogenic, luxuriant
decontamination of the specimen. RNA in the supplement acts as a stimulant and
smooth to rough
helps to increase the isolation rate of mycobacteria. Procedure
Formula* 1. The test procedures are those recommended by the Centers for Disease
Ingredients in grams per 600 ml
Control and Prevention (CDC) for primary isolation of specimens containing
Potato Starch, Soluble 30.0
mycobacteria. N-Acetyl-L-Cysteine-Sodium Hydroxide solution is
L-Asparagine 3.6
Monopotassium Phosphate 2.4 recommended as a gentle but effective digesting and decontaminating
Magnesium Citrate 0.60 agent.
Malachite Green 0.4 2. Following inoculation, the test containers should be kept away from light
Magnesium Sulphate 0.24 and placed in a suitable system providing an aerobic atmosphere enriched
* Formula adjusted to suit performance parameters
with CO2.
Directions
3. Incubate at 35-370C. Slanted and bottled media should be incubated in a
1. Suspend 37.24 gms of the powder in 600 ml distilled water containing 12
horizontal plane until the inoculum is absorbed. The screw caps of containers
ml glycerol and mix thoroughly. (Do not add glycerol if bovine tubercle
should be loose for the first three weeks to allow circulation of CO2.
bacilli or other glycerophobic organisms are to be cultivated).
Thereafter, to prevent dehydration, the caps should be tightened and
2. Boil to dissolve the powder completely.
loosened briefly once a week.
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. Cool to
Note: Cultures from skin lesions suspected to be M.marinum or M.ulcerans
approximately 500C. 0
should be incubated at 25-33 C for primary isolation; cultures suspected to
4. Add 1000 ml sterile whole egg emulsion (AS010). Gruft Mycobacterial contain M.avium or M.xenopi exhibit optimum growth at 40-420C.
Supplement (AS 013) may be added if desired, 1 vial per 400 ml of above Interpretation of Results
medium and mix gently to obtain a uniform mixture. 1. Cultures should be read within 5-7 days after inoculation and once a week
5. Dispense in sterile screw-capped tubes. thereafter for up to 8 weeks.
6. Arrange tubes in slanted position. 2. Record the following observations: -
7. Coagulate and inspissate the medium in an inspissator water bath or a) Number of days required for colonies to become macroscopically
autoclave at 850C for 45 minutes. visible.
Quality Control Rapid growers have mature colonies within 7 days; slow growers
Dehydrated Appearance
require more than 7 days.
Greenish blue coloured, homogeneous, free flowing powder.
Prepared Appearance b) Pigment production
Basal medium with whole egg emulsion, on inspissation, coagulates to yield White, cream to buff Non-chromogenic.
pale bluish green coloured, opaque, smooth slants.
Lemon, yellow, orange, red Chromogenic.

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3. Stained smears may show acid fast bacilli, which are reported only as “acid zones indicates a decrease in pH by gram-positive contaminants like
fast bacilli” unless definitive tests are performed. streptococci and yellow zones of dye destruction by gram-negative bacilli.
Precautions / Limitations Proteolytic contaminants cause localized or complete digestion of the
1. Biosafety Level 2 practices, containment equipment and facilities are medium.
required for non-aerosol producing activities such as preparation of acid- 6. Negative culture results do not rule out active infection by mycobacteria. Some
fast smears. factors responsible for unsuccessful cultures are:
2. All aerosol generating activities must be conducted in a class 1 or 2 The specimen was not representative of the infectious material i.e. saliva
biological safety cabinet. Level 3 practices, containment equipment and instead of sputum.
facilities are required for laboratory activities in the propagation and The mycobacteria were destroyed during digestion and decontamination of the
manipulation of cultures of M.tuberculosis and M.bovis. specimen.
3. Lowenstein Jensen Medium Base requires incubation in a 5-10% CO2 Gross contamination interfered with the growth of the mycobacteria. Proper
atmosphere in order to recover mycobacteria. aerobic conditions and increased CO2 tension were not provided during
4. The medium should be protected from all sources of light, as malachite incubation.
Storage
green is highly photosensitive.
Store at 22-300C and prepared medium at 2-80C.
5. Do not use media that have turned yellow, as it will interfere with
Shelf Life
interpretation of the pigmentation of mycobacteria. Formation of blue
Use before expiry date as mentioned on the label.

Luria Agar AM10571/AM50571

Luria Broth AM10572/AM50572
Use Luria Agar - 35 grams
Luria Agar and Luria Broth are recommended for the cultivation and maintenance Luria Broth - 20 grams
of recombinant strains of Escherichia coli and may be used for routine cultivation 2. Heat with frequent agitation to dissolve the powder completely.
of not particularly fastidious microorganisms.
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Summary
4. Dispense as desired.
Luria Agar / Luria Broth as described by Lennox is used for the cultivation and
Quality Control
maintenance of recombinant strains of Escherichia coli.
Dehydrated Appearance
Principle
Light yellow coloured, homogeneous, free flowing powder.
Tryptone provides a source of peptones. Yeast Extract is a source of Vitamin B Prepared Appearance
complex. Sodium Chloride maintains the osmotic equilibrium. Agar is the Yellow to amber coloured clear to slightly opalescent gel.
solidification agent. Cultural Response
Formula* Cultural response after 18 - 24 hours at 350 C.
Ingredients in grams per liter Agar Broth Organisms (ATCC) Growth RGI
Tryptone 10.0 10.0 Escherichia coli (25922) Luxuriant More than 70%
Yeast Extract 5.0 5.0 For growth RGI should be more than 70%
Sodium Chloride 5.0 5.0 RGI- Relative Growth Index
Agar 15.0 -- Storage
Final pH (at 250 C) 7.0 ± 0.2 Store at 22-300C and prepared medium at 2-80C.
* Formula adjusted to suit performance parameters Shelf Life
Directions
Use before expiry date as mentioned on the label.
1. Suspend the powder in 1000 ml distilled water.

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Luria Bertani Agar, Miller AM50573
Luria Bertani Broth, Miller AM50574
Use Directions
Luria Bertani Agar, Miller and Luria Bertani Broth, Miller are used for 1. Suspend the powder in 1000 ml distilled water
maintenance and cultivation of Escherichia coli in molecular microbiology Luria Bertani Agar, Miller - 40.00gms
procedures. Luria Bertani Broth, Miller 25.00gms
Summary
2. Mix thoroughly.
Luria Bertani Agar, Miller and Luria Bertani Broth, Miller (1.1) are nutritionally
3. Heat with frequent agitation and boil for 1 minute to dissolve the powder
rich media recommended for growth of pure cultures of recombinant strains of E.
completely.
coli. The media are nutritionally rich suitable for the growth of pure cultures like
recombinant strains. For example Escherichia coli K12 and derived strains which 4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes
are deficient in Vitamin B synthesis and modified by specific mutation to create Quality Control
Dehydrated Appearance
auxotrophic organisms, that means they are not able to grow on nutritionally poor
Light yellow coloured, homogeneous free flowing powder.
media. Luria Bertani Agar, Miller and Luria Bertani Broth, Miller contain double
Prepared Appearance
amount of sodium chloride of the Luria Agar (AM10571) (69.1) and Luria Luria Bertani Agar, Miller - Yellow to amber coloured, slightly opalescent gel.
Broth(AM10572). This allows the researcher to select the optimal salt Luria Bertani Broth, Miller- Yellow to amber, slightly opalescent solution.
concentration for a specific strain. Cultural Response
Principle Cultural characteristics after 18-24 hours at 35-370C.
Tryptone and Yeast extract serve as a source of nitrogen, sulfur and carbon while Organisms(ATCC) Growth Luria Bertani Luria Bertani
Yeast extract also contains Vitamin B complex. Sodium chloride provides sodium Broth, (RGI) Broth (RGI)
Escherichia coli (25922) Luxuriant More than 70% Luxuriant
ions for the membrane transport and maintains osmotic equilibrium of the
Escherichia coli (23724) Luxuriant More than 70% Luxuriant
medium. Agar is the solidifying agent.
Procedure
Formula*
Ingredients in Luria Bertani Agar, Luria Bertani Broth, Consult appropriate references for recommended test procedures.
grams per liter Miller Miller Interpretation of Results
Tryptone 10.0 10.0 Growth is results in the form of isolated colonies and/or a confluent lawn on the
Yeast Extract 5.0 5.0 surface of the agar medium or the appearance of turbidity in the broth medium.
Sodium Chloride 10.0 10.0 Storage
Agar 15.0 -
Store at 22-300C and prepared medium at 2-80C.
Final pH (at 250C) 7.5 ±0.2
Shelf Life
* Formula adjusted to suit performance parameters
Use before expiry date as mentioned on the label.

LPM Agar Base AM10575

Use metabolism. Glycine anhydride, lithium chloride and phenylethyl alcohol
LPM Agar is recommended for isolation and cultivation of Listeria monocytogenes suppress gram – positive cocci and gram – negative rods. Moxalactam inhibits
from food and dairy products. both gram-positive and gram-negative bacteria including Staphylococci, Proteus
Summary and Pseudomonas species. Listeria monocytogenes show blue-green iridescence
LPM Agar is a modified McBride agar developed by Lee and McClain. This medium when examined with oblique transmitted light.
is recommended by APHA for the isolation of Listeria monocytogenes. Formula*
Principle Ingredients in grams per liter
Casein enzymic hydrolysate 5.00
Casein enzymic hydrolysate and beef extract provides all essential nutrients for
Peptic digest of animal tissue 5.00

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Beef extract 3.00 Quality Control
Glycine anhydride 10.00 Dehydrated Appearance
Lithium chloride 5.00 Light yellow coloured, homogeneous, free flowing powder.
Sodium chloride 5.00 Prepared Appearance
Phenylethyl alcohol 2.50 Light yellow coloured, clear to slightly opalescent gel forms in petriplates.
Agar 15.00 Cultural Response
Final pH (at 250C) 7.3 ±0.2 Cultural characteristics after 24-48 hours at 35-370C.
* Formula adjusted to suit performance parameters Organisms (ATCC) Growth RGI
Directions Listeria monocytogenes (19112) Good - luxuriant More than 70%
1. Suspend the 50.5 gms of powder in 1000 ml distilled water. Staphylococcus aureus (25923) Inhibited 0%
Pseudomonas aeruginosa (27853) Inhibited 0%
2. Mix thoroughly.
Escherichia coli (25922) Inhibited 0%
3. Heat to boiling to dissolve the medium completely. For growth RGI should be more than 70%
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 12 minutes. For Inhibition RGI should be 0%
RGI- Relative Growth Index
5. Cool to 500C and aseptically add rehydrated contents of 1 vial of
Storage
Moxalactam Supplements (AS0182).
Store at 22-300C and prepared medium at 2-80C.
6. Mix well before pouring into sterile petri plates. Shelf Life
Caution: Lithium chloride is harmful. Avoid bodily contact and
inhalation of vapours. On contact with skin, wash with plenty of water Use before expiry date as mentioned on the label.
immediately.

Lysine Iron Agar AM10576/AM50576

Use Formula*
Lysine Iron Agar is used for differentiation of enteric bacteria on the basis of Ingredients in grams per liter
Peptic digest of animal tissue 5.0
hydrogen sulphide production and the decarboxylation or deamination of lysine.
Yeast extract 3.0
Summary
Dextrose 1.0
Edwards P.R. and Mary A. Fife designed Lysine Iron Agar in 1961 (86.1). A Lysine L-Lysine 10.0
Iron Agar is described and recommended for the detection of Arizona strains, Ferric ammonium citrate 0.50
which ferment lactose rapidly. Salmonella and Arizona cultures produce a Sodium thiosulphate 0.04
distinctive reaction since they are only recognized groups of enteric bacteria, Bromocresol purple 0.02
which regularly produce lysine decarboxylase rapidly and form large amount of Agar 15.0
hydrogen sulphide. Final pH (at 250C) 6.7 ±0.2
Principle * Formula adjusted to suit performance parameters
Directions
Peptic digest of animal tissue and yeast extract serves as a source of carbon,
1. Suspend the 34.56 gms of powder in 1000 ml distilled water.
nitrogen, vitamins and minerals. Bromocresol purple act as a indicator. An
alkaline reaction is seen by the presence of a purple colour, and an acidic reaction 2. Mix thoroughly.
is indicated by the appearance of a yellow colour. Sodium thiosulphate is the 3. Boil with frequent agitation to dissolve the powder completely. DO NOT
source of hydrogen sulphide, and ferric ammonium citrate as the indicator, which OVERHEAT.
turns the butt black in the presence of free hydrogen sulphide gas. Lysine is added 4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
to show the decarboxylation reaction, which causes an alkaline situation to occur, Quality Control
seen as a purple butt. The yellow colour is seen only if lysine decarboxylation does Dehydrated Appearance
not occur, as this reaction overcomes any acidic (yellow) conditions. If lysine is Yellow coloured, homogeneous, free flowing powder.
deaminated in the presence of oxygen (the reaction seen in the presence of Prepared Appearance
Proteus and Providencia species), a red colour change is seen on the slant. Purple coloured, slightly opalescent gel forms in tubes.

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Cultural Response 3. Incubate at 350C with caps loosened and examine after 18-24 hours for
0
Cultural characteristics after 18-24 hours at 35-37 C. carbohydrate fermentation, gas production and hydrogen sulphide
Organisms (ATCC) Growth Butt Slant H2 S
production.
Citrobacter freundii (8090) Luxuriant A K +
Escherichia coli (25922) Luxuriant K K _
Interpretation of Results
Proteus mirabilis (25933) Luxuriant A R + Lysine Decarboxylation is detected in the butt by an alkaline (Purple) reaction.
S.serotype typhimurium Luxuriant K K + Lysine deamination is detected by a red slant. Hydrogen sulphide production is
(14028) detected by the formation of a black precipitate. A negative reaction (purple slant
Shigella flexneri (12022) Luxuriant A K _ and yellow butt) indicates fermentation of dextrose only.
S. serotype arizonae Luxuriant K K +
(13314)
Hydrogen sulphide may not be detected in this medium by organisms that are
Key: negative for lysine decarboxylase activity since acid production in the butt may
A= Acid (yellow) suppress its formation. Because of this, hydrogen sulphide producing Proteus
K= Alkaline (red purple, no change in colour) species do not blacken this medium.
R= Red (lysine deaminase) Precautions / Limitations
H2 S + = Black precipitate It is important to stab the butt of the medium. Failure to stab the butt invalidates
H2 S _ = No black precipitate
this test. Do not use an inoculating loop to inoculate a tube of Lysine Iron Agar
Procedure
because while stabbing the butt, mechanical splitting of the medium occurs,
1. Touch only the center of an isolated colony on an enteric plated medium with causing a false positive result for gas production. Caps must be loosened during
a cool and sterile needle, stab into the medium and then streak back and this test or erroneous results will occur.
forth along the surface of the slant. Storage
2. Several colonies from each primary plate should be studied separately, since Store at 22-300C and prepared medium at 2-80C.
mixed infections may occur. Shelf Life
Use before expiry date as mentioned on the label.
Lysine Medium Base AM10577/AM50577
Use Adenine 0.00178
Lysine Medium Base is recommended for isolation and enumeration of wild DL-Methionine 0.000891
L-Histidine 0.000891
yeasts in pitching yeasts.
DL-Tryptophan 0.000891
Summary
Boric acid 0.0000089
Lysine Medium Base is used for the isolation and enumeration of wild yeasts Zinc sulphate 0.0000356
encountered in brewing. On this medium pitching yeasts are suppressed. Morris & Ammonium molybdate 0.0000178
Eddy first described this complex medium (80.1). Walters and thiselton Manganese sulphate 0.0000356
developed a liquid synthetic medium containing lysine as the sole nitrogen source Ferrous sulphate 0.0002225
for yeasts (117.1). Later Morris & Eddy formulated a solid lysine medium. L-Lysine 1.00
Principle Inositol 0.02
Calcium pantothenate 0.002
Dextrose is the source of energy. Lysine serve as sole nitrogen source. Different
Aneurine 0.0004
salts and amino acids support the growth of bacteria. Pyridoxine 0.0004
Formula* p-Amino benzoic acid 0.0002
Ingredients in grams per liter Nicotinic acid 0.0004
Dextrose 44.50 Riboflavin 0.0002
Monopotassium phosphates 1.78 Biotin 0.000002
Magnesium phosphates 0.89 Folic acid 0.000001
Calcium chloride 0.178 Agar 17.80
Sodium chloride 0.089 Final pH (at 250C) 5.0 ±0.2

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* Formula adjusted to suit performance parameters Cultural Response
Directions Cultural characteristics at 250C for 7 days.
1. Suspend 6.6 gm of the powder in 100 ml-distilled water containing 1ml Organisms (ATCC) Growth
potassium lactate (50%) (AS0211). Pichia fermentans (10651) Luxuriant
Procedure
2. Mix thoroughly.
Refer to appropriate references for specific procedures.
3. Boil to dissolve the powder completely. DO NOT AUTOCLAVE. Interpretation of Results
4. Cool the medium to 500C, adjust pH to 5.0 with 10% lactic acid and pour Refer to appropriate references and procedures for results.
into sterile petri dishes. Storage
Quality Control
Store at 22-300C and prepared medium at 2-80C.
Dehydrated Appearance
Shelf Life
White coloured, homogeneous, free flowing powder.
Prepared Appearance Use before expiry date as mentioned on the label.
Colourless, clear to slightly opalescent gel forms in petri plates.

M 17 Agar AM50578
Use Final pH ( at 25°C) 7.1±0.2
M 17 Agar used for cultivation of lactic Streptococci and plaque assay of lactic * Formula adjusted to suit performance parameters
Directions
bacteriophages.
Summary 1. Suspend 33.25 gms powder in 1000 ml distilled water.
M17 media are based on the formulation described by Terzaghi and Sandine 2. Add 19grams of Disodium ß-Glycerophosphate. Heat to boiling to dissolve
(109.1) for the cultivation and enumeration of lactic Streptococci and their the medium completely.
bacteriophages. It is possible to study plaque morphology and lysogeny. M17 3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Agar is recommended by the International Dairy Federation (46.1.2) for selective 4. Mix well and dispense as desired.
enumeration of Streptococcus thermophilus from yoghurt. M17 Agar is Quality Control
recommended by APHA for the cultivation of lactic Streptococci (20). Dehydrated Appearance
Principle Cream to yellow homogeneous free flowing powder.
Peptic digest of animal tissue, papaic digest of soyabean meal, yeast extract, beef Prepared Appearance
extract provide carbonaceous, nitrogenous compounds, vitamin B complex and Light yellow coloured clear to slightly opalescent gel forms in Petri plates.
Cultural Response
other essential growth factors. Lactose is the fermentable carbohydrate and
Cultural characteristics after 24-48 hours at 35-370C with added Disodium ß-
ascorbic acid is stimulatory for the growth of lactic Streptococci. Magnesium Glycerophosphate..
sulphate provides essential ions to the organisms. Disodium-beta- Organisms (ATCC) Growth RGI
glycerophosphate maintains the pH above 5.7. The maintenance of pH is very Enterococcus faecalis (29212) Luxuriant More than 70%
important as lower pH results in injury and reduced recovery of lactic Streptococci. Lactobacillus bulgaricus (11842) None-poor 0%
Disodium glycerophosphate suppresses Lactobacillus bulgaricus . Lactobacillus leichmannii (4797) Luxuriant More than 70%
Formula* Lactobacillus plantarum (8014) Luxuriant More than 70%
Ingredients in grams per liter Streptococcus thermophilus (14485) Luxuriant More than 70%
Peptic digest of animal tissue 5.0 For growth RGI should be more than 70%
Papaic digest of soyaben meal 5.0 For Inhibition RGI should be 0%
Yeast extract 2.5 RGI- Relative Growth Index
Beef extract 5.0 Storage
Ascorbic acid 0.5 Store at 22-300C and prepared medium at 2-80C.
Magnesium sulphate 0.25 Shelf Life
Lactose 5.0
Use before expiry date as mentioned on the label.
Agar 10.0

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M 17 Broth AM50579
Use Lactose 5.0
MM 17 Broth used for cultivation of lactic Streptococci and their bacteriophages. Ascorbic acid 0.5
Disodium - ß - glycerophosphate 19.0
Summary
Magnesium sulphate 0.25
M17 broth is based on the formulation described by Terzaghi and Sandine Final pH (at 250C) 7.1 ± 0.2
(109.1) for the cultivation and enumeration of lactic Streptococci and their * Formula adjusted to suit performance parameters
bacteriophages. M17 Broth is a modification of M16 Medium (64.1). Lactic Directions
Streptococci are nutritionally fastidious and require complex media for optimal 1. Suspend 42.25 gms powder in 1000 ml distilled water and mix thoroughly.
growth (1.4 & 91.3)). Disodium glycerophosphate maintains the pH above 5.7. 2. Heat if necessary to dissolve the powder completely. AVOID OVERHEATING.
The maintenance of pH is very important as the lower pH results in injury and
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
reduced recovery of lactic Streptococci. Glycerophosphate does not form
4. Mix well and dispense as desired.
precipitate with calcium which is needed for the plaque assay of lactic
Quality Control
bacteriophages.
Dehydrated Appearance
Principle
Cream to yellow homogeneous free flowing powder.
Peptic digest of animal tissue, casein enzymix hydrolysate, papaic digest of Prepared Appearance
soyabean meal, yeast extract, beef extract, provide carbonaceous, nitrogenous Light yellow coloured clear to slightly opalescent gel forms in Petri plates.
compounds, vitamin B complex and other essential growth factors. Lactose is the Cultural Response
fermentable carbohydrate and ascorbic acid is stimulatory for the growth of lactic Cultural characteristics after 24-48 hours at 35-370C
Streptococci. Magnesium sulphate provides essential ions to the organisms. Organisms (ATCC) Growth
Enterococcus faecalis (29212) Luxuriant
Disodium - ß – glycerophosphate maintains the pH above 5.7. The maintenance
Lactobacillus bulgaricus (11842) None-poor
of pH is very important as lower pH results of in injury and reduced recovery of
Lactobacillus leichmannii (4797) Luxuriant
lactic Streptococci. Lactobacillus plantarum (8014) Luxuriant
Formula* Streptococcus thermophilus (14485) Luxuriant
Ingredients in grams per liter Storage
Peptic digest of animal tissue 2.5 0 0
Store at 22-30 C and prepared medium at 2-8 C.
Casein enzymic hydrolysate 2.5
Papaic digest of soyabean meal 5.0 Shelf Life
Yeast extract 2.5 Use before expiry date as mentioned on the label.
Beef extract 5.0

MacConkey Agar Base AM1058/AM5058

MacConkey Agar with Crystal Violet, NaCl and AM1059/AM5059
0.15% Bile Salts
MacConkey Agar without Crystal Violet and AM1060/AM5060
with 0.15% Bile Salts
MacConkey Agar without Crystal Violet, NaCl and AM1061/AM5061
with 0.5% Sodium Taurocholate
Use Violet, NaCl and 0.15% Bile Salts is a slightly selective and differential medium
MacConkey Agar Base is used for studying carbohydrate fermentation reactions of for the detection of coliforms and enteric pathogens. MacConkey Agar without
coliforms by adding the desired carbohydrate. MacConkey Agar with Crystal Crystal Violet and with 0.15% Bile Salts is used for selective isolation and

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differentiation of lactose fermenting and non-lactose fermenting enteric bacteria. The lack of crystal violet in MacConkey Agar without Crystal Violet, NaCl & with
MacConkey Agar without Crystal Violet, NaCl & with Sodium Taurocholate is used Sodium Taurocholate permits the growth of Staphylococcus and Enterococcus. It is
for the cultivation and differentiation of enteric bacteria and potentially a differential medium used for isolating and cultivating gram-negative enteric
pathogenic gram-positive organisms, while restricting swarming of Proteus microorganisms and gram-positive cocci. Swarming of Proteus is reduced due to the
species. lack of salt in this medium.
Summary The above-mentioned MacConkey Agars except MacConkey Agar Base are
MacConkey Agar is the earliest selective and differential medium for cultivation of recommended for use in microbiological examination of clinical specimens,
enteric microorganisms from a variety of specimens like water, faeces and other foodstuffs (20) and for direct plating and inoculation of water samples (36) for
sources suspected of containing these microorganisms (76, 77). The original coliform counts. These media are included in the Standard Methods for the
MacConkey Agar was based on the bile salt-neutral red-lactose agar of Examination of Milk and Dairy Products (39), pharmaceutical preparations (46,
MacConkey, which was used to differentiate strains of Salmonella typhosa from 114) and industrial sources. They are also included in the Official Methods of
members of the coliform group. The formula was further modified to be more Analysis of AOAC International as well as the Bacteriological Analytical Manual
selective. The presence of bile salts allows the growth of enteric gram-negative (113).
organisms. Principle
MacConkey Agar Base is used with added carbohydrates in differentiating Peptone, proteose peptone and tryptone provide nitrogen and other nutrients, while
coliforms based on fermentation reactions. lactose is the carbohydrate source. Bile salts and crystal violet are selective agents
MacConkey Agar with Crystal Violet, NaCl and 0.15% Bile Salts is designed to that inhibit the growth of gram-positive bacteria but allow enteric gram-negative
achieve more differentiation of lactose fermenters and non-lactose fermenters, for bacteria to grow. Sodium taurocholate is also a selective agent that inhibits gram-
the promotion of superior growth of enteric pathogens and to improve the positive bacteria except staphylococci and enterococci. Neutral red is the pH
inhibition of swarming Proteus species. indicator.
Directions
MacConkey Agar without Crystal Violet and with 0.15% Bile Salts though less
selective than the original MacConkey Agar is used for isolating and cultivating 1. Suspend the powder in 1000 ml distilled water and mix thoroughly.
gram-negative enteric microorganisms. MacConkey Agar Base - 40 gms
Formula*
Ingredients in grams per liter AM1058/AM5058 AM1059/AM5059 AM1060/AM5060 AM1061/AM5061
Peptone 17.0 1.5 17.0 20.0
Proteose Peptone 3.0 - 3.0 -
Tryptone - 1.5 - -
Pancreatic Digest of Gelatin - 17.0 - -
Lactose - 10.0 10.0 10.0
Bile Salts 1.5 1.5 1.5 -
Sodium Chloride 5.0 5.0 5.0 -
Crystal Violet 0.001 0.001 - -
Neutral Red 0.03 0.03 0.03 0.04
Sodium Taurocholate - - - 5.0
Agar 13.5 15.0 15.0 20.0
Final pH (at 250C) 7.1 ± 0.2 7.1 ± 0.2 7.1 ± 0.2 7.4 ± 0.2
* Formula adjusted to suit performance parameters

MacConkey Agar with Crystal Violet, NaCl and 0.15% Bile Salts - 51.5 gms 2. Boil with frequent agitation to dissolve the powder completely. AVOID
MacConkey Agar without Crystal Violet and with 0.15% Bile Salts - 51.53 OVERHEATING.
gms 3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
MacConkey Agar without Crystal Violet, NaCl & with Sodium Taurocholate 4. Cool to 45-500C and (For MacConkey Agar Base add sterile 1% w/v desired
55 gms carbohydrate solution) pour into sterile petri plates.

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Quality Control Salmonella serotype Enteritidis (13076) Luxuriant Colourless
Dehydrated Appearance Shigella flexneri (12022) Luxuriant Colourless
Pinkish beige coloured, homogeneous, free flowing powder. Staphylococcus aureus (25923) Fair to good Pale pink to red
Prepared Appearance Procedure
MacConkey Agar Base & MacConkey Agar with Crystal Violet, NaCl and 1. The surface of the medium should be dry when inoculated.
0.15% Bile Salts Red with purplish tinge, clear to slightly opalescent gel.
MacConkey Agar without Crystal Violet and with 0.15% Bile Salts & 2. Use standard procedures to obtain isolated colonies from specimens.
MacConkey Agar without Crystal violet, NaCl & with Sodium Taurocholate 3. A non-selective medium should also be streaked to increase the chances of
Orange red coloured, clear to slightly opalescent gel.
recovery when the population of gram-negative organisms is low and to
Cultural Response
provide an indication of other organisms present in the specimen.
Cultural characteristics after 18-24 hours at 350C.
Interpretation of Results
MacConkey Agar with Crystal Violet, NaCl and 0.15% Bile Salts
Organisms (ATCC) Growth Colour of For MacConkey Agar with Crystal Violet, NaCl and 0.15% Bile Salts & MacConkey
Colony Agar without Crystal Violet and with 0.15% Bile Salts.
Enterobacter aerogenes (13048) Luxuriant Pink to red 1. Lactose fermenting bacteria produce pink to brick-red colonies and may be
Enterococcus faecalis (29212) Inhibited -
surrounded by a zone of bile precipitation.
Escherichia coli (25922) Luxuriant Pink to red with
bile precipitate 2. Non-Lactose fermenting bacteria produce colourless colonies.
Proteus vulgaris (13315) Luxuriant Colourless For MacConkey Agar without Crystal violet, NaCl & with Sodium Taurocholate.
Salmonella serotype Enteritidis (13076) Luxuriant Colourless
1. Lactose fermenting organisms grow as pink to brick red colonies with or
Shigella flexneri (12022) Fair to good Colourless
without a zone of precipitated bile.
Staphylococcus aureus (25923) Inhibited -
Cultural characteristics after 18-24 hours at 350C. 2. Non-Lactose fermenters grow as colourless or clear colonies.
MacConkey Agar without Crystal Violet and with 0.15% Bile Salts 3. Staphylococci produce pale pink to red colonies.
Organisms (ATCC) Growth Colour of
4. Enterococci produce tiny red colonies.
Colony
Enterobacter aerogenes (13048) Luxuriant Pink 5. Rapid growers of the M.fortuitum complex usually grow in 5-11days, while
Enterococcus faecalis (29212) Inhibited - the commonly saphyrophytic species are inhibited.
Escherichia coli (25922) Luxuriant Pink to red 6. The swarming of Proteus is reduced on this medium.
Proteus vulgaris (13315) Luxuriant Colourless Precautions / Limitations
Salmonella serotype Enteritidis (13076) Luxuriant Colourless
1. Incubation of plates under increased CO2 has been reported to reduce the
Shigella flexneri (12022) Fair to good Colourless
Staphylococcus aureus (25923) Inhibited - growth and recovery of a number of strains of gram-negative bacilli.
Cultural characteristics after 18-24 hours at 35-370C. 2. Some strains of M.smegmatis from humans may grow on MacConkey Agar
MacConkey Agar without Crystal Violet, NaCl & with 0.5% Sodium without Crystal Violet, NaCl & with Sodium Taurocholate, but these strains
Taurocholate
can be differentiated from M.fortuitum complex by the 3-day arylsulphatase
Organisms (ATCC) Growth Colour of
test.
Colony
Enterobacter aerogenes (13048) Luxuriant Pink to red 3. Not all strains of E.coli ferment lactose.
Enterococcus faecalis (29212) Fair to good Pale pink to red Storage
Escherichia coli (25922) Luxuriant Pink to red with Store at 22-300C and prepared medium at 2-80C.
bile precipitate Shelf Life
Proteus vulgaris (13315) Luxuriant Colourless Use before expiry date as mentioned on the label.

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MacConkey Agar (Harmonized) AMH5059
MacConkey Agar Base Medium USP AM10581/AM50581
Use Quality Control
MacConkey Agar Medium is a selective and differential medium for the detection Dehydrated Appearance
Pinkish beige coloured, homogeneous, free flowing powder.
of coliforms and enteric pathogens, in compliance with USP.
Prepared Appearance
Summary
Red with purplish tinge, clear to slightly opalescent gel.
MacConkey Agar is the earliest selective and differential medium for cultivation of Cultural Response
enteric microorganisms from a variety of specimens like water, faeces and other Cultural characteristics after 18-24 hours at 350C.
sources suspected of containing these microorganisms. The original MacConkey Organisms (ATCC) Growth Colour of RGI
Agar was based on the bile salt-neutral red-lactose agar of MacConkey, which Colony
was used to differentiate strains of Salmonella typhosa from members of the Enterobacer aerogenes luxuriant Pink to red More than 70%
coliform group. MacConkey Agar with Crystal Violet, NaCl and 0.15% Bile Salts is (13048)
designed to achieve more differentiation of lactose fermenters and non-lactose Enterococcus faecalis Fair to good Colourless 0%
(29212) to pink
fermenters, for the promotion of superior growth of enteric pathogens. It is Escherichia coli Luxuriant pink to red More than 70%
recommended by USP for microbial limit tests. (25922) with bile
Principle precipitate
Pancreatic digest of casein and peptic digest of animal tissue provide nitrogen and
other nutrients, while lactose is the carbohydrate source. Bile salts and crystal Proteus vulgaris Luxuriant Colourless More than 70%
(13315)
violet are selective agents that inhibit the growth of gram-positive bacteria but
Salmonella serotype Luxuriant Colourless More than 70%
allow enteric gram-negative bacteria to grow. Neutral red is the pH indicator.
Enteritidis (13076)
Formula*
Shigella flexneri Fair to good Colourless More than 70%
Ingredients in grams MacConkey Agar MacConkey Agar (12022)
per liter Medium USP (Harmonized) Staphylococcus aureus Inhibited - 0%
Pancreatic digest of casein 1.5 – (25923)
Peptic digest of animal tissue 1.5 – Procedure
Peptone (Meat and casein) – 3.0
Bile salts mixture 1.5 1.5
1. The surface of the medium should be dry when inoculated.
Pancreatic digest of gelatin 17.0 17.0 2. Use standard procedures to obtain isolated colonies from specimens.
Lactose 10.0 10.0 3. A non-selective medium should also be streaked to increase the chances of
Sodium chloride 5.0 5.0
recovery when the population of gram-negative organisms is low and to
Crystal violet 0.001 0.001
provide an indication of other organismspresent in the specimen.
Neutral red 0.03 0.03
Interpretation of Results
Agar 13.5 13.5
Final pH (at 250C) 7.1 ±0.2 1. Lactose fermenting bacteria produce pink to brick-red colonies and may be
* Formula adjusted to suit performance parameters surrounded by a zone of bile precipitation.
Directions 2. Non-Lactose fermenting bacteria produce colourless colonies.
1. Suspend 50.03 gms powder in 1000 ml distilled water and mix Precautions / Limitations
thoroughly. 1. Incubation of plates under increased CO2 has been reported to reduce the
2. Boil with frequent agitation to dissolve the powder completely. AVOID growth and recovery of a number of strains of gramnegative bacilli.
OVERHEATING. 2. Some strains of M.smegmatis from humans may grow on MacConkey Agar
0
3. Sterilize by autoclaving at 121 C (15 lbs pressure) for 15 minutes. without Crystal Violet, NaCl & with Sodium Taurocholate, but these strains
4. Cool to 45-500C and (For MacConkey Agar Base add sterile 1% w/v can be differentiated from M.fortuitum complex by the 3-day arylsulphatase
desired carbohydrate solution) pour into sterile petri plates. test.

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3. Not all strains of E.coli ferment lactose. Shelf Life
Storage Use before expiry date as mentioned on the label.
0 0
Store at 22-30 C and prepared medium at 2-8 C.

MacConkey Agar with CV, NaCl and 0.15% Bile Salts IP AM50591
MacConkey Agar with CV, NaCl and 0.15% Bile Salts USP AM50592
MacConkey Agar with CV, NaCl and 0.15% Bile Salts BP AM50594
Use 3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
MacConkey Agar Medium is a selective and differential medium for the detection 4. Cool to 45-500C and (For MacConkey Agar Base add sterile 1% w/v desired
of coliforms and enteric pathogens. carbohydrate solution) pour into sterile petri plates.
Summary Quality Control
MacConkey Agar is the earliest selective and differential medium for cultivation of Dehydrated Appearance
enteric microorganisms from a variety of specimens like water, faeces and other Pinkish beige coloured, homogeneous, free flowing powder.
sources suspected of containing these microorganisms. The original MacConkey Prepared Appearance
Agar was based on the bile salt-neutral red-lactose agar of MacConkey, which Red with purplish tinge, clear to slightly opalescent gel.
Cultural Response
was used to differentiate strains of Salmonella typhosa from members of the
Cultural characteristics after 18-24 hours at 350C.
coliform group. MacConkey Agar with Crystal Violet, NaCl and 0.15% Bile Salts is
Organisms (ATCC) Growth Colour of Colony RGI
designed to achieve more differentiation of lactose fermenters and non-lactose Enterobacter aerogenes Luxuriant Pink to red More than 70%
fermenters, for the promotion of superior growth of enteric pathogens. It is (13048)
recommended by USP for microbial limit tests. Enterococcus faecalis Fair to good Colourless to pink More than 70%
Principle (29212)
Pancreatic digest of casein and peptic digest of animal tissue provide nitrogen and Escherichia coli Luxuriant Pink to red with bile More than 70%
other nutrients, while lactose is the carbohydrate source. Bile salts and crystal (25922) precipitate
Proteus vulgaris Luxuriant Colourless More than 70%
violet are selective agents that inhibit the growth of gram-positive bacteria but
(13315)
allow enteric gram-negative bacteria to grow. Neutral red is the pH indicator.
Salmonella serotype Luxuriant Colourless More than 70%
Formula*
Enteritidis (13076)
Ingredients in grams per liter
Shigella flexneri (12022) Fair to good Colourless More than 70%
Pancreatic digest of casein 1.5
Staphylococcus aureus Inhibited - 0%
Peptic digest of animal tissue 1.5
(25923)
Bile salts mixture 1.5
For growth RGI should be more than 70%
Pancreatic digest of gelatin 17.0
For Inhibition RGI should be 0%
Lactose 10.0
RGI- Relative Growth Index
Sodium chloride 5.0
Procedure
Crystal violet 0.001
Neutral red 0.03
1. The surface of the medium should be dry when inoculated.
Agar 13.5 2. Use standard procedures to obtain isolated colonies from specimens.
Final pH (at 250C) 7.1 ±0.2 3. A non-selective medium should also be streaked to increase the chances of
* Formula adjusted to suit performance parameters
recovery when the population of gram-negative organisms is low and to
Directions
provide an indication of other organisms present in the specimen.
1. Suspend 50.03 gms powder in 1000 ml distilled water and mix thoroughly. Interpretation of Results
2. Boil with frequent agitation to dissolve the powder completely. AVOID 1. Lactose fermenting bacteria produce pink to brick-red colonies and may be
OVERHEATING. surrounded by a zone of bile precipitation.

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2. Non-Lactose fermenting bacteria produce colourless colonies. can be differentiated from M.fortuitum complex by the 3-day arylsulphatase
Precautions / Limitations test.
1. Incubation of plates under increased CO2 has been reported to reduce the 3. Not all strains of E.coli ferment lactose.
growth and recovery of a number of strains of gramnegative bacilli. Storage
2. Some strains of M.smegmatis from humans may grow on MacConkey Agar Store at 22-300C and prepared medium at 2-80C.
without Crystal Violet, NaCl & with Sodium Taurocholate, but these strains Shelf Life
Use before expiry date as mentioned on the label.

MacConkey Agar with Crystal Violet, NaCl and AM50593

0.15% Bile Salts (Agar Medium H)EP
Use 2. Boil with frequent agitation to dissolve the powder completely.
MacConkey Agar Medium is a selective and differential medium for the detection 3. Sterilize by autoclaving at 121ºC (15 lbs pressure) for 15 minutes.
of coliforms and enteric pathogens, in compliance with EP. Quality Control
Summary Dehydrated Appearance
MacConkey Agar is the earliest selective and differential medium for cultivation of Pinkish beige coloured, homogeneous, free flowing powder.
enteric microorganisms from a variety of specimens like water, faeces and other Prepared Appearance
sources suspected of containing these microorganisms. The original MacConkey Red with purplish tinge, clear to slightly opalescent gel.
Cultural Response
Agar was based on the bile salt-neutral red-lactose agar of MacConkey, which
Cultural characteristics after 18-24 hours at 35ºC.
was used to differentiate strains of Salmonella typhosa from members of the
Organisms Growth Colour of RGI
coliform group. (ATCC) Colony
MacConkey Agar with Crystal Violet, NaCl and 0.15% Bile Salts is designed to Enterobacter aerogenes Luxuriant Pink to red More than 70%
achieve more differentiation of lactose fermenters and non-lactose fermenters, for (13048)
the promotion of superior growth of enteric pathogens. EP recommends it for tests Enterococcus faecalis Inhibited Colourless 0%
(29212) to pink
for microbial contaminations.
Escherichia coli (25922) Luxuriant Pink to red More than 70%
Principle
with bile
Pancreatic digest of gelatin and peptones provide nitrogen and other nutrients, precipitate
while lactose is the carbohydrate source. Bile salts and crystal violet are selective Proteus vulgaris (13315) Luxuriant Colourless More than 70%
agents that inhibit the growth of gram-positive bacteria but allow enteric gram- Salmonella enteritidis Luxuriant Colourless More than 70%
negative bacteria to grow. Neutral red is the pH indicator. (13076)
Formula* Shigella flexneri Fair to good Ccolourless More than 70%
Ingredients in grams per liter (12022)
Peptones (meat and casein) 3.0 Staphylococcus aureus Inhibited - 0%
Bile salts 1.5 (25923)
Pancreatic digest of gelatin 17.0 For growth RGI should be more than 70%
Lactose monohydrate 10.0 For Inhibition RGI should be 0%
Sodium chloride 5.0 RGI- Relative Growth Index
Crystal violet 0.001 Procedure
Neutral red 0.03 1. The surface of the medium should be dry when inoculated.
Agar 13.5
2. Use standard procedures to obtain isolated colonies from specimens.
Final pH (at 25ºC) 7.1 ± 0.2
* Formula adjusted to suit performance parameters 3. A non-selective medium should also be streaked to increase the chances of
Directions recovery when the population of gram-negative organisms is low and to
1. Suspend 50.03 gms powder in 1000 ml distilled water and mix thoroughly. provide an indication of other organisms present in the specimen.

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187
188
185
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Interpretation of Results smegmatis from humans may grow on MacConkey Agar without Crystal Violet,
1. Lactose fermenting bacteria produce pink to brick-red colonies and may be NaCl & with Sodium Taurocholate, but these strains can be differentiated from M.
surrounded by a zone of bile precipitation. fortuitum complex by the 3-day arylsulphatase test. Not all strains of E. coli
2. Non-Lactose fermenting bacteria produce colourless colonies. ferment lactose.
Precautions / Limitations Storage

Incubation of plates under increased CO2 has been reported to reduce the growth Store at 22-300C and prepared medium at 2-80C.
Shelf Life
and recovery of a number of strains of gram-negative bacilli. Some strains of M.
Use before expiry date as mentioned on the label.

MacConkey Agar without Crystal Violet and AM50601

with 0.5% Bile Salts
Use Quality Control
MacConkey Agar without CV and with 0.5% Bile salts is used for isolation and Dehydrated Appearance
Pinkish beige coloured, homogeneous, free flowing powder.
differentiation of lactose fermenting and non-lactose fermenting enteric bacteria.
Prepared Appearance
Summary
Orange red coloured, clear to slightly opalescent gel forms in petri plates.
MacConkey Agar is the earliest selective and differential medium for cultivation of Cultural Response
enteric microorganisms from a variety of specimens like water, faeces and other Cultural characteristics after 18-24 hours at 350C.
sources suspected of containing these microorganisms. The original MacConkey Organisms (ATCC) Growth Colour of Colony
Agar was based on the bile salt-neutral red-lactose agar of MacConkey, which was Enterobacter aerogenes (13048) Luxuriant Ppink to red
used to differentiate strains of Salmonella typhosa from members of the coliform Enterococcus faecalis (29212) Fair to good Pale pink to red
group. MacConkey Agar without Crystal Violet and with 0.5% Bile Salts is less Escherichia coli (25922) Luxuriant Pink to red
Proteus vulgaris (13315) Luxuriant Colourless
selective than the original MaConkey Agar is used for isolating and cultivating
Salmonella serotypeEnteritidis (13076) Luxuriant Colourless
gram-negative enteric microorganisms.
Shigella flexneri (12022) Luxuriant Colourless
Principle
Staphylococcus aureus (25923) Fair to Good Pale pink to red
Peptone provide nitrogen and other nutrients, while lactose is the carbohydrate Procedure
source. Bile salts is selective agents that inhibit the growth of gram-positive 1. The surface of the medium should be dry when inoculated.
bacteria but allow enteric gram-negative bacteria to grow. Neutral red is the pH
2. Use standard procedures to obtain isolated colonies from specimens.
indicator.
Formula* 3. A non-selective medium should also be streaked to increase the chances of
Ingredients in grams per liter recovery when the population of gram-negative organisms is low and to
Peptone 20.0 provide an indication of other organisms present in the specimen.
Bile salts mixture 5.00 Interpretation of Results
Lactose 10.0 1. Lactose fermenting bacteria produce pink to brick-red colonies and may be
Sodium chloride 5.0
surrounded by a zone of bile precipitation.
Neutral red 0.075
Agar 12.0 2. Non-Lactose fermenting bacteria produce colourless colonies.
0
Final pH (at 25 C) 7.4 ±0.2 Precautions / Limitations
* Formula adjusted to suit performance parameters 1. Incubation of plates under increased CO2 has been reported to reduce the
Directions growth and recovery of a number of strains of gram-negative bacilli.
1. Suspend 52 gms powder in 1000 ml distilled water and mix thoroughly. 2. Some strains of M.smegmatis from humans may grow on MacConkey Agar
2. Boil with frequent agitation to dissolve the powder completely. AVOID without Crystal Violet, NaCl & with Sodium Taurocholate, but these strains
OVERHEATING. can be differentiated from M.fortuitum complex by the 3-day arylsulphatase
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. test.

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3. Not all strains of E.coli ferment lactose. Shelf Life
Storage Use before expiry date as mentioned on the label.
0 0
Store at 22-30 C and prepared medium at 2-8 C.

MacConkey Broth Double Strength with AM1062/AM5062

Neutral Red
MacConkey Broth Purple with AM1063/AM5063
Bromocresol Purple
MacConkey Broth with Neutral Red AM1064/AM5064
Use Lactose 20.0 10.0 10.0
MacConkey Broth Double Strength with Neutral Red is used for the primary Bile Salts 10.0 - 5.0
Sodium Chloride 10.0 5.0 5.0
isolation of coliforms from large samples such as water and wastewater.
Neutral Red 0.15 - 0.075
MacConkey Broth Purple with Bromocresol Purple is used for the presumptive Sodium Taurocholate - 5.0 -
identification of coliforms and for cultivating gram-negative, lactose fermenting Bromocresol Purple - 0.01 -
bacilli from a variety of samples like water, milk and food. Final pH (at 250C) 7.4 ± 0.2 7.4 ± 0.2 7.4 ± 0.2
* Formula adjusted to suit performance parameters
MacConkey Broth with Neutral Red is a standard medium for the primary isolation
Directions
as well as presumptive identification of coli-aerogenes group in food and water.
1. Suspend the powder in 1000 ml distilled water.
Summary
MacConkey Broth Double Strength with Neutral Red, MacConkey Broth Purple MacConkey Broth Double Strength with Neutral Red - 80.15 gms.
with Bromocresol Purple and MacConkey Broth with Neutral Red are all MacConkey Broth Purple with Bromocresol Purple - 40 gms.
modifications of the original bile salt broth recommended by MacConkey, which MacConkey Broth with Neutral Red - 40 gms.
contained 0.5% sodium taurocholate and litmus as the indicator. In later 2. Mix thoroughly.
publications, MacConkey suggested variations of this formula using neutral red as
3. Boil with frequent agitation to dissolve the powder completely.
the indicator instead of litmus. Bile salts in the medium replaces the original
4. Dispense into tubes containing inverted Durham's tubes.
sodium taurocholate to inhibit growth of gram-positive organisms.
5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
The above mentioned MacConkey Broths are recommended for use in
Quality Control
microbiological examination of clinical specimens, foodstuffs and for direct
Dehydrated Appearance
plating and inoculation of water samples for coliform counts. These media are Yellow coloured, homogeneous, free flowing powder.
also included in the Official Methods of Analysis as well as pharmaceutical Prepared Appearance
preparations (46, 114) and industrial products. MacConkey Broth Double Strength with Neutral Red & MacConkey Broth with
Principle Neutral Red - Red coloured, clear solution without any precipitate.
Peptone provides amino acids and other growth factors. Lactose is a carbon and MacConkey Broth Purple with Bromocresol Purple - Purple coloured, clear
solution without any precipitate.
energy source. Bile salts inhibit the growth of gram-positive organisms. Neutral Cultural Response
red and bromocresol purple are the pH indicators. Sodium chloride maintains the Cultural characteristics after 18-24 hours at 35-370C.
osmotic balance. MacConkey Broth Double Strength with Neutral Red, MacConkey Broth
Formula* Purple with Bromocresol Purple & MacConkey Broth with Neutral Red
Ingredients MacConkey MacConkey MacConkey Organisms (ATCC) Growth Acid Gas
in grams Broth Double Broth Purple Broth with Enterobacter aerogenes (13048) Luxuriant + +
per liter Strength with with Neutral Red
Neutral Red Bromocresol Escherichia coli (25922) Luxuriant + +
Purple Salmonella serotype Choleraesuis (12011) Fair to good - -
Peptone 40.0 20.0 20.0 Staphylococcus aureus (25923) Inhibited - -

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Interpretation of Results Storage
1. Lactose fermenting organisms grow well producing acid, causing the Store at 22-300C and prepared medium at 2-80C.
medium to turn yellow. Gas is also produced which collects in the inverted Shelf Life
Durham tubes. Use before expiry date as mentioned on the label.
2. Non-lactose fermenting organisms produce good growth but will not
produce acid or gas.

MacConkey Broth Double Strength with AM10621/AM50621

Neutral Red BIS
Use Directions
MacConkey Broth Double Strength with Neutral Red is used for the primary 1. Suspend 80.15 of the powder in 1000 ml distilled water.
isolation of coliforms from large samples such as water and wastewater. 2. Mix thoroughly.
Summary
3. Boil with frequent agitation to dissolve the powder completely.
MacConkey Broth Double Strength with Neutral Red is a modifications of the
4. Dispense into tubes containing inverted Durham's tubes.
original bile salt broth recommended by MacConkey, which contained 0.5%
sodium taurocholate and litmus as the indicator. In later publications, 5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Quality Control
MacConkey suggested variations of this formula using neutral red as the indicator
Dehydrated Appearance
instead of litmus. Bile salts in the medium replaces the original sodium
Yellow coloured, homogeneous, free flowing powder.
taurocholate to inhibit growth of gram-positive organisms. The above mentioned Prepared Appearance
MacConkey Broths are recommended for use in microbiological examination of Red coloured, clear solution without any precipitate.
clinical specimens, foodstuffs and for direct plating and inoculation of water Cultural Response
samples for coliform counts. These media are also included in the Official Cultural characteristics after 18-24 hours at 35-370C.
Methods of Analysis as well as pharmaceutical preparations and industrial Organisms (ATCC) Growth Acid Gas
products. Enterobacter aerogenes (13048) Luxuriant + +
Principle Escherichia coli (25922) Luxuriant + +
Salmonella serotype Choleraesuis (12011) Fair to good - -
Peptone provides amino acids and other growth factors. Lactose is a carbon and
Staphylococcus aureus (25923) Inhibited - -
energy source. Bile salts inhibit the growth of grampositive organisms. Neutral Interpretation of Results
red the pH indicators. Sodium chloride maintains the osmotic balance.
1. Lactose fermenting organisms grow well producing acid, causing the
Formula*
medium to turn yellow. Gas is also produced which collects in the inverted
Ingredients in grams per liter
Peptone 40.0
Durham tubes.
Lactose 20.0 2. Non-lactose fermenting organisms produce good growth but will not
Bile salts 10.0 produce acid or gas.
Sodium chloride 10.0 Storage
Neutral red 0.15
Store at 22-300C and prepared medium at 2-80C.
Final pH (at 250C) 7.5 ±0.2
Shelf Life
* Formula adjusted to suit performance parameters
Use before expiry date as mentioned on the label.

MacConkey Broth (Harmonized) AMH5063

MacConkey Broth USP AM50636
Use variety of specimens like water, milk and food.
MacConkey Broth is used for the presumptive identification of coliforms from a

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Summary 5. Sterilize by autoclaving at 121ºC (15 lbs pressure) for 15 minutes.
MacConkey Broth is recommended for use in microbiological examination of Quality Control
clinical specimens, foodstuffs and for direct plating and inoculation of water Dehydrated Appearance
samples for coliform counts. This medium is also included in the Official Methods Yellow coloured, homogeneous, free flowing powder.
Prepared Appearance
of Analysis as well as pharmaceutical preparations and industrial products.
Purple coloured, clear solution without any precipitate.
Principle
Cultural Response
Pancreatic digest of gelatin provides amino acids and other growth factors. Cultural characteristics after 18-24 hours at 35-37ºC.
Lactose is a carbon and energy source. Bile salts inhibit the growth of Gram Organisms (ATCC) Growth Acid Gas
positive organisms. Bromocresol purple are the pH indicators. Enterobacter aerogenes (13048) Luxuriant + +
Formula* Escherichia coli (25922) Luxuriant + +
Ingredients in grams per liter Salmonella choleraesuis (12011) Fair to good - -
Pancreatic digest of gelatin 20.0 Staphylococcus aureus (25923) Inhibited - -
Lactose monohydrate 10.0 Interpretation of Results
Dehydrated Ox bile 5.0 1. Lactose fermenting organisms grow well producing acid, causing the
Bromocresol purple 0.01
medium to turn yellow. Gas is also produced which collects in the inverted
Final pH (at 25ºC) 7.3 ± 0.2
Durham tubes.
* Formula adjusted to suit performance parameters
Directions 2. Non-lactose fermenting organisms produce good growth but will not produce
1. Suspend 35.01 gms of the powder in 1000 ml distilled water. acid or gas.
Storage
2. Mix thoroughly.
Store at 22-300C and prepared medium at 2-80C.
3. Boil with frequent agitation to dissolve the powder completely. Shelf Life
4. Dispense into tubes containing inverted Durham's tubes. Use before expiry date as mentioned on the label.

MacConkey Broth Purple with Bromocresol Purple IP AM10631/AM50631

Use Formula*
MacConkey Broth Purple with Bromocresol Purple is used for the presumptive Ingredients in grams per liter
Bromo cresol purple 0.01
identification of coliforms and for cultivating gramnegative, lactose fermenting
Pancreatic digest of gelatin 20.0
bacilli from a variety of samples like water, milk and food in compliance with IP.
Lactose 10.0
Summary
Dehydrated Ox bile 5.0
MacConkey Broth Purple with Bromocresol Purple is a modification of the original Final pH (at 250C) 7.3 ±0.2
bile salt broth recommended by MacConkey, which contained 0.5% sodium * Formula adjusted to suit performance parameters
taurocholate and litmus as the indicator. In later publications, MacConkey Directions
suggested variations of this formula using neutral red as the indicator instead of 1. Suspend 35.01 gms of the powder in 1000 ml distilled water.
litmus. It is recommended for use in microbiological examination of clinical 2. Mix thoroughly.
specimens, foodstuffs and for direct plating and inoculation of water samples for
3. Boil with frequent agitation to dissolve the powder completely.
coliform counts. This media is also included in the Official Methods of Analysis as
well as pharmaceutical preparations and industrial products. 4. Dispense into tubes containing inverted Durham's tubes.
Principle 5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Pancreatic digest of gelatin provides amino acids and other growth factors. Quality Control
Dehydrated Appearance
Lactose is a carbon and energy source. Dehydrated Ox bile inhibit the growth of
Yellow coloured, homogeneous, free flowing powder.
gram-positive organisms. Bromocresol purple is the pH indicator. Sodium
Prepared Appearance
chloride maintains the osmotic balance. Purple coloured, clear solution without any precipitate.

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Cultural Response medium to turn yellow. Gas is also produced which collects in the inverted
Cultural characteristics after 18-24 hours at 35-370C. Durham tubes.
Organisms (ATCC) Growth Acid Gas
Enterobacter aerogenes (13048) Luxuriant + +
2. Non-lactose fermenting organisms produce good growth but will not
Escherichia coli (25922) Luxuriant + + produce acid or gas.
Salmonella serotype Choleraesuis (12011) Fair to good - - Storage
Staphylococcus aureus (25923) Inhibited - - Store at 22-300C and prepared medium at 2-80C.
Interpretation of Results Shelf Life
1. Lactose fermenting organisms grow well producing acid, causing the Use before expiry date as mentioned on the label.

MacConkey Broth Purple with BCP (Broth Medium G) EP AM10632/AM50632

MacConkey Broth Purple with BCP (Broth Medium G) BP AM10633/AM50633
Use 2. Mix thoroughly.
MacConkey Broth Purple with Bromocresol Purple is used for the presumptive 3. Boil with frequent agitation to dissolve the powder completely.
identification of coliforms and for cultivating gram-negative, lactose fermenting
4. Dispense into tubes containing inverted Durham's tubes.
bacilli from a variety of samples like water, milk and food.
Summary
5. Sterilize by autoclaving at 121ºC (15 lbs pressure) for 15 minutes.
Quality Control
MacConkey Broth Purple with Bromocresol Purple is a modification of the original
Dehydrated Appearance
bile salt broth recommended by MacConkey, which contained 0.5% sodium Yellow coloured, homogeneous, free flowing powder.
taurocholate and litmus as the indicator. Prepared Appearance
It is recommended for use in microbiological examination of clinical specimens, Purple coloured, clear solution without any precipitate.
foodstuffs and for direct plating and inoculation of water samples for coliform Cultural Response
Cultural characteristics after 18-24 hours at 35-37ºC.
counts. This media is also included in the Official Methods of Analysis as well as
Organisms (ATCC) Growth Acid Gas
pharmaceutical preparations and industrial products.
Enterobacter aerogenes (13048) Luxuriant + +
Principle
Escherichia coli (25922) Luxuriant + +
Pancreatic digest of gelatin provides amino acids and other growth factors. Salmonella serotype choleraesuis (12011) Fair to good - -
Lactose is a carbon and energy source. Dehydrated Ox bile inhibits the growth of Staphylococcus aureus (25923) Inhibited - -
gram-positive organisms. Bromocresol purple is the pH indicator. Sodium Interpretation of Results
chloride maintains the osmotic balance. 1. Lactose fermenting organisms grow well producing acid, causing the
Formula*
medium to turn yellow. Gas is also produced which collects in the inverted
Ingredients in grams per liter
Durham tubes.
Pancreatic digest of gelatin 20.0
Lactose monohydrate 10.0 2. Non-lactose fermenting organisms produce good growth but will not
Bromo cresol purple 0.01 produce acid or gas.
Dehydrated Ox bile 5.0 Storage
Final pH (at 25ºC) 7.3 ± 0.2
Store at 22-300C and prepared medium at 2-80C.
* Formula adjusted to suit performance parameters
Shelf Life
Directions
Use before expiry date as mentioned on the label.
1. Suspend 35.01 gms of the powder in 1000 ml distilled water.

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MacConkey Broth Purple with BCP BIS AM10634/AM50634
Use 2. Mix thoroughly.
MacConkey Broth Purple with Bromocresol Purple is used for the presumptive 3. Boil with frequent agitation to dissolve the powder completely.
identification of coliforms and for cultivating gram-negative, lactose fermenting
4. Dispense into tubes containing inverted Durham's tubes.
bacilli from a variety of samples like water, milk and food.
Summary 5. Sterilize by autoclaving at 121ºC (15 lbs pressure) for 15 minutes.
Quality Control
MacConkey Broth Purple with Bromocresol Purple is a modification of the original
Dehydrated Appearance
bile salt broth recommended by MacConkey, which contained 0.5% sodium Yellow coloured, homogeneous, free flowing powder.
taurocholate and litmus as the indicator. Prepared Appearance
It is recommended for use in microbiological examination of clinical specimens, Purple coloured, clear solution without any precipitate.
foodstuffs and for direct plating and inoculation of water samples for coliform Cultural Response
counts. This media is also included in the Official Methods of Analysis as well as Cultural characteristics after 18-24 hours at 35-37ºC.
Organisms (ATCC) Growth Acid Gas
pharmaceutical preparations and industrial products.
Enterobacter aerogenes (13048) Luxuriant + +
Principle
Escherichia coli (25922) Luxuriant + +
Pancreatic digest of animal tissue provides amino acids and other growth factors. Salmonella serotype Choleraesuis (12011) Fair to good - -
Lactose is a carbon and energy source. Sodium taurocholate inhibits the growth of Staphylococcus aureus (25923) Inhibited - -
gram-positive organisms. Bromocresol purple is the pH indicator. Sodium Interpretation of Results
chloride maintains the osmotic balance. 1. Lactose fermenting organisms grow well producing acid, causing the
Formula* medium to turn yellow. Gas is also produced which collects in the inverted
Ingredients in grams per liter Durham tubes.
Peptic digest of animal tissue 20.00
Lactose 10.00
2. Non-lactose fermenting organisms produce good growth but will not
Sodium taurocholate 5.00 produce acid or gas.
Sodium chloride 5.00 Storage
Bromocresol Purple 0.02 Store at 22-300C and prepared medium at 2-80C.
Final pH (at 25ºC) 7.3±0.2 Shelf Life
* Formula adjusted to suit performance parameters Use before expiry date as mentioned on the label.
Directions
1. Suspend 40.02 gms of the powder in 1000 ml distilled water..

MacConkey Broth Purple with BCP ISO AM10635/AM50635

Use well as pharmaceutical preparations and industrial products.
MacConkey Broth Purple with Bromocresol Purple is used for the presumptive Principle
identification of coliforms and for cultivating gramnegative, lactose fermenting Peptone provides amino acids and other growth factors. Lactose is a carbon and
bacilli from a variety of samples like water, milk and food. energy source. Sodium Taurocholate inhibit the growth of gram-positive
Summary organisms. Bromocresol purple is the pH indicator. Sodium chloride maintains
MacConkey Broth Purple with Bromocresol Purple is a modification of the original the osmotic balance.
bile salt broth recommended by MacConkey, which contained 0.5% sodium Formula*
taurocholate and litmus as the indicator. In later publications, MacConkey Ingredients in grams per liter
suggested variations of this formula using neutral red as the indicator instead of Bromo cresol purple 0.01
litmus. It is recommended for use in microbiological examination of clinical Peptone 20.0
Lactose 10.0
specimens, foodstuffs and for direct plating and inoculation of water samples for
Sodium Chloride 5.0
coliform counts. This media is also included in the Official Methods of Analysis as

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Sodium Taurocholate 5.0 Organisms (ATCC) Growth Acid Gas
Final pH (at 250C) 7.4±0.2 Enterobacter aerogenes (13048) Luxuriant + +
* Formula adjusted to suit performance parameters Escherichia coli (25922) Luxuriant + +
Directions Salmonella serotype Choleraesuis (12011) Fair to good - -
1. Suspend 40 gms of the powder in 1000 ml distilled water. Staphylococcus aureus (25923) Inhibited - -
Interpretation of Results
2. Mix thoroughly.
1. Lactose fermenting organisms grow well producing acid, causing the
3. Boil with frequent agitation to dissolve the powder completely.
medium to turn yellow. Gas is also produced which collects in the inverted
4. Dispense into tubes containing inverted Durham's tubes. Durham tubes.
5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. 2. Non-lactose fermenting organisms produce good growth but will not produce
Quality Control acid or gas.
Dehydrated Appearance Storage
Yellow coloured, homogeneous, free flowing powder.
Prepared Appearance
Store at 22-300C and prepared medium at 2-80C.
Purple coloured, clear solution without any precipitate. Shelf Life
Cultural Response Use before expiry date as mentioned on the label.
0
Cultural characteristics after 18-24 hours at 35-37 C.

MacConkey Broth with Neutral Red BIS AM10641/AM50641

Use Neutral Red 0.075
MacConkey Broth with Neutral Red is a standard medium for the primary isolation Final pH (at 250C) 7.5 ±0.2
* Formula adjusted to suit performance parameters
as well as presumptive identification of coliaerogenes group in food and water in
Directions
compliance with BIS.
Summary 1. Suspend 40.07 gms of the powder in 1000 ml distilled water. MacConkey
MacConkey Broth with Neutral Red are all modifications of the original bile salt Broth Double Strength
broth recommended by MacConkey, which contained 0.5% sodium taurocholate 2. Mix thoroughly.
and litmus as the indicator. In later publications, MacConkey suggested 3. Boil with frequent agitation to dissolve the powder completely.
variations of this formula using neutral red as the indicator instead of litmus. Bile 4. Dispense into tubes containing inverted Durham's tubes.
salts in the medium replaces the original sodium taurocholate to inhibit growth of 5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
gram-positive organisms. The above mentioned MacConkey Broths are Quality Control
recommended for use in microbiological examination of clinical specimens, Dehydrated Appearance
foodstuffs and for direct plating and inoculation of water samples for coliform Yellow coloured, homogeneous, free flowing powder.
counts. These media are also included in the Official Methods of Analysis as well Prepared Appearance
as pharmaceutical preparations and industrial products. Red coloured, clear solution without any precipitate.
Principle Cultural Response
Cultural characteristics after 18-24 hours at 35-370C.
Peptone provides amino acids and other growth factors. Lactose is a carbon and
Organisms (ATCC) Growth Acid Gas
energy source. Bile salts inhibit the growth of grampositive organisms. Neutral
Enterobacter aerogenes (13048) Luxuriant + +
red and bromocresol purple are the pH indicators. Sodium chloride maintains the Escherichia coli (25922) Luxuriant + +
osmotic balance. Salmonella serotype Choleraesuis (12011) Fair to good - -
Formula* Staphylococcus aureus (25923) Inhibited - -
Peptone 20.0 Interpretation of Results
Lactose 10.0
1. Lactose fermenting organisms grow well producing acid, causing the
Bile Salts 5.0
Sodium chloride 5.0
medium to turn yellow. Gas is also produced which collects in the inverted

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Durham tubes. Storage

2. Non-lactose fermenting organisms produce good growth but will not Store at 22-300C and prepared medium at 2-80C.
Shelf Life
produce acid or gas.
Use before expiry date as mentioned on the label.

MacConkey Sorbitol Agar AM50642

Use Directions
MacConkey Sorbitol Agar is recommended for isolation and identification of 1. Suspend 50 gms of the powder in 1000ml of distilled water.
enteropathogenic Escherichia coli strains associated with infant diarrhea. 2. Mix thoroughly.
Summary
3. Heat gently with frequent agitation to dissolve the powder completely.
Escherichia coli O157:H7 is a human pathogen associated with hemorrhagic
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
colitis (78.2). MacConkey Sorbitol Agar is a variant of traditional MacConkey Agar
Quality Control
used in the detection of E. coli O157:H7. Escherichia coli O157:H7 differs from Dehydrated Appearance
most other strains of E. coli in being unable to ferment sorbitol. In MacConkey Light yellow to pink coloured homogeneous free flowing powder.
Sorbitol Agar, lactose is replaced by sorbitol. Most strains of E. coli ferment Prepared Appearance
sorbitol to produce acid: E. coli O157:H7 cannot ferment sorbitol, so this strain Purplish red coloured clear to slightly opalescent gel forms in petri plates.
uses peptone to grow. This raises the pH of the medium allowing the O157:H7 Cultural Response
strain to be differentiated from other E. coli strains through the action of the pH Cultural characteristics after 18-24 hours at 37°C.
indicator in the medium. Organisms (ATCC) Growth Colour of RGI
colony
Principle
Escherichia coli O157:H7 Luxuriant Colourless More than 70%
Peptic digest of animal tissues and proteose peptone provide the carbon and Escherichia coli (25922) Luxuriant Pink More than 70%
nitrogen while sodium chloride maintains the osmotic balance. Sorbitol is the Escherichia coli serotype Luxuriant Colourless More than 70%
source of energy. Bile salt mixture and crystal violet inhibit the Gram- positive 011 & 055
organisms. Neutral red is a pH indicator. S. serotype Typhi (6539) Luxuriant Pink More than 70%
Formula* Shigella flexneri (12022) Luxuriant Colourless More than 70%
Ingredients in grams per liter For growth RGI should be more than 70%
Peptic digest of animal tissue 17.0 RGI- Relative Growth Index
Proteose peptone 3.0 Procedure
D- Sorbitol 10.0 Refer to appropriate references for specific procedures.
Sodium chloride 5.0 Interpretation of Results
Bile salt mixture 1.5
Refer to appropriate references and procedures for results.
Neutral red 0.03
Storage
Crystal violet 0.001
Agar 13.5 Store at 22-300C and prepared medium at 2-80C.
0
Final pH (at 25 C) 7.1 ± 0.2 Shelf Life
* Formula adjusted to suit performance parameters Use before expiry date as mentioned on the label.

Malonate Broth Ewing Modified AM1065/AM5065

Use Aerobacter (Enterobacter) from Escherichia species based on their ability to
Malonate Broth Ewing Modified is used for the differentiation of utilize malonate. Ewing et al (28) devised the modification, in which dextrose and
Enterobacteriaceae on the basis of malonate utilization. yeast extract were incorporated. The addition of yeast extract, a source of vitamins,
Summary and a relatively small amount of dextrose, a minimal carbon source, was included
Leifson (66) developed a synthetic liquid medium, which differentiated in Ewing's modification to stimulate the growth of some organisms. Hence, the

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medium supports the growth of organisms that cannot utilize malonate or Quality Control
ammonium salt, but any alkalinization produced by such organisms is buffered Dehydrated Appearance
Light green coloured, homogeneous, free flowing powder.
by the phosphate system and counteracted by the acid produced in the
Prepared Appearance
fermentation of small amount of dextrose. Organisms capable of utilizing
Bluish green coloured, clear solution without any precipitate.
malonate and ammonium sulphate produce an alkaline result (blue colour) in Cultural Response
this medium. Malonate Broth Ewing Modified is recommended by APHA for the Cultural characteristics after 18-48 hours at 35-370C.
examination of foods (20) and milk (39) and is included in the Bacteriological Organisms (ATCC) Growth Malonate
Analytical Manual for testing food and cosmetics (113). Utilization
Principle Enterobacter aerogenes (13048) Luxuriant +
Organisms that simultaneously utilize malonate as its carbon source and Escherichia coli (25922) Poor to fair -
Klebsiella pneumoniae (13883) Luxuriant +
ammonium sulphate as its nitrogen source produce an alkalinity due to the
Salmonella serotype Typhimurium (14028) Fair to good -
formation of sodium hydroxide. The alkali changes the colour of the bromothymol
Procedure
blue indicator in the medium from green to blue. The colour of the medium
1. Inoculate tubes, using a light inoculum, with growth from an 18 to 24 hour
remains unchanged in the presence of organisms that cannot utilize these
pure culture.
substances. Yeast extract and the small amount of dextrose provides vitamins and
carbon respectively stimulating the growth of organisms that are unable to utilize 2. Incubate the tubes with loosened caps for 18-48 hours at 350C ± 20C in an
malonate or ammonium salts. Some malonate negative strains produce a yellow aerobic atmosphere.
colour due to the fermentation of dextrose only, which results in increased acidity Interpretation of Results

causing the pH indicator (bromothymol blue) to change to yellow at pH 6.0. 1. The bacterial genera in which the majority of species produce a positive
Dipotassium and Monopotassium phosphate act as buffering agents. Sodium alkaline reaction i.e. light blue to Prussian blue colour throughout the
chloride maintains the osmotic balance. medium are Enterobacter, Klebsiella and Citrobacter.
Formula* 2. The bacterial genera in which the majority of species produce a negative
Ingredients in grams per liter reaction i.e. colour of the medium remains unchanged, green or yellow are
Sodium Malonate 3.0 Escherichia, Salmonella, Shigella, Edwardsiella, Yersinia, Serratia,
Ammonium Sulphate 2.0
Morganella, Proteus and Providencia.
Sodium Chloride 2.0
Precautions / Limitations
Yeast Extract 1.0
Dipotassium Phosphate 0.60 1. Some malonate-positive organisms produce only slight alkalinity.
Monopotassium Phosphate 0.40 2. Compare the tubes with an un-inoculated malonate tube.
Dextrose 0.25
3. Any trace of blue colour after a 48-hour incubation period denotes a positive
Bromothymol Blue 0.025
Final pH (at 250C) 6.7 ± 0.2
reaction.
* Formula adjusted to suit performance parameters 4. Before making a final negative interpretation, be sure that test tubes have
Directions been incubated for 48 hours.
1. Suspend 9.28 gms of the powder in 1000 ml distilled water. Storage
2. Mix thoroughly. Store at 22-300C and prepared medium at 2-80C.
3. Dispense in desired containers as per requirements. Shelf Life
0 Use before expiry date as mentioned on the label.
4. Sterilize by autoclaving at 121 C (15 lbs pressure) for 15 minutes.

Malt Agar AM1066/AM5066

Use Summary
Malt Agar is used for isolating and cultivating yeasts and moulds from foods and Malt Agar is recommended by APHA (20) for use in antibiotic and acidified
dairy products and carrying stock cultures of yeasts and moulds. standard methods for the determination of yeast and mould counts in food and

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also for maintaining stock cultures of fungi. Malt Agar is included in the Quality Control
Dehydrated Appearance
Bacteriological Analytical Manual for food testing (113).
Light yellow coloured, homogeneous, free flowing powder.
Principle
Prepared Appearance
Malt extract provides nutrients required for growth of microorganisms. The acidic Light amber coloured, slightly opalescent gel.
pH of the medium allows the optimal growth of yeasts and moulds while Cultural Response
restricting bacterial growth. Cultural characteristics after 40-48 hours at 300C.
Formula* Organisms (ATCC) Growth RGI
Ingredients in grams per liter Aspergillus niger (16404) Luxuriant More than 70%
Malt Extract 30.0 Candida albicans (10231) Luxuriant More than 70%
Agar 15.0 Saccharomyces cerevisiae (9763) Luxuriant More than 70%
Final pH (at 250C) 5.5 ± 0.2 For growth RGI should be more than 70%
* Formula adjusted to suit performance parameters RGI- Relative Growth Index
Directions Precautions / Limitations
1. Suspend 45 gms of the powder in 1000 ml distilled water. 1. Do not reheat the medium after the addition of acid, as this will hydrolyze the
2. Mix thoroughly. agar and reduce its solidifying properties.
3. Boil with frequent agitation to dissolve the powder completely. Storage

4. Sterilize by autoclaving at 1180C (12 lbs pressure) for 15 minutes. Store at 22-300C and prepared medium at 2-80C.
Shelf Life
5. DO NOT OVERHEAT, as it will give a softer and darker agar.
Use before expiry date as mentioned on the label.
6. To lower the pH, add sterile 1:10 lactic acid, do not reheat the medium.

Malt Extract Agar AM1067/AM5067

Malt Extract Broth AM1068/AM5068
Use lactic acid or antibiotics as sterile solutions to the molten medium immediately
Malt Extract Agar is used for the enumeration, cultivation and isolation of yeasts before pouring into sterile petri plates. The acidic pH of Malt Extract Agar allows
and moulds while Malt Extract Broth is used for detection of yeasts, moulds and the optimal growth of moulds and yeasts while restricting the growth of bacteria.
aciduric organisms. Formula*
Summary Ingredients in grams per liter Malt Extract Agar Malt Extract Broth
Malt Extract 30.0 17.0
Malt Extract Agar is similar to the formula of Galloway and Burgess (33) used for
Mycological Peptone 5.0 3.0
the detection, isolation and enumeration of yeasts and moulds. The use of malt Agar 15.0 -
extract for the propagation of yeasts and moulds is quite common. Reddish (91) Final pH (at 250C) 5.4 ± 0.2 5.4 ± 0.2
described a culture medium prepared from malt extract that was a satisfactory * Formula adjusted to suit performance parameters
substitute for wort. Thom and Church (112), following the formula of Reddish, Directions
used malt extract as a base from which they prepared Malt Extract Agar and Malt 1. Suspend the powder in 1000 ml distilled water.
Extract Broth. This media are included in the Bacteriological Analytical Manual for Malt Extract Agar - 50 gms
food and cosmetics testing (113) and are recommended by APHA in examination
Malt Extract Broth - 20 gms
of foods (20).
Principle 2. Mix thoroughly. Soak for 15 minutes.
Malt Extract provides the energy source. Mycological peptone serves as the 3. Sterilize by autoclaving at 1150C (10 lbs pressure) for 10 minutes. DO NOT
nitrogen source and gives rapid luxuriant growth with typical morphology and OVERHEAT.
pigmentation. Agar is the solidifying agent. For mycological count it is advisable 4. Mix well before dispensing.
to adjust the reaction of the medium to more acidic with the addition of 10% 5. To adjust acidic pH use 10% lactic acid.

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Quality Control Candida albicans (10231) Luxuriant More than 70%
Dehydrated Appearance Saccharomyces cerevisiae Luxuriant More than 70%
Yellow coloured, homogeneous, free flowing powder. (9763)
Prepared Appearance For growth RGI should be more than 70%
Malt Extract Agar - Light brown coloured, slightly opalescent gel. RGI- Relative Growth Index
Malt Extract Broth - Light brown coloured, slightly opalescent solution. Precautions / Limitations
Cultural Response 1. Avoid overheating Malt Extract Agar as it results in a softer gel.
Cultural characteristics after 48-72 hours at 25-300C. Storage
Organisms (ATCC) Growth on RGI
Store at 22-300C and prepared medium at 2-80C
Malt Extract
Shelf Life
Agar & in Malt
Extract Broth
Use before expiry date as mentioned on the label.
Aspergillus niger (16404) Luxuriant More than 70%

Mannitol Motility Test Medium AM10681/AM50681

Use 3. Dispense into sterile test tubes.
Mannitol Motility Test Medium is a semisolid medium suitable for determining 4. Sterilize by autoclaving at 1210 C (15 lbs pressure) for 15 minutes.
motility and mannitol fermentation.
5. Cool the tubed medium in an upright position.
Summary
Quality Control
Semi-solid media have been employed for many years in the study of bacterial Dehydrated Appearance
motility. Tittsler and Sandholzer in their study have sited its importance in Light pink coloured, homogeneous, free flowing powder.
detecting bacterial motility (111). Mannitol Motility Test Medium is a semi-solid Prepared Appearance
medium that has been developed for rapid identification of Enterobacteriaceae Pinkish red slightly opalescent gel.
on the basis of motility and mannitol utilization. Cultural Response
0
Principle Cultural response after 18 - 24 hours at 35 ± 2 C.
Organisms (ATCC) Motility Mannitol
Peptic digest of animal tissue provides the nitrogen, minerals and amino acid
Escherichia coli (25922) + +
nutrients essential for bacterial growth. Mannitol is the fermentable carbohydrate Klebsiella pneumoniae - +
for energy source. Potassium nitrate provides additional nutrients to the medium. Proteus mirabilis (25933) + -
Phenol red is the pH indicator and agar is the solidification agent. Procedure
Formula* 1. Inoculate the medium using standard procedure like stab inoculation.
Ingredients in grams per liter
Peptic Digest of animal Tissue 20.0
2. Incubate the tubes at 35 ± 20 C for 18-24 hours.
Mannitol 2.0 Interpretation of Results
Potassium Nitrate 1.0 1. Motility is observed as diffused growth away from the stab inoculation line.
Phenol Red 0.04 2. Non-motile organism growth is seen along the stab line.
Agar 3.0
Final pH (at 250 C) 7.6 ± 0.2
3. Mannitol fermentation is indicated by a change in colour of the medium from
* Formula adjusted to suit performance parameters red to yellow.
Directions Storage

1. Suspend 26 grams of the powder in 1000 ml distilled water. Store at 22-300C and prepared medium at 2-80C.
Shelf Life
2. Boil with frequent agitation to dissolve the powder completely.
Use before expiry date as mentioned on the label.

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D-Mannitol, A. R. Sterile (g
irradiated) AM506811
Use Principle
D-Mannitol, A. R. Sterile (g
irradiated) used for the media fill runs in dry filling During Media Fill Run for validation of dry injectable g irradiated D-Mannitol is
injectable. dispersed into individual vial/ampules. After completion of filling process
Summary individual vial is reconstituted with sterile distilled water (As per label claim of
Routine sampling for sterility testing is not sensitive enough to detect any low injection). Reconstituted vials an incubated at 35-370C and monitored till 14
level contamination in sterile pharmaceutical formulations. Sample numbers are days.
too small and only gross contamination is likely to be detected. Pharmaceutical Formula*
manufactures therefore need other means of guaranteeing the quality of their Quality Control
Dehydrated AppearanceLight yellow coloured, homogeneous, free flowing
product. This is why process stimulations (Media Fill Run) supported by
powder.
environmental monitoring is must in pharmaceutical industry. Prepared AppearanceColourless solution without any precipitate.
The FDA guidelines have recommended using SCDM for liquid injectable and D- Cultural Response
Mannitol for dry injectable. Regular dehydrated culture media or D.Mannitol is Storage
usually supplied in non sterile form which carries high bioburden and should not Store at 22-300C and prepared medium at 2-80C.
be directly taken into a controlled area therefore irradiated sterile SCDM/D- Shelf Life
Mannitol is used for Media Fill Run. g irradiation also assumes that sterile Use before expiry date as mentioned on the label.
products is free from Mycoplasma.

Mannitol Salt Agar (Harmonized) AMH5069

Mannitol Salt Agar AM1069/AM5069
Mannitol Salt Agar Medium IP AM10693/AM50693
Mannitol Salt Agar Medium USP AM10694/AM50694
Use which helps in the differentiation of staphylococcal species. Coagulase-negative
Mannitol Salt Agar is a selective medium for the isolation and identification of species of staphylococci and micrococci do not ferment mannitol and grow as
Staphylococcus aureus from clinical and non-clinical specimens. small red colonies surrounded by red or purple zones. Yellow coloured colonies
Summary should be tested for production of coagulase. Addition of 5% v/v Egg Yolk
Koch (60) reported that only staphylococci grow on agar media containing 7.5% Emulsion (AS009) enables the detection of lipase activity of staphylococci along
sodium chloride. Chapman (14) studied this phenomenon in detail and with mannitol fermentation. The salt clears the egg yolk emulsion and lipase
concluded that the addition of 7.5% salt to phenol red mannitol agar results in an production is detected as yellow opaque zone around the colonies. Coagulase
improved medium for the isolation of plasma coagulating staphylococci. positive staphylococci produce colonies surrounded by bright yellow zones while
Mannitol Salt Agar is recommended by the USP (114) and IP (46) for use in non-pathogenic staphylococci produce colonies with reddish purple zones.
Microbial Limit Tests. It is used for the detection and enumeration of coagulase Formula*
positive staphylococci in milk, food and other specimens. This medium is also Ingredients in grams per liter
included in the Bacteriological Analytical Manual for cosmetics testing (113). Mannitol Mannitol Mannitol Mannitol
salt agar salt agar USP salt agar IP salt agar
Principle
(Harmonized)
Proteose peptone and beef extract supplies essential growth factors such as Beef extract 1.0 1.0 1.0 1.0
nitrogen, carbon, sulphur and trace nutrients. The 7.5% salt concentration results Proteose peptone 10.0 – – –
in partial or complete inhibition of bacteria other than staphylococci. Mannitol Sodium chloride 75.0 75.0 75.0 75.0
fermentation, results in change in the phenol red indicator, (from red to yellow) D-Mannitol 10.0 10.0 10.0 10.0

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Phenol red 0.025 0.025 0.025 0.025 Staphylococcus Fair to good Red More than 70%
Agar 15.0 15.0 15.0 15.0 epidermidis (12228)
Pancreatic digest – 5.0 5.0 5.0 For growth RGI should be more than 70%
of casein For Inhibition RGI should be 0%
Peptic digest of – 5.0 5.0 5.0 RGI- Relative Growth Index
animal tissue Procedure
Final pH (at 250C) 7.4 ±0.2 1. Use standard procedures to obtain isolated colonies from specimens.
* Formula adjusted to suit performance parameters
2. Incubate plates for 24-48 hours at 35 ±20C in an aerobic atmosphere.
Directions
Interpretation of Results
1. Suspend 111 gms of the powder in 1000 ml distilled water and mix well.
Typical colony morphology on Mannitol Salt Agar is as follows:
2. Boil with frequent agitation to dissolve the powder completely.
Staphylococcus aureus--------------------- Small to large with yellow zones.
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Staphylococci other than S.aureus--------- Small to large with red zones.
4. OPTIONAL: Add 5% v/v Egg Yolk Emulsion (AS010).
Streptococci------------------------------- No growth to trace growth.
5. Mix well, dispense as desired.
Micrococci-------------------------------- Large white to orange.
Quality Control
Dehydrated Appearance Gram-negative bacteria------------------ No growth to trace growth.
Light pink coloured, homogeneous, free flowing powder. Precautions / Limitations
Prepared Appearance 1. Negative plates should be re-incubated overnight before discarding.
Red coloured, clear to slightly opalescent gel.
2. Presumptive Staphalococcus aureus should be confirmed with a coagulase
Cultural Response
test.
Cultural characteristics after 18-48 hours at 35-370C.
Organisms (ATCC) Growth Colour of RGI 3. A few strains of S.aureus may exhibit delayed fermentation of mannitol.
the Colony Storage
Escherichia coli (25922) Inhibited - 0% Store at 22-300C and prepared medium at 2-80C.
Staphylococcus aureus Good to Good to More than 70%
Shelf Life
(25923) luxuriant Yellow
Use before expiry date as mentioned on the label.

Mannitol Salt Broth AM10695/AM50695

Use Organisms capable of fermenting mannitol e.g. Staphylococcus aureus, cause a
Mannitol Salt Broth is a selective medium for the isolation and enumeration of pH change in the media. With phenol red as the pH indicator the colonies appear
Staphylococcus aureus from clinical and non-clinical specimens. with a yellow coloration
Summary Principle
Koch, reported that only staphylococci grow on media containing 7.5% sodium Proteose peptone and beef extract supplies essential growth factors such as
chloride. Chapman, studied this phenomenon in detail and concluded that the nitrogen, carbon, sulphur and trace nutrients. The 7.5% salt concentration results
addition of 7.5% salt to phenol red mannitol salt results in an improved medium in partial or complete inhibition of bacteria other than staphylococci. Mannitol
for the isolation of plasma coagulating staphylococci. Mannitol Salt is fermentation, results in change in the phenol red indicator, (from red to yellow)
recommended by the USP and IP for use in Microbial Limit Tests. It is used for the which helps in the differentiation of staphylococcal species. Coagulase-negative
detection and enumeration of coagulase positive staphylococci in milk, food and species of staphylococci and micrococci do not ferment mannitol and grow as
other specimens. This medium is also included in the Bacteriological Analytical small red colonies surrounded by red or purple zones. Yellow coloured colonies
Manual for cosmetics testing. Because of the amount of peptones and beef should be tested for production of coagulase.
extract, Mannitol Salt is a nutrient rich medium. Most bacteria (other than Formula*
staphylococci) are inhibited by the high concentration of sodium chloride. Ingredients in grams per liter
Beef extract 1.0

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Pancreatic digest of casein 5.0 Procedure
Peptic digest of animal tissue 5.0 1. Use standard procedures for isolation of Staphylococcus aureus from
Sodium chloride 75.0
specimens.
D-Mannitol 10.0
Phenol red 0.025 2. Incubate plates for 24-48 hours at 35-370C in an aerobic atmosphere.
Final pH (at 250C) 7.4 ±0.2 Interpretation of Results
* Formula adjusted to suit performance parameters Typical colony morphology on Mannitol Salt Agar is as follows:
Directions Staphylococcus aureus--------------------- Small to large with yellow zones.
1. Suspend 96 gms of the powder in 1000 ml distilled water and mix well. Staphylococci other than S.aureus---------- Small to large with red zones.
2. Boil with frequent agitation to dissolve the powder completely. Streptococci-------------------------------- No growth to trace growth.
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. Micrococci---------------------------------- Large white to orange.
4. Mix well, dispense as desired. Gram-negative bacteria-------------------- No growth to trace growth.
Quality Control
Precautions / Limitations
Dehydrated Appearance
Light pink coloured, homogeneous, free flowing powder.
1. Negative tubes should be re-incubated overnight before discarding.
Prepared Appearance 2. Presumptive Staphalococcus aureus should be confirmed with a coagulase
Red coloured, clear transparent solution. test.
Cultural Response
3. A few strains of S.aureus may exhibit delayed fermentation of mannitol.
Cultural characteristics after 18-24 hours at 35-370C and then recovery on
Storage
Mannitol Salt Agar.
Organisms Growth Colour of Recovery on Store at 22-300C and prepared medium at 2-80C.
(ATCC) the medium Mannitol Salt Agar Shelf Life
Staphylococcus Good to Yellow or Yellow colonies Use before expiry date as mentioned on the label.
aureus (25923) luxuriant Orange
E. coli Inhibited Red Inhibited

Marine Agar 2216 (Zobell Marine Agar) AM10691/AM50691

Use Peptone 5.0
Marine Agar 2216 (Zobell Marine Agar) is used for isolation and enumeration of Ferric citrate 0.10
Sodium chloride 19.45
heterotrophic marine bacteria.
Magnesium chloride 8.80
Summary
Sodium sulphate 3.24
Zobell Marine media are designed, based on the formula suggested by Zobell Calcium chloride 1.80
(122.1). Marine bacteria are very much essential to the life cycle of nearly all Potassium chloride 0.55
marine flora and fauna. Marine bacteria are of prime importance in food industry Sodium bicarbonate 0.16
and marine life conservation and hence activity of these bacteria can be studied Potassium bromide 0.08
by the use of this medium. Strontium chloride 0.034
Principle Boric acid 0.022
Sodium silicate 0.004
This medium contain the nutrients which are required for the growth of marine
Sodium fluorate 0.0024
bacteria. Medium is consist not only of minerals as sea water (122.2) but also has Ammonium nitrate 0.0016
peptic digest of animal tissue and yeast extract as the better sources of nutrients Dipotassium phosphate 0.008
for the marine bacteria as reported by Jones (49.2). Agar 15.00
Formula* Final pH (at 250C) 7.6 ±0.2
Ingredients in grams per liter * Formula adjusted to suit performance parameters
Yeast extract 1.0

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Directions Cultural Response
1. Suspend 55.25 gms of the powder in 1000 ml distilled water and mix well. Cultural characteristics after 2 – 3 days (longer if required) at 200C .
Organisms (ATCC) Growth RGI
2. Boil with frequent agitation to dissolve the powder completely.
Vibrio fischeri (7744) Good-luxuriant More than 70%
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. Vibrio harveyi (14126) Good-luxuriant More than 70%
Quality Control For growth RGI should be more than 70%
Dehydrated Appearance RGI- Relative Growth Index
Light beige with a few dark particles, free flowing powder. Storage
Prepared Appearance Store at 22-300C and prepared medium at 2-80C.
Light amber, slightly opalescent may have a slight precipitate, may contain
Shelf Life
dark particle.
Use before expiry date as mentioned on the label.

Marine Broth 2216 (Zobell Marine Broth) AM10692/AM50692

Use Strontium chloride 0.034
Marine Broth 2216 (Zobell Marine Broth) is used for isolation and enumeration Boric acid 0.022
Sodium silicate 0.004
of heterotrophic marine bacteria.
Sodium fluorate 0.0024
Summary
Ammonium nitrate 0.0016
Zobell Marine media are designed, based on the formula suggested by Zobell. Dipotassium phosphate 0.008
Marine bacteria are very much essential to the life cycle of nearly all marine flora Final pH (at 250C) 7.6 ±0.2
and fauna. Marine bacteria are of prime importance in food industry and marine * Formula adjusted to suit performance parameters
life conservation and hence activity of these bacteria can be studied by the use of Directions
this medium. 1. Suspend 40.25 gms of the powder in 1000 ml distilled water and mix well.
Principle
2. Boil with frequent agitation to dissolve the powder completely.
This medium contain the nutrients which are required for the growth of marine
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
bacteria. Medium is consist not only of minerals as sea water but also has peptic Quality Control
digest of animal tissue and yeast extract as the better sources of nutrients for the Dehydrated Appearance
marine bacteria as reported by Jones. Light beige with a few dark particles, free flowing powder.
Formula* Prepared Appearance
Ingredients in grams per liter Light amber, with slight precipitate.
Yeast extract 1.0 Cultural Response
Peptone 5.0 Cultural characteristics after 2 – 3 days (longer if required) at 200C .
Ferric citrate 0.10 Organisms (ATCC) Growth
Sodium chloride 19.45 Vibrio fischeri (7744) Good-luxuriant
Magnesium chloride 8.80 Vibrio harveyi (14126) Good-luxuriant
Sodium sulphate 3.24 Storage
Calcium chloride 1.80
Store at 22-300C and prepared medium at 2-80C.
Potassium chloride 0.55
Shelf Life
Sodium bicarbonate 0.16
Use before expiry date as mentioned on the label.
Potassium bromide 0.08

M-Endo Agar LES AM106921/AM506921

Use Summary
M Endo Agar LES is recommended for enumeration of coliforms in water using a The membrane filter (MF) been an method detection and enumeration coliform
two step membrane filter technique. organisms water. , Delaney and GrassoEndo Agar LES (Lawrence Experimental

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Station) for testing water for coliform bacteria by a two-step membrane filter Warning: Basic fuchsin is a potential carcinogen and care must be taken
to avoid inhalation and contamination of the skin.
procedure (79.3). High numbers of coliforms are recovered by this method
Quality Control
compared with the one step technique using M Endo Broth. In two-step
Dehydrated Appearance
membrane filter procedure lauryl tryptose broth is used as a preliminary Pink coloured, homogeneous free flowing powder.
enrichment medium. The American Public Health Association specifies using M Prepared Appearance
Endo Agar LES in the standard total coliform membrane filtration procedure for Red coloured, slightly opalescent gel with precipitate.
testing drinking waterbottled water (17.1 & 55.3). Cultural Response
Principle Cultural characteristics after 20-24 hours at 35ºC.
Peptic digest of animal tissue, casein enzymic hydrolysate and yeast extract act as Organisms Growth Colour of RGI
(ATCC) Colony
a source of carbon, nitrogen, vitamins and minerals. Lactose and tryptose are the
Escherichia coli Luxuriant Red – black with More than 70%
source of energy. Sodium chloride maintains the osmotic balance.
metallic Sheen
Monopotassium phosphate and dipotassium phosphate are the buffering agents. Enterobacter aerogenes Luxuriant Red – black with More than 70%
Sodium deoxycholate and sodium lauryl sulphate are added as inhibitors for the (13048) metallic Sheen
Gram positive organisms. Basic fuchsin is a pH indicator. Sodium sulfite is added S. serotypeTyphi Luxuriant Colourless More than 70%
to decolorize the basic fuchsin solution. Agar is the solidifying agent. Lactose- (6539)
fermenting bacteria produce acetaldehyde that reacts with the sodium sulfite and Staphylococcus aureus Inhibited - 0%
fuchsin to form red colonies. The development of a metallic sheen occurs when the (25923)
Procedure
organism produces aldehydes with the rapid fermentation of lactose.
Formula* 1. Place a membrane filter absorbent pad inside the cover of a petridish.
Ingredients in grams per liter 2. Add 1.8-2 ml of Lauryl Tryptose Broth (AM 1053/ AM 5053) to each pad.
Peptic digest of animal tissue 3.7
3. Run the water sample through a membrane filter.
Casein enzymic hydrolysate 3.7
Lactose 9.4 4. Place the filter, topside up, onto the pad containing Lauryl Tryptose Broth.
Sodium chloride 3.7 5. Incubate at 350C for 1.5-2.5 hours. Transfer the membrane from the pad to
Tryptose 7.5 the surface of the M Endo Agar LES medium I the petri dish bottom, keeping
Dipotassium phosphate 3.3 the side on which the bacteria have been collected facing upward.
Monopotassium phosphate 1.0
Sodium sulphite 1.6
6. Leave the filter pad in the lid and incubate the plates in the inverted position
Yeast extract 1.2 at 350C for 20-24 hours.
Basic fuchsin 0.8 Interpretation of Results
Sodium deoxycholate 0.1 Red colour colonies with characteristic metallic sheen are considered as coliform.
Sodium lauryl sulphate 0.05 Precautions / Limitations
Agar 15.0
1. Non-coliform colonies may produce typical colonies with metallic sheen.
Final pH (at 25ºC) 7.2 ± 0.2
* Formula adjusted to suit performance parameters 2. Coliform organisms may also occasionally produce atypical colonies without
Directions metallic sheen. It is advisable to verify both the colony types.
1. Suspend 51 gms of powder in 980ml of distilled water. Storage

2. Warm slightly with frequent agitation to dissolve the powder completely. DO Store at 22-300C and prepared medium at 2-80C.
Shelf Life
NOT AUTOCLAVE.
Use before expiry date as mentioned on the label.
3. Cool to 450C and aseptically add 20 ml of 95% ethanol.
4. Dispense adequate amount into petri plates. DO NOT EXPOSE PLATES TO
DIRECT SUNLIGHT.

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M-Endo Broth AM106922/AM506922
Use 4. Dispense in tubes or adequate containers and sterilize by autoclaving at
M- Endo Broth is used for estimation of coliforms in water using a two step 15lbs pressure (121ºC) for 15 minutes.
membrane filter. Warning: Basic fuchsin is a potential carcinogen and care must be taken
Summary to avoid inhalation and contamination of the skin.
Quality Control
The membrane filter (MF) been an method detection and enumeration coliform
Dehydrated Appearance
organisms water. numbers of coliforms are recovered by using this medium .
Purple coloured, homogeneous, free flowing powder.
Principle
Prepared Appearance
Peptic digest of animal tissue and yeast extract act as a source of carbon, nitrogen, Pinkish orange solution without any precipitate.
vitamins and minerals. Lactose is the source of energy. Sodium chloride Cultural Response
maintains the osmotic balance. Basic fuchsin is a pH indicator. Sodium sulfite is Cultural characteristics after 18-48 hours at 370C.
added to decolorize the basic fuchsin solution. Organisms (ATCC) Growth Colour of
Formula* colony
Ingredients in grams per liter Escherichia coli (25922) Good-luxuriant Pink with
metallic sheen
Peptic digest of animal tissue 20.0
S. s erotype Typhimurium (14028) Good-luxuriant Colourless to
Yeast extract 6.0
very light pink
Lactose 25.0
Staphylococcus aureus (25923) Inhibited -
Dipotassium phosphate 7.0
Procedure
Basic fuchsin 1.0
Sodium sulphite 2.5 Refer to appropriate references for specific procedures.
0
Final pH (at 25 C) 7.5 ± 0.2 Interpretation of Results
* Formula adjusted to suit performance parameters Refer to appropriate references and procedures for results.
Directions Storage
1. Suspend 61.50 gms powder in 1000ml distilled water. Store at 22-300C and prepared medium at 2-80C.
2. Mix thoroughly. Shelf Life
3. Heat with frequent agitation to dissolve the powder completely. Do not boil. Use before expiry date as mentioned on the label.

M-FC Agar Base AM506923

Use Formula*
M-FC Agar media is used for the detection and enumeration of faecal coliforms Ingredients in grams per liter
Tryptose 10.00
using membrane filter technique at higher temperature.
Proteose peptone 5.00
Summary
Yeast extract 3.00
M-FC Agar was designed by Geldreich, clark, Huff and Bert (33.3). It is Lactose 12.50
recommended by APHA for the detection and enumeration of faecal coliforms Bile salt mixture 1.50
using membrane filter technique. Sodium chloride 5.00
Principle Aniline blue 0.10
Proteose peptone, tryptose and yeast extract provide necessary nutrients for the Agar 15.00
Final pH (at 250C) 7.4 ± 0.2
growth of faecal coliforms. Lactose is the carbon source as well as fermentable
* Formula adjusted to suit performance parameters
carbohydrate in the medium. Bile salts inhibit the growth of contaminating gram-
Directions
positive microorganisms. Aniline blue is a triphenyl methane dye which
1. Suspend 52.1 gms powder in 1000ml distilled water containing 10 ml 1%
suppressive the growth of many gram-positive microorganisms.
Rosolic Acid.

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2. Mix thoroughly. S. serotype Luxuriant Inhibited Pinkish More than 70% 0%
Typhimurium
3. Heat to boiling to dissolve the medium completely. DO NOT AUTOCLAVE. (14028)
Shigella flexneri Luxuriant Inhibited Pinkish More than 70% 0%
4. Cool to 450C and add 2 ml of M-FC Broth on sterile absorbent pad placed in a
(12022)
sterile petri plate. Enterococcus Inhibited Inhibited -- 0% 0%
Quality Control faecalis (29212)
Dehydrated Appearance For growth RGI should be more than 70%
Yellow coloured, homogeneous, free flowing powder. For Inhibition RGI should be 0%
Prepared Appearance RGI- Relative Growth Index
With addition of rosolic acid, red coloured slightly opalescent gel forms in petri Procedure
plates. Refer to appropriate references for specific procedures.
Cultural Response Storage
Cultural characteristics after 22-24 hours at …....
Organisms Growth Growth Colour of RGI at 350C RGI at 45.50C
Store at 22-300C and prepared medium at 2-80C.
(ATCC) at 350C at 45.50C the colony Shelf Life
Escherichia coli Luxuriant luxuriant Light blue More than 70% More than 70% Use before expiry date as mentioned on the label.
(25922)

M-FC Broth Base AM506924

Use Rosolic Acid (AS0232).
M-FC Broth media is used for the detection and enumeration of faecal coliforms 2. Mix thoroughly.
using membrane filter technique at higher temperature.
3. Heat to boiling to dissolve the medium completely. DO NOT AUTOCLAVE.
Summary
Quality Control
M-FC Broth was designed by Geldreich, clark, Huff and Bert (33.3). It is Dehydrated Appearance
recommended by APHA for the detection and enumeration of faecal coliforms Yellow coloured, homogeneous, free flowing powder.
using membrane filter technique. Prepared Appearance
Principle With addition of rosolic acid, red coloured slightly opalescent solution forms in
tubes.
Proteose peptone, tryptose and yeast extract provide necessary nutrients for the
Cultural Response
growth of faecal coliforms. Lactose is the carbon source as well as fermentable Cultural characteristics after 22-24 hours at …....
carbohydrate in the medium. Bile salts inhibit the growth of contaminating gram- Organisms (ATCC) Growth Growth Colour of
positive microorganisms. Aniline blue is a triphenyl methane dye which at 350C at 45.50C the colony
suppressive the growth of many gram-positive microorganisms. Escherichia coli (25922) Luxuriant Luxuriant Light blue
Formula* S. serotype Typhimurium Luxuriant Inhibited Pinkish
Ingredients in grams per liter (14028)
Tryptose 10.00 Shigella flexneri (12022) Luxuriant Inhibited Pinkish
Proteose peptone 5.00 Enterococcus faecalis (29212) Inhibited Inhibited --
Yeast extract 3.00 Procedure
Lactose 12.50 Refer to appropriate references for specific procedures.
Bile salt mixture 1.50 Storage
Sodium chloride 5.00
Aniline blue 0.10
Store at 22-300C and prepared medium at 2-80C.
Shelf Life
Final pH (at 250C) 7.4 ± 0.2
* Formula adjusted to suit performance parameters Use before expiry date as mentioned on the label.
Directions
1. Suspend 37.1 gms powder in 1000ml distilled water containing 10 ml 1%

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M-(HPC) Heterotrophic Plate Count Agar Base AM506925
Use 2. Heat to boiling to dissolve the medium completely.
M-(HPC) Heterotrophic Plate Count Agar is used for enumeration of heterotrophic 3. Sterilize by autoclaving at 15lbs pressure (121ºC) for 10 minutes.
microorganisms from water samples using membrane filter technique. Quality Control
Summary Dehydrated Appearance
MM-(HPC) Agar Base, with added glycerol is recommended for heterotrophic Light yellow coloured, homogeneous, free flowing powder.
plate counts of potable water, swimming pool and other waters (108.1). It is used Prepared Appearance
as an alternative medium to Standard Methods Agar and R2A Agar. Light yellow coloured, clear to slightly opalescent gel forms in petri plates.
Cultural Response
Principle
Cultural characteristics after 18-24 hours at 350C
Peptic digest of animal tissue is the source of nutrients for organisms which are not Organisms (ATCC) Growth RGI
highly fastidious. Gelatin is utilized by microorganisms through a proteolytic Escherichia coli(25922) Luxuriant More than 70%
mechanism. The addition of glycerol to the basal medium provides a source of Pseudomonas aeruginosa (27853) Luxuriant More than 70%
carbon and energy. Enterococcus faecalis (29212) Luxuriant More than 70%
Formula* For growth RGI should be more than 70%
Ingredients in grams per liter RGI- Relative Growth Index
Peptic digest of animal tissue 20.00 Procedure
Gelatin 25.00 Refer to appropriate references for specific procedures.
Agar 15.00 Storage
Final pH (at 250C) 7.1 ± 0.2
Store at 22-300C and prepared medium at 2-80C.
* Formula adjusted to suit performance parameters
Shelf Life
Directions
Use before expiry date as mentioned on the label.
1. Suspended 60 grams in 1000 ml distilled water containing 10 ml glycerol.

M-(HPC) Heterotrophic Plate Count Broth Base AM506926

Use Directions
M-(HPC) Heterotrophic Plate Count Broth is used for enumeration of heterotrophic 1. Suspend 45 grams in 1000 ml distilled water containing 10 ml glycerol.
microorganisms from water samples using membrane filter technique. 2. Heat to boiling to dissolve the medium completely.
Summary
3. Sterilize by autoclaving at 15lbs pressure (121ºC) for 5 minutes.
M-(HPC) Broth Base, with added glycerol is recommended for heterotrophic plate Quality Control
counts of potable water, swimming pool and other waters (108.1). It is used as an Dehydrated Appearance
alternative medium to Standard Methods Agar and R2A Agar. Light yellow coloured, homogeneous, free flowing powder.
Principle Prepared Appearance
Peptic digest of animal tissue is the source of nutrients for organisms which are not Light yellow coloured, clear to slightly opalescent solution forms in tubes.
Cultural Response
highly fastidious. Gelatin is utilized by microorganisms through a proteolytic
Cultural characteristics after 18-24 hours at 350C
mechanism. The addition of glycerol to the basal medium provides a source of
Organisms (ATCC) Growth
carbon and energy. Escherichia coli (25922) Luxuriant
Formula* Pseudomonas aeruginosa (27853) Luxuriant
Ingredients in grams per liter Enterococcus faecalis (29212) Luxuriant
Peptic digest of animal tissue 20.00 For growth RGI should be more than 70%
Gelatin 25.00 RGI- Relative Growth Index
Final pH (at 250C) 7.1 ± 0.2 Procedure
* Formula adjusted to suit performance parameters
Refer to appropriate references for specific procedures.

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Storage Shelf Life
Store at 22-300C and prepared medium at 2-80C. Use before expiry date as mentioned on the label.

Middlebrook 7H9 Agar Base AM506927

Use Agar 15.00
Middlebrook 7H9 Agar Base enriched with OADC enrichment are recommended Final pH (at 250C) 6.6 ± 0.2
* Formula adjusted to suit performance parameters
for isolation, cultivation and sensitivity testing of Mycobacterium tuberculosis.
Directions
Summary
Middlebrook 7H9 Agar Base developed developed by Middlebrook et al., (80.2) 1. Suspend 19.6 gms powder in 1000ml distilled water.
is used for cultivating Mycobateria and for assaying INH content of Patients sera. 2. Add either 2 ml glycerol or 0.5 g polysorbate 80.
The medium can also be used for preparing inocula for antimicrobial assays, as a 3. Mix thoroughly.
basal medium for biochemical tests and for the subculture of stock strains. 4. Heat if necessary to dissolve the medium completely.
Principle
5. Cool to 450C and add 2 ml of M-FC Broth on sterile absorbent pad placed in a
This medium contains many inorganic salts which support the growth of sterile petri plate.
Mycobacteria. Sodium citrate becomes citric acid in the medium which retains
6. Sterilize by autoclaving at 15lbs pressure (121ºC) for 10 minutes.
certain inorganic cations in the solution. This medium is supplemented for the
growth of Mycobacteria. Middlebrook OADC Growth Supplement (AS0181) 7. Cool to 450C or below and aseptically add 1 vial of reconstituted Middlebrook
contains oleic acid, bovine albumin, dextrose, catalase and sodium chloride. Oleic OADC Growth Supplement. Mix well before dispensing.
Quality Control
acid and other long chain fatty acids are essential for metabolism of
Dehydrated Appearance
Mycobacteria. Some free fatty acids are toxic to Mycobacteria but albumin binds
Light yellow coloured, homogeneous, free flowing powder.
to those fatty acids and prevents toxic action on Mycobacteria. Toxic peroxides Prepared Appearance
present in the medium are destroyed by catalase. Dextrose supplies energy while Light amber coloured, clear gel with slight precipitate.
sodium chloride maintains osmotic equilibrium. Glycerol enhances the growth. Cultural Response
Formula* Cultural characteristics after 2-4 week at 35-370C
Ingredients in grams per liter Organisms (ATCC) Growth RGI
Ammonium sulphate 0.50 Mycobacterium tuberculosis H37 RV Good-luxuriant More than 70%
Disodium phosphate 2.50 (25618)
Monopotassium phosphate 1.00 Mycobacterium smegmatis ( 14468) Good-luxuriant More than 70%
Sodium citrate 0.10 Mycobacterium fortuitum (6841) Good-luxuriant More than 70%
Magnesium sulphate 0.05 For growth RGI should be more than 70%
Calcium chloride 0.0005 RGI- Relative Growth Index
Zinc sulphate 0.001 Procedure
Copper sulphate 0.001
Refer to appropriate references for specific procedures.
Ferric ammonium citrate 0.04
Storage
L-Glutamic acid 0.50
Pyridoxine 0.001 Store at 22-300C and prepared medium at 2-80C.
Shelf Life
Biotin 0.0005
Use before expiry date as mentioned on the label.

M-TEC Agar AM5069261

Use Summary
M-TEC Agar is recommended for isolation, differentiation and rapid enumeration M-TEC Agar is recommended for rapid isolation, differentiation and rapid
of thermotolerant Escherichia coli from water by membrane filtration enumeration of thermotolerant E. coli from water by membrane filtration. TEC
stands for thermotolerant E. coli, the presence of which is widely used as an

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indicator of faecal contamination in water. Cultural Response
Principle Cultural characteristics observed after an incubation at 35-37°C for 2 hours
and at 44.5°±0.5°C for 22 hours.
Proteose peptone and yeast extract act as source of nitrogen, carbon, amino acids
Organisms (ATCC) Growth RGI
and vitamins. Potassium phosphate salts help in buffering the medium. Lactose is Escherichia coli (25922) Good (further testing More than 70%
the source of fermentable carbohydrate. Bromocresol purple and bromophenol red usingurease substrate
serve as indicator. Sodium lauryl sulphate and sodium deoxycholate inhibit gram- should be performed)
positive bacteria. For growth RGI should be more than 70%
Formula* RGI- Relative Growth Index
Ingredients in grams per liter Procedure
Proteose peptone 5.00 1. Follow applicable membrane filter procedures.
Yeast extract 3.00
2. Incubate inoculated plates for 2 hours at 35 ± 2°C to resuscitate injured
Lactose 10.00
cells.
Sodium chloride 7.50
Potassium dihydrogen phosphate 1.00 3. Transfer the plates to a 44.5 ± 0.5°C waterbath or incubator and incubate
Dipotassium hydrogen phosphate 3.30 for 22 ± 2 hours.
Sodium lauryl sulphate 0.20
4. Transfer countable filters to pads saturated with urea substrate. Prepare urea
Sodium deoxycholate 0.10
Bromocresol purple 0.08 substrate by combining 2 g urea and 10 mg phenol red in 100 mL of purified
Bromphenol red 0.08 water and adjust the pH to 5.0 ± 0.2. Store at 2-8°C and use within 1
Agar 15.00 week.
Final pH ( at 25°C) 7.3±0.2 NOTE: Other methods may recommend an alternative pH. Prepare substrate
* Formula adjusted to suit performance parameters
according to recommended guidelines.
Directions
5. After 15-20 minutes, count all yellow to yellow-brown colonies with the aid
1. Suspend 45.26 grams in 1000 ml distilled water.
of a stereoscopic microscope.
2. Heat to boiling to dissolve the medium completely. Expected Results
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Yellow to yellow-brown colonies (urease negative) may be presumptively
Cool to 45°C and pour into sterile Petri plates. identified as E. coli.
Quality Control Storage
Dehydrated Appearance Store at 22-300C and prepared medium at 2-80C.
Cream to greenish yellow homogeneous free flowing powder Shelf Life
Prepared Appearance Use before expiry date as mentioned on the label.
Dark purple coloured with red cast clear to slightly opalescent gel forms in Petri
plates

M 7 Hr FC Agar AM5069262
Use water during emergencies involving water treatment plant failure or line breaks in
M7 Hr FC Agar is recommended for examination of water and wastewater. a distribution network. It is reliable and has sensitivity levels equal to those of the
Summary standard tests routinely used.
M7 Hr FC Agar is a modified method of Van Donsel et al (115.5) and Reasoner et Principle
al (90.4), which is recommended by APHA (22.1) for the examination of water Biopeptone and yeast extract provide nutritional requirement to a wide variety of
and wastewater for the presence of faecal coliforms by the membrane filter organisms. Lactose and mannitol are energy sources and sodium chloride
technique. This medium has an advantage over other media to yield results in 7 maintains osmotic equilibrium of the medium. Sodium lauryl sulphate and
hours that generally are comparable to those obtained by the standard coliform sodium deoxycholate help to restrict the gram-positive and gram-negative
method. Thus this medium is accepted for assessment of the sanitary quality of bacterial flora present in water. Bromocresol purple and phenol red help as

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indicators in the detection of organisms. This is a solid culture medium for the 4. Mix well and pour into sterile Petri plates.
rapid detection of faecal coliforms by membrane filtration method. After filtering Quality Control
a suitable or desired volume of water, the membrane is placed on the surface of Dehydrated Appearance
plate and then incubated at 41.5°C for 7 hours. Faecal coliform form yellow Beige to purple homogeneous free flowing powder.
Prepared Appearance
colonies, indicating lactose fermentation.
Dark pinkish purple coloured clear to slightly opalescent gel forms in Petri
Formula*
plates
Ingredients in grams per liter
Cultural Response
Biopeptone 5.00
Cultural characteristics observed after an incubation at 41.5°C for 7-18 hours
Yeast extract 3.00
Organisms Growth Colour RGI
Lactose 10.00
(ATCC) of Colony
D-Mannitol 5.00
Escherichia coli (25922) Luxuriant yellow More than 70%
Sodium chloride 7.50
Staphylococcus aureus Inhibited - 0%
Sodium lauryl sulphate 0.20
(25923)
Sodium deoxycholate 0.10
Enterococcus faecalis Inhibited - 0%
Bromo cresol purple 0.35
(29212)
Phenol red 0.30
For growth RGI should be more than 70%
Agar 15.00
For Inhibition RGI should be 0%
Final pH ( at 25°C) 7.3±0.2
RGI- Relative Growth Index
* Formula adjusted to suit performance parameters
Storage
Directions
Store at 22-300C and prepared medium at 2-80C.
1. Suspend 46.45 grams in 1000 ml distilled water. Shelf Life
2. Heat to boiling to dissolve the medium completely. Use before expiry date as mentioned on the label.
3. DO NOT AUTOCLAVE.

MLCB Agar AM5069263

(Mannitol Lysine Crystal Violet Brilliant Green)
Use sodium deoxycholate help to restrict the gram-positive and gram-negative
MLCB (Mannitol Lysine Crystal Violet Brilliant Green Agar) is used for the isolation bacterial flora present in water. Bromocresol purple and phenol red help as
of salmonellae (not Salmonella typhi or Salmonella paratyphi A.). indicators in the detection of organisms. This is a solid culture medium for the
Summary rapid detection of faecal coliforms by membrane filtration method. After filtering
Mannitol Lysine Crystal Violet Brilliant Green Agar (MLCB Agar) is based on the a suitable or desired volume of water, the membrane is placed on the surface of
formula of Inoue et al (106.2) for the selective isolation of salmonellae from plate and then incubated at 41.5°C for 7 hours. Faecal coliform form yellow
faeces and foods. Visual detection of very small numbers of hydrogen sulphide- colonies, indicating lactose fermentation.
producing strains is easy because of the distinctive colonial appearance. The Formula*
concentration of Magnesium ions appears to be critical for maximum growth of Ingredients in grams per liter
salmonellae on MLCB Agar. van Schothorst et al. (114.1) showed that MLCB Yeast Extract 5.0
Pepton 10.0
Agar did not inhibit any of the salmonellae species investigated.
''Lab-Lemco' Powder 2.0
MLCB Agar is not suitable for Salmonella typhi and Salmonella paratyphi A Sodium chloride 4.0
because of the inhibitory concentration of brilliant green. Mannitol 3.0
Principle L-lysine hydrochloride 5.0
Biopeptone and yeast extract provide nutritional requirement to a wide variety of Sodium thiosulphate 4.0
organisms. Lactose and mannitol are energy sources and sodium chloride Ammnium iron (III) citrate 1.0
Brilliant green 0.0125
maintains osmotic equilibrium of the medium. Sodium lauryl sulphate and

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Crystal violet 0.01 Cultural Response
Agar 15 Cultural characteristics after 18-24 hours at 35-370C
Final pH (at 250C) 6.8±0.2 Organisms Growth Colour RGI
* Formula adjusted to suit performance parameters (ATCC) of Colony
Directions Salmonella typhimurium Luxuriant Purple colour More than 70%
1. Suspend 49.0 gms powder in 1000 ml distilled water. (23564) with black center
Escherichia coli Inhibition - 0%
2. Mix and bringgently to the boil with frequent agitation to dissolve the
(25922)
medium completely.
Salmonella enteritidis Luxuriant Purple colour More than 70%
3. Cool to 50°C and pour approximately 20ml into sterile Petri dishes. (13076) with black center
4. DO NOT AUTOCLAVE OR OVERHEAT. For growth RGI should be more than 70%
Quality Control For Inhibition RGI should be 0%
Dehydrated Appearance RGI- Relative Growth Index
Green/straw coloured, homogeneous, free flowing powder. Storage
Prepared Appearance Store at 22-300C and prepared medium at 2-80C.
Purple coloured, gel forms in Petri plates Shelf Life
Use before expiry date as mentioned on the label.

Minimal Broth AM506927

Use Yeast Extract 5.0
Minimal media are recommended for isolation and enumeration of Bacillus Potassium dihydrogen phosphate 5.0
cereus. Final pH (at 250C) 7.0 ±0.2
Summary * Formula adjusted to suit performance parameters
Directions
Minimal media described by Lederberg for the isolation of nutritional mutants of
E. coli and B. subtilis. The minimal media contains the necessary nutrients only 1. Suspend 25.0 gms of the powder in 1000 ml distilled.
for the growth of wild type E. coli and B. subtilis strains. The random isolation 2. Mix thoroughly.
and delayed enrichment method, described by Lederberg, can be used to isolate 3. Warm slightly with frequent agitation to dissolve the powder completely.
nutritional mutants derived from irradiated cultures of wild type E. coli (65.1). 4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
With added dextrose it also supports the growth of nutritional mutants of Bacillus Quality Control
subtilis. B. subtilis mutants can be isolated by the same techniques as E. coli or Dehydrated Appearance
by the penicillin method described by Nester et al., (84.3). Yellow coloured, homogeneous, free flowing powder.
Principle Prepared Appearance
Peptone and yeast extract provide nitrogenous nutrients. Glucose is the Dark yellow coloured clear solution.
Cultural Response
fermentable carbohydrate and carbon source in the medium. Acetyl methyl
Cultural characteristics after 18-24 hours at 350C
carbinol is produced from glucose by the members of Bacillus cereus groups. After
Organisms (ATCC) Growth RGI
the inoculation and incubation at 350C for 48 hours, the presence of acetyl methyl
Bacillus cereus (10876) luxuriant More than 70%
carbinol is determined by adding 0.2 ml of 40% potassium hydroxide and 0.6 ml For growth RGI should be more than 70%
of 5% alcoholic alpha-naphthol solution to 1 ml of culture tube. This reaction is RGI- Relative Growth Index
hastened by the addition of a few crystals of creatine by which the purple colour Procedure
development take place within 15 minutes. Refer to appropriate references for specific procedures.
Formula* Storage
Ingredients Gms/Liter
Store at 22-300C and prepared medium at 2-80C.
Glucose 10.0
Shelf Life
Peptone 5.0
Use before expiry date as mentioned on the label.

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Milk Agar AM10692711/AM50692711
Use Prepared Appearance
Milk Agar is recommended for enumeration of microorganisms in milk and milk Light yellow coloured slightly opalescent gel forms in petri plates.
Cultural Response
products.
Cultural characteristics after 18-48 hours at 35ºC.
Summary
Organisms (ATCC) Growth RGI
Milk Agar is formulated as per the official medium described by Dept. of Health Bacillus subtilis (6633) Good to luxuriant More than 70%
Memo (18.3). EEC Commission also recommends it for the examination of ice Lactobacillus casei (9595) Good to luxuriant More than 70%
creams (57.1). Pseudomonas aeruginosa (27853) Good to luxuriant More than 70%
Principle Staphylococcus aureus (25923) Good to luxuriant More than 70%
Peptic digest of animal tissue and yeast extract provide the essential nutrient for Serratia marcescens (8100) Good to luxuriant More than 70%
For growth RGI should be more than 70%
the growth of the microorganisms. Milk solids are the source of carbohydrate.
RGI- Relative Growth Index
Agar is used as an solidifying agent.
Procedure
Formula*
Ingredients in grams per liter Using either pour plates or surface counting techniques may carry out bacterial
Peptic digest of animal tissue 5.0 enumeration.
Yeast extract 3.0 Prepare milk dilutions of 1/10, 1/100, 1/1000 in ¼ strength Ringer’s salt (AM
Milk solids 1.0 108531) solution.
Agar 15.0
(a) Pour plate: 1 ml of each dilution is pipetted aseptically into sterile petri plates
Final pH (at 25ºC) 7.2 ± 0.2
*Formula adjusted to suit performance parameters and 10 ml of sterile milk agar is added to it and mixed thoroughly.
Directions (b) Spread plate: Spread 1 ml of milk dilution over the surface of the solidified
1. Suspend 24gms of the powder in 1000 ml distilled water. medium in a petri plate. (C)Incubate the plates at 35ºC for 18-48 hours.
Storage
2. Mix thoroughly.
Store at 22-300C and prepared medium at 2-80C.
3. Boil with frequent agitation to dissolve the powder completely.
Shelf Life
4. Sterilize by autoclaving at 121ºC (15 lbs pressure) for 15 minutes. Use before expiry date as mentioned on the label.
Quality Control
Dehydrated Appearance
Light yellow coloured, homogeneous, free flowing powder.

Middlebrook 7H9 Broth Base AM5069272

Use Principle
Middlebrook 7H9 Broth Base with added enrichment is recommended for Middlebrook media consists of many inorganic salts, which help, in growth of
cultivation and sensitivity testing of Mycobacterium tuberculosis . Mycobacteria. Citric acid formed from sodium citrate helps in retaining inorganic
Summary cations in solution. Glycerol supplies carbon and energy. Oleic acid and other long
Middlebrook 7H9 Broth Base was formulated by Middlebrook (80.2.1) and chain fatty acids are essential for metabolism of Mycobacteria. Middlebrook ADC
Middlebrook et al and Schaeffer (80.2, 80.2.2). This medium with Growth Supplement (AS01811) contains bovine albumin, dextrose, catalase and
Middlebrook ADC Growth Supplement (AS01811)) and glycerol or polysorbate sodium chloride. Some free fatty acids are toxic to Mycobacteria but albumin
80 is also recommended for cultivation of Mycobacteria and for assaying the INH binds to those fatty acids and prevents toxic action on Mycobacteria. Dextrose
content of the patients sera. The medium can also be used for preparing inocula serves as an energy source.
for antimicrobial assays, as a basal medium for biochemical tests and for the Catalase neutralizes toxic peroxides. Mycobacteria grow more rapidly in broth
subculture of stock strains. media; therefore primary isolation of all specimens can be done in Middlebrook
7H9 Broth Base. After processing the sample as required, inoculate the media

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with the test specimen. Mycobacteria are strict aerobes and therefore increased 3. Heat if necessary to dissolve the medium completely.
CO2 tension and aerobic conditions must be provided during incubation. 4. Sterilize by autoclaving at 15 lbs pressure (121°C) for 10 minutes.
Formula*
5. Cool to 45°C or below and aseptically add contents of 1 vial of Middlebrook
Ingredients in grams per liter
ADC Growth Supplement (AS01811).
Ammonium sulphate 0.50
Disodium phosphate 2.50 6. Mix well before dispensing.
Monopotassium phosphate 1.00 Quality Control
Sodium citrate 0.10 Dehydrated Appearance
Magnesium sulphate 0.05 Cream to beige homogeneous free flowing powder
Calcium chloride 0.0005 Prepared Appearance
Zinc sulphate 0.001 Light amber coloured clear solution in tubes
Copper sulphate 0.001 Cultural Response
Ferric ammonium citrate 0.040 Cultural characteristics observed with added Middlebrook ADC Growth
L-Glutamic acid 0.50 Supplement (AS01811) and glycerol or Polysorbate 80 after an incubation at
Pyridoxine 0.001 35-37°C for 2-4 weeks
Biotin 0.0005 Organisms (ATCC) Growth
Final pH ( at 25°C) 6.6±0.2 Mycobacterium fortuitum (6841) Good-luxuriant
* Formula adjusted to suit performance parameters Mycobacterium smegmatis (14468) Good-luxuriant
Directions Mycobacterium tuberculosis (25618) Good-luxuriant
Storage
1. Suspend 2.35 grams in 450 ml distilled water.
Store at 22-300C and prepared medium at 2-80C.
2. Add either 2 ml glycerol or 0.5 g polysorbate 80.
Shelf Life
Use before expiry date as mentioned on the label.

Middlebrook 7H11 Agar Base AM5069273

Use Catalase neutralizes toxic peroxides. Malachite green partially inhibits other
Middlebrook 7H11 Agar Base with the addition of supplement is recommended bacteria.
for isolation, cultivation and sensitivity testing of Mycobacterium Formula*
Summary Ingredients in grams per liter
Casein enzymic hydrolysate 1.00
Middlebrook 7H11 Agar is a modification of Middlebrook 7H10 Agar (80.2)
Ammonium sulphate 0.50
used for the isolation, cultivation and sensitivity testing of M. tuberculosis. It was
Monopotassium phosphate 1.50
shown by Cohn et al (17.4) that the addition of casein enzymic hydrolysate Disodium phosphate 1.50
enhanced the growth of more fastidious M. tuberculosis strains, which in turn was Sodium citrate 0.40
helpful in drug susceptibility testing (77.1). The media is enriched by the Magnesium sulphate 0.05
addition of Middlebrook OADC Growth Supplement (AS0181) and glycerol. L-Glutamic acid 0.50
Principle Ferric ammonium citrate 0.04
Pyridoxine 0.001
Middlebrook media contain a variety of inorganic salts, which help, in growth of Biotin 0.0005
Mycobacteria. Citric acid formed fromsodium citrate helps in retaining inorganic Malachite green 0.001
cations in solution. Glycerol supplies carbon and energy. Middlebrook OADC Agar 15.000
Growth Supplement (AS0181) contains oleic acid, bovine albumin, sodium Final pH ( at 25°C) 6.6±0.2
chloride, dextrose and catalase. Oleic acid and other long chain fatty acids are *Formula adjusted to suit performance parameters
Directions
essential for metabolism of Mycobacteria. Some free fatty acids are toxic to
Mycobacteria but albumin binds to those fatty acids and prevents toxic action on 1. Suspend 10.25 gms of the powder in 450 ml distilled water containing
Mycobacteria. Dextrose serves as an energy source. 2.5 ml Glycerol.

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2. Heat to boiling to dissolve the medium completely. fortuitum (6841)
Mycobacterium Good-luxuriant >=50% More than 70%
3. Sterilize by autoclaving at 121ºC (15 lbs pressure) for 15 minutes.
smegmatis (14468)
4. Cool to 50°C. Aseptically add contents of 1 vial of Middlebrook OADC Growth Mycobacterium Good-luxuriant >=50% More than 70%
Supplement (AS0181). Mix thoroughly before dispensing. tuberculosis H37RV
Quality Control (25618)
Dehydrated Appearance For growth RGI should be more than 70%
Light yellow to light green homogeneous free flowing powder RGI- Relative Growth Index
Prepared Appearance Storage
Light amber coloured clear to slightly opalescent gel with greenish tinge forms Store at 22-300C and prepared medium at 2-80C
in Petri plates
Shelf Life
Cultural Response
Use before expiry date as mentioned on the label.
Cultural characteristics after 2-4 weeks at 35-37ºC.
Organisms (ATCC) Growth Growth RGI
Recovery
Mycobacterium Good-luxuriant >=50% More than 70%

Modified Rappaport Vassiliadis Medium AM506928

Modified Rappaport Vassiliadis Medium for Water Testing BIS AM506929
Modified Rappaport Vassiliadis Medium for Water Testing ISO AM506930
Use Papaic digest of soyabean meal 5.00
Modified Rappaport Vassiliadis Medium is used for the selective enrichment of Sodium chloride 8.00
Salmonella from environmental and foods specimens. Mono-Potassium phosphate 1.60
Summary Magnesium chloride.6H2O 40.00
Malachite green 0.04
Rappaport F. developed a culture media for the selective enrichment of
Final pH (at 250C) 5.2 ± 0.2
Salmonella, which enable the salmonella to multiply freely and inhibit
* Formula adjusted to suit performance parameters
accompanying coliform organism like Proteus and pseudomonas etc (90.1).
Directions
Vassiliadis modified Rappaport Broth by lowering the concentration of malachite
1. Suspend the 30 gms (the equivalent weight of dehydrated medium per liter)
green and raising the incubation temperature to 43°C (115.3). This modified
of powder in 1110 ml distilled water and mix thoroughly.
Rappaport Enrichment Broth is RV or Rappaport-Vassiliadis Medium and has
been found to be superior to other salmonella selective enrichment media, 2. Boil with frequent agitation to dissolve the powder completely.
especially with small inoculum. 3. Sterilize by autoclaving at 1150C (10 lbs pressure) for 15 minutes.
Principle Quality Control
Dehydrated Appearance
Papaic digest of soyabean-meal provide nitrogen compounds, carbon compounds
Yellow coloured, homogeneous, free flowing powder.
and nutrients. Magnesium chloride increases the osmotic pressure in the medium.
Prepared Appearance
Malachite green makes the medium selective for Salmonella. chloride provides Blue coloured, clear solution without any precipitate.
sodium ions for the membrane transport and maintains osmotic equilibrium of Cultural Response
the medium. Cultural characteristics after 24-48 hours at 370C and 430C.
The ingredients magnesium chloride & malachite green and the low pH of the Organisms (ATCC) Growth Recovery RGI
medium inhibit the growth of coliform contaminants and permit unrestricted on XLD Agar
development of salmonellae. (Colour of colony)
S.serotype Typhimurium Luxuriant Red colonies with More than 70%
Formula*
(23564) black center
Ingredients in grams per liter
Salmonella abony Luxuriant Red colonies with More than 70%

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NCTC (6017) black center 2. Inoculate 0.1 ml of the pre-enrichment Buffered peptone water culture in to
Salmonella serotype Luxuriant Red colonies with More than 70% the 10 ml of Modified Rappaport Vassiliadis and incubate at 30-350C for
typhi NCTC (780) black center
18-24 hours.C for 24 to 48 hours.
Salmonella enterica Luxuriant Red colonies with More than 70%
(13076) black center 3. Subculture the broth by streaking on to plates of X.L.D. Agar (AM1112 /
Escherichia coli Fair yellow More than 70% 5112) and Brilliant Green Agar, Modified (AM1018 / 5018). Incubate at
(25922) 30-350C for 18-48 hours.
Staphylococcus aureus Inhibited - 0% Interpretation of Results
(6538)
Examine cultured plates for typical Salmonella colonies.
For growth RGI should be more than 70%
Storage
For Inhibition RGI should be 0%
RGI- Relative Growth Index Store at 22-300C and prepared medium at 2-80C.
Procedure Shelf Life

1. For pre-enrichment, aseptically add the test specimen to Buffered Peptone Water Use before expiry date as mentioned on the label.
(AM10211/50211) and incubate at 370C for 18-24 hours.

Modified Teepol Broth (Twin Pack) ISO AM506931

Use Lactose 30.00
Modified Teepol Broth (Twin Pack) ISO is used for selective isolation and Phenol red 0.2
Part B:
identification of enteric lactose fermenting bacteria.
Teepol 4.00
Summary
Final pH (at 250C) 7.4 ± 0.2
Modified Teepol Broth is formulated as described by Burman (11.3), where he * Formula adjusted to suit performance parameters
substituted Teepol in place of bile salts in the formulation of Membrane Directions
Enrichment Teepol Broth. The use of Teepol in place of bile salts was previously 1. Suspend 76.2 grams of Part A in 1000 ml distilled water containing 4 grams
recommended by Jameeson and Emberley (47.1). Burman (11.4) showed that if of Part B.
a preliminary incubation is carried out at a lower temperature resuscitation is not
2. Heat if necessary to dissolve the medium completely.
required. Non-chlorinated organisms benefit from 4 hour incubation at 300C but
chlorinated organisms require 6 hours incubation at 250C. 3. Dispense in tubes containing inverted Durham's tubes.
Principle 4. Sterilize by autoclaving at 15 lbs pressure (1210C) for 15 minutes.
The coliform and Escherichia coli count are made on separate volumes of water. Quality Control
Dehydrated Appearance
The water samples are filtered through membrane filter and this filter is placed
Part A: Pink coloured, homogeneous, free flowing powder.
face upwards on an absorbent pad saturated with Modified Teepol Broth. The
Part B: Colourless viscous solution.
yellow colonies formed are further identified. Prepared Appearance
Presumptive coliform organisms: Yellow colonies from membranes incubated at Red coloured, clear to slightly opalescent solution.
350C, when subcultured in Lactose Peptone Water produce gas at 350C after 43 Cultural Response
hours. Cultural characteristics after 24-48 hours at …........
Organisms (ATCC) Growth at 350C Growth at 440C
Presumptive Escherichia coli : Yellow colonies from membrane at 440C produce
Escherichia coli (25922) + +
gas and indole after 24 hours. Enterobacter aerogenes (13048) + –
Formula* Storage
Ingredients in grams per liter
Store at 22-300C and prepared medium at 2-80C.
Part A :
Shelf Life
Peptic digest of animal tissue 40.00
Yeast extract 6.00 Use before expiry date as mentioned on the label.

214 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
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Modified Tergitol 7 Agar Base ISO AM506932
Use 4. Cool to 45-500C.
Modified Tergitol 7 Agar Base is used for selective isolation and enumeration of 5. Add 2.5 ml of 1 % 2,3,5 Triphenyl Tetrazolium Chloride (AS0271).
coliform organisms in water by membrane filteration method.
6. Mix well and pour into sterile petri plates.
Summary
Quality Control
Tergitol 7 Agar is a selective and differential medium for the detection and Dehydrated Appearance
enumeration of coliforms in water. Chapman, (16.2 & 16.3) modified his original Yellow coloured, homogeneous, free flowing powder.
formula of Tergitol 7 Agar by addition of Triphenyl Tetrazolium Chloride (TTC). It is Prepared Appearance
now recommended by ISO Committee (46.4). Green coloured, clear to slightly opalescent gel forms in petriplates.
Principal Cultural Response
Cultural characteristics after 18-24 hours at 350C
Tergitol 7 is a selective agent (89.3) which inhibits gram positive organisms and
Organisms Growth Colour Colour RGI
minimizes swarming of Proteus species enabling better coliform recovery.
(ATCC) or colony* of colony**
Lactose fermentation is observed by change in colour of bromothymol blue, the Escherichia coli Luxuriant Yellow Yellow with More than
pH indicator. Triphenyl Tetrazolium Chloride (TTC) allows earlier recognition and (25922) yellow zone 70%
identification of Escherichia coli and Enterobacter aerogenes in water and food Enterobacter Luxuriant Yellow Greenish More than
(81.3). TTC is rapidly reduced by coliforms except Escherichia coli and aerogenes yellow 70%
Enterobacter aerogenes to insoluble fromazan which gives red colour to the Klebsiella Luxuriant Yellow Greenish More than
colonies. The lactose fermenters show greenish yellow colonies with yellow zones pneumoniae yellow 70%
while lactose non fermenters show red colonies surrounded by blue zones. (13883)
S. serotype Luxuriant Colurless Red with More than
Formula*
Typhimurium bluish zone 70%
Ingredients in grams per liter
(14028)
Peptic digest of animal tissue 10.00
Pseudomonas Good Colourless Red with More than
Yeast extract 6.00
aeruginosa bluish zone 70%
Meat extract 5.00
(27853)
Lactose 20.00
Proteus vulgaris Good Colourless Red with More than
Bromo thymol blue 0.05
(13315) bluish zone 70%
Tergitol 0.10
Staphylococcus Inhibited – – 0%
Agar 16.00
aureus (25923)
Final pH (at 250C) 7.2 ± 0.2
Key: *= on plain medium
* Formula adjusted to suit performance parameters
**= on medium with TTC
Directions
Storage
1. Suspend 57.15 in 1000 ml distilled water.
Store at 22-300C and prepared medium at 2-80C.
2. Boil to dissolve the medium completely. Shelf Life
3. Sterilize by autoclaving at 15 lbs pressure (1210C) for 15 minutes. Use before expiry date as mentioned on the label.

Motility Test Medium AM506933

Use formulated Motility Test medium (23.3).
Motility Test Medium is recommended for detection of bacterial motility. Principle
Summary Tryptose serves as a source of carbon, nitrogen, vitamins and minerals. Sodium
Bacterial motility is an important determinant in making a final species chloride provides sodium ions for the membrane transport and maintains osmotic
identification. In 1936, Tittsler and Sandholzer first introduced the use of semi equilibrium of the medium. In motility media agar concentrations is used higher
solid agar for the detection of bacterial motility (90.2). Edward and Ewing than 0.3% to produce semi solid media. In semi solid media motility is detected

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 215
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more easily. Motile organisms spread out from the line of inoculation, while non- Prepared Appearance
motile organisms grow only along the stab line. Light yellow coloured, clear to slightly opalescent gel form in tubes as butts.
Cultural Response
Formula*
Cultural characteristics after 18- 48 hours at 35-37°C.
Ingredients in grams per liter
Organisms (ATCC) Growth Motility
Tryptose 10.0
Escherichia coli (25922) Luxuriant +
Sodium chloride 5.0
Enterobacter aerogenes (13048) Luxuriant +
Agar 5.0
Klebsiella pneumoniae (13883) Luxuriant -
Final pH (at 250C) 7.2 ± 0.2
Staphylococcus aureus (25923) Luxuriant -
*Formula adjusted to suit performance parameters
Salmonella enteritidis (13076) Luxuriant +
Directions
Procedure
1. Suspend 20 gms of powder in 1000ml distilled water.
1. Tubes are inoculated by stabbing with a straight wire.
2. Mix thoroughly.
2. Incubate tubes for 24-48 hours at 35 ± 2°C in an aerobic atmosphere.
3. Warm slightly with frequent agitation to dissolve the powder completely.
Interpretation of Results
4. Dispense in test tubes.
Motile organisms spread out from the line of inoculation. But the non-motile
5. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes. Allow organisms grow only along the line of inoculation.
tubed medium to cool in an upright position. Storage
Quality Control
Store at 22-300C and prepared medium at 2-80C.
Dehydrated Appearance
Shelf Life
Yellow coloured, homogeneous free flowing powder.
Use before expiry date as mentioned on the label.

Motility Test Medium (Edwards and Ewing) BIS AM506934

Use Sodium chloride 5.0
Motility Test Medium (Edwards and Ewing) BIS is used for testing motility of Agar 4.0
enteric bacteria in compliance with BIS specifications IS: 5887 (Part 1 and Part 5) Final pH (at 250C) 7.5 ± 0.2
*Formula adjusted to suit performance parameters
1976 reaffirmed, 1986.
Directions
Summary
1. Suspend 22 gms of powder in 1000ml distilled water.
Bacterial motility is an important determinant in making a final species
identification. In 1936, Tittsler and Sandholzer first introduced the use of semi 2. Mix thoroughly.
solid agar for the detection of bacterial motility. Edward and Ewing formulated 3. Warm slightly with frequent agitation to dissolve the powder completely.
Motility Test medium. 4. Dispense 8ml amounts in test tubes.
Principle
5. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Peptone serves as a source of carbon, nitrogen, vitamins and minerals. Meat Quality Control
extract also serves as a good source of nutrition. Sodium chloride provides sodium Dehydrated Appearance
ions for the membrane transport and maintains osmotic equilibrium of the Yellow coloured, homogeneous free flowing powder.
medium. In motility media agar concentrations is used higher than 0.3% to Prepared Appearance
produce semi solid media. In semi solid media motility is detected more easily. Yellow coloured, clear gel form in tubes as butts.
Cultural Response
Motile organisms spread out from the line of inoculation, while non-motile
Cultural characteristics after 18- 24 hours at 35-37°C.
organisms grow only along the stab line.
Organisms (ATCC) Growth Motility
Formula*
Escherichia coli (25922) Luxuriant +
Ingredients in grams per liter
Enterobacter aerogenes (13048) Luxuriant +
Peptone 10.0
S. serotype Enteritidis (13076) Luxuriant +
Meat extract 3.0

216 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
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Klebsiella pneumoniae (13883) Luxuriant - organisms grow only along the line of inoculation.
Staphylococcus aureus (25923) Luxuriant - Storage
Procedure
Store at 22-300C and prepared medium at 2-80C.
1. Tubes are inoculated by stabbing with a straight wire. Shelf Life
2. Incubate tubes for 24-48 hours at 35 ± 2°C in an aerobic atmosphere. Use before expiry date as mentioned on the label.
Interpretation of Results
Motile organisms spread out from the line of inoculation. But the non-motile

MR-VP Medium AM1070/AM5070

Use 3. Dispense 10 ml amounts in test tubes.
MR-VP Medium also known as Buffered Peptone Glucose Broth is used for the 4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
differentiation of coli-aerogenes group by means of the Methyl Red and Voges- Quality Control
Proskauer reactions. Dehydrated Appearance
Summary Cream coloured, homogeneous, free flowing powder.
Clark and Lubs found that the addition of methyl red to cultures of E.coli resulted Prepared Appearance
in a red colour due to the high acidity produced during dextrose fermentation. Light yellow coloured, clear solution without any precipitate.
Cultural Response
Voges-Proskauer reported red colouration after the addition of potassium
Cultural characteristics after 48 hours at 300C.
hydroxide to specific culture media with organisms in it. Thus, the investigators
Organisms (ATCC) Growth MR Test VP Test
developed MR-VP Medium, which enables both tests to be performed in the same Enterobacter aerogenes (13048) Luxuriant - (yellow) + (red)
medium in different tubes. ISO has recommended this medium for the detection Escherichia coli (25922) Luxuriant + (red) - (no change)
of coli-aerogenes group. MR-VP Medium is included in the Bacteriological Klebsiella pneumoniae (23357) Luxuriant -(yellow) +(red)
Analytical Manual for food and cosmetics testing (113) and is also recommended Procedure
by APHA for the examination of foods (20) and milk (39). 1. Using a light inoculum, inoculate tubes of MR-VP Medium with an 18-24
Principle hour pure cultures.
Methyl red positive organisms produce high levels of acid during fermentation of 2. Incubate tubes aerobically at 300C for a minimum of 48 hours for Voges-
dextrose, overcoming the phosphate buffering system and produce a red colour on Proskaeur test and preferably for 5 days for Methyl Red test.
the addition of methyl red pH indicator.
3. Prepare the methyl red indicator by dissolving 0.1 gm of methyl red in 300
In Voges-Proskauer test, the red colour produced by the addition of potassium ml of 95% ethyl alcohol. Add sufficient water to make 500 ml.
hydroxide to cultures of certain microbial species is due to their ability to produce a
4. After the appropriate incubation period, aseptically remove aliquots of the
neutral end product, acetoin (acetylmethylcarbinol), from the fermentation of
medium and conduct the tests.
dextrose. The acetoin is oxidized in the presence of oxygen and alkali to produce
diacetyl, which reacts with creatine to give a red colour, which is a positive VP test. l Methyl Red Test- Add 5 drops of methyl red indicator to an aliquot of the
Formula*
broth. Interpret the result immediately.
Ingredients in grams per liter l Voges-Proskaeur Test- Add 15 drops of reagent A (5% w/v a -
Buffered Peptone 7.0 naphthol in absolute alcohol) and 5 drops of reagent B (40 % w/v
Dextrose 5.0 Potassium hydroxide in distilled water) to 1 ml of broth culture. Shake
Dipotassium Phosphate 5.0 well after the addition of each reagent to aerate the sample.
Final pH (at 250C) 6.9 ± 0.2
Interpretation of Results
* Formula adjusted to suit performance parameters
Directions
1. Methyl Red Test
1. Suspend 17 gms of the powder in 1000 ml distilled water and mix well. l Positive test - red colour at the surface of the medium.
2. If necessary, heat to dissolve the medium. l Negative test - yellow colour at the surface of the medium.

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 217
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2. Voges-Proskauer Test precipitate has not been shown to reduce the effectiveness of the reagent.
l Positive test - development of a distinct red colour within 5 minutes. 5 Most members of the family Enterobacteriaceae give either a positive MR
l Negative test - appearance of a yellow colour or copper like colour on test or a positive VP test. However, certain organisms such as Hafnia alvei
the surface of the medium. and Proteus mirabilis may give a positive result for both.
3. Certain species within Enterobacteriaceae genera may react differently or 6 Incubation time for the methyl red test cannot be shortened by increasing the
give variable results. concentration of dextrose in the medium or by heavily inoculating the broth.
Precautions / Limitations 7 Incubate MR negative tests for more than 48 hours and test again.
1. While adding the VP reagents to the medium, it is important that the a- 8 Read the VP test at 48 hours. Increased incubation may produce acid
naphthol be added first and the KOH added second. A change in the order conditions in the broth that will interfere with reading the results.
may produce invalid test results. 9 Due to the possible presence of acetoin, diacetyl or related substances in
2 False positive VP results may occur if VP tests are read beyond one hour certain raw materials, the use of media, low in these substances (MR-VP
following the addition of reagents. Medium) is recommended for this test.
3 Results of the MR and VP tests need to be used in conjunction with other Storage
biochemical tests to differentiate genus and species within the Store at 22-300C and prepared medium at 2-80C.
Enterobacteriaceae. Shelf Life

4 A precipitate may form in the potassium hydroxide reagent solution. The Use before expiry date as mentioned on the label.

MR-VP Medium BIS AM10701/AM50701

Use Formula*
MR-VP Medium is used for the differentiation of coli-aerogenes group by means Ingredients in grams per liter
Peptone 5.0
of the Methyl Red and Voges-Proskauer reactions in compliance with BIS.
Dextrose 5.0
Summary
Dipotassium hydrogen phosphate 5.0
Clark and Lubs (12.1) found that the addition of methyl red to cultures of E.coli Final pH (at 250C) 7.5 ±0.2
resulted in a red colour due to the high acidity produced during dextrose * Formula adjusted to suit performance parameters
fermentation. Voges-Proskauer (116.1) reported red colouration after the Directions
addition of potassium hydroxide to specific culture media with organisms in it. 1. Suspend 15 gms of the powder in 1000 ml distilled water and mix well.
Thus, the investigators developed MR-VP Medium, which enables both tests to be
2. If necessary, heat to dissolve the medium.
performed in the same medium in different tubes. ISO has recommended this
3. Dispense 10 ml amounts in test tubes.
medium for the detection of coli-aerogenes group. MR-VP Medium is included in
the Bacteriological Analytical Manual for food and cosmetics testing and is also 4. Sterilize by autoclaving at 1150C (10lbs pressure) for 10 minutes.
Quality Control
recommended by APHA for the examination of foods and milk .
Dehydrated Appearance
Principle
Cream coloured, homogeneous, free flowing powder.
Methyl red positive organisms produce high levels of acid during fermentation of A Differential Dehydrated Culture Medium
dextrose, overcoming the phosphate buffering system and produce a red colour on Prepared Appearance
the addition of methyl red pH indicator. In Voges-Proskauer test, the red colour Light yellow coloured, clear solution without any precipitate.
produced by the addition of potassium hydroxide to cultures of certain microbial Cultural Response
species is due to their ability to produce a neutral end product, acetoin Cultural characteristics after 48 hours at 300C.
(acetylmethylcarbinol), from the fermentation of dextrose (77.1). The acetoin is Organisms (ATCC) Growth MR Test VP Test
Enterobacter aerogenes (13048) Luxuriant - (Yellow) + (Red)
oxidized in the presence of oxygen and alkali to produce diacetyl, which reacts
Escherichia coli (25922) Luxuriant + (Red) - (No change)
with creatine to give a red colour, which is a positive VP test.
Klebsiella pneumoniae (23357) Luxuriant -(Yellow) +(Red)

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Procedure Precautions / Limitations
1. Using a light inoculum, inoculate tubes of MR-VP Medium with an 18-24 1. While adding the VP reagents to the medium, it is important that the a-
hour pure cultures. naphthol be added first and the KOH added second. A change in the order
2. Incubate tubes aerobically at 300C for a minimum of 48 hours for Voges- may produce invalid test results.
Proskaeur test and preferably for 5 days for Methyl Red test. 2. False positive VP results may occur if VP tests are read beyond one hour
3. Prepare the methyl red indicator by dissolving 0.1 gm of methyl red in 300 following the addition of reagents.
ml of 95% ethyl alcohol. Add sufficient water to make 500 ml. 3. Results of the MR and VP tests need to be used in conjunction with other
4. After the appropriate incubation period, aseptically remove aliquots of the biochemical tests to differentiate genus and species within the
medium and conduct the tests. enterobacteriaceae.
?
Methyl Red Test- Add 5 drops of methyl red indicator to an aliquot of 4. A precipitate may form in the potassium hydroxide reagent solution. The
the broth. Interpret the result immediately. precipitate has not been shown to reduce the effectiveness of the reagent.
?
Voges-Proskaeur Test- Add 15 drops of reagent A (5% w/v naphthol in 5. Most members of the family enterobacteriaceae ive either a positive MR test
absolute alcohol) and 5 drops of reagent B (40 % w/v Potassium or a positive VP test. However, certain organisms such as Hafnia alvei and
hydroxide in distilled water) to 1 ml of broth culture. Shake well after Proteus mirabilis ay give a positive result for both.
the addition of each reagent to aerate the sample. 6. Incubation time for the methyl red test cannot be shortened by increasing the
Interpretation of Results concentration of dextrose in the medium or by heavily inoculating the
1. Methyl Red Test broth.
?
Positive test - red colour at the surface of the medium. 7. Incubate MR negative tests for more than 48 hours and test again.
?
Negative test - yellow colour at the surface of the medium. 8. Read the VP test at 48 hours. Increased incubation may produce acid
2. Voges-Proskauer Test conditions in the broth that will interfere with reading the results.
?
Positive test - development of a distinct red colour within 5 minutes. 9. Due to the possible presence of acetoin, diacetyl or related substances in
certain raw materials, the use of media, low in these substances (MR-VP
?
Negative test - appearance of a yellow colour or copper like colour on
Storage
the surface of the medium.
Store at 22-300C and prepared medium at 2-80C.
3. Certain species within enterobacteriaceae genera may react differently or
Shelf Life
give variable results. Use before expiry date as mentioned on the label.

MR-VP Medium ISO AM10702/AM50702

Use Principle
MR-VP Medium used for the differentiation of coli-aerogenes group by means of Methyl red positive organisms produce high levels of acid during fermentation of
the Methyl Red and Voges-Proskauer reactions in compliance with ISO. dextrose, overcoming the phosphate buffering system and produce a red colour on
Summary the addition of methyl red pH indicator. In Voges-Proskauer test, the red colour
Clark and Lubs found that the addition of methyl red to cultures of E.coli resulted produced by the addition of potassium hydroxide to cultures of certain microbial
in a red colour due to the high acidity produced during dextrose fermentation. species is due to their ability to produce a neutral end product, acetoin
Voges-Proskauer reported red colouration after the addition of potassium (acetylmethylcarbinol), from the fermentation of dextrose. The acetoin is oxidized
hydroxide to specific culture media with organisms in it. Thus, the investigators in the presence of oxygen and alkali to produce diacetyl, which reacts with
developed MR-VP Medium, which enables both tests to be performed in the same creatine to give a red colour, which is a positive VP test.
medium in different tubes. ISO has recommended this medium for the detection Formula*
of coli-aerogenes group. MR-VP Medium is included in the Bacteriological Ingredients in grams per liter
Pancreatic digest of casein 3.5
Analytical Manual for food and cosmetics testing and is also recommended by
Peptic digest of animal tissue 3.5
APHA for the examination of foods and milk .

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 219
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Dextrose 5.0 Positive test - red colour at the surface of the medium.
Dipotassium phosphate 5.0
Negative test - yellow colour at the surface of the medium.
Final pH (at 250C) 6.9 ±0.2
* Formula adjusted to suit performance parameters 2. Voges-Proskauer Test
Directions Positive test - development of a distinct red colour within 5 minutes.
1. Suspend 17 gms of the powder in 1000 ml distilled water and mix well. Negative test - appearance of a yellow colour or copper like colour on the
2. If necessary, heat to dissolve the medium. surface of the medium.
3. Dispense 10 ml amounts in test tubes. 3. Certain species within Enterobacteriaceae genera may react differently or
0
4. Sterilize by autoclaving at 121 C (15 lbs pressure) for 15 minutes. give variable results.
Quality Control Precautions / Limitations
Dehydrated Appearance 1. While adding the VP reagents to the medium, it is important that the a-
Cream coloured, homogeneous, free flowing powder. naphthol be added first and the KOH added second. A change in the order
Prepared Appearance may produce invalid test results.
Light yellow coloured, clear solution without any precipitate.
2. False positive VP results may occur if VP tests are read beyond one hour
Cultural Response
0
Cultural characteristics after 48 hours at 30 C.
following the addition of reagents.
Organisms (ATCC) Growth MR Test VP Test 3. Results of the MR and VP tests need to be used in conjunction with other
Enterobacter aerogenes (13048) Luxuriant - (Yellow) + (Red) biochemical tests to differentiate genus and species within the
Escherichia coli (25922) Luxuriant + Red) - (No change) Enterobacteriaceae.
Klebsiella pneumoniae (23357) Luxuriant -(Yellow) +(Red)
4. A precipitate may form in the potassium hydroxide reagent solution. The
Procedure
precipitate has not been shown to reduce the effectiveness of the reagent.
1. Using a light inoculum, inoculate tubes of MR-VP Medium with an 18-24
5. Most members of the family Enterobacteriaceae give either a positive MR
hour pure cultures.
test or a positive VP test. However, certain organisms such as Hafnia alvei
2. Incubate tubes aerobically at 300C for a minimum of 48 hours for Voges-
and Proteus mirabilis may give a positive result for both.
Proskaeur test and preferably for 5 days for Methyl Red test.
6. Incubation time for the methyl red test cannot be shortened by increasing the
3. Prepare the methyl red indicator by dissolving 0.1 gm of methyl red in 300
concentration of dextrose in the medium or by heavily inoculating the broth.
ml of 95% ethyl alcohol. Add sufficient water to make 500 ml.
7. Incubate MR negative tests for more than 48 hours and test again.
4. After the appropriate incubation period, aseptically remove aliquots of the
8. Read the VP test at 48 hours. Increased incubation may produce acid
medium and conduct the tests.
conditions in the broth that will interfere with reading the results.
?
Methyl Red Test- Add 5 drops of methyl red indicator to an aliquot of
9. Due to the possible presence of acetoin, diacetyl or related substances in
the broth. Interpret the result immediately.
certain raw materials, the use of media, low in these substances (MR-VP
?
Voges-Proskaeur Test- Add 15 drops of reagent A (5% w/v a-naphthol
Medium) is recommended for this test.
in absolute alcohol) and 5 drops of reagent B (40 % w/v Potassium
Storage
hydroxide in distilled water) to 1 ml of broth culture. Shake well after
Store at 22-300C and prepared medium at 2-80C.
the addition of each reagent to aerate the sample.
Shelf Life
Interpretation of Results
Use before expiry date as mentioned on the label.
1. Methyl Red Test

Mueller Hinton Agar AM1071/AM5071

Use Summary
Mueller Hinton Agar is used for antimicrobial disc diffusion susceptibility testing Mueller Hinton Agar was originally developed for the cultivation of Neisseria
of common, rapidly growing bacteria by the Bauer-Kirby method. (82). These organisms are now isolated on selective media. Since clinical

220 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
 Exploring... Accumix
laboratories were using a wide variety of procedures for determining the Prepared Appearance
susceptibility of bacteria to antibiotic and chemotherapeutic agents, Bauer, Kirby Light amber coloured, slightly opalescent gel.
Cultural Response
and others (6) developed a standardized procedure in which Mueller Hinton Agar
Cultural characteristics after 18-24 hours at 370C.
was selected as the test medium. Subsequently, international collaborative study
Organisms (ATCC) Growth RGI
confirmed the value of Mueller Hinton Agar for this purpose due to its relatively Escherichia coli (25922) Luxuriant More than 70%
good reproducibility, the simplicity of its formula, and the wealth of experimental Neisseria gonorrhoeae (49226) Luxuriant More than 70%
data that had been accumulated using this medium. Mueller Hinton Agar Pseudomonas aeruginosa (27853) Luxuriant More than 70%
complies with the requirements of World Health Organization and is specified in Staphylococcus aureus (25923) Luxuriant More than 70%
the FDA's Bacteriological Analytical Manual for food testing (113). Streptococcus faecalis (19433) Luxuriant More than 70%
For growth RGI should be more than 70%
For additional details refer to The National Committee for Clinical Laboratory
RGI- Relative Growth Index
Standards (NCCLS) which contains the performance standard for the Baeur-Kirby
Procedure
procedure (84). This procedure is recommended for testing rapidly growing Standard Method
aerobic or facultative anaerobic bacterial pathogens, such as staphylococci,
1. Gram staining is done before starting susceptibility testing to confirm the
members of the Enterobacteriaceae, aerobic gram-negative rods, e.g.
culture purity and to determine appropriate battery of tests.
Pseudomonas species and Acinetobacter species, enterococci and Vibrio cholerae.
The procedure is modified for testing fastidious species; i.e. H.influenza, 2. 3-5 well-isolated colonies should be selected and transferred to 4-5 ml of a
N.gonorrhoeae, S.pneumoniae and other streptococci. The NCCLS Document M2, suitable broth using an inoculating needle.
Performance for Antimicrobial Disc Susceptibility Tests, recommends Mueller 3. Incubate the broth (usually 2-6 hours) at 350C until it achieves or exceeds
Hinton Agar supplemented with 5% defibrinated sheep blood for fastidious the turbidity of the 0.5 McFarland's barium sulphate standard. This results
organisms. in a suspension containing approximately 1 - 2 X 108 CFU/ml.
Principle 4. Adjust the turbidity to the barium sulphate standard. For the diluents use
Casein acid hydrolysate and beef infusion supply amino acids and other sterile broth or sterile saline. The turbidity of the standard and the test
nitrogenous substances, minerals, vitamins, carbon and other nutrients to inoculums should be compared by holding both tubes in front of a white
support the growth of microorganisms. Starch acts as a protective colloid against background with finely divided lines or by use of a photometric device.
toxic substances that may be present in the medium. Hydrolysis of starch during 5. Within 15 minutes of adjusting the turbidity of the inoculum, immerse a
autoclaving provides a small amount of dextrose, which is a source of energy. sterile cotton swab into the properly diluted inoculum and rotate it firmly
Formula* several times against the upper inside wall of the tube to express excess
Ingredients in grams per liter fluid.
Casein Acid Hydrolysate 17.5
Beef, Infusion from 300.0
6. Inoculate the entire agar surface of the plate three times, rotating the plate
Starch 1.5 600 between streaking to obtain even inoculation. Swab the rim of the agar
Agar 17.0 bed too.
Final pH (at 250C) 7.3 ± 0.2 7. The lid may be left ajar for 3-5 minutes and the plate held at room
* Formula adjusted to suit performance parameters temperature for not more than 15 minutes to allow the surface moisture to
Directions
be absorbed before applying the antibiotic discs.
1. Suspend 38 gms of the powder in 1000 ml distilled water and mix well.
8. Apply discs by means of an antimicrobial dics dispenser, aseptically, at least
2. Boil with frequent agitation to dissolve the powder completely. DO NOT 24 mm apart. Preferably, deposit Penicillin and Cephalosporin discs not
OVER HEAT. more than 10 mm from the edge of the Petri dish, and their centers at least
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. 30 mm apart. Avoid placing such discs adjacent to one another. Tap the
4. Mix well before pouring. discs with a sterile needle or forceps after placing them on the agar for
Quality Control complete contact with the medium surface.
Dehydrated Appearance 9. Within 15 minutes of applying the discs, invert the plates and incubate at
Yellow coloured, homogeneous, free flowing powder. 370C. With non-fastidious organisms the plates should not be incubated

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under an increased concentration of carbon dioxide. Precautions / Limitations

10. Examine plates after 16-18 hours of incubation. A full 24 hours is 1. Unsupplemented Mueller Hinton Agar, although adequate for susceptibility
recommended for S.aureus with oxacillin to detect methicillin-resistant testing of rapidly growing aerobic pathogens, is not adequate for more
S.aureus (MRSA) and for Enterococcus species when tested with vancomycin fastidious organisms such as S.pneumoniae.
to detect vancomycin resistant strains. Growth within the apparent zone of 2. Numerous factors can affect the result: inoculum size, rate of growth, media
inhibition is indicative of resistance. formulation, pH, length of incubation, disc content, drug diffusion rates,
Interpretation of Results and measurement of endpoints. Hence, strict adherence to protocol is
1. A confluent “lawn” of growth should be obtained. Too light inoculum gives required to ensure reliable results.
isolated colonies and the test should be repeated. Measure the diameter of 3. Mueller Hinton Agar deeper than 4 mm may cause false resistant results,
the zones of complete inhibition, including the diameter of the disc, to the and agar less than 4 mm deep may be associated with a false-susceptibility
nearest whole millimeter, using calipers, a ruler, or a template prepared for report.
this purpose. The measuring device is held on the back of the inverted plate 4. pH outside the range of 7.3 ± 0.2 may adversely affect susceptibility test
over a black, non-reflecting background, and illuminated from above. The results. If the pH is too low, aminoglycosides and macrolides will appear to
endpoint should be taken as the area showing no obvious visible growth lose potency; others may appear to have excessive activity. The opposite
that can be detected with the unaided eye. Disregard faint growth of tiny effects are possible if the pH is too high.
colonies, which can be detected with difficulty near the edge of the obvious
5. The following technical and human errors may occur which compromise on
zone of inhibition. The zone diameters measured around the discs should be
reliability and accuracy and must be avoided:-
compared with those in the NCCLS Document M100 (M2).
·Improper disc storage.
2. S.aureus when tested with oxacillin discs is an exception, as are enterococci
when tested with vancomycin. In these cases, transmitted light should be ·Inoculum not properly adjusted (too light or too heavy).
used to detect a haze of growth around the disc, which is shown, by “occult ·Incubation temperature deviating from 35-370C.
resistant” MRSA strains or vancomycin-resistant enterococci. With Proteus ·Use of an increased CO2 atmosphere.
species, if the zone of inhibition is distinct enough to measure, disregard any
·Reading plates before or after the full 16-18 hours of incubation.
swarming inside the zone. With trimethoprim and sulphonamides,
antagonistics in the medium may allow some slight growth; therefore, ·Transcribing errors.
disregard slight growth (20% or less of the lawn of growth) and measure the ·Reading error while measuring zone diameter.
more obvious margin to determine the zone diameter. ·Deterioration of the McFarland Turbidity Standard.
3. The results obtained with specific organisms may be reported as resistant, ·Contamination or mutation in the control strain.
intermediate or susceptible. Storage
NOTE: Informational supplements to NCCLS Document M2, containing Store at 22-300C and prepared medium at 2-80C.
revised tables of antimicrobial discs and interpretive standards are Shelf Life
published periodically. The latest tables should be consulted for current Use before expiry date as mentioned on the label.
recommendations.

Mueller Hinton Broth AM1072/AM5072

Use liquid medium, usually by serial dilution. The mixture, consisting of
Mueller Hinton Broth is used to determine the antimicrobial susceptibility of microorganisms, nutrient medium and antimicrobial agent, is incubated at 350C
bacteria by the tube dilution method. for 16-20 hours. The lowest concentration of antimicrobial agent at which no
Summary visible growth occurs is defined as the Minimal Inhibitory Concentration (MIC).
Mueller Hinton Broth is used for determining the Minimal Inhibitory The quantitative disc diffusion antimicrobial susceptibility procedure has been
Concentration (MIC) of antimicrobials for aerobic bacteria. The tube dilution test standardized. The rationale for an MIC susceptibility test rather than the disc
involves exposing bacteria to decreasing concentrations of antimicrobial agents in diffusion test is that it gives quantitative information. It provides a relationship

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between the amount of antimicrobial agent required to inhibit the growth of an 2. Consult appropriate references for procedures for broth dilution
organism in vitro and the achievable concentration in the blood, urine, antimicrobial susceptibility testing.
cerebrospinal fluid or bile, under various dosage conditions. It has been Interpretation of Results
suggested that in the treatment of systemic infections the drug dosage should 1. The minimum inhibitory concentration (MIC) of an antimicrobial agent for a
yield a peak concentration at the site of infection that is two to four times greater specific organism is the lowest concentration, which will inhibit the growth
than the MIC value, while for urinary tract infections, a peak urine concentration of the organism.
of 10-20 times the MIC value should be achieved. However, effective 2. Growth is indicated by turbidity or sediment.
antimicrobial therapy also depends on many other factors. 3. Some microorganisms when tested against trimethoprim /
Principle sulphamethoxazole or sulphonamides alone do not always give clear-cut
Casein acid hydrolysate and beef infusion supply amino acids and other end points.
nitrogenous substances, minerals, vitamins, carbon and other nutrients. Starch 4. In the case of doubling dilutions of trimethoprim / sulphamethoxazole,
acts as a protective colloid against toxic substances that may be present in the there may be a “trailing” of growth. Such a pattern typically shows an
medium. Hydrolysis of starch during autoclaving provides a small amount of obvious reduction in the amount of growth and, then, either small pellets
dextrose, which is a source of energy. (usually less than 1 mm in diameter) in the rest of the wells, or an obvious
Formula* reduction in the amount of growth and then a slight but detectable
Ingredients in grams per liter graduation in the size of the pellets. In these cases, the MIC end point should
Beef, Infusion from 300.0 be identified as the lowest concentration of antimicrobial agent beyond
Casein Acid Hydrolysate 17.5 which there is no further reduction in the size of the pellet or the amount of
Starch 1.5
turbidity.
Final pH (at 250C) 7.4 ± 0.2
* Formula adjusted to suit performance parameters
5. An organism may be susceptible, intermediate or resistant for a given
Directions antimicrobial agent depending on the MIC value.
1. Suspend 21 gms of the powder in 1000 ml distilled water. 6. The interpretive standards for MIC values with various drugs may be found in
NCCLS document M100 (M7) obtained from the manufacturer.
2. Mix thoroughly.
Precautions / Limitations
3. Boil with frequent agitation to dissolve the powder completely.
1. Muller Hinton Broth should be inoculated within15 minutes after preparing
4. Dispense in desired test tubes as per requirements. the inoculum suspension.
5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. 2. Inoculum size, rate of growth, medium formulation and pH, length of
Quality Control incubation and incubation environment, drug diffusion rate and
Dehydrated Appearance measurement of end points can affect results, hence care should be taken to
Yellow coloured, homogeneous, free flowing powder.
follow procedures accurately to ensure reliable results.
Prepared Appearance
Light amber coloured, clear solution without any precipitate.
3. Some strains may be encountered that fail to grow or grow poorly in this
Cultural Response broth.
Cultural characteristics after 18-24 hours at 370C. 4. A pH outside the range of 7.4 ± 0.2 may adversely affect susceptibility test
Organisms (ATCC) Growth results. If the pH is too low, aminoglycosides and macrolides will appear to
Escherichia coli (25922) Luxuriant lose potency; others may appear to have excess activity. The opposite effects
Neisseria gonorrhoeae (49226) Luxuriant
are possible if the pH is too high.
Pseudomonas aeruginosa (27853) Luxuriant
Storage
Staphylococcus aureus (25923) Luxuriant
Streptococcus faecalis (19433) Luxuriant Store at 22-300C and prepared medium at 2-80C.
Procedure Shelf Life
1. Inoculate as per recognized practices for the cultivation of microorganisms. Use before expiry date as mentioned on the label.

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MUG Lauryl Sulphate Broth AM50721
Use Dipotassium phosphate 2.75
MUG Lauryl Sulphate Broth is recommended for detection of coliform organisms Monopotassium phosphate 2.75
Sodium lauryl sulphate 0.10
in water, food by fluorogenic method.
4- Methylumbelliferyl â –D- glucuronide (MUG) 0.05
Summary
Final pH (at 25°C) 6.8±0.2
Lauryl Sulphate Broth was formulated by Mallmann and Darby (78) and is * Formula adjusted to suit performance parameters
recommended by APHA for the detection and enumeration of coliform organisms Directions
in foods, water and wastewater (103.2). MUG is added in Lauryl sulphate Broth 1. Suspend the 35.65 gms of powder in 1000 ml distilled water and mix
as the fluorogenic compound which permits the rapid detection of Escherichia coli thoroughly.
when observed under UV light where further confirmation is not required (93.1).
2. Boil with frequent agitation to dissolve the powder completely.
MUG detects anaerogenic strains which may not be detected in the conventional
procedure. Feng and Hartman (30.1) used MUG- containing medium for studying 3. Dispense into tubes with inverted Durham's tube.
â-glucuronidase activity and found Escherichia coli has 96-1000C activity, 4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Salmonella species with 17% and Shigella species 40% activity and other Quality Control
Dehydrated Appearance
genera were negative. For weakly positive strains incubation should be carried out
Light yellow coloured, homogeneous, free flowing powder.
overnight. Robison reported that no false negative results and about 5% false
Prepared Appearance
positive results.
Light amber coloured clear solution without any precipitate.
Principle
Cultural Response
Casein enzymic hydrolysate provides nutrients while lactose act as energy source. Cultural characteristics after 4-24 hours at 350C.
Sodium Lauryl sulphate inhibits many organisms other than coliforms. 4- Organisms (ATCC) Growth Fluorescence
methylumbelliferyl-â-D-glucuronide is hydrolyzed by an enzyme â- Enterobacter aerogenes Luxuriant -
glucuronidase possessed by organisms to yield 4-methylumbelliferone, a (13048)
fluorescent end product. Escherichia coli (25922) Luxuriant +
Key: +=Fluorescence under UV light at 366nm.
Formula*
Storage
Ingredients in grams per liter
Casein enzymic hydrolysate 20.0 Store at 22-300C and prepared medium at 2-80C.
Lactose 5.0 Shelf Life
Sodium chloride 5.0 Use before expiry date as mentioned on the label.

Muller Kauffmann Tetrathionate Broth Base (Novobiocin Broth) AM50722

Use isolating Salmonella from meat and meat products and from poultry and poultry
Muller Kauffmann Tetrathionate Broth Base (Novobiocin Broth) is used for products (46.1.3). Muller Kauffmann Tetrathionate Broth Base conforms with ISO
selective and differential isolation of Salmonella species. Standards.
Summary Principle
Muller (82.4) recommended Tetrathionate Broth as a selective medium for the Enzymatic Digest of Casein and Meat Peptone provide nitrogen, carbon, vitamins,
recovery of Salmonella and demonstrated the effectiveness of Tetrathionate and amino acids. Sodium Chloride maintains the osmotic balance of the medium.
Broth for enriching typhoid and paratyphoid bacilli while inhibiting coliform Calcium Carbonate neutralizes and absorbs toxic metabolites. Selectivity is
organisms. Kauffmann (51 & 52) modified this formula to include Oxbile and accomplished by the combination of Sodium Thiosulfate and tetrathionate, which
Brilliant Green for their selective properties. Using modified Muller's broth, suppresses commensal intestinal organisms. Tetrathionate is formed in the
Kauffmann increased the number of rapid screening of Salmonella in food medium upon addition of the iodine and potassium iodide solution. Organisms
(22.3). containing the enzyme tetrathionate reductase will proliferate in the medium. Ox
The British Standard Specification specifies Brilliant Green Tetrathionate Broth for Bile and Brilliant Green are additional selective agents used to suppress coliform

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bacteria and inhibit Gram-positive organisms. The addition of Novobiocin was Quality Control
described by Jeffries11 and use to suppress the growth of Proteus species Dehydrated Appearance
White to cream coloured, homogeneous, free flowing powder.
Formula*
Prepared Appearance
Ingredients in grams per liter
Milky pale green to slightly yellow-green and opaque solution.
Enzymatic Digest of Casein 8.6
Cultural Response
Meat Peptone 4.3
Sodium Chloride 2.6 Culture characteristics after 18-24hours at 35-370C. After incubation in Muller
Kauffmann Tetrathionate Broth Base (With Novobiocin), organisms were
Calcium Carbonate 38.7
subcultured to XLD Agar (AM1112/AM5112), incubated at 35 -370C 2 for 18 -
Sodium Thiosulfate, anhydrous 30.5 24 hours.
Ox Bile 4.78 Microorganisms(ATCC) Recovery on XLD Agar
Brilliant Green 0.0096 Growth Colour of colony
Final pH (at 250C) 8.0 ± 0.2 Salmonella typhimurium (14028) Good Red colonies
* Formula adjusted to suit performance parameters with black center
Directions Salmonella enteritidis (13076) Good Red colonies
1. Suspend 89.5 gms in 1000 ml distilled water and bring to a boil. with black center
Escherichia coli (25922) None to poor Yellow
2. Cool to less than 45°C before adding supplements.
Pseudomonas aeruginosa (27853) None to poor Yellow
3. Supplement with 40 mg per liter of Novobiocin. Enterococcus faecalis (29212) Inhibited -
4. Immediately before use aseptically add 20 mL of an iodine-iodide solution Staphylococcus aureus (25923) Inhibited -
prepared by dissolving 25 g of potassium iodide in 10 mL sterile water, Storage
adding 20 g of iodine and then diluting to 100 mL with sterile water. Store at 22-300C and prepared medium at 2-80C.
5. Mix well and dispense into sterile containers. Shelf Life
Use before expiry date as mentioned on the label.

Mycoplasma Agar Base (PPLO Agar) AM1073/AM5073

Use microorganisms. Crystal violet and potassium tellurite inhibits many gram-
Mycoplasma Agar Base (PPLO Agar) when supplemented with nutritive negative and gram-positive bacteria. Most Mycoplasma species are aerobic or
enrichments is used for isolation and cultivation of Mycoplasma species. facultatively anaerobic while some are microaerophilic. Anaerobic saprophytic
Summary mycoplasmas grows best at 22-250C while pathogenic strains grow at 350C.
Mycoplasma was first recognized from a case of pleuropneumonia in a cow. The Mycoplasmas do not grow without serum, but when grown in the agar medium it
organism was designated “pleuropneumonia-like organism,” or PPLO. Although shows typical colony morphology, and forms subsurface colonies.
some species are normal human respiratory tract flora, M.pneumoniae is a major Formula*
cause of respiratory disease (primary atypical pneumoniae, sometimes called Ingredients in grams per liter
“walking pneumonia”). M.hominis, M.genitalium and Ureaplasma urealyticum Beef Heart, Infusion from 250.0
Peptone 10.0
are important pathogens of the human genital tract. Morton, Smith and
Sodium Chloride 5.0
Leverman (81) formulated Mycoplasma Agar. It was used in the study of the
Agar 15.0
growth requirements as well as for the identification and cultivation of Final pH (at 250C) 7.8 ± 0.2
Mycoplasma. * Formula adjusted to suit performance parameters
Principle Directions
Peptone and beef heart infusion provides nitrogen, vitamins, amino acids and 1. Suspend 36 gms of the powder in 700 ml distilled water and mix well.
carbon. Sodium chloride maintains the osmotic balance. Mycoplasma cultivation 2. Boil with frequent agitation to dissolve the powder completely.
requires all ingredients of medium and supplement to be free from even small
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
amounts of toxic substances. Many mycoplasmas require serum and antibiotics
for their good growth as well as to prevent the growth of contaminating 4. Cool to 450C and aseptically add 300 ml of Horse Serum (AS015) or 10 vials

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of Mycoplasma Enrichment Supplement (AS019). liquid inoculum or by swab inoculation technique.
5. Mix well before dispensing. 2. Incubate the plates at 350C for up to 21 days in a moist atmosphere
6. 25% ascitic fluid can be used instead of Horse Serum. containing 5-10% carbon dioxide or anaerobically if the presence of
Quality Control M.buccale, M.faucium, M.orale, or M.salivarium is suspected.
Dehydrated Appearance Interpretation of Results
Yellow coloured, homogeneous, free flowing powder. 1. The colonies on Mycoplasma Agar Base are round with a dense center and a
Prepared Appearance less dense periphery, giving a “fried egg” appearance.
Yellow coloured, clear to slightly opalescent gel.
Cultural Response
2. Vacuoles and large bodies are characteristic of mycoplasma species and are
0
Cultural characteristics after 48 hours at 35 C with 10% carbon dioxide. seen in the periphery.
Organisms (ATCC) Growth RGI 3. The colonies vary in diameter from 10 to 500 microns (0.01-0.5 mm) and
Mycoplasma gallinarium Good to luxuriant More than 70% penetrate into the medium.
(19708) Precautions / Limitations
Mycoplasma pneumoniae Good to luxuriant More than 70%
1. Thallium acetate is toxic, proper care should be taken while handling the
(15531)
Mycoplasma Enrichment Supplement.
Streptococcus pneumoniae Good to luxuriant More than 70%
(6303) 2. Some mycoplasmas may be inhibited by thallium acetate.
For growth RGI should be more than 70% Storage
RGI- Relative Growth Index Store at 22-300C and prepared medium at 2-80C.
Procedure Shelf Life
1. Inoculate the surface of the plates containing the medium by adding drops of Use before expiry date as mentioned on the label.

Nitrate Broth BIS AM10731/AM50731

Nitrate Broth ISO AM10732/AM50732
Use Final pH (at 250C) 7.0 ± 0.2
Nitrate Broth is used for the detection of nitrate reduction by bacteria. It is also *Formula adjusted to suit performance parameters
recommended for the enumeration of Bacillus cereus in compliance with BIS Directions

specification IS: 5887 (Part 4), 1976 and compliance with ISO specification 1. Suspend 9 gms in 100 ml distilled water. Soak for 5 minutes.
ISO/DIS 7932, 1993. 2. Warm slightly with frequent agitation to dissolve the powder completely. DO
Summary NOT OVERHEAT.
Reduction of nitrate is generally an anaerobic respiration in which an organism 3. Dispense in tubes or adequate containers and sterilize by autoclaving at
derives its oxygen from nitrate. In presence of nitrate reductase enzyme nitrate 15lbs pressure (121ºC) for 15 minutes.
reduces to nitrite, which can be tested for by an appropriate colorimetric reagent Quality Control
(77.2). Dehydrated Appearance
Principle Yellow coloured, homogeneous, free flowing powder.
Prepared Appearance
Nitrate Broth is recommended for the detection of nitrate reduction. Peptone and
Light to medium amber coloured clear solution.
beef extract provide the essential nutrient for growth of bacteria while potassium
Cultural Response
nitrate is the source of nitrate.
Culture characteristics after 18-24hours at 350C.
Formula*
Organisms (ATCC) Growth Nitrate Reduction
Ingredients in grams per liter
Bacillus cereus (6633) Good +
Peptone 5.0
Enterobacter aerogenes (13048) Good +
Beef extract 3.0
Escherichia coli (25922) Luxuriant +
Potassium nitrate 1.0
Salmonella typhimurium (14028) Luxuriant +

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Procedure Storage
Refer to appropriate references for specific procedures. Store at 22-300C and prepared medium at 2-80C.
Interpretation of Results Shelf Life

Refer to appropriate references and procedures for results. Use before expiry date as mentioned on the label.

Nitrofurantoin Broth Base AM50733

Use Nitrofurantoin solution prepared by dissolving 1 gm Nitrofurantoin in 500 ml
Nitrofurantoin Broth Base is used for the selective enrichment and cultivation of polyethylene glycol 30.
Pseudomonas species from food. 5. Mix well and dispense in tubes or flasks as desired.
Principle Quality Control
Casein enzymic hydrolysate and peptic digest of animal tissue provides the Dehydrated Appearance
essential nutrients especially nitrogenous sources. Nitrofurantoin, (17.2) [(5- Cream to yellow coloured, homogeneous, free flowing powder.
nitrofurfurylidene)amino] hydantoin, is a synthetic antibacterial agent which is Prepared Appearance
effective against most common gram-negative and grampositive urinary tract Fluorescent yellow coloured with added Nitrofurantoin, clear solution without any
precipitate.
pathogenic bacteria (16.4).
Cultural Response
Formula*
Cultural characteristics after 18-24 hours at 35-37°C.
Ingredients in grams per liter
Organisms (ATCC) Growth
Peptic digest of animal tissue 7.5
Escherichia coli (25922) Inhibited
Casein enzymic hydrolysate 7.5
Pseudomonas aeruginosa (27853) Good-luxuriant
Sodium chloride 5.0
Staphylococcus aureus (25923) Inhibited
Final pH (at 25°C) 7.2 ± 0.2
For growth RGI should be more than 70%
*Formula adjusted to suit performance parameters
For Inhibition RGI should be 0%
Directions RGI- Relative Growth Index
1. Suspend 20 grams in 1000 ml distilled water. Storage
2. Heat to boiling to dissolve the medium completely. Store at 22-300C and prepared medium at 2-80C.
Shelf Life
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
Use before expiry date as mentioned on the label.
4. Cool to room temperature and aseptically add 50 ml sterile 0.2%

Nutrient Agar AM1074/AM5074

Nutrient Broth AM1077/AM5077
Use nutritious medium.
Nutrient Agar and Nutrient Broth are used as general-purpose culture media for Nutrient Broth is a basic non-selective culture medium used for the routine
the cultivation of bacteria, which may also be used as enrichment media by cultivation of microorganisms. Nutrient Agar and Nutrient Broth can be used for
incorporating 10% v/v sterile blood or other biological fluids. the cultivation of more exacting bacteria by incorporating biological fluids like
Summary horse or sheep blood, serum, ascitic fluid, egg yolk, etc. Nutrient Agar is included
Nutrient Agar is a basic culture medium used to subculture organisms for in the Bacteriological Analytical Manual for food testing (113).
maintenance purpose or to check the purity of sub-cultures from isolation plates Principle
prior to biochemical or serological testing. It is used for the cultivation and Peptone and beef extract provide water-soluble substances including
enumeration of organisms in water, sewage, faeces and other materials, which carbohydrates, vitamins, organic nitrogen compounds and salts. Peptone is the
are not particularly fastidious. Nutrient Agar is suitable for teaching purpose and principle source of organic nitrogen, particularly amino acids and long chained
maintenance of cultures, where a prolonged survival of organisms at an ambient peptides.
temperature is required without risk of the overgrowth that can occur with a more

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Formula* Streptococcus pyogenes Good to luxuriant More than 70%
Ingredients in grams per liter Nutrient Agar Nutrient Broth (19615)
Peptone 5.0 5.0 For growth RGI should be more than 70%
Sodium Chloride 5.0 5.0 RGI- Relative Growth Index
Beef Extract 1.5 1.5 Procedure
Yeast Extract 1.5 1.5 For Nutrient Agar
Agar 15.0 -
1. Use standard procedures like streak plate method to obtain isolated
Final pH (at 250C) 7.4 ± 0.2 7.4 ± 0.2
* Formula adjusted to suit performance parameters
colonies.
Directions 2. If the specimen to be cultured is on a swab, roll the swab on a small agar
1. Suspend the powder in 1000 ml distilled water and mix thoroughly. surface and streak for isolation with a sterile loop.
Nutrient Agar - 28 gms 3. Incubate plates at 35-370C for 18-24 hours and 42-48 hours, if required.
Nutrient Broth - 13 gms For Nutrient Broth
2. Boil with frequent agitation to dissolve the powder completely. 1. Inoculate tubes of medium with the test samples.
0
3. Sterilize by autoclaving at 121 C (15 lbs pressure) for 15 minutes. 2. Incubate for 18-24 hours at 35-370C in an aerobic atmosphere.
Quality Control Interpretation of Results
Dehydrated Appearance Nutrient Agar
Yellow coloured, homogeneous, free flowing powder.
1. Examine plates for growth.
Prepared Appearance
Nutrient Agar - Light amber coloured, clear to slightly opalescent gel. 2. Growth from tubes inoculated with pure cultures can be used for biochemical
Nutrient Broth - Light amber coloured, clear solution without any precipitate. and serological testing.
Cultural Response Nutrient Broth
Cultural characteristics after 18-48 hours at 35-370C.
1. Growth is seen as turbidity in the medium.
Organisms (ATCC) Growth on Nutrient RGI
Agar 2. Aliquots of the medium can be used for sub-culturing onto a solid medium
and in Nutrient Broth for isolation and identification of pure cultures.
Escherichia coli (25922) Good to luxuriant More than 70% Storage
Pseudomonas aeruginosa Good to luxuriant More than 70% Store at 22-300C and prepared medium at 2-80C.
(27853) Shelf Life
Staphylococcus aureus Good to luxuriant More than 70% Use before expiry date as mentioned on the label.
(25923)

Nutrient Agar IP AM10741/AM50741

Use nutritious medium. Nutrient Agar can be used for the cultivation of more exacting
Nutrient Agar is used as general purpose culture media for the cultivation of bacteria by incorporating biological fluid like horse or sheep blood, serum, ascitic
bacteria in compliance with IP, which may also be used as enrichment media by fluid, egg yolk, etc.
incorporating 10% v/v sterile blood or other biological fluid. Principle
Summary Peptone and beef extract provide water-soluble substances including
Nutrient Agar is a basic culture medium used to subculture organisms for carbohydrates, vitamins, organic nitrogen compounds and salts. Peptone is the
maintenance purpose or to check the purity of sub-cultures from isolation plates principle source of organic nitrogen, particularly amino acids and long chained
prior to biochemical or serological testing. It is used for the cultivation and peptides.
enumeration of organisms in water, sewage, faeces and other materials, which Formula*
are not particularly fastidious. Nutrient Agar is suitable for teaching purpose and Ingredients in grams per liter
maintenance of cultures, where a prolonged survival of organisms at an ambient Peptone 10.00
temperature is required without risk of the overgrowth that can occur with a more Beef extract 10.00

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Sodium chloride 5.00 Escherichia coli (25922) Good to luxuriant More than 70%
Agar 15.00 Streptococcus pyogenes Good to luxuriant More than 70%
Final pH (at 250C) 7.3 ± 0.2 (19615)
*Formula adjusted to suit performance parameters For growth RGI should be more than 70%
Directions RGI- Relative Growth Index
Procedure
1. Suspend 40 gms powder in 1000 ml distilled water and mix thoroughly.
1. Use standard procedures like streak plate method to obtain isolated
2. Boil with frequent agitation to dissolve the powder completely.
colonies.
3. Sterilize by autoclaving at 121ºC (15 lbs pressure) for 15 minutes.
2. If the specimen to be cultured is on a swab, roll the swab on a small agar
Quality Control
Dehydrated Appearance surface and streak for isolation with a sterile loop.
Yellow coloured, homogeneous, free flowing powder. 3. Incubate plates at 35-37ºC for 18-24 hours and 42-48 hours, if required.
Prepared Appearance Interpretation of Results
Light amber coloured, clear to slightly opalescent gel forms in petri plates. Refer to appropriate references and procedures for results.
Cultural Response
Storage
Cultural characteristics after 18-48 hours at 35-37°C.
Organisms (ATCC) Growth RGI
Store at 22-300C and prepared medium at 2-80C.
Pseudomonas aeruginosa Good to luxuriant More than 70% Shelf Life
(27853) Use before expiry date as mentioned on the label.
Staphylococcus aureus Good to luxuriant More than 70%
(25923)

Nutrient Agar 1.5% ISO AM50742

Use digest of animal tissue is the principle source of organic nitrogen, particularly
Nutrient Agar 1.5% ISO is a general purpose culture medium for the cultivation of amino acids and long chained peptides.
fastidious bacteria after enrichment by incorporating 10% v/v sterile blood or Formula*
other biological fluids, in compliance with ISO specification ISO/DIS Ingredients in grams per liter
13720:1995. Peptic digest of animal tissue 5.00
Summary Beef extract 3.00
Sodium chloride 5.00
Nutrient Agar is a basic culture medium used to subculture organisms for
Agar 15.00
maintenance purpose or to check the purity of sub-cultures from isolation plates Final pH (at 250C) 7.0± 0.2
prior to biochemical or serological testing. It is used for the cultivation and *Formula adjusted to suit performance parameters
enumeration of organisms in water, sewage, faeces and other materials, which Directions
are not particularly fastidious. Nutrient Agar is suitable for teaching purpose and 1. Suspend 28gms powder in 1000 ml distilled water and mix thoroughly.
maintenance of cultures, where a prolonged survival of organisms at an ambient
2. Boil with frequent agitation to dissolve the powder completely.
temperature is required without risk of the overgrowth that can occur with a more
3. Sterilize by autoclaving at 121ºC (15 lbs pressure) for 15 minutes.
nutritious medium. Nutrient Agar is recommended by APHA for cultivation and
Quality Control
maintenance of nonfastidious microorganisms. Recently ISO Commended it with
Dehydrated Appearance
a slight modification (AM50742) for sub cultivation of Pseudomonas species Creamish yellow coloured, homogeneous, free flowing powder.
isolated from meat and meat products. Prepared Appearance
Principle Yellow coloured, clear gel forms in petri plates. With the addition of blood,
Peptic digest of animal tissue and beef extract provide water-soluble substances cherry red coloured, opaque gel forms in petri plates.
including carbohydrates, vitamins, organic nitrogen compounds and salts. Peptic Cultural Response
Cultural characteristics after 18-24 hours at 35°C.

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Organisms (ATCC) Growth RGI 2. If the specimen to be cultured is on a swab, roll the swab on a small agar
Pseudomonas aeruginosa (27853) Luxuriant More than 70% surface and streak for isolation with a sterile loop.
Staphylococcus aureus (25923) Luxuriant More than 70%
Escherichia coli (25922) Luxuriant More than 70%
3. Incubate plates at 35-37ºC for 18-24 hours and 42-48 hours, if required.
For growth RGI should be more than 70% Interpretation of Results
RGI- Relative Growth Index Refer to appropriate references and procedures for results.
Procedure Storage
1. Use standard procedures like streak plate method to obtain isolated Store at 22-300C and prepared medium at 2-80C.
colonies. Shelf Life
Use before expiry date as mentioned on the label.

Nutrient Agar pH 6.8 AM1075/AM5075

Use Prepared Appearance
Nutrient Agar pH 6.8 is used for the cultivation of a wide variety of bacteria and Yellow coloured, clear to slightly opalescent gel.
Cultural Response
for the enumeration of microorganisms in water, sewage, faeces and other
Cultural characteristics after 18-24 hours at 370C.
materials.
Organisms (ATCC) Growth RGI
Summary
Enterococcus faecalis (29212) Luxuriant More than 70%
Nutrient Agar pH 6.8 is a basic culture media used to subculture organisms for Escherichia coli (25922) Luxuriant More than 70%
maintenance purposes or to check the purity of sub-cultures prior to biochemical Salmonella serotype Enteritidis Luxuriant More than 70%
or serological tests from water, dairy, etc. (13076)
Principle Salmonella serotype Typhimurium Luxuriant More than 70%
Peptone and beef extract provide water-soluble substances including (14028)
Staphylococcus aureus (25923) Luxuriant More than 70%
carbohydrates, vitamins, organic nitrogen compounds and salts. Agar is the
For growth RGI should be more than 70%
solidifying agent. RGI- Relative Growth Index
Formula*
Procedure
Ingredients in grams per liter
Peptone 5.0
1. Use standard procedures like streak plate method to obtain isolated
Beef Extract 3.0 colonies.
Agar 15.0 2. If the specimen to be cultured is on a swab, roll the swab on a small agar
Final pH (at 250C) 6.8 ± 0.2 surface and streak for isolation with a sterile loop.
* Formula adjusted to suit performance parameters
3. Incubate plates at 370C for 18-24 hours and 42-48 hours, if required.
Directions
Interpretation of Results
1. Suspend 23 gms of the powder in 1000 ml distilled water.
1. Examine plates for growth.
2. Mix thoroughly.
2. Growth from tubes inoculated with pure cultures can be used for biochemical
3. Boil with frequent agitation to dissolve the powder completely. and serological testing.
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. Storage
5. If required, enrich with 5-10% v/v sterile defibrinated blood. Store at 22-300C and prepared medium at 2-80C.
Quality Control Shelf Life
Dehydrated Appearance Use before expiry date as mentioned on the label.
Yellow coloured, homogeneous, free flowing powder.

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Nutrient Agar pH 7.0 AM50751
Use Quality Control
Nutrient agars are used for the cultivation of bacteria and for the enumeration of Dehydrated Appearance
Yellow coloured, homogeneous, free flowing powder
organisms in water, sewage, faeces and other materials.
Prepared Appearance
Summary
Yellow coloured clear to slightly opalescent gel forms in Petri plates
Nutrient agar is a basic culture medium used for maintenance or to check purity of Cultural Response
subcultures prior to biochemical or serological tests from water, dairy etc. Cultural characteristics after 18-24 hours at 37°C.
Principle Organisms (ATCC) Growth RGI
Peptone and beef extract provide water – soluble substance including Enetrococcus faecalis (29212) Luxuriant More than 70%
carbohydrates, vitamins, organic nitrogen compound and salts. Agar is the Escherichia coli (25922) Luxuriant More than 70%
Salmonella serotype Enteritidis Luxuriant More than 70%
solidifying agent.
(1307 6)
Formula*
Salmonella serotype Luxuriant More than 70%
Ingredients in grams per liter
Typhimurium (14028)
Peptic digest of animal tissue 5.00
Staphylococcus aureus Luxuriant More than 70%
Beef extract 3.00
(25923)
Agar 15.00
For growth RGI should be more than 70%
Final pH ( at 25°C) 7.0±0.2
RGI- Relative Growth Index
*Formula adjusted to suit performance parameters
Storage
Directions
Store at 22-300C and prepared medium at 2-80C.
1. Suspend 23 grams in 1000 ml distilled water.
Shelf Life
2. Heat to boiling to dissolve the medium completely. Use before expiry date as mentioned on the label.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. If desired, the medium can be enriched with 5-10% v/v sterile defibrinated
blood.

Nutrient Agar with 1% Peptone AM1076/AM5076

Nutrient Broth with 1% Peptone AM1078/AM5078
Use organisms. Nutrient Agar with 1% Peptone and Nutrient Broth with 1% Peptone
Nutrient Agar with 1% Peptone and Nutrient Broth with 1% Peptone are general- are used for the examination of water, wastewater and dairy products.
purpose media used for the examination of water and dairy products. Principle
Summary Peptone and beef extract provide water-soluble substances including
Nutrient Agar with 1% Peptone is a basic culture medium used to subculture carbohydrates, vitamins, organic nitrogen compounds and salts. Sodium chloride
organisms for maintenance purpose or to check the purity of sub-cultures from maintains the osmotic balance.
isolated plates prior to biochemical or serological testing and is also used for the Formula*
cultivation and enumeration of organisms in water, sewage, faeces and other Ingredients in grams per liter Nutrient Agar Nutrient Broth
materials which are not particularly fastidious. with 1% Peptone with 1% Peptone
Peptone 10.0 10.0
Nutrient Broth with 1% Peptone is a basic non-selective medium used for the
Beef Extract 5.0 10.0
routine cultivation of microorganisms. Nutrient Agar with 1% Peptone and Sodium Chloride 5.0 5.0
Nutrient Broth with 1% Peptone can be enriched by the addition of 10% v/v Agar 15.0 -
sterile blood or other biological fluids for the cultivation of more fastidious Final pH (at 250C) 7.4 ± 0.2 7.4 ± 0.2

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* Formula adjusted to suit performance parameters 1. Use standard procedures like streak plate method to obtain isolated
Directions colonies.
1. Suspend the powder in 1000 ml distilled water. 2. If the specimen to be cultured is on a swab, roll the swab on a small agar
Nutrient Agar with 1% Peptone - 35 gms surface and streak for isolation with a sterile loop.
Nutrient Broth with 1% Peptone - 25 gms 3. Incubate plates at 35-370C for 18-24 hours and 42-48 hours, if required.
2. Mix thoroughly. For Nutrient Broth with 1% Peptone
3. Boil with frequent agitation to dissolve the powder completely. 1. Inoculate medium with the test samples.
0
4. Sterilize by autoclaving at 121 C (15 lbs pressure) for 15 minutes. 2. Incubate for 18-24 hours at 35-370C in an aerobic atmosphere.
Quality Control Interpretation of Results
Dehydrated Appearance
Nutrient Agar with 1% Peptone
Yellow coloured, homogeneous, free flowing powder.
Prepared Appearance 1. Examine plates for growth.
Nutrient Agar with 1% Peptone: Basal medium - Light yellow coloured, clear to 2. Growth from tubes inoculated with pure cultures can be used for biochemical
slightly opalescent gel.
and serological testing.
With the addition of blood - Cherry red coloured, opaque gel.
Nutrient Broth with 1% Peptone - Light yellow coloured clear solution. Nutrient Broth with 1% Peptone
Cultural Response 1. Growth is seen as turbidity in the medium.
Cultural characteristics after 18-24 hours at 35-370C. 2. Aliquots of the medium can be used for sub-culturing onto a solid media for
Organisms (ATCC) Growth Growth Haemolysis
isolation and identification of pure cultures.
without Blood with Blood
Storage
Streptococcus pneumoniae (6303) Good Luxuriant Alpha
Streptococcus pyogenes (19615) Good Luxuriant Beta Store at 22-300C and prepared medium at 2-80C.
Staphylococcus aureus (25923) Luxuriant Luxuriant Beta Shelf Life
Procedure Use before expiry date as mentioned on the label.
For Nutrient Agar with 1% Peptone

Nutrient Broth IP AM10781/AM50781

Use Formula*
Nutrient Broth is used as general purpose culture media for the cultivation of Ingredients in grams per liter
Peptone 10.00
bacteria in compliance with IP, which may also be used as enrichment media by
Beef extract 10.00
incorporating 10% v/v sterile blood or other biological fluid.
Sodium chloride 5.00
Summary
Final pH (at 250C) 7.3 ± 0.2
Nutrient Broth is a basic non- selective culture medium used for the routine *Formula adjusted to suit performance parameters
cultivation of microorganisms. Nutrient Broth can be used for the cultivation of Directions
more exacting bacteria by incorporating biological fluid like horse or sheep blood, 1. Suspend 25gms powder in 1000 ml distilled water and mix thoroughly.
serum, ascitic fluid, egg yolk, etc.
2. Boil with frequent agitation to dissolve the powder completely.
Principle
3. Sterilize by autoclaving at 115ºC (15 lbs pressure) for 30minutes.
Peptone and beef extract provide water-soluble substances including
Quality Control
carbohydrates, vitamins, organic nitrogen compounds and salts. Peptone is the Dehydrated Appearance
principle source of organic nitrogen, particularly amino acids and long chained Yellow coloured, homogeneous, free flowing powder.
peptides. Prepared Appearance
Light amber coloured, clear to slightly opalescent solution forms in tubes.

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Cultural Response 2. If the specimen to be cultured is on a swab, roll the swab on a small agar
Cultural characteristics after 18-48 hours at 35-37°C.
surface and streak for isolation with a sterile loop.
Organisms(ATCC) Growth
Pseudomonas aeruginosa (27853) Good to luxuriant 3. Incubate plates at 35-37ºC for 18-24 hours and 42-48 hours, if required.
Staphylococcus aureus (25923) Good to luxuriant Interpretation of Results
Escherichia coli (25922) Good to luxuriant Refer to appropriate references and procedures for results.
Streptococcus pyogenes (19615) Good to luxuriant Storage
Procedure
Store at 22-300C and prepared medium at 2-80C.
1. Use standard procedures like streak plate method to obtain isolated Shelf Life
colonies. Use before expiry date as mentioned on the label.

Oxytetracyclin Glucose Yeast Extract Agar Base AM507811

Use 2. Heat to boiling to dissolve the medium completely.
Oxytetra Glucose Yeast Agar Base (OGYE Agar Base) is recommended for isolation 3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
and enumeration of yeasts and moulds from foods.
4. Cool to 50°C and aseptically add reconstituted contents of one vial of
Summary
Oxytetra Selective Supplement (AS0202).
Acidified agar may be used for enumerating yeasts and molds in foods and diary
5. Mix well and pour into sterile Petri plates.
products. But Acidic media are not completely suitable for counting yeasts and
Quality Control
moulds in foods since yeast cells, stressed by heat do not tolerate the acidic
Dehydrated Appearance
conditions necessary to inhibit bacterial contamination. Yeast and mould growth Cream to light yellow homogeneous free flowing powder
is often limited by the presence of acid-tolerant bacterial flora. Therefore it is Prepared Appearance
evident that more active media and different selective agents are needed in order Light amber coloured clear to slightly opalescent gel forms in Petri plates
to deal with various kinds of foodstuffs, incubation conditions and types of Cultural Response
microorganisms to be studied. Cultural characteristics after 2-5 days at 25-30°C.
Organisms (ATCC) Growth RGI
Mossel et al., described Oxytetra Glucose Yeast Agar Base (OGYE Agar Base) for
the selective isolation and enumeration of yeast and moulds in foods (81.5 & Aspergillus niger (16404) Good - luxuriant More than 70%
81.6). Candida albicans (10231) Good – luxuriant More than 70%
Principle Escherichia coli (25922 inhibited 0%
OGYE Agar Base contains yeast extract to supply B-complex vitamins which Saccharomyces cerevisiae Good – luxuriant More than 70%
stimulate bacterial growth. Dextrose is the energy source. Oxytetracycline inhibits (9763)
the growth of bacteria. Saccharomyces uvarum Good – luxuriant More than 70%
(9080)
Formula*
For growth RGI should be more than 70%
Ingredients in grams per liter
For Inhibition RGI should be 0%
Yeast extract 5.00
RGI- Relative Growth Index
Glucose 20.00
Storage
Agar 12.00
Final pH ( at 25°C) 7.0±0.2 Store at 22-300C and prepared medium at 2-80C.
*Formula adjusted to suit performance parameters Shelf Life
Directions Use before expiry date as mentioned on the label.
1. Suspend 18.5 grams in 500 ml distilled water.

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Orange Serum Agar AM50782
Use Directions
Orange Serum Agar is used for cultivation and enumeration of microorganisms 1. Suspend 45.5gms powder in 1000 ml distilled water and mix thoroughly.
associated with the spoilage of citrus products, cultivation of Lactobacilli, other 2. Heat to boiling to dissolve the powder completely.
aciduric organisms and pathogenic fungi. 3. Sterilize by autoclaving at 121ºC (15 lbs pressure) for 15 minutes. AVOID
Summary
OVER HEATING
Orange Serum Agar was originally developed by Murdock et al.,(82.2) and Hays Quality Control
(45.3) for examining citrus concentrates. Hays and Riester (45.4) further used Dehydrated Appearance
this medium for studying spoilage of orange juice. Dehydrated agar media Yellow coloured, homogeneous, free flowing powder.
containing orange serum was reported by Stevens (103.3) and these media are Prepared Appearance
recommended by APHA (103.4). Medium to dark amber coloured, clear to slightly opalescent gel forms in petri
plates.
Principle
Cultural Response
Casein enzymic hydrolysate and yeast extract provide essential nitrogenous Cultural characteristics after 40-48 hours at 37°C.
nutrients while dextrose serves as the fermentable carbohydrates and energy Organisms (ATCC) Growth RGI
sources. Orange Serum gives an optimal environment for the recovery of acid Aspergillus niger (16404) Good to luxuriant More than 70%
tolerant microorganisms from citrus fruit products. Lactobacillus fermentum (9338) Good to luxuriant More than 70%
Formula* Lactobacillus acidophilus (4356) Good to luxuriant More than 70%
Ingredients in grams per liter Leuconostoc mesenteroides Good to luxuriant More than 70%
Casein enzymic hydrolysate 10.00 (12291)
Yeast extract 3.00 Saccharomyces cerevisiae Good to luxuriant More than 70%
Dextrose 4.00 (9763)
Dipotassium phosphate 2.50 For growth RGI should be more than 70%
Orange serum (solid from 200ml) 9.00 RGI- Relative Growth Index
Agar 17.00 Storage
Final pH (at 250C) 5.5± 0.2 Store at 22-300C and prepared medium at 2-80C.
*Formula adjusted to suit performance parameters Shelf Life
Use before expiry date as mentioned on the label.

Orange Serum Broth AM50783

Use Formula*
Orange Serum Broth is used for cultivation of microorganisms associated with the Ingredients in grams per liter
Casein enzymic hydrolysate 10.00
spoilage of citrus products, cultivation of Lactobacilli, other aciduric organisms
Yeast extract 3.00
and pathogenic fungi.
Dextrose 4.00
Summary
Dipotassium phosphate 2.50
Murdock and Brokaw employed Orange Serum Broth for studies of sanitary Orange serum (Solids from 200 ml) 9.00
control of the processing of citrus concentrates. Hays and Riester recommended Final pH (at 250C) 5.5± 0.2
Orange serum Broth, pH 5.5 which is accepted as a control medium is most *Formula adjusted to suit performance parameters
productive for the growth of spoilage organisms. Directions
Principle 1. Suspend 28.5 gms powder in 1000 ml distilled water and mix thoroughly.
Casein enzymic hydrolysate and yeast extract provide essential nitrogenous 2. Heat to boiling to dissolve the powder completely.
nutrients while dextrose serves as the fermentable carbohydrates and energy 3. Sterilize by autoclaving at 121ºC (15 lbs pressure) for 15 minutes. AVOID
sources. OVER HEATING

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Quality Control Lactobacillus acidophilus (4356) Good to luxuriant
Dehydrated Appearance Leuconostoc mesenteroides (12291) Good to luxuriant
Yellow coloured, homogeneous, free flowing powder. Saccharomyces cerevisiae (9763) Good to luxuriant
Prepared Appearance Storage
Medium to dark amber coloured, clear to slightly opalescent solution forms in Store at 22-300C and prepared medium at 2-80C.
tubes.
Shelf Life
Cultural Response
Use before expiry date as mentioned on the label.
Cultural characteristics after 40-48 hours at 37°C.
Organisms (ATCC) Growth
Aspergillus niger (16404) Good to luxuriant
Lactobacillus fermentum (9338) Good to luxuriant

PA Broth AM507831
Use * Formula adjusted to suit performance parameters
PA Broth is used for the detection of presence or absence of coliform bacteria from Directions
water samples. 1. Suspend 30.5 gms in 1000 ml distilled water.
Summary 2. Warm slightly with frequent agitation to dissolve the powder completely. DO
The coliform group of bacteria is the principle indicator of suitability of water for NOT OVERHEAT.
domestic, industrial or other uses. Presence-absence procedures can be effectively 3. Dispense in tubes or adequate containers and sterilize by autoclaving at
used to monitor water from treatment plants and distribution systems. The 15lbs pressure (121ºC) for 12 minutes.
presence-absence test for the coliform group is a simple modification of the Quality Control
multiple tube procedure. Simplification, by use of one large test portion (100 ml) Dehydrated Appearance
in a single culture bottle to obtain qualitative information on the presence or Light yellow coloured with green tinge, homogeneous, free flowing powder.
absence of coliforms, is justified on the theory that no coliforms should be present Prepared Appearance
in 100 ml of a drinking water sample. Purple coloured, clear solution without any precipitate.
Principle Cultural Response
Cultural characteristics observed after 18-24 hours at 35-370C.
The medium contains nutrients required for the growth of coliforms. Lactose is the
Organisms (ATCC) Growth Colour of medium
fermentable carbohydrate. Phosphates provide buffering action while sodium
Escherichia coli (25922 Good-Luxuriant Yellow
lauryl sulphate inhibits organisms other than coliforms. Bromocresol purple is the Enterobacter aerogenes (13048) Good-Luxuriant Light yellow
pH indicator, which turns yellow at acidic pH. Klebsiella pneumoniae (13883) Good-Luxuriant Yellow
Formula* S. serotype Typhimurium (14028) Good-Luxuriant No change (purple)
Ingredients in grams per liter Enterococcus faecalis (29212) Inhibited -
Peptic digest of animal tissue 5.0 Procedure
Tryptose 9.83
Refer to appropriate references for specific procedures.
Beef extract 3.00
Interpretation of Results
Lactose 7.46
Sodium chloride 2.46 Refer to appropriate references and procedures for results.
Dipotassium phosphate 1.35 Storage
Monopotassium phosphate 1.35 Store at 22-300C and prepared medium at 2-80C.
Sodium lauryl sulphate 0.05 Shelf Life
Bromo cresol purple 0.0085 Use before expiry date as mentioned on the label.
Final pH (at 250C) 6.8 ± 0.2

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PE-2 Medium AM507832

Use * Formula adjusted to suit performance parameters
PE-2 Medium is used for detection and cultivation of mesophilic anaerobic spore- Directions

formers in specimens collected from food processing plants. 1. Suspend 23.04 grams in 1000 ml distilled water.
Summary 2. Heat if necessary to dissolve the medium completely.
PE-2 Medium is prepared as per the formulation described by Folinazzo and Troy 3. Dispense 18-20 ml aliquots into 18 x 150 mm screw capped test tubes.
(31.1.2) and recommended by APHA (20) for detection and cultivation of 4. Add 8-10 untreated Alaska seed peas and let the tubes stand for 1 hour to
mesophilic anaerobic spore-formers in specimens from food processing plants. effect hydration.
These organisms mainly include the genus Clostridium.
5. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
Clostridial species are highly heat resistant and are able to grow in the absence of Quality Control
oxygen. Clostridial growth range covers the temperature of the normal storage of Dehydrated Appearance
canned and other processed foods including refrigerated storage of cured meats Light yellow coloured without green tinge, homogeneous, free flowing powder.
and hence these anaerobes are important in the spoilage of low-acid foods Prepared Appearance
packed in hermetically sealed containers. Purple coloured, clear to slightly opalescent solution over alaska seeds.
Principle Cultural Response
Cultural characteristics observed after 18-24 hours at 35-370C.
Peptic digest of animal tissue and yeast extract provide nitrogenous compounds,
Organisms (ATCC) Growth
vitamin B complex and trace ingredients required for the growth of clostridia. Clostridium sporogenes (11437) Luxuriant
Addition of untreated alaska seed peas creates anaerobic conditions in the Clostridium botulinum (25763) Luxuriant
medium. Storage
Formula* Store at 22-300C and prepared medium at 2-80C.
Ingredients in grams per liter
Shelf Life
Peptic digest of animal tissue 20.00
Use before expiry date as mentioned on the label.
Yeast extract 3.00
Bromocresol purple 0.04

Pantothenate Assay Medium AM10784

Use Formula*
Pantothenate Assay Medium is recommended for the microbiological assay of Ingredients in grams per liter
Casein acid hydrolysate 10.0
Pantothenic acid or its salts using Lactobacillus plantarum.
Dextrose 40.0
Summary
Sodium acetate 20.0
Pantothenate Culture Agar is formulated according to United States L- Cystine 0.40
Pharmacopoeia (USP) (111.1) and is recommended for culturing Lactobacillus DL- Tryptophan 0.20
plantarum ATCC 8014 used in the microbiological assay of pantothenate. Adenine sulphate 0.02
Lactobacillus plantarum requires pantothenate for the growth but the medium Guanine hydrochloride 0.02
lacks pantothenate. The growth of the organism will occur if the materials being Uracil 0.02
assayed contain pantothenate. Thiamine hydrochloride 0.0002
Riboflavin (Vitamin B2) 0.0004
Principle
Niacin 0.001
Casein acid hydrolysate supplies the nitrogen and other essential elements for the Pyridoxin 0.0008
growth of the bacteria. Dextrose is the source of energy. Sodium acetate inhibits p- Amino benzoic acid 0.0002
the growth of some organisms including Gram negative bacteria and moulds. Biotin 0.0000008
Monopotassium phosphate 1.0

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Dipotassium phosphate 1.0 Quality Control
Magnesium sulphate 0.40 Dehydrated Appearance
Sodium chloride 0.02 Light yellow coloured, homogeneous, free flowing powder.
Ferrous sulphate 0.02 Prepared Appearance
Manganese sulphate 0.02 Light yellow coloured, clear solution which may have a slight precipitate.
Final pH (at 25ºC) 6.8 ± 0.2 Cultural Response
* Formula adjusted to suit performance parameters Cultural characteristics after 18-24 hours at 35-37ºC.
Directions Organisms (ATCC) Growth
1. Suspend 73.12 gms of the powder in 1000 ml distilled water. Lactobacillus plantarum (8014) Good to luxuriant
Storage
2. Boil to dissolve the medium completely. Mix well.
Store at 2-80C and prepared medium at 2-80C.
3. Dispense in 5ml amounts to assay tube in increasing amounts of the
Shelf Life
standard or unknown and total volume 10ml per tube is adjusted by
Use before expiry date as mentioned on the label.
addition of distilled water.
4. Sterilize by autoclaving at 15 lbs pressure (121ºC) for 10 minutes.

Pantothenate Medium AOAC AM10785

Use Manganese sulfate 20.0 mg
Pantothenate Medium AOAC is used for determining the concentration of Adenine sulfate 20.0 mg
Guanine hydrochloride 20.0 mg
pantothenic acid or its salts using Lactobacillus platarum ATCC8014.
Uracil 20.0 mg
Summary
Riboflavin 400.0 ìg
Pantothenate Medium AOAC is prepared for use in the microbiological assay of Thiamine hydrochloride 200.0 ìg
pantothenic acid and pantothenate according to the procedures of Calcium Biotin 0.8 ìg
Pantothenate Assay in the USP and Pantothenate Acid Assay in the Official p-Aminobenzoic acid 200.0 ìg
Methods of Analysis of AOAC International (AOAC). Lactobacillus plantarum Niacin 1.0 mg
ATCC 8014 is the test organism used in this assay (85.1). Pyridoxine hydrochloride 800.0 ìg
Principle Final pH (at 25ºC) 6.8 ± 0.2
Pantothenate Medium AOAC is a pantothenic acid/pantothenatefree dehydrated * Formula adjusted to suit performance parameters
medium containing all other nutrients and vitamins essential for the cultivation Directions
of Lactobacillus plantarum ATCC 8014. The addition of calcium pantothenate in 1. Suspend 73 g of the powder in 1000 mL of purified water.
specified increasing concentrations gives a growth response that can be 2. Boil to dissolve the medium completely. Mix well.
measured turbidimetrically or titrimetrically.
3. Dispense in 5ml amounts to assay tube in increasing amounts of the
Formula*
Ingredients in grams per liter
standard or unknown and total volume 10ml per tube is adjusted by
Dextrose 40.0 g addition of distilled water.
Sodium acetate 20.0 g 4. Sterilize by autoclaving at 15 lbs pressure (121ºC) for 10 minutes.
Casein acid hydrolysate 10.0 g Quality Control
Dipotassium phosphate 1.0 g Dehydrated Appearance
Monopotassium phosphate 1.0 g White to very light beige, homogeneous, tendency to clump.
L-Cystine 0.4 g Prepared Appearance
L-Tryptophan 0.2 g (Single strength) light amber, clear, may have a very slight precipitate.
Magnesium sulfate 0.4 g Cultural Response
Sodium chloride 20.0 mg Cultural characteristics after 18-24 hours at 35-37ºC.
Ferrous sulfate 20.0 mg

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Organisms (ATCC) Growth Shelf Life
Lactobacillus plantarum (8014) Good to luxuriant Use before expiry date as mentioned on the label.
Storage
Store below 2-80C and prepared medium at 2-80C.

Pantothenate Culture Agar AM10786

Use 2. Boil with the frequent agitation to dissolve the powder completely.
Pantothenate Culture Agar is used for culturing Lactobacillus plantarum ATCC 3. Distribute in tubes and sterilize by autoclaving at 121ºC (15 lbs pressure) for
8014 15 minutes.
Summary Quality Control
Pantothenate Culture Agar is formulated according to United States Dehydrated Appearance
Pharmacopoeia (USP) and is recommended for culturing Lactobacillus Yellow coloured, homogeneous, free flowing powder.
plantarum ATCC 8014 used in the microbiological assay of pantothenate. Prepared Appearance
Lactobacillus plantarum requires pantothenate for the growth but the medium Light yellow coloured, clear to very slightly opalescent gel forms in tubes as
butts.
lacks pantothenate. The growth of the organism will occur if the materials being Cultural Response
assayed contain pantothenate. Cultural characteristics after 18-48 hours at 35-370C.
Principle Organisms (ATCC) Growth RGI
Yeast Extract supplies the nitrogen and other essential elements for the growth of Lactobacillus plantarum (8014) Good-luxuriant More than 70%
the bacteria. Dextrose is the source of energy. Sodium acetate inhibits the growth Escherichia coli (25922) Inhibited 0%
of some organisms including Gram negative bacteria and moulds. For growth RGI should be more than 70%
Formula* For Inhibition RGI should be 0%
Ingredients in grams per liter RGI- Relative Growth Index
Yeast extract 20.0 Procedure
Dextrose 5.0 Refer to appropriate references for specific procedures.
Sodium acetate 5.0 Interpretation of Results
Agar 15.0 Refer to appropriate references and procedures for results.
Storage
Final pH (at 25ºC) 6.8 ± 0.2
Store at 22-300C and prepared medium at 2-80C.
* Formula adjusted to suit performance parameters Shelf Life
Directions Use before expiry date as mentioned on the label.
1. Suspend 45 gms of powder in 1000ml distilled water and mix thoroughly.

Pantothenate Inoculum Broth AM10787

Use extract provides vitamin B complex, nitrogenous complex and trace elements.
Pantothenate Inoculum Broth is used in preparation of inoculum for pantothenate Dextrose is the source of energy. Tomato juice makes the environment favorable
assay. for acidophilic bacteria like Lactobacillus, inhibiting other microorganisms.
Summary Polysorbate 80 provides fatty acids for the growth of bacteria.
Pantothenate Inoculum Broth is recommended for the preparation of inoculum for Formula*
pantothenate assay. Kulp and White have formulated the Pantothenate Inoculum Ingredients in grams per liter
Broth (63). Peptonized milk 15.0
Principle Yeast extract 5.0
Peptonized milk is a good source of carbon and nitrogenous compound. Yeast Dextrose 10.0

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Monopotassium phosphate 2.0 Cultural Response
Cultural characteristics after 18-24 hours at 35-37ºC.
Tomato juice (100ml) 5.0
Organisms (ATCC) Growth
Polysorbate 80 1.0 Lactobacillus casei (9595) Luxuriant
Final pH (at 25ºC)6.8 ± 0.2 Lactobacillus leichmannii (4797) Luxuriant
Lactobacillus plantarum (8014) Luxuriant
* Formula adjusted to suit performance parameters
For growth RGI should be more than 70%
Directions
For Inhibition RGI should be 0%
1. Suspend 38 gms of powder in 1000ml distilled water and mix thoroughly. RGI- Relative Growth Index
2. Boil with the frequent agitation to dissolve the powder completely. Refer to appropriate references for specific procedures.
Interpretation of Results
3. Distribute in tubes and sterilize by autoclaving at 121ºC (15 lbs pressure) for
Refer to appropriate references and procedures for results.
15 minutes.
Storage
Quality Control
Dehydrated Appearance
Store at 22-300C and prepared medium at 2-80C.
Light yellow coloured, homogeneous, free flowing powder. Shelf Life
Prepared Appearance Use before expiry date as mentioned on the label.
Medium amber coloured, clear solution without any precipitate.

Peptone Water BIS AM10791/AM50791

Use 3. Add 10 ml Andrade indicator & make the pH upto 7.5.
Peptone Water BIS is used for cultivating non-fastidious organisms and a base for 4. Warm slightly with frequent agitation to dissolve the powder completely. DO
carbohydrate fermentation media in compliance with BIS specification IS: 5887 NOT OVERHEAT.
(Part 1) : 1976.
5. Dispense in tubes with or without Durham's tubes as desired and sterilize by
Summary
autoclaving at 15lbs pressure (121ºC) for 15 minutes.
Peptone Water BIS is used for biochemical tests such as determining carbohydrate
6. Aseptically add sterile carbohydrate solution to yield a 1% final
fermentation pattern, which help in differentiation of genera and species. With
concentration (10 gm of requisite sugar in 90 ml of water and steam for 30
the pH adjusted to 8.4 it is suitable for the cultivation and enrichment of Vibrio
minutes or sterilize by filtration)
cholerae. Adding Andrade indicator and the test carbohydrate to detect the
fermentation reactions may modify Peptone Water BIS. 7. Dispense in tubes with or without Durham's tubes as desired.
Quality Control
Principle
Dehydrated Appearance
Peptone provides the essential nutrients while sodium chloride maintains the Light yellow coloured, homogeneous, free flowing powder.
osmotic equilibrium. Andrade's indicator acts as a pH indicator, which shows a Prepared Appearance
colour change of the medium from yellow to pink in the presence of an acid. Light yellow coloured, clear solution without any precipitate.
Formula* Cultural Response
Ingredients in grams per liter Cultural characteristics after 18-24 hours at 35-370C.
Peptone 10.0 Organisms (ATCC) Growth
Sodium chloride 5.0 Escherichia coli (25922) Luxuriant
Andrade's Indicator 10 ml Salmonella serotype Typhimurium (23564) Luxuriant
Final pH (at 250C) 7.2± 0.2 Staphylococcus aureus (25923) Luxuriant
* Formula adjusted to suit performance parameters Procedure
Directions
1. Andrade indicator is a solution of acid fuchsin titrated with sodium hydroxide
1. Suspend the 25 gms of powder in 900 ml distilled water. until the colour changes from pink to yellow.
2. Mix thoroughly. 2. When the indicator is added to Peptone Water it is colourless to slightly pink

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at pH 7.2; it becomes pink at acid pH levels and yellow at alkaline pH levels. 4. Some sugar solutions may affect the pH of Peptone Water, which must be
3. Filter sterilized sugar solutions are added to the base medium after checked and corrected.
sterilization. 5. It may be required to make subcultures to ensure purity of the inoculum
4. Inoculate tubes with test organism. since mixed or contaminated cultures may give false reactions.
5. Incubate at 35-370C for 18-48 hours 6. Andrade indicator may fade on prolonged storage; do not use beyond expiry
period.
6. Observe for colour change.
Interpretation of Results 7. It is advisable to maintain cultures of organisms, which have known positive
1. Acid is produced when carbohydrates are fermented which is indicated by a and negative reactions in each sugar. Using fresh sub-cultures, test each
pink colour in the medium and gas production is detected by formation of batch of sugar media with the appropriate organisms.
gas bubbles in the Durham's tubes. 8. Vibrio species should not be incubated longer than 18-20 hours as it may
Precautions / Limitations lead to development of suppressed forms.
1. A pure culture in Peptone Water is a convenient inoculum. Storage

2. Each tube of Peptone Water with carbohydrate should be correctly coded for Store at 22-300C and prepared medium at 2-80C.
the contained carbohydrate. Shelf Life
Use before expiry date as mentioned on the label.
3. Peptone Water with Andrade indicator is reddish- pink when hot; and should
return to a colourless or slightly pink colour when cooled to room
temperature.

Peptone Water AM1079/AM5079

Peptone Water with Phenol Red AM1080/AM5080
Use * Formula adjusted to suit performance parameters
Peptone Water and Peptone Water with Phenol Red are non-selective media used Directions

for cultivating non-fastidious organisms and as a base for carbohydrate 1. Suspend the powder in 1000 ml distilled water.
fermentation media. Peptone Water - 15 gms
Summary Peptone Water with Phenol Red - 15.02 gms
Peptone Water is used for biochemical tests such as determining carbohydrate 2. Mix thoroughly.
fermentation pattern, which help in differentiation of genera and species. With
3. Warm slightly with frequent agitation to dissolve the powder completely.
the pH adjusted to 8.4 it is suitable for the cultivation and enrichment of Vibrio
cholerae. Peptone Water may be modified by adding Andrade indicator and the 4. Dispense in tubes with or without Durham's tubes as desired.
test carbohydrate to detect the fermentation reactions. 5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Principle 6. Aseptically add sterile carbohydrate solution to yield a 1% final
Peptone provides the essential nutrients while sodium chloride maintains the concentration. Rotate the tubes thoroughly to distribute the carbohydrate.
osmotic equilibrium. Phenol red is the pH indicator, which shows a colour change Quality Control
of the medium from red to yellow in the presence of an acid. Dehydrated Appearance
Formula* Peptone Water - Light yellow coloured, homogeneous, free flowing powder.
Ingredients in grams per liter Peptone Water Peptone Water Peptone Water with Phenol Red - Light pink coloured, homogeneous, free
flowing powder.
with Phenol Red
Prepared Appearance
Peptone 10.0 10.0
Peptone Water - Light yellow coloured, clear solution without any precipitate.
Sodium Chloride 5.0 5.0
Peptone Water with Phenol Red - Red coloured, clear solution without any
Phenol Red - 0.02
precipitate.
Final pH (at 250C) 7.2 ± 0.2 6.8 ± 0.2

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Cultural Response 1. Acid is produced when carbohydrates are fermented which is indicated by a
Cultural characteristics after 18-24 hours at 35-370C. pink colour in the medium and gas production is detected by formation of
Peptone Water
gas bubbles in the Durham's tubes.
Organisms (ATCC) Growth
Escherichia coli (25922) Luxuriant Peptone Water with Phenol Red
Salmonella serotype Typhimurium (23564) Luxuriant 1. Acid is produced when carbohydrates are fermented which is indicated by a
Staphylococcus aureus (25923) Luxuriant yellow colour in the medium and gas production is detected by formation of
Peptone Water with Phenol Red gas bubbles in the Durham's tubes.
Organisms (ATCC) Growth Sacrose L(+) Salicin
Precautions / Limitations
Acid Rhamnose Acid
Acid 1. A pure culture in Peptone Water is a convenient inoculum.
Yersinia enterocolitica Luxuriant + +* - 2. Each tube of Peptone Water with carbohydrate should be correctly coded for
(27729) the contained carbohydrate.
Key:
* = Moderate
3. Peptone Water with Andrade indicator is reddish- pink when hot; and should
Procedure return to a colourless or slightly pink colour when cooled to room
For Peptone Water temperature.

1. Andrade indicator is a solution of acid fuchsin titrated with sodium hydroxide 4. Some sugar solutions may affect the pH of Peptone Water, which must be
until the colour changes from pink to yellow. checked and corrected.

2. When the indicator is added to Peptone Water it is colourless to slightly pink 5. It may be required to make subcultures to ensure purity of the inoculum
at pH 7.2; it becomes pink at acid pH levels and yellow at alkaline pH levels. since mixed or contaminated cultures may give false reactions.

3. Filter sterilized sugar solutions are added to the base medium after 6. Andrade indicator may fade on prolonged storage; do not use beyond expiry
sterilization. period.

4. Inoculate tubes with test organism. 7. It is advisable to maintain cultures of organisms, which have known positive
and negative reactions in each sugar. Using fresh sub-cultures, test each
5. Incubate at 35-370C for 18-48 hours.
batch of sugar media with the appropriate organisms.
6. Observe for colour change.
8. Vibrio species should not be incubated longer than 18-20 hours as it may
For Peptone Water with Phenol Red lead to development of suppressed forms.
1. Inoculate tubes with test organism. Storage
0
2. Incubate at 35-37 C for 18-48 hours. Store at 22-300C and prepared medium at 2-80C.
Shelf Life
3. Observe for colour change.
Use before expiry date as mentioned on the label.
Interpretation of Results
Peptone Water

Peptone Water with Phenol Red ISO AM50801

Use the pH adjusted to 8.4 it is suitable for the
Peptone Water with Phenol Red medium for studying fermentation ability of cultivation and enrichment of Vibrio cholerae. Peptone Water may be modified by
Yersinia enterocolitica, In compliance with ISO specification ISO/DIS adding Andrade indicator and the test carbohydrate to detect the fermentation
10273:1994. reactions.
Summary Principle
Peptone Water is used for biochemical tests such as determining carbohydrate Peptone provides the essential nutrients while sodium chloride maintains the
fermentation pattern, which help in differentiation of genera and species. With osmotic equilibrium. Phenol red is the pH indicator, which shows a colour change

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of the medium from red to yellow in the presence of an acid. Cultural Response

Formula* Cultural characteristics after 18-24 hours at 35-370C.

Ingredients in grams per liter Peptone Water
Peptone 10.0 Organisms (ATCC) Growth Sacrose L (+) Salicin
Sodium chloride 5.0 Acid Rhamnose Acid
Phenol red 0.02 Acid
Yersinia enterocolitica Luxuriant + +* -
Final pH (at 250C) 6.8 ± 0.2
(27729)
* Formula adjusted to suit performance parameters
Key: * = Moderate
Directions
Procedure
1. Suspend 15.02 gms of the powder in 1000 ml distilled water.
1. Inoculate tubes with test organism.
2. Mix thoroughly.
2. Incubate at 35-370C for 18-48 hours.
3. Warm slightly with frequent agitation to dissolve the powder completely.
3. Observe for colour change.
4. Dispense in tubes with or without Durham's tubes as desired.
Interpretation of Results
5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. Acid is produced when carbohydrates are fermented which is indicated by a yellow
6. Aseptically add sterile carbohydrate solution to yield a 1% final colour in the medium and gas production is detected by formation of gas bubbles
concentration. Rotate the tubes thoroughly to distribute the carbohydrate. in the Durham's tubes.
Quality Control Storage
Dehydrated Appearance
Store at 22-300C and prepared medium at 2-80C.
Light pink coloured, homogeneous, free flowing powder.
Shelf Life
Prepared Appearance
Use before expiry date as mentioned on the label.
Red coloured, clear solution without any precipitate.

Pfizer Selective Enterococcus Agar AM508011

Use Peptic digest of animal tissue 3.00
Pfizer Selective Enterococcus Agar is used for selective isolation and cultivation of Yeast extract 5.00
Bile salts (ox gall) 10.00
Enterococci.
Sodium chloride 5.00
Summary
Sodium citrate 1.00
Pfizer Selective Enterococcus Agar is used for the selective isolation and Esculin 1.00
cultivation of Enterococci. This medium is formulated as per Isenberg, Goldberg Ferric ammonium citrate 0.50
and Sampson (46.1.1) by reducing the concentration of bile salts and sodium Sodium azide 0.25
azide from the original formulation. The importance of esculin hydrolysis in Agar 15.00
differentiating Enterococci and streptococci was first reported by Rochaix as Final pH ( at 25°C) 7.1±0.2
streptococci do not exhibit esculin hydrolysis (94). * Formula adjusted to suit performance parameters
Directions
Principle
Casein enzymic hydrolysate, peptic digest of animal tissue and yeast extract 1. Suspend 57.75 grams in 1000 ml distilled water.
provide nutrients like nitrogenous compounds, carbon, sulphur, vitamin B 2. Heat to boiling to dissolve the medium completely.
complex and trace ingredients for the growth of Enterococci. Esculin, a glycoside, 3. Dispense as desired and sterilize by autoclaving at 15 lbs pressure (121°C)
is hydrolyzed by Enterococci to esculetin and dextrose. Esculetin reacts with ferric for 15 minutes.
ammonium citrate to form a dark brown to black coloured complex. 4. Mix well and pour into Petri plates
Formula* Warning : Sodium azide has a tendency to form explosive metal azides
Ingredients in grams per liter with plumbing materials. It is advisable to use enough water to flush off
Casein enzymic hydrolysate 17.00 the disposables.

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Quality Control Staphylococcus Fair-good Negative reaction More than 70%
Dehydrated Appearance aureus (25923)
Light yellow to pale green homogeneous free flowing powder Enterococcus Good-luxuriant Positive reaction, More than 70%
Prepared Appearance faecalis (29212) blackening
Light amber coloured clear to slightly opalescent gel with a bluish tinge forms around the colony
in Petri plates. For growth RGI should be more than 70%
Cultural Response For Inhibition RGI should be 0%
Cultural characteristics after 18-24 hours at 35-370C. RGI- Relative Growth Index
Organism Growth Esculin RGI Storage
(ATCC) hydrolysis
Store at 22-300C and prepared medium at 2-80C.
Enterobacter Inhibited - 0% Shelf Life
aerogenes Use before expiry date as mentioned on the label.
(13048)
Escherichia coli Inhibited - 0%
(25922)

Phenol Red Dextrose Broth AM50802

Use 4. Dispense in tubes containing inverted Durham's tubes.
Phenol Red Dextrose Broth media is used for dextrose fermentation studies of 5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
microorganisms. Quality Control
Summary Dehydrated Appearance
Phenol Red Dextrose Broth media are recommended (77.1, 31.1 & 28.1) for Light pink coloured, homogeneous, free flowing powder.
determination of fermentation reactions in differentiation of microorganisms. Prepared Appearance
Ability of an organism to ferment specific carbohydrate added in a basal medium, Red coloured, clear solution without any precipitate. Petri plates.
Cultural Response
results in the production of acid and gas which helps in the differentiation
Cultural characteristics after 18-24 hours at 35-370C.
between genera and species.
Organism Growth Acid
Principle Gas
Proteose peptone and beef extract provide nitrogenous nutrients to the Citrobacter ferundii (8090) Luxuriant + +
organisms. Phenol red is the pH indicator which turns yellow at acidic pH. Sodium Enterobacter aerogenes (13048) Luxuriant + +
chloride maintains osmotic equilibrium. Gas formation is seen in Durham's tubes. Escherichia coli (25922) Luxuriant + +
Formula* Klebsiella pneumoniae (13883) Luxuriant + +
Ingredients in grams per liter Proteus vulgaris (13315) Luxuriant + +
Proteose peptone 10.00 S. serotype Typhimurium (14028) Luxuriant + +
Beef extract 1.00 S. serotype Typhi (6539) Luxuriant + -
Sodium chloride 5.00 Serratia marcescens (8100) Luxuriant + +
Dextrose 5.00 Shigella flexneri (12022) Luxuriant + -
Phenol red 0.018 Key: += Positive reaction yellow colour
-= Negative reaction, no colour change or red.
Final pH (at 250C) 7.4 ± 0.2
* Formula adjusted to suit performance parameters Storage
Directions Store at 22-300C and prepared medium at 2-80C.
Shelf Life
1. Suspend 21 gms of the powder in 1000 ml distilled water.
Use before expiry date as mentioned on the label.
2. Mix thoroughly.
3. Heat to dissolve the powder completely.

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Phenol Red Lactose Broth AM50803

Use 3. Heat to dissolve the powder completely.
Phenol Red Lactose Broth media is used for lactose fermentation studies of 4. Dispense in tubes containing inverted Durham's tubes.
microorganisms.
5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Summary
Quality Control
Phenol Red Lactose Broth media are recommended (77.1, 31.1 & 28.1) for Dehydrated Appearance
determination of fermentation reactions in differentiation of microorganisms. Light pink coloured, homogeneous, free flowing powder.
Ability of an organism to ferment specific carbohydrate added in a basal medium, Prepared Appearance
results in the production of acid and gas which helps in the differentiation Red coloured, clear solution without any precipitate.
between genera and species. Cultural Response
Principle Cultural characteristics after 18-24 hours at 35-370C.
Organism Growth Acid Gas
Proteose peptone and beef extract provide nitrogenous nutrients to the
Citrobacter ferundii (8090) Luxuriant + +
organisms. Phenol red is the pH indicator which turns yellow at acidic pH. Sodium Enterobacter aerogenes (13048) Luxuriant + +
chloride maintains osmotic equilibrium. Gas formation is seen in Durham's tubes. Escherichia coli (25922) Luxuriant + +
Formula* Klebsiella pneumoniae (13883) Luxuriant + +
Ingredients in grams per liter Proteus vulgaris (13315) Luxuriant - -
Proteose peptone 10.00 S. serotype Typhimurium (14028) Luxuriant - -
Beef extract 1.00 S. serotype Typhi (6539) Luxuriant - -
Sodium chloride 5.00 Serratia marcescens (8100) Luxuriant - -
Dextrose 5.00 Shigella flexneri (12022) Luxuriant - -
Phenol red 0.018 Key: += Positive reaction yellow colour
Final pH (at 250C) 7.4 ± 0.2 -= Negative reaction, no colour change or red.
* Formula adjusted to suit performance parameters Storage
Directions
Store at 22-300C and prepared medium at 2-80C.
1. Suspend 21 gms of the powder in 1000 ml distilled water. Shelf Life
2. Mix thoroughly. Use before expiry date as mentioned on the label.

Phenol Red Lactose Broth ISO AM50804

Use Sodium chloride 5.00
Phenol Red Lactose Broth media is used for lactose fermentation studies. It is Lactose 10.00
Phenol red 0.018
recommended by ISO Committee under the specification ISO 9308-1:1990.
Final pH (at 250C) 7.5 ± 0.2
Summary
* Formula adjusted to suit performance parameters
Phenol Red Lactose Broth media is formulated by as recommended by ISO Directions
committee for studying lactose fermentation by coliforms which is an important 1. Suspend 25 gms of the powder in 1000 ml distilled water.
differentiating characteristics for the members of Enterobacteriaceae.
2. Mix thoroughly.
Principle
3. Heat to dissolve the powder completely.
Peptic digest of animal tissue provide nitrogenous nutrients to the organisms.
Phenol red is the pH indicator which turns yellow at acidic pH. Sodium chloride 4. Dispense in tubes containing inverted Durham's tubes.
maintains osmotic equilibrium. 5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Formula* Quality Control
Ingredients in grams per liter Dehydrated Appearance
Peptic digest of animal tissue 10.00 Light pink coloured, homogeneous, free flowing powder.

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Prepared Appearance S. serotype Typhimurium (14028) Luxuriant - -
Red coloured, clear solution without any precipitate. Shigella flexneri (12022) Luxuriant - -
Cultural Response Key: += Positive reaction yellow colour
Cultural characteristics after 18-24 hours at 35-370C. -= Negative reaction, no colour change or red.
Organism Growth Acid Gas Storage
Enterobacter aerogenes (13048) Luxuriant + + Store at 22-300C and prepared medium at 2-80C.
Escherichia coli (25922) Luxuriant + + Shelf Life
Klebsiella pneumoniae (13883) Luxuriant + + Use before expiry date as mentioned on the label.
Proteus vulgaris (13315) Luxuriant - -

Phenol Red Maltose Broth AM50805

Use Directions
Phenol Red Maltose Broth media is used for maltose fermentation studies of 1. Suspend 21 gms of the powder in 1000 ml distilled water.
microorganisms. 2. Mix thoroughly.
Summary
3. Heat to dissolve the powder completely.
Phenol Red Maltose Broth media are recommended (77.1, 31.1 & 28.1) for
4. Dispense in tubes containing inverted Durham's tubes.
determination of fermentation reactions in differentiation of microorganisms.
Ability of an organism to ferment specific carbohydrate added in a basal 5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Quality Control
medium, results in the production of acid and gas which helps in the
Dehydrated Appearance
differentiation between genera and species.
Light pink coloured, homogeneous, free flowing powder.
Principle
Prepared Appearance
Proteose peptone and beef extract provide nitrogenous nutrients to the Red coloured, clear solution without any precipitate.
organisms. Phenol red is the pH indicator which turns yellow at acidic pH. Cultural Response
Sodium chloride maintains osmotic equilibrium. Gas formation is seen in Cultural characteristics after 18-24 hours at 35-370C.
Durham's tubes. Organism Growth Acid Gas
Formula* Citrobacter freundii (8090) Luxuriant + +
Ingredients in grams per liter Enterobacter aerogenes (13048) Luxuriant + +
Proteose peptone Escherichia coli (25922) Luxuriant + +
10.00 Klebsiella pneumoniae (13883) Luxuriant + +
Beef extract Proteus vulgaris (13315) Luxuriant + +
1.00 S. serotype Typhimurium (14028) Luxuriant + +
Sodium chloride S. serotype Typhi (6539) Luxuriant + -
5.00 Serratia marcescens (8100) Luxuriant + -
Maltose Shigella flexneri (12022) Luxuriant + -
5.00
Key: += Positive reaction yellow colour
Phenol red
-= Negative reaction, no colour change or red.
0.018
Storage
Final pH (at 250C) 7.4 ± 0.2
* Formula adjusted to suit performance parameters Store at 22-300C and prepared medium at 2-80C.
Shelf Life
Use before expiry date as mentioned on the label.

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Phenol Red Mannitol Broth AM50806

Use 2. Mix thoroughly.
Phenol Red Mannitol Broth media is used for mannitol fermentation studies of 3. Heat to dissolve the powder completely.
microorganisms.
4. Dispense in tubes containing inverted Durham's tubes.
Summary
5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Phenol Red Mannitol Broth media are recommended (77.1, 31.1 & 28.1) for
Quality Control
determination of fermentation reactions in differentiation of microorganisms.
Dehydrated Appearance
Ability of an organism to ferment specific carbohydrate added in a basal Light pink coloured, homogeneous, free flowing powder.
medium, results in the production of acid and gas which helps in the Prepared Appearance
differentiation between genera and species. Red coloured, clear solution without any precipitate.
Principle Cultural Response
Proteose peptone and beef extract provide nitrogenous nutrients to the Cultural characteristics after 18-24 hours at 35-370C.
organisms. Phenol red is the pH indicator which turns yellow at acidic pH. Organism Growth Acid Gas
Citrobacter freundii (8090) Luxuriant + +
Sodium chloride maintains osmotic equilibrium. Gas formation is seen in
Enterobacter aerogenes (13048) Luxuriant + +
Durham's tubes.
Escherichia coli (25922) Luxuriant + +
Formula*
Klebsiella pneumoniae (13883) Luxuriant + +
Ingredients in grams per liter
Proteus vulgaris (13315) Luxuriant - -
Proteose peptone 10.00
S. serotype Typhimurium (14028) Luxuriant + +
Beef extract 1.00
S. serotype Typhi (6539) Luxuriant + -
Sodium chloride 5.00
Serratia marcescens (8100) Luxuriant + -
Mannitol 5.00
Shigella flexneri (12022) Luxuriant + -
Phenol red
Key: += Positive reaction yellow colour
0.018
-= Negative reaction, no colour change or red.
Final pH (at 250C) 7.4 ± 0.2
Storage
* Formula adjusted to suit performance parameters
Directions Store at 22-300C and prepared medium at 2-80C.
Shelf Life
1. Suspend 21 gms of the powder in 1000 ml distilled water.
Use before expiry date as mentioned on the label.

Phenol Red Sorbitol Broth AM50807

Use chloride maintains osmotic equilibrium. Gas formation is seen in Durham's tubes.
Phenol Red Sorbitol Broth media is used for sorbitol fermentation studies of Formula*
microorganisms. Ingredients in grams per liter
Summary Proteose peptone 10.00
Beef extract 1.00
Phenol Red Sorbitol Broth media are recommended (77.1, 31.1 & 28.1) for
Sodium chloride 5.00
determination of fermentation reactions in differentiation of microorganisms.
Sorbitol 5.00
Ability of an organism to ferment specific carbohydrate added in a basal medium, Phenol red 0.018
results in the production of acid and gas which helps in the differentiation Final pH (at 250C) 7.4 ± 0.2
between genera and species. * Formula adjusted to suit performance parameters
Principle Directions
Proteose peptone and beef extract provide nitrogenous nutrients to the 1. Suspend 21 gms of the powder in 1000 ml distilled water.
organisms. Phenol red is the pH indicator which turns yellow at acidic pH. Sodium 2. Mix thoroughly.

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3. Heat to dissolve the powder completely. Escherichia coli (25922) Luxuriant + +
Klebsiella pneumoniae (13883) Luxuriant + +
4. Dispense in tubes containing inverted Durham's tubes.
Proteus vulgaris (13315) Luxuriant - -
5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. S. serotype Typhimurium (14028) Luxuriant + +
Quality Control S. serotype Typhi (6539) Luxuriant + -
Dehydrated Appearance Serratia marcescens (8100) Luxuriant + -
Light pink coloured, homogeneous, free flowing powder. Shigella flexneri (12022) Luxuriant + -
Prepared Appearance Key: += Positive reaction yellow colour
Red coloured, clear solution without any precipitate. -= Negative reaction, no colour change or red.
Cultural Response Storage
Cultural characteristics after 18-24 hours at 35-370C. Store at 22-300C and prepared medium at 2-80C.
Organism (ATCC) Growth Acid Gas
Shelf Life
Citrobacter freundii (8090) Luxuriant + +
Use before expiry date as mentioned on the label.
Enterobacter aerogenes (13048) Luxuriant + +

Phenol Red Sucrose Broth AM50808

Use 4. Dispense in tubes containing inverted Durham's tubes.
Phenol Red Sucrose Broth media is used for sucrose fermentation studies of 5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
microorganisms. Quality Control
Summary Dehydrated Appearance
Phenol Red Sucrose Broth media are recommended (77.1, 31.1 & 28.1) for Light pink coloured, homogeneous, free flowing powder.
determination of fermentation reactions in differentiation of microorganisms. Prepared Appearance
Ability of an organism to ferment specific carbohydrate added in a basal medium, Red coloured, clear solution without any precipitate.
Cultural Response
results in the production of acid and gas which helps in the differentiation
Cultural characteristics after 18-24 hours at 35-370C.
between genera and species.
Organism (ATCC) Growth Acid Gas
Principle
Citrobacter freundii (8090) Luxuriant [+] +
Proteose peptone and beef extract provide nitrogenous nutrients to the Enterobacter aerogenes (13048) Luxuriant + +
organisms. Phenol red is the pH indicator which turns yellow at acidic pH. Sodium Escherichia coli (25922) Luxuriant - -
chloride maintains osmotic equilibrium. Gas formation is seen in Durham's tubes. Klebsiella pneumoniae (13883) Luxuriant + +
Formula* Proteus vulgaris (13315) Luxuriant + +
Ingredients in grams per liter S. serotype Typhimurium (14028) Luxuriant - -
Proteose peptone 10.00 S. serotype Typhi (6539) Luxuriant - -
Beef extract 1.00 Serratia marcescens (8100) Luxuriant + +
Sodium chloride 5.00 Shigella flexneri (12022) Luxuriant - -
Sorbitol 5.00 Key: + = Positive reaction yellow colour
Phenol red 0.018 [+] = Weak/slight
Final pH (at 250C) 7.4 ± 0.2 - = Negative reaction, no colour change or red.
* Formula adjusted to suit performance parameters Storage
Directions Store at 22-300C and prepared medium at 2-80C
1. Suspend 21 gms of the powder in 1000 ml distilled water. Shelf Life

2. Mix thoroughly. Use before expiry date as mentioned on the label.

3. Heat to dissolve the powder completely.

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Phenol Red Xylose Broth AM50809
Use 3. Heat to dissolve the powder completely.
Phenol Red xylose Broth media is used for xylose fermentation studies of 4. Dispense in tubes containing inverted Durham's tubes.
microorganisms.
5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Summary
Quality Control
Phenol Red Xylose Broth media are recommended (77.1, 31.1 & 28.1) for Dehydrated Appearance
determination of fermentation reactions in differentiation of microorganisms. Light pink coloured, homogeneous, free flowing powder.
Ability of an organism to ferment specific carbohydrate added in a basal medium, Prepared Appearance
results in the production of acid and gas which helps in the differentiation Red coloured, clear solution without any precipitate.
Cultural Response
between genera and species.
Cultural characteristics after 18-24 hours at 35-370C.
Principle
Organism (ATCC) Growth Acid Gas
Proteose peptone and beef extract provide nitrogenous nutrients to the Citrobacter freundii (8090) Luxuriant + +
organisms. Phenol red is the pH indicator which turns yellow at acidic pH. Sodium Enterobacter aerogenes (13048) Luxuriant + +
chloride maintains osmotic equilibrium. Gas formation is seen in Durham's tubes. Escherichia coli (25922) Luxuriant + +
Formula* Klebsiella pneumoniae (13883) Luxuriant + +
Ingredients in grams per liter Proteus vulgaris (13315) Luxuriant + [+]
Proteose peptone 10.00 S. serotype Typhimurium (14028) Luxuriant + +
Beef extract 1.00 S. serotype Typhi (6539) Luxuriant + -
Sodium chloride 5.00 Serratia marcescens (8100) Luxuriant - -
Xylose 5.00 Shigella flexneri (12022) Luxuriant - -
Phenol red 0.018 Key: + = Positive reaction yellow colour
Final pH (at 250C) 7.4 ± 0.2 [+] = Weak/slight
* Formula adjusted to suit performance parameters - = Negative reaction, no colour change or red.
Directions Storage
1. Suspend 21 gms of the powder in 1000 ml distilled water. Store at 22-300C and prepared medium at 2-80C.
2. Mix thoroughly. Shelf Life
Use before expiry date as mentioned on the label.

Phenylalanine Agar AM108091/AM508091

Use Formula*
Phenylalanine Agar is used for differentiation of Proteus and Providencia from Ingredients in grams per liter
Yeast extract 3.0
other Enterobacteriaceae on the basis of deamination of phenylalanine.
Sodium chloride 5.0
Summary
DL- Phenylalanine 2.0
Phenylalanine Agar is a modification of the medium originally formulated by Disodium phosphate 1.0
Ewing et al. Proteus, Providencia and Morganella species are capable to Agar 15.0
deaminate phenylalanine and form phenylpyruvic acid by enzymatic action (23). Final pH (at 250C) 7.3 ± 0.2
Principle * Formula adjusted to suit performance parameters
Yeast extract provides the essential nutrients for the growth of the Directions
microorganisms. Sodium chloride maintains the osmotic balance. DL- 1. Suspend the 26 gms of powder in 1000 ml distilled water.
Phenylalanine is the substrate for the deaminase enzyme which converts 2. Mix thoroughly.
phenylalanine to phenylpyruvic acid. And addition of few drops of ferric chloride 3. Boil with frequent agitation to dissolve the powder completely. DO NOT
gives green colour, indicating a positive test result. OVERHEAT.

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4. Dispense in tubes and sterilize by autoclaving at 121°C (15 lbs pressure) for Proteus mirabilis Luxuriant + More than 70%
(25933)
15 minutes.
Providencia alcalifaciens Luxuriant - More than 70%
5. Allow the medium to cool in slanting position. (12013)
Quality Control For growth RGI should be more than 70%
Dehydrated Appearance RGI- Relative Growth Index
Light pink coloured, homogeneous, free flowing powder. Key:
Prepared Appearance + = Green colouration after addition of 10% ferric chloride
Light amber coloured, slightly opalescent gel forms in tubes and slants. Procedure
Cultural Response Tubes are inoculated with heavy inoculum and incubated aerobically at
Cultural characteristics after 18-24 hours at 35-370C. 35±20C for 18-24 hours.
Organism (ATCC) Growth Phenylalanine RGI Following the incubation period, add few drops of the 10% aqueous ferric
deaminase chloride reagent.
Enterobacter aerogenes Luxuriant + More than 70% Interpretation of Results
(13048) Appearance of green colour is an indication of positive test result.
Escherichia coli Luxuriant + More than 70% Storage
(25922) Store at 22-300C and prepared medium at 2-80C.
Proteus vulgaris Luxuriant + More than 70% Shelf Life
(13315) Use before expiry date as mentioned on the label.

Pikovskaya's Agar AM108092/AM508092

Use * Formula adjusted to suit performance parameters
Pikovskaya's Agar is used for the detection of phosphate solubilizing Directions

microorganisms. 1. Suspend 31.3 gms of the powder in 1000ml distilled water.

Summary 2. Mix thoroughly.
Phosphorus is one of major limiting factors for crop production on many tropical 3. Boil with frequent agitation to dissolve the powder completely. Do not
and subtropical soils as a result of high phosphorus fixation (42.2). Phosphate overheat.
dissolving soil microorganisms play part in correcting phosphorus balance of crop 4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
plants. Many fungi and bacteria are potential solubilizers of bound phosphates so Quality Control
they are used in phosphate dissolving culture preparations (84.4). Dehydrated Appearance
Principle Off white coloured, homogeneous, free flowing powder.
Dextrose serves as energy source. Yeast extract supplies the nitrogen for the Prepared Appearance
support of bacterial growth. Calcium phosphate is the source of phosphorus. White with flocculant precipitate, opaque gel forms in petri plates.
Various salts support the growth of the microorganism. Cultural Response
i) Cultural characteristics after 5 days at R.T.
Formula*
ii)Cultural characteristics after 48 hours at 35-37°C.
Ingredients in grams per liter
Organism (ATCC) Growth Phenylalanine RGI
Yeast extract 0.50
solubilization
Dextrose 10.00
i) a-- Aspergillus niger Luxuriant + More than 70%
Calcium phosphate 5.00
(16404)
Ammonium sulphate 0.50
b-- Penicillin notatum Luxuriant + More than 70%
Potassium chloride 0.20
ii) a-- Pseudomonas Luxuriant + More than 70%
Magnesium sulphate 0.10
aeruginosa (27853)
Manganese sulphate 0.0001
b-- Bacillus subtilis Good (+) More than 70%
Ferrous sulphate 0.0001
(6633)
Agar 15.0
For growth RGI should be more than 70%
Final pH (at 250C) 7.4 ± 0.2

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RGI- Relative Growth Index Storage
Key: + = clear zone surrounding the colony Store at 22-300C and prepared medium at 2-80C.
(+) = moderate clear zone surrounding the colony
Shelf Life
Procedure
Use before expiry date as mentioned on the label.
Refer to appropriate references for specific procedures for the cultivation of
phosphate solubilizing soil microorganisms.
Interpretation of Results
Refer to appropriate references and procedures for results.

Pikovskaya's Broth AM508093

Use 2. Mix thoroughly.
Pikovskaya's Broth is used for the detection of phosphate solubilizing 3. Boil with frequent agitation to dissolve the powder completely. Do not
microorganisms. overheat.
Summary
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Phosphorus is one of major limiting factors for crop production on many tropical Quality Control
and subtropical soils as a result of high phosphorus fixation. Phosphate dissolving Dehydrated Appearance
soil microorganisms play part in correcting phosphorus balance of crop plants. Off white coloured, homogeneous, free flowing powder.
Many fungi and bacteria are potential solubilizers of bound phosphates so they Prepared Appearance
are used in phosphate dissolving culture preparations (84.4). White with flocculant precipitate, opaque solutions in tubes.
Principle Cultural Response
i) Cultural characteristics after 5 days at R.T.
Dextrose serves as energy source. Yeast extract supplies the nitrogen for the
ii) Cultural characteristics after 48 hours at 35-37°C.
support of bacterial growth. Calcium phosphate is the source of phosphorus.
Organism (ATCC)
Various salts support the growth of the microorganism. Growth
Formula* i) a-- Aspergillus niger(16404) Luxuriant
Ingredients in grams per liter b-- Penicillin notatum Luxuriant
Yeast extract 0.50 ii) a-- Pseudomonas aeruginosa (27853) Luxuriant
Dextrose 10.00 b-- Bacillus subtilis (6633) Good
Calcium phosphate 5.00 For growth RGI should be more than 70%
Ammonium sulphate 0.50 RGI- Relative Growth Index
Potassium chloride 0.20 Procedure
Magnesium sulphate 0.10 Refer to appropriate references for specific procedures for the cultivation of
Manganese sulphate 0.0001 phosphate solubilizing soil microorganisms.
Ferrous sulphate 0.0001 Interpretation of Results
Final pH (at 250C) 6.2 ± 0.2 Refer to appropriate references and procedures for results.
* Formula adjusted to suit performance parameters Storage
Directions Store at 22-300C and prepared medium at 2-80C.
1. Suspend 16.3 gms of the powder in 1000ml distilled water. Shelf Life
Use before expiry date as mentioned on the label.

Plate Count Agar (Standard Methods Agar) AM1081/AM5081

Use Summary
Plate Count Agar (Standard Methods Agar) is used for obtaining microbial plate Plate Count Agar is formulated as described by Buchbinder et al (10). It is
counts from milk and dairy products, foods, water and other materials of sanitary equivalent to the medium recommended by APHA for the plate count of
importance. microorganisms in milk (39) and other dairy products and may also be used to

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determine sanitary quality of foods (20), water (36) and other materials. This Escherichia coli (25922) Luxuriant More than 70%
medium is suitable for obtaining bacterial counts of sterile rooms. It is included in Lactobacillus casei (9595) Luxuriant More than 70%
Staphylococcus aureus (25923) Luxuriant More than 70%
the Bacteriological Analytical Manual for food and cosmetics testing (113).
Streptococcus pyogenes (19615) Luxuriant More than 70%
Principle
For growth RGI should be more than 70%
Tryptone provides nitrogenous substances and other amino acids. Yeast extract RGI- Relative Growth Index
provides B complex vitamins while dextrose is the energy source. Procedure
Formula* 1. APHA recommends pour plate technique.
Ingredients in grams per liter
Tryptone 5.0
2. Samples are diluted and appropriate dilutions are pipetted into sterile petri
Yeast Extract 2.5 plates.
Dextrose 1.0 3. Sterile molten medium is added followed by gentle mixing to distribute the
Agar 15.0 sample throughout the agar.
Final pH (at 250C) 7.0 ± 0.2
4. Incubate plates for 48 hours at 320C (dairy products) or 350C (for foods) in an
* Formula adjusted to suit performance parameters
Directions
aerobic atmosphere.
1. Suspend 23.5 gms of the powder in 1000 ml distilled water. 5. For the enumeration of microorganisms with other temperature
requirements, plates may also be incubated for 7-10 days at 5-70C, for 3-5
2. Mix thoroughly.
days at 200C, for 2-3 days at 450C, or for 48 hours at 550C.
3. Boil with frequent agitation to dissolve the powder completely.
Interpretation of Results
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
1. Count the number of colonies and express as colony forming units (CFU) per
Quality Control
gram or ml of sample, taking into account the applicable dilution factor.
Dehydrated Appearance
Precautions / Limitations
Light yellow coloured, homogeneous, free flowing powder.
Prepared Appearance 1. After autoclaving, do not heat the medium longer than 3 hours at 45-500C.
Light yellow coloured clear to slightly opalescent gel. 2. Sterile solidified medium can be remelted only once.
Cultural Response Storage
Cultural characteristics after 18-24 hours at 35-370C.
Store at 22-300C and prepared medium at 2-80C.
Organisms (ATCC) Growth RGI
Shelf Life
Bacillus subtilis (6633) Luxuriant More than 70%
Enterococcus faecalis (29212) Luxuriant More than 70%
Use before expiry date as mentioned on the label.

Plate Count Agar BIS AM10811/AM50811

Use Principle

Plate Count Agar is used for obtaining microbial plate counts from milk and dairy Tryptone provides nitrogenous substances and other amino acids. Yeast extract
products, foods, water and other materials of sanitary importance, in compliance provides B complex vitamins while dextrose is the energy source.
with BIS. Formula*
Summary Ingredients in grams per liter
Tryptone 5.0
Plate Count Agar is formulated as described by Buchbinder et al., (10) It is
Dehydrated yeast extract 2.5
equivalent to the medium recommended by APHA for the plate count of Anhydrous glucose 1.0
microorganisms in milk and other dairy products and may also be used to Agar 15.0
determine sanitary quality of foods, water and other materials. This medium is Final pH (at 250C) 7.0 ± 0.2
suitable for obtaining bacterial counts of sterile rooms. It is included in the * Formula adjusted to suit performance parameters
Bacteriological Analytical Manual for food and cosmetics testing.

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Directions Procedure
1. Suspend 23.5 gms of the powder in 1000 ml distilled water. 1. APHA recommends pour plate technique.
2. Samples are diluted and appropriate dilutions are pipetted into sterile
2. Mix thoroughly. petri plates.
3. Boil with frequent agitation to dissolve the powder completely. 3. Sterile molten medium is added followed by gentle mixing to distribute
the sample throughout the agar.
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
4. Incubate plates for 48 hours at 320C (dairy products) or 350C (for foods)
Quality Control in an aerobic atmosphere.
Dehydrated Appearance 5. For the enumeration of microorganisms with other temperature
Light yellow coloured, homogeneous, free flowing powder. requirements, plates may also be incubated for 7-10 days at 5- 70C, for 3
Prepared Appearance 5 days at 200C, for 2-3 days at 450C, or for 48 hours at 550C.
Light yellow coloured clear to slightly opalescent gel.. Interpretation of Results
Cultural Response 1. Count the number of colonies and express as colony forming units (CFU)
Cultural characteristics after 18-24 hours at 35-370C. per gram or ml of sample, taking into account the applicable dilution
factor.
Organism (ATCC) Growth RGI
Precautions / Limitations
Bacillus subtilis (6633) Luxuriant More than 70%
1. After autoclaving, do not heat the medium longer than 3 hours at 45-
Enterococcus faecalis (29212) Luxuriant More than 70%
500C.
Escherichia coli (25922) Luxuriant More than 70%
2. Sterile solidified medium can be remelted only once.
Lactobacillus casei (9595) Luxuriant More than 70%
Storage
Staphylococcus aureus (25923) Luxuriant More than 70%
Streptococcus pyogenes (19615) Luxuriant More than 70% Store at 22-300C and prepared medium at 2-80C.
For growth RGI should be more than 70% Shelf Life
RGI- Relative Growth Index Use before expiry date as mentioned on the label.

PNY Medium AM10692711/AM50692711

Use Sodium chloride 0.01
PNY Medium is used for cultivation and isolation of Lactobacillus species. Copper sulphate 0.001
Cobalt sulphate 0.001
Summary
Zinc sulphate 0.001
Lactobacillus is a genus of Gram positive facultative anaerobic or microaerophilic Agar 15.0
bacteria. Some Lactobacillus species are used industrially for the production of Final pH (at 250C) 6.0±0.2
yogurt, cheese, sauerkraut, pickles, beer, wine and other fermented foods. PNY * Formula adjusted to suit performance parameters
Medium is recommended for cultivation and isolation of Lactobacillus species. Directions
Principle 1. Suspend 31.28 gms powder in 1000ml distilled water.
Peptic digest of animal tissue and yeast extract provide nitrogen compounds, 2. Mix thoroughly.
carbon compounds and nutrients. Dextrose is the source of energy. Different salts
3. Boil with frequent agitation to dissolve the powder completely. DO NOT
support the growth of the bacteria. OVERHEAT.
Formula*
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Ingredients in grams per liter
Warning: Cobalt sulphate is harmful. Avoid bodily contact and
Peptic digest of animal tissue 5.0
inhalation of vapours. On contact with skin, wash with plenty of water
Yeast extract 5.0 immediately.
Dextrose 5.0 Quality Control
Monopotassium phosphate 0.5 Dehydrated Appearance
Dipotassium phosphate 0.5 Cream to yellow coloured, homogeneous, free flowing powder.
Magnesium sulphate 0.25 Prepared Appearance
Ferrous sulphate 0.01 Yellow coloured, clear gel formed in petriplates.
Manganese sulphate 0.01

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Cultural Response Procedure
Cultural characteristics after 18-24 hours at 35-370C. Refer to appropriate references for specific procedures.
Organism (ATCC) Growth RGI Interpretation of Results:
Lactobacillus casei (9595) Luxuriant More than 70% Refer to appropriate references and procedures for results.
Lactobacillus leichmannii (4797) Luxuriant More than 70% Storage
Lactobacillus plantarum (8014) Luxuriant More than 70% Store at 22-300C and prepared medium at 2-80C.
For growth RGI should be more than 70% Shelf Life
RGI- Relative Growth Index
Use before expiry date as mentioned on the label.

Potato Dextrose Agar (Harmonized) AMH5082

Use Prepared Appearance
Potato Dextrose Agar Harmonized is used for the cultivation and enumeration of Light amber coloured, clear to slightly opalescent gel.
Cultural Response
yeasts and moulds from dairy and other food products.importance.
Cultural characteristics after 4-5 days at 20-250C.
Summary
Organisms Growth Ascospore RGI
Potato Dextrose Agar is recommended by the USP and IP for use in the (ATCC) Formation
performance of Microbial Limit Tests and by APHA. This medium is included in the Aspergillus niger (16404) Luxuriant - More than 70%
Bacteriological Analytical Manual for food and cosmetics testing and is also used Candida albicans (10231) Luxuriant - More than 70%
for stimulation of sporulation, maintenance of stock cultures of certain Saccharomyces Luxuriant + More than 70%
dermatophytes and in differentiating atypical varieties of dermatophytes on the cerevisiae (9763)
basis of pigment production. For growth RGI should be more than 70%
Principle RGI- Relative Growth Index
Procedure
Potato infusion and dextrose provide nutrients for luxuriant growth of fungi.
For Quantitative test
Acidifying the medium by lowering the pH to 3.5 with sterile tartaric acid inhibits
bacterial growth. 1. Prepare decimal dilutions of the sample in a sterile diluent to obtain 30-300
Formula* colony forming units per plate.
Ingredients in grams per liter 2. Isolate using spread plate or pour plate technique.
Potato, Infusion from 200.0
3. Incubate plates aerobically for 48 hours at 370C.
Dextrose 20.0
Agar 15.0 For the determination of yeasts and mould counts
0
Final pH (at 25 C) ) 5.6 ± 0.2 1. Adjust the pH to approximately 3.5 with sterile tartaric acid and use in pour
* Formula adjusted to suit performance parameters plate technique.
Directions
2. Incubate the plates at 20-250C in an inverted position with increased
1. Suspend 39 gms of the powder in 1000 ml distilled water. Mix thoroughly.
humidity.
2. Boil with frequent agitation to dissolve the powder completely.
For isolation of specimens from potentially contaminated specimens
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
1. Isolate using the streak plate method to get isolated colonies.
4. Mix well before dispensing.
2. A selective medium should be inoculated along with this nonselective
5. When pH 3.5 is required, cool the base to 450C and aseptically add an
medium.
appropriate amount of sterile 10% tartaric acid (approximately 1ml in 100
ml of medium) to each liter of the medium and mix well. 3. When used as a non-selective medium, do not add the acid.
6. Do not reheat the medium after addition of acid. 4. Alternatively, a general-purpose mycological medium such as Malt Extract
Quality Control Agar can be used.
Dehydrated Appearance For isolating fungi causing systemic mycosis
Cream coloured, homogeneous, free flowing powder.

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1. Inoculate two sets of media, with one set incubated at 25-300C and the other 3. Examine all cultures at least weekly for fungal growth and preserve for at
0
set at 35-37 C. least 4-6 weeks before being reported as negative.
Interpretation of Results Storage
1. Count the number of colonies and express as colony forming units (CFU) per Store at 22-300C and prepared medium at 2-80C.
gram or ml of sample, taking into account the applicable dilution factor. Shelf Life

2. If duplicate plates were set up, express the average for the two plates in terms Use before expiry date as mentioned on the label.
of number of micro-organisms per gram or ml of sample.

Potato Dextrose Agar USP AM10821/AM50821

Use Prepared Appearance
Potato Dextrose Agar USP is used for the cultivation and enumeration of yeasts Light amber coloured, clear to slightly opalescent gel.
Cultural Response
and moulds from dairy and other food products in compliance with USP.
Cultural characteristics after 4-5 days at 20-250C.
Summary
Organisms Growth Ascospore RGI
Potato Dextrose Agar is recommended by the USP and IP for use in the (ATCC) Formation
performance of Microbial Limit Tests and by APHA. This medium is included in the Aspergillus niger (16404) Luxuriant - More than 70%
Bacteriological Analytical Manual for food and cosmetics testing and is also used Candida albicans (10231) Luxuriant - More than 70%
for stimulation of sporulation, maintenance of stock cultures of certain Saccharomyces Luxuriant + More than 70%
dermatophytes and in differentiating atypical varieties of dermatophytes on the cerevisiae (9763)
basis of pigment production. For growth RGI should be more than 70%
Principle RGI- Relative Growth Index
Procedure
Potato infusion and dextrose provide nutrients for luxuriant growth of fungi.
For Quantitative test
Acidifying the medium by lowering the pH to 3.5 with sterile tartaric acid inhibits
bacterial growth. 1. Prepare decimal dilutions of the sample in a sterile diluent to obtain 30-300
Formula* colony forming units per plate.
Ingredients in grams per liter 2. Isolate using spread plate or pour plate technique.
Potato, Infusion from 300.0
3. Incubate plates aerobically for 48 hours at 370C.
Glucose 20.0
Agar 15.0 For the determination of yeasts and mould counts
Final pH (at 250C) 5.6 ± 0.2 1. Adjust the pH to approximately 3.5 with sterile tartaric acid and use in pour
* Formula adjusted to suit performance parameters plate technique.
Directions
2. Incubate the plates at 25-300C in an inverted position with increased
1. Suspend 41 gms of the powder in 1000 ml distilled water. Mix thoroughly.
humidity.
2. Boil with frequent agitation to dissolve the powder completely.
For isolation of specimens from potentially contaminated specimens
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
1. Isolate using the streak plate method to get isolated colonies.
4. Mix well before dispensing.
2. A selective medium should be inoculated along with this nonselective
5. When pH 3.5 is required, cool the base to 450C and aseptically add an medium.
appropriate amount of sterile 10% tartaric acid (approximately 1ml in 100
3. When used as a non-selective medium, do not add the acid.
ml of medium) to each liter of the medium and mix well.
4. Alternatively, a general-purpose mycological medium such as Malt Extract
6. Do not reheat the medium after addition of acid.
Agar can be used. For isolating fungi causing systemic mycosis
Quality Control
Dehydrated Appearance 1. Inoculate two sets of media, with one set incubated at 25-300C and the other
Cream coloured, homogeneous, free flowing powder. set at 35-370C.

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Interpretation of Results 4- 6 weeks before being reported as negative.
1. Count the number of colonies and express as colony forming units (CFU) per Storage
gram or ml of sample, taking into account the applicable dilution factor. Store at 22-300C and prepared medium at 2-80C.
2. If duplicate plates were set up, express the average for the two plates in terms Shelf Life
of number of micro-organisms per gram or ml of sample. Use before expiry date as mentioned on the label.
3. Examine all cultures at least weekly for fungal growth and preserve for at least

Potato Dextrose Agar AM1082/AM5082

Potato Dextrose Broth AM1083/AM5083
Use 5. When pH 3.5 is required, cool the base to 450C and aseptically add an
Potato Dextrose Agar and Potato Dextrose Broth are used for the cultivation and appropriate amount of sterile 10% tartaric acid (approximately 1ml in 100
enumeration of yeasts and moulds from dairy and other food products. ml of medium) to each liter of the medium and mix well.
Summary 6. Do not reheat the medium after addition of acid.
Potato Dextrose Agar is recommended by the USP (114) and IP (46) for use in the Quality Control
performance of Microbial Limit Tests and by APHA (20). This medium is included Dehydrated Appearance
in the Bacteriological Analytical Manual for food and cosmetics testing (113) and Cream coloured, homogeneous, free flowing powder.
is also used for stimulation of sporulation, maintenance of stock cultures of certain Prepared Appearance
dermatophytes and in differentiating atypical varieties of dermatophytes on the Potato Dextrose Agar - Light amber coloured, clear to slightly opalescent gel.
Potato Dextrose Broth - Light amber coloured, clear to slightly opalescent
basis of pigment production. Potato Dextrose Broth is a general-purpose medium solution.
used for the cultivation of yeasts and moulds. Cultural Response
Principle Cultural characteristics after 4-5 days at 22-250C.
Potato infusion and dextrose provide nutrients for luxuriant growth of fungi. Organisms (ATCC) Growth Ascospore RGI
Acidifying the medium by lowering the pH to 3.5 with sterile tartaric acid inhibits Formation
bacterial growth. Aspergillus niger (16404) Luxuriant - More than 70%
Formula* Candida albicans (10231) Luxuriant - More than 70%
Ingredients in grams Potato Dextrose Potato Dextrose Saccharomyces Luxuriant + More than 70%
per liter Agar Broth cerevisiae (9763)
Potato, Infusion from 200.0 200.0 Procedure
Dextrose 20.0 20.0 For Potato Dextrose Agar
Agar 15.0 - For Quantitative test
Final pH (at 250C) 5.6 ± 0.2 5.1 ± 0.2
1. Prepare decimal dilutions of the sample in a sterile diluent to obtain 30-300
* Formula adjusted to suit performance parameters
Directions colony forming units per plate.
1. Suspend the powder in 1000 ml distilled water. 2. Isolate using spread plate or pour plate technique.
Potato Dextrose Agar - 39 gms 3. Incubate plates aerobically for 48 hours at 370C.
Potato Dextrose Broth - 24 gms For the determination of yeasts and mould counts
Mix thoroughly. 1. Adjust the pH to approximately 3.5 with sterile tartaric acid and use in pour
plate technique.
2. Boil with frequent agitation to dissolve the powder completely.
2. Incubate the plates at 25-300C in an inverted position with increased
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
humidity.
4. Mix well before dispensing.

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For isolation of specimens from potentially contaminated specimens 2. If duplicate plates were set up, express the average for the two plates in
1. Isolate using the streak plate method to get isolated colonies. terms of number of micro-organisms per gram or ml of sample.
2. A selective medium should be inoculated along with this non-selective 3. Examine all cultures at least weekly for fungal growth and preserve for at
medium. least 4-6 weeks before being reported as negative.
3. When used as a non-selective medium, do not add the acid. Potato Dextrose Broth
4. Alternatively, a general-purpose mycological medium such as Malt Extract 1. Observe cultures for surface growth and pellicle formation.
Agar can be used. Precautions / Limitations

For isolating fungi causing systemic mycosis 1. Heating the medium after acidification hydrolyzes the agar and may destroy
the gelling properties.
1. Inoculate two sets of media, with one set incubated at 25-300C and the other
set at 35-370C. 2. This medium is not a primary isolation medium. Direct inoculation of
specimens will give wrong results.
For Potato Dextrose Broth
3. For proper identification of yeasts and moulds, microscopic examination and
1. Inoculate the broth tubes.
evaluation of morphological structures may be required.
2. Incubate for 40-48 hours at 22-250C. Storage
Interpretation of Results
Store at 22-300C and prepared medium at 2-80C.
Potato Dextrose Agar Shelf Life
1. Count the number of colonies and express as colony forming units (CFU) per Use before expiry date as mentioned on the label.
gram or ml of sample, taking into account the applicable dilution factor.

Preston Agar Base AM10831/AM50831

Use Agar 12.0
0
Preston Agar Base with added supplements is recommended for selective Final pH (at 25 C) 7.5 ± 0.2
* Formula adjusted to suit performance parameters
isolation of thermotolerant Campylobacter species especially Campylobacter
Directions
jejuni and Campylobacter coli.
Summary 1. Suspend 18.5 grams of the powder in 470 ml distilled water.
Campylobacter species can cause mild to severe diarrhea with loose watery stools 2. Boil with frequent agitation to dissolve the powder completely.
often followed by bloody diarrhea. These pathogens are highly infective and 3. Sterilize by autoclaving at 1210 C (15 lbs pressure) for 15 minutes.
transmitted by contaminated food or water. 4. Cool to 45-500 C.
Preston Agar Base is based on the formulation described by Bolton and Robertson 5. Aseptically add 25 ml sterile, lysed horse blood and reconstituted contents of
(1). This formula with the addition of Preston Selective Supplement is used to 1 vial of Preston Selective Supplement (Campylobacter Selective
isolate Campylobacter species from human, animal and environmental Supplement IV, Modified) (AS0231).
specimens.
6. Mix well and pour into sterile petriplates.
Principle
Quality Control
Peptic digest of animal tissue and beef extract provide nutritional sources of Dehydrated Appearance
nitrogen, vitamins, minerals and amino acids required for growth. Sodium Light yellow coloured, homogeneous, free flowing powder.
chloride maintains the osmotic equilibrium. Agar is the solidification agent. Prepared Appearance
Formula* Light yellow coloured clear to slightly opalescent gel. On addition of sterile
Ingredients in grams per liter Horse Blood, chocolate brown coloured opaque gel.
Peptic Digest of animal Tissue 10.0 Cultural Response
Beef Extract 10.0 Cultural response at 370 C after 18 - 48 hours incubation on 5% Horse Blood
plates in an atmosphere consisting of approximately 5-6% oxygen, 3-10%
Sodium Chloride 5.0
carbon dioxide and 84-85% nitrogen.

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Organisms (ATCC) Growth RGI Interpretation of Results
Campylobacter jejuni (29428) Luxuriant More than 70% 1. Campylobacter colonies are round to irregular with smooth edges. They may
Campylobacter coli (33559) Luxuriant More than 70%
exhibit translucent, white colonies to spreading, flat, transparent growth.
Escherichia coli (25922) Inhibited 0%
Some strains may appear tan or slightly pink.
Staphylococcus aureus (25923) Inhibited 0%
For growth RGI should be more than 70% 2. Normal enteric flora is completely to markedly inhibited.
For Inhibition RGI should be 0% Limitations
RGI- Relative Growth Index
Some strains of Campylobacter may grow poorly or may be inhibited due to
Procedure
variation in nutritional requirements.
1. Streak the isolated specimen directly onto the surface of the medium. Storage
2. Incubate at 370C in an atmosphere consisting of approximately 5-6% Store at 22-300C and prepared medium at 2-80C.
oxygen, 3-10% carbon dioxide and 84-85% nitrogen for 24-48 hours. Shelf Life
3. Observe for growth. Use before expiry date as mentioned on the label.

Pseudomonas Agar Base AM1084/AM5084

Use pharmaceuticals and cosmetics. The organism is resistant to many commonly
Pseudomonas Agar Base with added supplements is used for the selective used disinfectants. It has emerged as an important opportunistic pathogen in
isolation of Pseudomonas species. urinary, abdominal, respiratory and other infections and could be isolated from
Summary mixed cultures of cystic fibrosis patients.
Pseudomonas Agar Base is a modification of King's A medium (55) in which Formula*
magnesium chloride and potassium sulphate are incorporated to enhance Ingredients in grams per liter
pigment production and is recommended by USP (114) and IP (46) for detecting Pancreatic Digest of Gelatin 16.0
Potassium Sulphate 10.0
pyocyanin, a water soluble pigment produced by Pseudomonas species.
Tryptone 10.0
Principle
Magnesium Chloride 1.4
Pancreatic digest of gelatin and tryptone provides amino acids and other essential Agar 11.0
nutrients. Magnesium chloride and potassium sulphate enhance pigment Final pH (at 250C) 7.1 ± 0.2
production. The peptic digest of gelatin is low in phosphorus to minimize the * Formula adjusted to suit performance parameters
inhibitory action on pyocyannin production. This medium enhances the formation Directions
of pyocyanin but inhibits the formation of fluorescein pigment. The fluorescein 1. Suspend 24.2 gms of the powder in 500 ml distilled water containing 5 ml
pigment diffuses from the colonies of Pseudomonas into the agar to give a blue glycerol and mix well.
colouration. Some Pseudomonas species produce small amounts of fluorescein 2. Boil with frequent agitation to dissolve the powder completely.
resulting in a blue-green colouration. Addition of CFC Supplement (AS009)
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
makes the medium selective for Pseudomonas species. Mead and Adam's showed
that reducing the cetrimide content to 10 microgram per ml allowed the growth of 4. Cool to 450C and aseptically add sterile rehydrated contents of 1 vial each
all pigmented and non-pigmented psychrophilic Pseudomonas species. Addition either Cetrinix Supplement (AS008) or CFC Supplement (AS009) as desired.
of cephaloridine (50 microgram/ ml) and fucidin (10 microgram/ml) makes the 5. Mix well and pour into sterile petri plates.
medium more specific for isolating Pseudomonas species from chilled foods and Note: Do not keep the molten agar for longer than 4 hours.
processing plants. Goto and Enomoto formulated Cetrinix Supplement (AS008) Quality Control
for the selective isolation of Pseudomonas aeruginosa. Cetrinix Supplement Dehydrated Appearance
(AS008) suppresses Klebsiella, Proteus and Providencia species. Considerable Light yellow coloured, homogeneous, free flowing powder.
Prepared Appearance
importance is given to detection of Burkholderia cepacia (formerly P. cepacia) in
Yellow coloured, clear to slightly opalescent gel.
water systems, particularly where the water is to be used in the preparation of

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Cultural Response evidence of Pseudomonas species.
Cultural characteristics at 24-48 hours at 250C or 350C. Precautions / Limitations
Organisms (ATCC) Growth with Cetrinix Growth with CFC
1. Growth on Pseudomonas Agar Base with CFC supplement is usually limited to
Supplement (350C) Supplement (250C)
Proteus vulgaris (13315) - -
Pseudomonas species, but some members of Enterobacteriaceae may also
Pseudomonas aeruginosa (27853) Luxuriant - grow.
Pseudomonas cepacia (10661) - Luxuriant 2. Freshly prepared medium should be used as much as possible and should not
Staphylococcus aureus (25923) - -
be remelted. Molten agar should not be kept for more than 4 hours.
Procedure
3. If swarming of Proteus species is a problem in food samples then the
For food, water, and environmental samples.
incubation temperature can be lowered to 200C for a period of 3-5 days.
1. Prepare Pseudomonas Agar Base and add CFC supplement as directed.
4. Chilled foods can carry a wide range of Pseudomonas and the colonies on
2. Pour plates under aseptic conditions and dry the surface.
Pseudomonas Agar Base with CFC supplement, incubated at lower
3. Prepare food samples by diluting 1 in 5 or 1 in 10 with 1% (w/v) sterile temperatures, may include P.fluorescens, P.putida as well as P.aeruginosa.
peptone water and homogenize in a blender. Aeromonas species if present will also appear as pink / brown colonies,
4. Pipette 0.5 ml or 1 ml of the homogenate onto the plate and spread evenly particularly from fish products.
over the surface with a sterile glass spreader. Inoculate water and swab Storage
samples directly on the surface of the medium. Store at 22-300C and prepared medium at 2-80C.
5. Incubate at 250C and examine after 24 and 48 hours under both, white and Shelf Life
ultraviolet light. Use before expiry date as mentioned on the label. l.
Interpretation of Results
1. The presence of blue-green pigmentation, or fluorescence is a presumptive

Pseudomonas Agar (for Fluorescein) AM108411/AM508411

Use Magnesium sulphate enhance fluorescin production. Glycerin as an energy source
Pseudomonas Agar for Fluorescein is used for the detection of fluorescein also increases fluorescin production. Agar is the solidifying agent.
production by Pseudomonas species. Formula*
Summary Ingredients in grams per liter
Pseudomonas species may produce water-soluble pigments in culture media. Enzymetic hydrolysate of Casein 10.0
Proteose peptone 10.0
This property is sometimes used as a characteristic for the taxonomic classification
Dipotassium phosphate 1.5
of different species of Pseudomonas (33.1). Most strains of P. aeruginosa
Magnesium sulphate 1.5
produce pyocyanin( blue) or pyoverdin (Yellow) or both, as well as pyorubrin Agar 15.0
(red), pyomelanin (Brown), or various combinations of these pigments (49.3). P. Final pH (at 250C) 7.0± 0.2
aeruginosa and other Pseudomonas isolated from humans often produce water * Formula adjusted to suit performance parameters
soluble fluorescent pigments; pyoverdin is also one of these. Fluorescent Directions
pigment-producing strains fluoresce in under short-wave ultraviolet light. the 1. Suspend the 38 gms of powder in 990 ml distilled water.
fluorescence of pseudomonas is best observed at 254nm (19.3). Pseudomonas 2. Add10 ml of glycerin and mix thoroughly.
Agar (for fluorescein) is a modification of formulation described by King et al.,
3. Boil with frequent agitation to dissolve the powder completely.
This medium is recommended by USP for use in Microbial Limit Tests.
Principle 4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Quality Control
Pancreatic digest of casein and proteose peptone provide nutrients, carbon,
Dehydrated Appearance
sulphur and trace elements for growth. Equal proportion of enzymetic hydrolysate Light yellow coloured, homogeneous, free flowing powder.
of casein and pancreatic digest of animal tissue is helpful for fluorescin Prepared Appearance
production by pseudomonas. Dipotassium phosphate serve as the buffer. Light yellow coloured, homogeneous, free flowing powder.

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Prepared Appearance 3. Examine for growth and fluorescein production.
Light yellow coloured, clear to slightly opalescent gel. Interpretation of Results
Cultural Response
1. Examine growth under short wavelength UV light (254nm) for fluorescin.
Cultural characteristics after 18-24 hours at 35-370C.
Organisms (ATCC) Growth Colour of RGI 2. Presence of fluorescin is appear with a greenish yellow fluorescent pigment
medium in the colonies and surrounding medium.
Pseudomonas aeruginosa Good Greenish yellow More than 70% Limitations
(27853) Some strains of Campylobacter may grow poorly or may be inhibited due to
For growth RGI should be more than 70%
variation in nutritional requirements.
RGI- Relative Growth Index
Storage
Procedure
Store at 22-300C and prepared medium at 2-80C.
1. Obtain a pure culture of the organism to be tested.
Shelf Life
2. Inoculate and incubate plates aerobically at 35-370C for 18-48 hours.
Use before expiry date as mentioned on the label.

Pseudomonas Agar (For Fluorescein) IP AM108412/AM508412

Use Magnesium Sulphate (MgSO47H2O) 1.5
Pseudomonas Agar for Fluorescein is used for the detection of fluorescein Agar 15.0
production by Pseudomonas species in compliance with IP. Final pH (at 250C) 7.2± 0.2
* Formula adjusted to suit performance parameters
Summary
Directions
Pseudomonas species may produce water-soluble pigments in culture media.
1. Suspend the 38 gms of powder in 990 ml distilled water.
This property is sometimes used as a characteristic for the taxonomic
classification of different species of Pseudomonas. Most strains of P. aeruginosa 2. Add10 ml of glycerin and mix thoroughly.
produce pyocyanin( blue) or pyoverdin (Yellow) or both, as well as pyorubrin 3. Boil with frequent agitation to dissolve the powder completely.
(red), pyomelanin (Brown), or various combinations of these pigments. 4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
P. aeruginosa and other Pseudomonas isolated from humans often produce Quality Control
water soluble fluorescent pigments; pyoverdin is also one of these. Fluorescent Dehydrated Appearance
pigment-producing strains fluoresce in under short-wave ultraviolet light. the Light yellow coloured, homogeneous, free flowing powder.
Prepared Appearance
fluorescence of pseudomonas is best observed at 254nm. Pseudomonas Agar
Light yellow coloured, clear to slightly opalescent gel.
(for fluorescein) is a modification of formulation described by King et al., This
Cultural Response
medium is recommended by IP for use in Microbial Limit Tests. Cultural characteristics after 18-24 hours at 35-370C.
Principle Organisms (ATCC) Growth Colour of RGI
Pancreatic digest of casein and peptic digest of animal tissue provide nutrients, medium
carbon, sulphur and trace elements for growth. Pancreatic digest of animal tissue Pseudomonas aeruginosa Good Greenish yellow More than 70%
is helpful for fluorescin production by pseudomonas. Dipotassium phosphate (27853)
serve as the buffer. Magnesium sulphate enhance fluorescin production. Glycerin For growth RGI should be more than 70%
RGI- Relative Growth Index
as an energy source also increases fluorescin production. Agar is the solidifying
Procedure
agent.
Formula* 1. Obtain a pure culture of the organism to be tested.
Ingredients in grams per liter 2. Inoculate and incubate plates aerobically at 35-370C for 18-48 hours.
Pancreatic Digest of Casein 10.0 3. Examine for growth and fluorescein production.
Peptic Digest of animal tissue 10.0
Interpretation of Results
Anhydrous Dibasic Potassium Phosphate 1.5
1. Examine growth under short wavelength UV light (254nm) for fluorescin.

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2. Presence of fluorescin is appear with a greenish yellow fluorescent pigment Store at 22-300C and prepared medium at 2-80C.
in the colonies and surrounding medium. Shelf Life

Limitations Use before expiry date as mentioned on the label.

Some strains of Campylobacter may grow poorly or may be inhibited due to
variation in nutritional requirements.
Storage
Pseudomonas Agar (For Fluorescein) USP AM108413/AM508413
Use Directions
Pseudomonas Agar for Fluorescein is used for the detection of fluorescein 1. Suspend the 38 gms of powder in 990 ml distilled water.
production by Pseudomonas species in compliance with USP. 2. Add10 ml of glycerin and mix thoroughly.
Summary
3. Boil with frequent agitation to dissolve the powder completely.
Pseudomonas species may produce water-soluble pigments in culture media.
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
This property is sometimes used as a characteristic for the taxonomic classification
Quality Control
of different species of Pseudomonas. Most strains of P. aeruginosa produce Dehydrated Appearance
pyocyanin( blue) or pyoverdin (Yellow) or both, as well as pyorubrin (red), Light yellow coloured, homogeneous, free flowing powder.
pyomelanin (Brown), or various combinations of these pigments. Prepared Appearance
P. aeruginosa and other Pseudomonas isolated from humans often produce water Light yellow coloured, clear to slightly opalescent gel.
soluble fluorescent pigments; pyoverdin is also one of these. Fluorescent Cultural Response
Cultural characteristics after 18-24 hours at 35-370C.
pigment-producing strains fluoresce in under short-wave ultraviolet light. the
Organisms (ATCC) Growth Colour of RGI
fluorescence of pseudomonas is best observed at 254nm. Pseudomonas Agar (for
medium
fluorescein) is a modification of formulation described by King et al, This medium Pseudomonas aeruginosa Good Greenish yellow More than 70%
is recommended by USP for use in Microbial Limit Tests. (27853)
Principle For growth RGI should be more than 70%
Pancreatic digest of casein and proteose peptone provide nutrients, carbon, RGI- Relative Growth Index
sulphur and trace elements for growth. Equal proportion of enzymetic hydrolysate Procedure

of casein and pancreatic digest of animal tissue is helpful for fluorescin production 1. Obtain a pure culture of the organism to be tested.
by pseudomonas. Dipotassium phosphate serve as the buffer. Magnesium 2. Inoculate and incubate plates aerobically at 35-370C for 18-48 hours.
sulphate enhance fluorescin production. Glycerin as an energy source also 3. Examine for growth and fluorescein production.
increases fluorescin production. Agar is the solidifying agent.
Interpretation of Results
Formula*
Ingredients in grams per liter 1. Examine growth under short wavelength UV light (254nm) for fluorescin.
Pancreatic Digest of Casein 10.0 2. Presence of fluorescin is appear with a greenish yellow fluorescent pigment
Peptic Digest of animal tissue 10.0 in the colonies and surrounding medium.
Anhydrous Dibasic Potassium Phosphate 1.5 Storage
Magnesium Sulphate (MgSO47H2O) 1.5
Store at 22-300C and prepared medium at 2-80C.
Agar 15.0
Shelf Life
Final pH (at 250C) 7.2± 0.2
* Formula adjusted to suit performance parameters Use before expiry date as mentioned on the label.

Pseudomonas Agar (for Pyocyanin) AM108414/AM508414

Use Summary
Pseudomonas Agar (for Pyocyanin) is used for the detection of Pseudomonas Pseudomonas species may produce water-soluble pigments in culture media. This
species using pyocyanin pigment production. property is sometimes used as a characteristic for the taxonomic classification of

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different species of Pseudomonas. Most strains of P. aeruginosa produce Prepared Appearance
pyocyanin( blue) or pyoverdin (Yellow) or both, as well as pyorubrin(red), Light yellow coloured, clear to slightly opalescent gel.
Cultural Response
pyomelanin(Brown), or various combinations of these pigments. Pseudomonas
Cultural characteristics after 18-24 hours at 35-370C.
Agar (for Pyocyanin) is a modification of formulation described by King et al., This
Organisms (ATCC) Growth Colour of RGI
medium is recommended by USP and IP for use in Microbial Limit Tests. medium
Principle Pseudomonas aeruginosa Good Blue to blue -green More than 70%
Peptic digest of animal tissue provide nutrients, amino acids and trace elements (27853)
for growth. Magnesium chloride and Potassium sulfate enhance pyocyanin For growth RGI should be more than 70%
production. Glycerin as an energy source also increases pyocyanin production. RGI- Relative Growth Index
Agar is the solidifying agent. Procedure
Formula* 1. Obtain a pure culture of the organism to be tested.
Ingredients in grams per liter 2. Inoculate and incubate plates aerobically at 35-370C for 18-48 hours.
Peptic Digest of Animal Tissue 20.0
3. Examine for growth and pigment production.
Anhydrous Magnesium Chloride 1.4
Interpretation of Results
Anhydrous Potassium Sulfate 10.0
Agar 15.0 1. Presence of pyocyanin is appear with a blue to blue green pigment in the
Final pH (at 250C) 7.0 ± 0.2 colonies and surrounding medium.
* Formula adjusted to suit performance parameters 2. Confirm the presence of pyocyanin by adding several drops of chloroform
Directions
and observe for a blue colour in chloroform.
1. Suspend the 46.4 gms of powder in 990 ml distilled water. Storage
2. Add10 ml of glycerin and mix thoroughly. Store at 22-300C and prepared medium at 2-80C.
3. Boil with frequent agitation to dissolve the powder completely. Shelf Life
0
4. Sterilize by autoclaving at 121 C (15 lbs pressure) for 15 minutes. Use before expiry date as mentioned on the label.
Quality Control
Dehydrated Appearance
Light yellow coloured, homogeneous, free flowing powder.

Pseudomonas Agar (for Pyocyanin) IP AM108415/AM508415

Pseudomonas Agar (for Pyocyanin) USP AM108416/AM508416
Use Principle
Pseudomonas Agar (for Pyocyanin) is used for the detection of Pseudomonas Peptic digest of gelatin provide nutrients, amino acids and trace elements for
species using pyocyanin pigment production. growth. Magnesium chloride and Potassium sulfate enhance pyocyanin
Summary production. Glycerin as an energy source also increases pyocyanin production.
Pseudomonas species may produce water-soluble pigments in culture media. Agar is the solidifying agent.
This property is sometimes used as a characteristic for the taxonomic Formula*
classification of different species of Pseudomonas. Most strains of P. aeruginosa Ingredients in grams per liter
Pancreatic Digest of gelatin 20.0
produce pyocyanin( blue) or pyoverdin (Yellow) or both, as well as
Anhydrous Magnesium Chloride 1.4
pyorubrin(red), pyomelanin(Brown), or various combinations of these pigments.
Anhydrous Potassium Sulfate 10.0
Pseudomonas Agar (for Pyocyanin) is a modification of formulation described by Agar 15.0
King et al. This medium is recommended by USP and IP for use in Microbial Limit Final pH (at 250C) 7.2 ± 0.2
Tests. * Formula adjusted to suit performance parameters

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Directions Procedure
1. Suspend the 46.4 gms of powder in 990 ml distilled water. 1. Obtain a pure culture of the organism to be tested.
2. Add10 ml of glycerin and mix thoroughly. 2. Inoculate and incubate plates aerobically at 35-370C for 18-48 hours.
3. Boil with frequent agitation to dissolve the powder completely. 3. Examine for growth and pigment production.
0
4. Sterilize by autoclaving at 121 C (15 lbs pressure) for 15 minutes. Interpretation of Results
Quality Control 1. Presence of pyocyanin is appear with a blue to blue green pigment in the
Dehydrated Appearance colonies and surrounding medium.
Light yellow coloured, homogeneous, free flowing powder.
2. Confirm the presence of pyocyanin by adding several drops of chloroform
Prepared Appearance
Light yellow coloured, clear to slightly opalescent gel. and observe for a blue colour in chloroform.
Cultural Response Storage
Cultural characteristics after 18-24 hours at 35-370C. Store at 22-300C and prepared medium at 2-80C.
Organisms (ATCC) Growth Colour of RGI Shelf Life
medium Use before expiry date as mentioned on the label.
Pseudomonas aeruginosa Good Blue to More than 70%
(27853) blue -green
For growth RGI should be more than 70%
RGI- Relative Growth Index

Pseudomonas Isolation Agar AM108417/AM508417

Use Directions
Pseudomonas Isolation Agar Base is recommended for selective isolation and 1. Suspend 45.03 gms of the powder in 1000ml distilled water. Containing
identification of Pseudomonas aeruginosa from clinical and non-clinical 20 ml of glycerol.
specimens.
2. Mix thoroughly.
Summary
3. Boil with frequent agitation to dissolve the powder completely. DO NOT
Pseudomonas aeruginosa is a classic opportunist pathogen and causes
OVERHEAT.
nosocomial infection. Pseudomonas Isolation Agar is formulated according to a
slight modification of the medium A formulation of King, Ward and Raney (55). 4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Quality Control
Pseudomonas Isolation Agar incorporates triclosan, a potent broad-spectrum
Dehydrated Appearance
antimicrobial that makes the medium selective for Pseudomonas aeruginosa.
Light yellow coloured, homogeneous, free flowing powder.
Principle
Prepared Appearance
Peptic digest of animal tissue provides the carbon and nitrogen. Magnesium Yellow coloured, slightly opalescent gel forms in petriplates.
chloride and potassium sulfate support the bacterial growth. Triclosan is an Cultural Response
antimicrobial agent selectively inhibits gram positive and gram negative bacteria Cultural characteristics after 18-48 hours at 35-37°C.
other than Pseudomonas species. Organisms Growth Colour of RGI
Formula* (ATCC) medium
Ingredients in grams per liter Pseudomonas Luxuriant green More than 70%
Peptic digest of animal tissue 20.0 aeruginosa (10145)
Magnesium chloride 1.40 Pseudomonas Luxuriant blue – blue-green More than 70%
Potassium sulphate 10.0 aeruginosa (27853)
Triclosan(Irgasan) 0.025 Proteus mirabilis Inhibited - 0%
Agar 13.6 (25933)
Final pH (at 250C) 7.0 ± 0.2 Escherichia Inhibited - 0%
* Formula adjusted to suit performance parameters coli (25922)

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For growth RGI should be more than 70% and turn blue-green as incubation continues up to 24-48hours, with diffusion of
For Inhibition RGI should be 0% the pigment into the medium.
RGI- Relative Growth Index
Limitations
Procedure
Some strains of Pseudomonas aeruginosa may fail to produce pyocyanin.
Inoculate the medium using streak plate method to obtain isolated colonies.
Storage
Incubate for 18-48 hours at 35 ± 2°C.
Store at 22-300C and prepared medium at 2-80C.
Interpretation of Results
Shelf Life
Examine the plates for the presence of Pseudomonas aeruginosa colonies.
Use before expiry date as mentioned on the label.
Pseudomonas aeruginosa colonies may be greenish after incubation for 18 hours

Pseudomonas Asparagine Broth AM108418/AM508418

Use Magnesium sulphate 0.50
Pseudomonas Asparagine Broth used for presumptive determination of Final pH (at 25°C) 7.0±0.2
* Formula adjusted to suit performance parameters
Pseudomonas aeruginosa from water samples.
Directions
Summary
1. Suspend 4.5gms of the powder in 1000ml-distilled water and mix
Pseudomonas Asparagine Broth is formulated as recommended by APHA (36.1)
thoroughly.
for presumptive detection of Pseudomonas aeruginosa from recreational or
natural waters. Recreational water like from swimming pool is a body of water in 2. Boil with frequent agitation to dissolve the powder completely.
a holding structure. Microorganisms of concern are those causing infection of ear, 3. Pour into adequate containers.
skin and upper respiratory tract etc. Pseudomonas aeruginosa is one of those 4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
organisms which account for a large percentage of swimming pool associated Quality Control
illness. Dehydrated Appearance
Principle White coloured, homogeneous, free flowing powder.
This medium is a relatively simple medium containg an amino acid DL- Prepared Appearance
Colourless clear solution with slight precipitate.
Asparagine and two salts like dipotassium phosphate and magnesium sulphate.
Cultural Response
Asparagine is the amino acid source while phosphate and sulphate provide the
Cultural characteristics after 20-24 hours at 35°C.
ions for the growth of pseudomonas aeruginosa. Dipotassium phosphate also Organisms(ATCC) Growth
helps in maintaining the buffering condition of the medium. This medium is only Pseudomonas aeruginosa (27853) Luxuriant
a presumptive medium for Pseudomonas aeruginosa, and further confirmatory Storage
tests are necessary for the identification. Store at 22-300C and prepared medium at 2-80C.
Formula* Shelf Life
Ingredients in grams per liter
Use before expiry date as mentioned on the label.
DL- Asparagine 3.0
Dipotassium phosphate 1.0

Purple Broth Base AM508419

Use recommended by FDA (2.1) for fermentation studies of sugars.
Purple Broth Base is recommended for the preparation of carbohydrate media Principle
used in fermentation studies for the culture identification of pure culture of enteric Peptone special supply the essential nutrients especially nitrogenous to the
and other microorganisms. growing organisms. Sodium chloride maintains the osmotic balance to the
Summary medium. Bromo cresol purple is the pH indicator which turns yellow at acidic pH.
Purple media is originally formulated by Vera (115.4). These media are

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Gas production is evident by its collection in Durham's tube. The acid produced Prepared Appearance
Purple coloured clear solution form in tubes.
during the fermentation of carbohydrate causes bromo cresol purple, the pH
Cultural Response
indicator to turn yellow.
Cultural characteristics after 18-48 hours at 35-37°C.
Formula* Organisms Growth Without With 1%
Ingredients in grams per liter (ATCC) Carbohydrate Carbohydrate
Peptone, special 10.00 Acid Gas Acid Gas
Sodium chloride 5.00 Neisseria meningitidis Good- luxuriant – – + –
Bromo cresol purple 0.02 (13090)
Final pH (at 25°C) 6.8±0.2 Esherichia coli Luxuriant – – + +
* Formula adjusted to suit performance parameters (25922)
Directions Staphylococcus Luxuriant – – + –
aureus (25923)
1. Suspend 15.02 gms of the powder in 1000ml-distilled water.
Listeria Luxuriant – – + –
2. Add 5-10 grams of the carbohydrate to be tested.
monocytogenes*
3. Boil to dissolve the powder completely. (19112)
4. Dispense in tubes as desired and sterilize by autoclaving at 121°C (15 lbs Key: Acid + = Yellow colur
pressure) for 15 minutes. * = Fermentative metabolism
Storage
5. Alternatively sterilize the basal medium prepared using 900 ml distilled
Store at 22-300C and prepared medium at 2-80C.
water and add 100 ml separately sterilized 5-10% solution of the desired
Shelf Life
carbohydrate to it.
Quality Control Use before expiry date as mentioned on the label.
Dehydrated Appearance
Greenish yellow coloured, homogeneous, free flowing powder.

R-2A Agar AM10841/AM50841

R-2A Agar (Agar Medium S) EP AM50842
Use R2A is useful in heterotrophic plate count analyses and for subculture of bacteria
R-2A Agar is used for obtaining heterotrophic plate count from treated potable isolated from potable water samples. It is used for the recovery of stressed and
water. chlorine-tolerant bacteria from drinking water. It is recommended by APHA for
Summary enumeration of heterotrophic bacteria in water and wastewater (17.1 & 32.1).
Reasoner and Geldreich (90.3) developed R2A medium to check the bacterial Principle
count in treated potable water. They found that plate count agar does not permit Since media contains low concentration of nutrients, it allows the growth of slow
the growth of many bacteria that may be present in treated potable water growing bacteria without suppressed by fast growing bacteria. Yeast extract
supplies. Results from parallel studies with spread, membrane filter, and pour provides a source of trace elements and vitamins. Proteose peptone provides
plate procedures showed that R2A medium yielded significantly higher bacterial nitrogen, vitamins, amino acids, carbon and minerals. Dextrose serves as a
counts than did plate count agar. carbon source. Soluble starch aids in the recovery of injured organisms by
Low nutritional content, longer incubation time, yielded higher counts and absorbing toxic metabolic by-products. Sodium pyruvate increases the recovery
increased detection of heterotrophic bacteria. As a tool to monitor heterotrophic of stressed cells. Potassium phosphate is used to balance the pH and provide
bacterial populations in water treatment processes and in treated distribution phosphate. Magnesium sulfate is a source of divalent cations and sulfate. Agar is
water, R2A spread or membrane filter plates incubated at 28°C for 5 to 7 days is the solidifying agent.
recommended. These conditions provide adequate time for growth of slow- Formula*
growing bacteria. Ingredients in grams per liter
Casein hydrolysate 0.50

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Proteose peptone 0.50 Procedure
Yeast extract 0.50 1. Water samples should be collected as described in Standard Methods for the-
Glucose 0.5
Soluble starch 0.5
Examination of Water and Wastewater, Section 9060A. Initiate analysis as
Sodium pyruvate . 0.3 soon as possible after collection to minimize changes in bacterial population.
Dipotassium hydrogen phosphate 0.3 The recommended maximum elapsed time between collection and analysis
Magnesium sulfate, anhydrous 0.024 of samples is 8h (maximum transit time 6h, maximum processing time
Agar 15.00 2h).when analysis cannot begin within 8h, maintain sample at a
Final pH: 7.2 ± 0.2 at 25°C emperature below 40C but do not freeze. Maximum elapsed time between
* Formula adjusted to suit performance parameters
collection and analysis must not exceed 24h.
Directions
2. Prepare test dilutions for heterotrophic plate count.
1. Suspend 18.12 gms of the powder in 1000 ml distilled water
3. Plate the test sample and dilutions by the spread plate, pour plate or
2. Mix thoroughly.
membrane filter method. Do not exceed 1 mL of sample or dilution per spread
3. Heat with frequent agitation and boil for 1 minute to dissolve the powder or pour plate. The volume of test sample to be filtered for the membrane filter
completely. technique will vary.
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes. 4. Maintain proper humidity during prolonged incubation.
5. Cool the medium to approximately 45-500C, pour in to sterile petriplates. Interpretation of Results
Quality Control
Count colonies promptly on spread or pour plates showing 30-300 colonies per
Dehydrated Appearance
Light yellow coloured, homogeneous, free flowing powder. plate using the membrane filter method Compute bacterial count per milliliter by
Prepared Appearance the following equation:
Light amber, slightly opalescent gel forms in petri plates. Colonies counted
Cultural Response
CFU/mL =___________________________
Cultural characteristics after 5-7 days at 350C.
Organisms Growth RGI Actual volume of sample in dish, mL
Candia albicans (10231) Good to luxuriant More than 70% Limitations
Enterococcus faecalis Good to luxuriant More than 70% 1. Fast growing bacteria may produce smaller size colonies on R2A agar than on
(29212)
nutritionally rich media.
Escherichia coli Good to luxuriant More than 70%
(25922) 2. Pour plates do not give satisfactory results.
S. serotype Enteritidis Good to luxuriant More than 70% Storage
(13076) Store at 22-300C and prepared medium at 2-80C.
S. serotype Typhi Good to luxuriant More than 70% Shelf Life
For growth RGI should be more than 70%
Use before expiry date as mentioned on the label.
RGI- Relative Growth Index
(6539)

R-2A Agar(Agar Medium S) BP AM50843

Use the growth of many bacteria that may be present in treated potable water
R-2A Agar is used for obtaining heterotrophic plate count from treated potable supplies. Results from parallel studies with spread, membrane filter, and pour
water in compliance with BP. plate procedures showed that R2A medium yielded significantly higher bacterial
Summary counts than did plate count agar.
Reasoner and Geldreich (90.3) developed R2A medium to check the bacterial Low nutritional content, longer incubation time, yielded higher counts and
count in treated potable water. They found that plate count agar does not permit increased detection of heterotrophic bacteria. As a tool to monitor heterotrophic

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bacterial populations in water treatment processes and in treated distribution Cultural Response
water, R2A spread or membrane filter plates incubated at 28°C for 5 to 7 days is Cultural characteristics after 5-7 days at 350C.
Organisms Growth RGI
recommended. These conditions provide adequate time for growth of slow-
Candia albicans (10231) Good to luxuriant More than 70%
growing bacteria.
Enterococcus faecalis (29212) Good to luxuriant More than 70%
R2A is useful in heterotrophic plate count analyses and for subculture of bacteria Escherichia coli (25922) Good to luxuriant More than 70%
isolated from potable water samples. It is used for the recovery of stressed and S. serotype Enteritidis (13076) Good to luxuriant More than 70%
chlorine-tolerant bacteria from drinking water. It is recommended by APHA for S. serotype Typhi (6539) Good to luxuriant More than 70%
enumeration of heterotrophic bacteria in water and wastewater (17.1 & 32.1). For growth RGI should be more than 70%
Principle RGI- Relative Growth Index
Procedure
Since media contains low concentration of nutrients, it allows the growth of slow
growing bacteria without suppressed by fast growing bacteria. Yeast extract 1. Water samples should be collected as described in Standard Methods for
provides a source of trace elements and vitamins. Tryptone and peptone provides the-Examination of Water and Wastewater, Section 9060A. Initiate
nitrogen, vitamins, amino acids, carbon and minerals. Dextrose serves as a analysis as soon as possible after collection to minimize changes in bacterial
carbon source. Soluble starch aids in the recovery of injured organisms by population. The recommended maximum elapsed time between collection
absorbing toxic metabolic by-products. Sodium pyruvate increases the recovery of and analysis of samples is 8h (maximum transit time 6h, maximum
stressed cells. Potassium phosphate is used to balance the pH and provide processing time 2h).when analysis cannot begin within 8h, maintain
phosphate. Magnesium sulfate is a source of divalent cations and sulfate. Agar is sample at a temperature below 40C but do not freeze. Maximum elapsed
the solidifying agent. time between collection and analysis must not exceed 24h.
Formula* 2. Prepare test dilutions for heterotrophic plate count.
Ingredients in grams per liter 3. Plate the test sample and dilutions by the spread plate, pour plate or
Casein hydrolysate 0.50 membrane filter method. Do not exceed 1 mL of sample or dilution per
Proteose peptone 0.50
spread or pour plate. The volume of test sample to be filtered for the
Yeast extract 0.50
membrane filter technique will vary.
Glucose 0.5
Soluble starch 0.5 4. Maintain proper humidity during prolonged incubation.
Sodium pyruvate . 0.3 Interpretation of Results
Dipotassium hydrogen phosphate 0.3 Count colonies promptly on spread or pour plates showing 30-300 colonies per
Magnesium sulfate, anhydrous 0.024 plate using the membrane filter method Compute bacterial count per milliliter by
Agar 15.00
the following equation:
Final pH: 7.2 ± 0.2 at 25°C
* Formula adjusted to suit performance parameters Colonies counted
Directions CFU/mL =___________________________
1. Suspend 18.12 gms of the powder in 1000 ml distilled water Actual volume of sample in dish, mL
2. Mix thoroughly. Limitations

3. Heat with frequent agitation and boil for 1 minute to dissolve the powder 1. Fast growing bacteria may produce smaller size colonies on R2A agar than
completely. on nutritionally rich media.
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes. 2. Pour plates do not give satisfactory results.
0 Storage
5. Cool the medium to approximately 45-50 C, pour in to sterile petriplates.
Quality Control Store at 22-300C and prepared medium at 2-80C.
Dehydrated Appearance Shelf Life
Light yellow coloured, homogeneous, free flowing powder. Use before expiry date as mentioned on the label.
Prepared Appearance
Light yellow coloured, slightly opalescent gel forms in petri plates.

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Raka-Ray Agar, Base AM10844

(Lactic Acid Bacteria Selective Agar, Base)
Use Glucose 5.0
Raka-Ray Agar, Base medium used for the isolation of lactic acid bacteria in beer Betaine hydrochloride 2.0
Diammonium hydrogen-citrate 2.0
and brewing processes.
Potassium aspartate 2.5
Summary
Potassium glutamate 2.5
Raka-Ray Agar, was developed of Saha, Sondag and Middlekauff for the Magnesium sulphate 2.0
detection of lactic acid bacteria in beer and brewing processes. The European Manganese sulphate 0.66
Brewing Convention (EBC) and the American Society of Brewing Chemists (ASBC) Monopotassium phosphate 2.0
recommend using this Agar. Diverse members of the family of Lactobacillaceae N-acetyl glucosamine 0.5
are important spoilage organisms in the brewing process. This medium was Agar 17.0
optimized to meet the natural requirements of the Lactobacillaceae. The original Final pH: 5.4± 0.2 at 25°C
* Formula adjusted to suit performance parameters
Medium Raka-Ray Medium No. 3 was developed to enable brewers to monitor
Directions
in-process beer quickly and accurately for a wide range of organisms including
pediococci. Originally diverse combinations of stimulating agents were added to 1. Suspend 77.1 g in 1000 ml of distilled water.
Universal Beer Agar to get an optimized media with improved colony size, colony 2. Bring to the boil and dissolve the medium completely.
numbers and incubation time. This was then the base for Raka-Ray Medium No. 3. Distribute into tubes or bottles and sterilize by autoclaving at 121°C for 15
3. Van Keer et al., found that Raka-Ray Medium No. 3 yielded the highest colony minutes.
count of divers media and allowed the enumeration of the greatest number of 4. Cool to 50-55°C and add contents of 1vial of lactic acid bacteria selective
strains. 30 strains of Lactobacillus have been taken from different origins and supplement (AS0151).
was incubated 48 hours under semi-anaerobic conditions.
5. Mix well and pour into sterile petri dishes or dispense as desired.
Principle
Quality Control
Casein enzymic hydrolysate, liver concentrate, yeast extract provide carbon, Dehydrated Appearance
nitrogen, amino acids, minerals, vitamins, trace elements and other essential Light yellow coloured, homogeneous, free flowing powder.
nutrients for growth. Potassium aspartate and potassium glutamate are Prepared Appearance
additional sources of amino acids. Maltose is to detect lactobacilli, which cannot Light yellow coloured, clear to slightly opalescent gel.
utilise glucose as a carbon source and fructose is the carbon source of Cultural Response
Lactobacillus fructivorans. Glucose is needed as the fermentative energy source Cultural characteristics after 18-24 hours at 35-37°C
for the pediococci. Sorbitan mono-oleate act as a stimulant for lactic acid Organisms Growth RGI
Leuconostoc mesenteroides (8293) Luxuriant More than 70%
bacteria in general. Also N-acetyl glucosamine, yeast extract and betaine
Lactobacillus acidophilus (11506) Luxuriant More than 70%
hydrochloride are used as growth stimulating agents. Diammonium hydrogen-
Lactobacillus bulgaricus (11842) Luxuriant More than 70%
citrate and Monopotassium phosphate are the buffering agents while Lactobacillus casei (7469) Luxuriant More than 70%
Magnesium and Manganese are important trace elements for Lactobacillaceae. Lactobacillus leichmannii (7830) Luxuriant More than 70%
The addition of 3 g/l of phenylethanol inhibits Gram-negative organisms, 5 Lactobacillus plantarum (8014) Luxuriant More than 70%
mg/l amphotericin B and 7mg/l of cycloheximide to inhibit yeasts and moulds. Lactobacillus fermentans (9338) Luxuriant More than 70%
Formula* Escherichia coli (25922) None-Poor 0%
Ingredients in grams per liter Sacch. cerevisae (9763) Inhibited 0%
Yeast extract 5.0 For growth RGI should be more than 70%
Casein enzymic hydrolysate 20.0 For Inhibition RGI should be 0%
Liver concentrate 1.0 RGI- Relative Growth Index
Maltose 10.0 Incubation:
Fructose 5.0 An incubation period of 4 days is generally sufficient. The test strains needs no

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longer than 24 hours incubation but slower growing organisms may require up to Storage
7 days. Depending on the species of lactic acid bacteria a semi-anaerobic Store at 22-300C and prepared medium at 2-80C.
atmosphere may be recommendable. Shelf Life
Use before expiry date as mentioned on the label.

Raka Ray No 3 Broth Base AM10845

(Lactic Acid Bacteria Selective Broth Base)
Use Liver concentrate 1.0
Raka Ray No 3 Broth Base is a selective medium for the isolation of lactic acid Maltose 10.0
Fructose 10.0
bacteria in beer and brewing processes.
Betaine hydrochloride 2.0
Summary
Diammonium citrate 2.0
Raka Ray No 3 Broth, was developed of Saha, Sondag and Middlekauff for the Potassium aspartate 2.5
detection of lactic acid bacteria in beer and brewing processes. Diverse members Potassium glutamate 2.5
of the family of Lactobacillaceae are important spoilage organisms in the Magnesium sulfate heptahydrate 0.98
brewing process. This medium was optimised to meet the natural requirements of Manganese sulfate monohydrate 0.42
the Lactobacillaceae. This formulation was developed to enable brewers to Dipotassium hydrogen phosphate 2.0
monitor in-process beer quickly and accurately for a wide range of organisms N-Acetylglucosamine 0.5
Final pH: 5.4 ± 0.2 at 37°C
including pediococci. Originally diverse combinations of stimulating agents were
* Formula adjusted to suit performance parameters
added to Universal Beer Agar to get an optimized media with improved colony
Directions
size, colony numbers and incubation time. This was then the base for Raka-Ray
1. Dissolve 58.9 gms. in 1000 ml distilled water and add 2 ml Tween 80.
Broth No. 3. Van Keer et al., found that Raka-Ray Broth No. 3 yielded the highest
colony count of divers media and allowed the enumeration of the greatest 2. Bring to the boil and dissolve the medium completely.
number of strains. 30 strains of Lactobacillus have been taken from different 3. Distribute into tubes and sterilize by autoclaving at 121°C for 15 minutes.
origins and was incubated 48 hours under semi-anaerobic conditions. 4. Cool to 50-55°C and add contents of 1vial of lactic acid bacteria selective
Principle supplement (AS0151).
Casein peptone, yeast extract provide carbon, nitrogen, amino acids, minerals, 5. Mix well and pour into sterile petri dishes or dispense as desired.
vitamins, trace Quality Control
elements and other essential nutrients for growth. Potassium aspartate and Dehydrated Appearance
potassium glutamate are additional sources of amino acids. Maltose is to detect Beige coloured, homogeneous, free flowing powder.
Prepared Appearance
lactobacilli, which cannot utilise glucose as a carbon source and fructose is the
Brown-yellow coloured, clear solution.
carbon source of Lactobacillus fructivorans. N-acetyl glucosamine, liver
Cultural Response
concentrate yeast extract and betaine hydrochloride are used as growth Cultural characteristics after 18-24 hours at 27-30°C under anaerobic
stimulating agents. Diammonium citrate and potassium hydrogen phosphate atmosphere.
are buffering agents while Magnesium and Manganese are important trace Organisms Growth
elements for Lactobacillaceae. Tween 80 acts as a fatty acid source. The addition Escherichia coli (25922) -/+
of 3 g/l of phenylethanol inhibits Gramnegative organisms, 5 mg/l amphotericin Lactobacillus brevis (367) +++
Lactobacillus buchneri (11307) +++
B and 7mg/l of cycloheximide to inhibit yeasts and moulds.
Storage
Formula*
Ingredients in grams per liter Store at 22-300C and prepared medium at 2-80C.
Yeast extract 5.0 Shelf Life
Casein peptone 20.0 Use before expiry date as mentioned on the label.

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Rapid Accu Coliform Broth AM1084511/AM5084511
Use Final pH (at 250C) 6.8± 0.2
Rapid Accu Coliform Broth is used for detection and confirmation of Escherichia * Formula adjusted to suit performance parameters
Directions
coli and total coliforms on the basis of enzyme substrate reaction from water
samples, using a combination of chromogenic and fluorogenic substrate. 1. Suspend 16gms of the powder in 1000ml distilled water and mix
Summary thoroughly.
Rapid Accu Coliform Broth is the modification of LMX Broth described by Manafi 2. Boil with frequent agitation to dissolve the powder completely.
and Kneifel (78.3). Rapid Accu Coliform Broth is recommended for the 3. Pour into adequate containers.
simultaneous detection of total coliforms and Escherichia coli. 4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Principle Quality Control
Peptone, special provides the essential nutrient for the growth of the Dehydrated Appearance
microorganisms and it is rich in tryptophan, useful for the simultaneous indole Light yellow coloured, homogeneous, free flowing powder.
production. Sorbitol is the source of carbon. Phosphate salts acts as buffering Prepared Appearance
agent while sodium chloride maintains the osmotic balance. Sodium lauryl Light yellow coloured, clear solution forms in tubes.
Cultural Response
sulphate makes the medium selective by inhibiting the Gram - positive
Cultural characteristics after 18-24 hours at 35-37ºC.
microorganisms. The fluorogenic substrate is split by enzyme â-D-
Organisms Colour change Fluorescence* Indole
glucuronidase, which is specifically found in Escherichia coli. The reaction is in medium reaction
indicated by a blue fluorescence under UV light. The presence of total coliforms is Enterobacter aerogenes Blue-green - -
indicated by a blue-green colour of the broth due to the cleavages of the (13048)
chromogenic substrate. IPTG amplifies enzyme synthesis and increases the Escherichia coli Blue-green + +
activity of â-D-galactosidase. To confirm the presence of Escherichia coli in (25922)
broth medium by indole reaction overlay the medium with Kovac's reagent. The Key: + = Positive reaction
layers turn red within 2 minutes in case of positive reaction. - = negative reaction
* = Fluorescence as under UV light.
Formula*
Procedure
Ingredients in grams per liter
Chromogenic substrate 0.08 Refer to appropriate references for specific procedures.
Dipotassium hydrogen phosphate 2.70 Interpretation of Results
Fluorogenic substrate 0.05 Refer to appropriate references and procedures for results.
IPTG (Isopropyl-b-D-thiogalactopyranoside) 0.10 Storage
Peptone, Special 5.0
Store below 80C and prepared medium at 2-80C.
Potassium dihydrogen phosphate 2.0
Shelf Life
Sodium chloride 5.0
Sodium lauryl sulphate 0.10 Use before expiry date as mentioned on the label.
Sorbitol 1.0

Rappaport Vassiliadis Salmonella Enrichment Broth AMH508453

(Harmonized)
Use Summary
Rappaport Vassiliadis Salmonella Enrichment Broth is recommended for Rappaport et al., formulated an enrichment medium for Salmonella that was
selective enrichment of Salmonella species under conditions of high osmotic modified by Vassiliadis et al., Rappaport Vassiliadis (90.1 & 115.3) Salmonella
pressure and low pH with modest nutritional requirements. Enrichment Broth is a selective enrichment for Salmonella species. This medium

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is selective for Salmonella species because they are typically resistant to Prepared Appearance
malachite green, high osmotic pressure and low pH. S. typhi and S. choleraesuis Blue coloured clear solution without any precipitate
Cultural Response
are sensitive to malachite green and may be inhibited. This medium is also
Cultural characteristics observed after an incubation of 18-24 hours at 30-35°C
recommended by United States of Pharmacopeia. and subcultured on XLD Agar, incubated at 30-350C for 18-48 hours..
Principle Organisms Growth Colour RGI
Soya peptone provides the essential nutrients for the growth of the bacteria. of colony
Phosphate salts act as buffer to maintain the pH. Magnesium chloride maintains Salmonella typhimurium Luxuriant Red colonies More than 70%
the high osmotic pressure and Salmonella generally survive at little high osmotic (23564) with black center
pressure. Malachite green inhibits other microorganisms other than Salmonella. Salmonella typhi Luxuriant Red colonies with More than 70%
(6539) black center
Formula*
Escherichia coli Fair yellow 0%
Ingredients in grams per liter
(25922)
Soya peptone 4.5
Salmonella abony Luxuriant Red colonies More than70%
Magnesium chloride hexahydrate 29.0
NCTC (6017) with black center
Sodium chloride 8.0
Salmonella enterica Luxuriant Red colonies More than 70%
Dipotassium phosphate 0.4
(13076) with black center
Potassium dihydrogen phosphate 0.6
Salmonella typhi Luxuriant Red colonies More than 70%
Malachite green 0.036
NCTC (786) with black center
Final pH (at 25ºC) 5.2±0.2
Staphylococcus aureus Inhibited - 0%
* Formula adjusted to suit performance parameters
(6538)
Directions
For growth RGI should be more than 70%
1. Suspend 42.54 gms of the powder in 1000ml distilled water and mix For Inhibition RGI should be 0%
thoroughly. RGI- Relative Growth Index
2. Boil with frequent agitation to dissolve the powder completely. Storage

3. Dispense in tubes as desired. Store at 22-300C and prepared medium at 2-80C.

Shelf Life
4. Sterilize by autoclaving at 115°C (10 lbs pressure) for 15 minutes.
Quality Control Use before expiry date as mentioned on the label.
Dehydrated Appearance
Pale blue coloured, homogeneous, free flowing powder.

Reddy's Differential Agar, Modified AM50846

(Lactic Streak Agar)
Use hydrolysis and citrate utilization. Lactose fermenters produce acid and are seen as
Reddy's Differential Agar, Modified is recommended for qualitative and yellow colonies. Lactococcus lactis initially produces acid but later on turns to
quantitative differentiation of lactic Streptococci. violet – purple subspecies diacetylactis produces a more intense purple colour
Summary than Lactococcus lactis.
Reddy's Differential Agar, Modified was originally described by Reddy et al., Citrate utilization is seen as clear zone around the colony. For quantitative
(91.1) and further modified by Mullan and Walker (82.3) and recommended by determination, decimal dilution of cultures are prepared and spread on agar
APHA (115.1) for the differentiation of lactic Streptococci. This modification gives plates. After incubation at 36 to 40 hours at 320C yellow colonies of subspecies
faster results and there is no need of incubation in CO2 enrichment environment. cremoris are counted. The plates are further incubated for 4 days and then total
Principle count is taken as well as colonies with clearing zones of subspecies diacetylactis
Lactococcus lactis and its subspecies cremoris and diacetylactis are used as are counted and subtracted from total count to get Lactococcus lactis population
starter cultures in dairy products. They are differentiated on the basis of arginine in the mixture.

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Formula* Part B: Yellow coloured, homogeneous, free flowing powder.
Ingredients in grams per liter Prepared Appearance
Part A: Sodium carboxymethyl cellulose 10.00 Light yellow coloured, opalescent gel having greenish tinge forms in petri
Calcium citrate 10.00 plates.
Part B: Peptic digest of animal tissue 5.00 Cultural Response
Peptic digest of soyabean meal 5.00 Cultural characteristics after 4 hours at 32°C.
Yeast extract 5.00 Organisms Growth Colour Citrate RGI
of colony Utilization
Beef extract 5.00
Lactose 1.50 Lactococcus lactis Good-luxuriant Yellow – More than 70%
L-Arginine Hydrochloride 1.50 (8000)
Bromo cresol purple 0.002 Lactococcus lactis Good-luxuriant Purple – More than 70%
Agar 15.00 (19527)
Final pH: at 25°C6.0 ± 0.2 subsp. Cremoris
Lactococcus lactis Good-luxuriant Purple + More than 70%
* Formula adjusted to suit performance parameters
subsp. diacetylactis
Directions
Key: + = Positive, clearing zone around colony
1. Suspend 38 gms of Part B in 800 ml distilled water. – = Negative
2. Heat to boiling to dissolve the powder completely. For growth RGI should be more than 70%
RGI- Relative Growth Index
3. Suspended 20 grams of Part A in 200 ml distilled water.
Storage
4. Mix Part A and Part B.
Store at 22-300C and prepared medium at 2-80C.
5. Sterilize by autoclaving at 115°C (10 lbs pressure) for 10 minutes. Shelf Life
Quality Control
Use before expiry date as mentioned on the label.
Dehydrated Appearance
Part A: Yellow coloured, homogeneous, free flowing powder.

Reinforced Clostridial Agar AM1085/AM5085

Use Formula*
Reinforced Clostridial Agar is used for the cultivation and enumeration of Ingredients in grams per liter
Peptone 10.00
clostridia and other anaerobes.
Yeast extract 3.00
Summary
Beef extract 10.00
Reinforced Medium For Clostridia is formulated by Hirsch and Grinsted (43.2). It Glucose monohydrate 5.00
can be used to initiate growth from small inocula and to obtain the highest viable Sodium chloride 5.00
count of Clostridia. Barnes and Ingrams used the broth medium for diluting an Sodium acetate 3.00
inoculum of vegetative cells of Clostridium perfringens (5). It can be used in Starch, soluble 1.00
studies of spore forming anaerobes, especially Clostridium butyrium in cheese, Cysteine hydrochloride 0.50
for enumeration of clostridia in tube dilution counts or for preparation of plates for Agar 0.50
Final pH: 6.8 ± 0.2 at 25°C*
isolation (71.1). Other spore forming anaerobes, streptococci and Lactobacilli
Formula adjusted to suit performance parameters
also grow in these media. These are enriched but non selective media.
Directions
Principle
1. Suspend 38 gms in 1000 ml distilled water.
Casein enzymic hydrolysate, yeast extract, beef extract, starch, L-cysteine and
sodium acetate provide all the necessary nutrients for the growth of Clostridia. 2. Boil to dissolve the medium completely.
Dextrose is a fermentable carbohydrate in the medium while sodium chloride 3. Sterilize by autoclaving at 115°C (10 lbs pressure) for 15 minutes.
maintain osmotic equilibrium. These media can be made selective by addition of Quality Control
Dehydrated Appearance
15-20 mg Polymyxin B per liter of media.
Light yellow coloured, homogeneous, free flowing powder.

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Prepared Appearance Clostridium butyricum (9690) Good to luxuriant
Light yellow coloured, Clear to slightly opalescent solution in tubes. Clostridium perfringens (13124) Good to luxuriant
Cultural Response Storage
Cultural characteristics after 40-48 hours at 35-37°C. Store at 22-300C and prepared medium at 2-80C.
Organisms (ATCC) Growth
Shelf Life
Bacteroides Fragilis (23745) Good to luxuriant
Bacteroides vulgatus (8482) Good to luxuriant
Use before expiry date as mentioned on the label.

Reinforced Clostridial Broth (Harmonized) AMH50851

Reinforced Clostridial Broth USP AM10851/AM50851
Reinforced Clostridial Broth (Medium P) EP AM50852
Reinforced Clostridial Broth (Medium P) BP AM50853
Use Soluble starch 1.0
Reinforced Clostridial Broth is used for the cultivation and enumeration of Cysteine hydrochloride 0.5
Agar 0.5
clostridia and other anaerobes.
Final pH (at 25ºC) 6.8 ± 0.2
Summary
Formula adjusted to suit performance parameters
Reinforced Clostridial Broth is based on the formulation of Hirsch and Grinsted Directions
(43.2). The medium supports growth of clostridia from small inocula and
1. Suspend 38.0 gms of the powder in 1000 ml distilled water.
produces higher viable cell counts. Barne's et al., used this medium to enumerate
2. Mix thoroughly.
clostridia in food. Attenborough and Scarr used this medium in conjunction with
membrane filters, for the count of C.thermosaccharolyticum in sugar. This is a 3. Boil with frequent agitation to dissolve the powder completely.
non-selective enriched medium, which allows growth of various anaerobic and 4. Sterilize by autoclaving at 121ºC (15 lbs pressure) for 15 minutes.
facultative bacteria when incubated anaerobically. Reinforced Clostridial Broth is Quality Control
recommended by APHA for the examination of milk . Reinforced Clostridial Broth Dehydrated Appearance
Light yellow coloured, free flowing, homogeneous powder.
EP/USP/BP is recommended for the cultivation and enumeration of clostridia and
Prepared Appearance
other anaerobes in compliance with EP/USP/BP.
Light yellow coloured, clear to slightly opalescent solution in tubes.
Principle
Cultural Response
Peptone and beef extract provide sources of nitrogen, carbon and other growth Cultural characteristics after 40-48 hours at 35-37ºC, in an anaerobic
factors. Yeast extract provides B complex vitamins while dextrose is the atmosphere.
carbohydrate source. In low concentrations, soluble starch detoxifies metabolic Organisms (ATCC) Growth
byproducts and Cysteine hydrochloride apart from being a nutrient acts as a Bacteroides fragilis (23745) Good to luxuriant
Bacteroides vulgatus (8482) Good to luxuriant
reducing agent. Sodium acetate is the buffering agent while sodium chloride
Clostridium perfringens (13124) Good to luxuriant
maintains the osmotic balance. This medium can be made selective by the
Procedure
addition of 15-20 mg of polymixin B per liter of medium.
Refer to appropriate references for specific procedures.
Formula*
Interpretation of Results
Ingredients in grams per liter
Beef extract 10.0 Refer to appropriate references and procedures for results.
Peptone 10.0 Precautions / Limitations
Sodium chloride 5.0 1. This is a non-selective medium. Other spore forming anaerobes, streptococci
Glucose monohydrate 5.0 and lactobacilli also grow on this medium.
Sodium acetate 3.0
Yeast extract 3.0
2. Biosafety Level 2 practices, containment equipment and facilities are

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recommended for activities with clinical specimens of human or animal Storage

origin containing C.botulinum, C.tetani or their toxins. Store at 22-300C and prepared medium at 2-80C
Shelf Life
3. Biosafety Level 3 practices, containment equipment and facilities are
recommended for all manipulations of cultures of these organisms and for Use before expiry date as mentioned on the label.
activities with a high potential for aerosol or droplet production.

Ringer Salt Solution Powder AM108531

Use Directions
Ringer Salt Solution Powder is a recommended isotonic diluent for food, milk and 1. Suspend 8.91 gms powder in 1000ml of distilled water.
dairy products during microbiological examinations. 2. Mix thoroughly.
Summary
3. Heat gently with frequent agitation to dissolve the powder completely.
Ringer Salt Solution is a balanced salt solution contains an array of inorganic
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
ionic species that are essential for critical functions when cells are removed from
Quality Control
their in vivo milieu. Dehydrated Appearance
Principle White coloured, homogeneous, free flowing powder.
Ringer Salt Solution Powder is used as isotonic solution. Different types of salts Prepared Appearance
maintain the osmotic balance and make the medium isotonic. These include Colourless clear solution without any precipitate.
sodium and potassium to regulate toxicity and permeability, calcium to maintain Cultural Response
the integrity of cell membranes and internal structures, and bicarbonate to control Satisfactory results are obtained when used as a diluent during bacteriological
examination of foods, dairy products as well as for serial dilutions of pure
the hydrogen ion concentration through their buffering effect. cultures of bacteria.
Formula* Procedure
Ingredients in grams per liter
Refer to appropriate references for specific procedures.
Sodium chloride 8.50
Interpretation of Results
Potassium chloride 0.20
Calcium chloride 0.20 Refer to appropriate references and procedures for results.
Sodium bicarbonate 0.01 Storage
Final pH (at 25°C) 7.0 ± 0.2 Store at 22-300C and prepared medium at 2-80C.
Formula adjusted to suit performance parameters Shelf Life
Use before expiry date as mentioned on the label.

Rogosa SL Agar AM50854

Use fatty acids. Ammonium citrate and sodium acetate inhibit moulds, Streptococci
Rogosa SL Agar is used for selective solid medium for cultivation of oral and faecal and many other organisms. Low pH of the medium selective for Lactobacilli
Lactobacilli. inhibiting other bacterial flora.
Summary Formula*
Rogosa SL media are the modification of the medium described by Rogosa et al. Ingredients in grams per liter
(95), and give excellent results when used in qualitative and quantitative studies Tryptose 10.0
Yeast Extract 5.0
of Lactobacilli in faeces, saline and in dairy products.
Dextrose 10.0
Principle
Arabinose 5.0
Tryptose, yeast extarct provides nitrogenous compounds, sulphur, trace elements Saccharose 5.0
and vitamin B complex, essential for growth of Lactobacilli . Dextrose, arabinose, Sodium acetate 15.0
saccharose are the fermentable carbohydrates. Polysorbate 80 is the source of Ammonium citrate 2.0

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Monopotassium phosphate 6.0 Prepared Appearance:
Magnesium sulphate 0.57 Light yellow coloured slightly opalescent gel forms in petri plates.
Manganese sulphate 0.12 Cultural Response
Ferrous sulphate 0.03 Cultural characteristics after 40-48 hours at 35-37°C, in 5% CO2 and 95% H2.
Polysorbate 80 1.00 Organisms (ATCC) Growth RGI
Agar 15.00 Lactobacillus casei (9595) Good to luxuriant More than 70%
Final pH (at 25ºC) 5.4 ± 0.2 Lactobacillus fermentum (9338) Good to luxuriant More than 70%
Formula adjusted to suit performance parameters Lactobacillus leichmanni (4797) Good to luxuriant More than 70%
Directions Lactobacillus plantarum (8014) Good to luxuriant More than 70%
1. Suspend 75 gms of the powder in 1000 ml distilled water. Staphylococcus aureus (25923) Inhibited 0%
For growth RGI should be more than 70%
2. Boil to dissolve the medium completely.
For Inhibition RGI should be 0%
3. Add 1.32 ml glacial acetic acid. Mix thoroughly. Heat to 90-100ºC for 2-3
RGI- Relative Growth Index.
minutes. Cool to 45ºC for direct inoculation. DO NOT AUTOCLAVE. Storage
Quality Control
Dehydrated Appearance
Store at 2- 80C and prepared medium at 2-80C.
Light yellow coloured, homogeneous, powder containing soft lumphs. Shelf Life
Use before expiry date as mentioned on the label.

Rogosa SL Broth AM50855

Use Ferrous sulphate 0.03
Rogosa SL Broth is used for selective medium for cultivation of oral and faecal Polysorbate 80 1.00
Final pH (at 25ºC) 5.4 ± 0.2
Lactobacilli.
Formula adjusted to suit performance parameters
Summary
Directions
Rogosa SL media are the modification of the medium described by Rogosa et al.
1. Suspend 60 gms of the powder in 1000 ml distilled water.
(95), and give excellent results when used in qualitative and quantitative studies
of Lactobacilli in faeces, saline and in dairy products. 2. Boil to dissolve the medium completely.
Principle 3. Add 1.32 ml glacial acetic acid. Mix thoroughly. Heat to 90-100ºC for 2-3
Casein enzyme hydrolysate, yeast extract provides nitrogenous compounds, minutes. Cool to 45ºC for direct inoculation. DO NOT AUTOCLAVE.
sulphur, trace elements and vitamin B complex, essential for growth of Quality Control
Dehydrated Appearance
Lactobacilli. Dextrose, arabinose, saccharose are the fermentable carbohydrates.
Light yellow coloured, homogeneous, powder containing soft lumphs.
Polysorbate 80 is the source of fatty acids. Ammonium citrate and sodium acetate
Prepared Appearance:
inhibit moulds, Streptococci and many other organisms. Low pH of the medium Light yellow coloured slightly opalescent solution forms in tubes.
selective for Lactobacilli inhibiting other bacterial flora. Cultural Response
Formula* Cultural characteristics after 40-48 hours at 35-37°C, in 5% CO2 and 95% H2.
Ingredients in grams per liter Organisms (ATCC) Growth
Casein enzyme hydrolysate 10.0 Lactobacillus casei (9595) Good to luxuriant
Yeast extract 5.0 Lactobacillus fermentum (9338) Good to luxuriant
Dextrose 10.0 Lactobacillus leichmanni (4797) Good to luxuriant
Arabinose 5.0 Lactobacillus plantarum (8014) Good to luxuriant
Saccharose 5.0 Staphylococcus aureus (25923) Inhibited
Sodium acetate 15.0 Storage
Ammonium citrate 2.0
Store at 2- 80C and prepared medium at 2-80C.
Monopotassium phosphate 6.0
Shelf Life
Magnesium sulphate 0.57
Manganese sulphate 0.12 Use before expiry date as mentioned on the label.

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Rose Bengal Agar Base AM50856
Use 3. Boil with frequent agitation to dissolve the powder completely. Do not
Rose Bengal Agar Base is recommended for selective isolation and enumeration overheat.
of yeasts and moulds from environmental materials and food stuffs. 4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Summary
5. Cool to 45°C and add 2 ml of rehydrated Chloramphenicol Selective
Yeasts and moulds can cause various degrees of food decomposition. Rose- Supplement (AS00911) for each 500ml of Rose Bengal Agar Base.
Bengal Chloramphenicol Agar is a selective medium for the enumeration of yeasts
6. Mix thoroughly and pour into sterile petri plates.
and moulds from a wide variety of foodstuffs. Rose Bengal Chloramphenicol Agar
Quality Control
was formulated originally by Jarvis (47.2) and further modified by Overcast and
Dehydrated Appearance
Weakly (85.2). Rose-Bengal is taken up by mould and yeast colonies thereby Pink coloured, homogeneous, free flowing powder.
assisting enumeration of small colonies. Prepared Appearance
Principle Deep pink coloured, clear to very slightly opalescent gel forms in petri plates.
Papaic digest of soyabean meal serves as an essential source of nutrients. Cultural Response
Dextrose is the fermentable carbohydrate. Monopotassium phosphate acts as a Cultural characteristics after 5 days at 20-25°C for fungi and 24-48 hrs at 35-
370C for bacteria.
buffering agents and magnesium is a trace element important for the growth of
Organisms (ATCC) Growth RGI
yeasts and moulds. Rose bengal dye suppresses the development of bacteria and Aspergillus niger (16404) Good More than 70%
reduces the excessive mycelial growth of moulds. Candida albicans (10231) Good More than 70%
Formula* Escherichia coli (25922) Inhibited 0%
Ingredients in grams per liter Micrococcus luteus (10240) Inhibited 0%
Papaic digest of soyabean meal 5.0 For growth RGI should be more than 70%
Dextrose 10.0 For Inhibition RGI should be 0%
Monopotassium phosphate 1.0 RGI- Relative Growth Index
Magnesium sulphate 0.50 Limitations
Rose Bengal 0.05 It is essential to store plates of media containing Rose-Bengal in the dark to
Agar 15.0 prevent toxic photo-oxidation of the dye.
Final pH (at 250C) 7.2 ± 0.2 Colonies of bacteria on this medium may be mistaken for those of yeasts and
Formula adjusted to suit performance parameters thus should be examined microscopically to confirm their identity.
Directions Storage

1. Suspend 31.55 gms of the powder in 1000ml distilled water. Store at 22-300C and prepared medium at 2-80C.
Shelf Life
2. Mix thoroughly.
Use before expiry date as mentioned on the label.

Rose Bengal Chloramphenicol Agar AM10857/AM50857

Use Principle
Rose Bengal Chloramphenicol Agar is used for selective isolation and Mycological peptone serves as an essential source of nutrients. Dextrose is the
enumeration of yeasts and moulds from environmental materials and foodstuffs. fermentable carbohydrate. Monopotassium phosphate acts as a buffering agents
Summary and magnesium is a trace element important for the growth of yeasts and moulds.
Yeasts and molds can cause various degrees of food decomposition (1.3). Rose- Rose bengal dye suppresses the development of bacteria and reduces the
Bengal Chloramphenicol Agar is a selective medium for the enumeration of yeasts excessive mycelial growth of moulds. Chloramphenicol has inhibitory action on
and moulds from a wide variety of foodstuffs. Rose Bengal Chloramphenicol Agar gram-negative bacteria.
was formulated originally by Jarvis (47.2) and further modified by Overcast and Formula*
Weakly (85.2). Rose-Bengal is taken up by mould and yeast colonies thereby Ingredients in grams per liter
Mycological peptone 5.0
assisting enumeration of small colonies.

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Dextrose 10.0 Mucor racemosus (42647) Luxuriant More than 70%
Monopotassium phosphate 1.0 Penicillium notatum (10108) Luxuriant More than 70%
Magnesium sulphate 0.50 Saccharomyces cerevisiae (9763) Luxuriant More than 70%
Rose Bengal 0.05 Enterococcus faecalis (29212) Inhibited 0%
Chloramphenicol 0.10 Escherichia coli (25922) Inhibited 0%
Agar 15.5 Bacillus subtilis (6633) Inhibited 0%
Final pH (at 250C) 7.2 ± 0.2 For growth RGI should be more than 70%
Formula adjusted to suit performance parameters For Inhibition RGI should be 0%
Directions RGI- Relative Growth Index
1. Suspend 32.15 gms of the powder in 1000ml distilled water. Procedure
1. Inoculate the agar plates directly using surface spreading technique with
2. Mix thoroughly. serial dilutions.
3. Boil with frequent agitation to dissolve the powder completely. Do not 2. Incubate the plates at 25°C for 5 days.
overheat. 3. Examine the results of the plates.
Interpretation of Results
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
The number of yeasts or moulds is calculated per 1 g or 1 ml of sample being
Quality Control tested by multiplying the number of colonies observed by the dilution factor.
Dehydrated Appearance Limitations
Pink coloured, homogeneous, free flowing powder. 1. It is essential to store plates of media containing Rose-Bengal in the dark to
Prepared Appearance prevent toxic photo- oxidation of the dye.
Deep pink coloured, clear to very slightly opalescent gel forms in petri plates. 2. Colonies of bacteria on this medium may be mistaken for those of yeasts
and thus should be examined microscopically to confirm their identity.
Cultural Response
Storage
Cultural characteristics after 5 days at 20-25°C for fungi and 24-48 hrs at 35-
370C for bacteria.. Store at 22-300C and prepared medium at 2-80C.
Organisms (ATCC) Growth RGI Shelf Life
Aspergillus niger (16404) Luxuriant More than 70% Use before expiry date as mentioned on the label.
Cladosporium cladosporoides (45534) Luxuriant More than 70%

Sabouraud Chloramphenicol Agar AM1086/AM5086

Sabouraud Chloramphenicol Agar IP AM50861
Sabouraud Chloramphenicol Agar EP AM50862
Sabouraud Chloramphenicol Agar BP AM50863
Use Principle
Sabouraud Chloramphenicol Agar is used for selective cultivation of yeasts and Tryptone and peptone provide nitrogenous compounds, carbon and other growth
moulds. factors. Dextrose is the carbohydrate source. The low pH of approximately 5.6 is
Summary favorable for the growth of fungi, especially dermatophytes and slightly
Sabouraud Chloramphenicol Agar is Carliers modification (12) of the formulation inhibitory to contaminating bacteria. Chloramphenicol inhibits a wide range of
described by Sabouraud (99) with the addition of chloramphenicol, for the gram-positive and gram-negative bacteria making the medium selective for
cultivation of fungi, particularly those associated with skin infections. It is used for fungi (7.1).
the isolation of pathogenic fungi from materials containing large number of Formula*
saprophytic fungi or bacteria. It is also recommended by the IP/EP/BP in Ingredients in grams AM1086/AM5086 AM50862 AM50863
per liter AM50861
Microbial Limit Tests for performing total yeast and mould count.
Peptic digest of animal tissue 5.0 - -

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Pancreatic digest of casein 5.0 - - 4. Once inoculated, the medium should be protected from light and incubated
Peptone (meat+casein) - 10.0 10.0 aerobically at 20-250C with increased humidity for four weeks or longer.
Glucose monohydrate - 40.0 40.0
Dextrose 40.0 - -
For Quantitative test
Chloramphenicol 0.05 0.05 0.05 1. Prepare decimal dilutions of the sample in a sterile diluent to obtain 30-300
Agar 15.0 15.0 5.0 colony forming units per plate.
Final pH (at 250C) 5.6 ± 0.2
2. Isolate using pour plate or streak plate technique.
* Formula adjusted to suit performance parameters
Directions 3. Incubate plates aerobically for 7 days at 20-250C.
0
1. Suspend 65 gms of the powder in 1000 ml distilled water. Note: After autoclaving, do not heat medium longer than 3 hours at 45-50 C.

2. Mix thoroughly. Sterile solid medium can be remelted only once.

Interpretation of Results
3. Boil with frequent agitation to dissolve the powder completely.
1. Identification of fungi is done by observing colony morphology, characteristic
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. microscopic structures, rate of growth, etc.
Quality Control
Dehydrated Appearance
2. Yeasts are identified by various biochemical tests. For spread plate and pour
Light yellow coloured, homogeneous, free flowing powder. plate method
Prepared Appearance 3. Count the number of colonies and express as colony forming units (CFU) per
Light amber coloured, clear to slightly opalescent gel. gram or ml of sample, taking into account the applicable dilution factor.
Cultural Response Precautions / Limitations
Cultural characteristics after 48-72 hours at 20-250C for fungi and 24-48 hours
at 35-370C for bacteria.
1. Some of the pathogenic fungi may produce infective spores, which can be
Organisms (ATCC) Growth RGI easily dispersed in the laboratory. Examine such organisms only within a
Aspergillus niger (16404) Luxuriant More than 70% protective cabinet.
Candida albicans (10231) Luxuriant More than 70% 2. When used for selective isolation, antimicrobials like chloramphenicol and
Escherichia coli (25922) Inhibited 0% cycloheximide may inhibit some pathogenic fungi. However, the mycelial
Lactobacillus casei (9595) Inhibited 0%
phase of Histoplama capsulatum, Paracoccidioides brasiliensis, Sporothrix
Saccharomyces cerevisiae (9763) Luxuriant More than 70%
schoenckii and Blastomyces dermatidis is not inhibited by these antibiotics
For growth RGI should be more than 70%
For Inhibition RGI should be 0% when incubated at 20-250C .
RGI- Relative Growth Index 3. A non-selective and selective medium should be inoculated for isolation of
Procedure fungi from potentially contaminated specimens.
1. Allow the agar surface to dry before inoculation. Storage

2. Inoculate and streak the specimen as soon as possible after collection. Store below 80C and prepared medium at 2-80C.
Shelf Life
3. If the specimen to be cultured is on a swab, roll the swab over a small area of
the agar surface. Use before expiry date as mentioned on the label.

Sabouraud Chloramphenicol Agar AM50864

with Lecithin and Tween 80
Use described by Sabouraud with the addition of chloramphenicol, for the cultivation
Sabouraud Chloramphenicol Agar with Lecithin and Tween 80 medium used as a of fungi, particularly those associated with skin infections. It is used for the
selective cultivation of yeasts and moulds. isolation of pathogenic fungi from materials containing large number of
Summary saprophytic fungi or bacteria.
Sabouraud Chloramphenicol Agar is Carliers modification of the formulation

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Principle 2. Mix thoroughly.
Tryptone and peptone provide nitrogenous compounds, carbon and other growth 3. Boil with frequent agitation to dissolve the powder completely.
factors. Dextrose is the carbohydrate source. The low pH of approximately 5.6 is
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
favorable for the growth of fungi, especially dermatophytes and slightly inhibitory Quality Control
to contaminating bacteria. Chloramphenicol inhibits a wide range of gram- Dehydrated Appearance
positive and gram-negative bacteria making the medium selective for fungi. Light yellow coloured, homogeneous, free flowing powder.
Tween 80 and lecithin act as neutralizers to inactivate the residual disinfectants Prepared Appearance
where the samples are collected. Lecithin inactivates quaternary ammonium Light amber coloured, clear to slightly opalescent gel.
compounds whereas tween 80 neutralizes formalin, phenolic disinfectants, Cultural Response
hexachlorophene etc. Cultural characteristics after 48-72 hours at 20-250C for fungi and 24-48 hours
at 35-370C for bacteria..
Formula*
Organisms (ATCC) Growth RGI
Ingredients in grams per liter
Aspergillus niger (16404) Luxuriant More than 70%
Tryptone 5.00
Candida albicans (10231) Luxuriant More than 70%
Peptone 5.00
Escherichia coli (25922) Inhibited 0%
Dextrose 40.00
Lactobacillus casei (9595) Inhibited 0%
Chloramphenicol 0.05
Saccharomyces cerevisiae (9763) Luxuriant More than 70%
Agar 15.00
For growth RGI should be more than 70%
Lecithin 0.70
For Inhibition RGI should be 0%
Tween 80 5.00
RGI- Relative Growth Index
Final pH (at 250C) 5.6 ± 0.2
Storage
* Formula adjusted to suit performance parameters
Directions Store at 2-80C and prepared medium at 2-80C.
1. Suspend 70.7 gms of the powder in 1000 ml distilled water. Shelf Life
Use before expiry date as mentioned on the label.

Sabouraud Dextrose Agar (Harmonized ) AMH5087

Sabouraud Dextrose Agar IP AM10871/AM50871
Sabouraud Dextrose Agar USP AM10872/AM50872
Sabouraud Dextrose Agar EP AM10873/AM50873
Use Microbial Limit Tests for performing total yeast and mould count and is included in
Sabouraud Dextrose Agar is general-purpose media used for the cultivation of the Bacteriological Analytical Manual for food testing. It is also recommended by
yeasts, moulds and aciduric bacteria. APHA for the examination of foods. Sabouraud Dextrose Agar can be made
Summary inhibitory to most pathogenic fungi and bacteria by the addition of antibiotics.
Sabouraud Dextrose Agar is Carliers (12) modification of the formulation Gentamycin is an amino glycoside that inhibits the growth of gram-negative
described by Sabouraud for the cultivation of fungi, particularly those associated bacteria. Chloramphenicol is inhibitory to a wide range of gram-positive and
with skin infections. It is used in qualitative procedures for cultivation of gram-negative bacteria, cycloheximide is an antifungal agent that inhibits
pathogenic and non-pathogenic fungi, particularly dermatophytes. Carlier saprophytic fungi while allowing the growth of yeasts or dermatophytes. George
showed that this medium gives reliable results with Microsporum audouini, et al aseptically added 0.5 gm cycloheximide, 20000 units penicillin and 40000
M.canis, Trichophyton mentagrophytes, T.flavum, T.rubrum and Candida units streptomycin to each liter of autoclaved, cooled medium. Cryptococcus
albicans. The fungi maintain their typical cultural appearance and thus may be neoformans, Aspergillus fumigatus and Allescheria boydii were found to be
readily identified according to the standard macroscopic characters described by sensitive to cycloheximide; Actinomyces bovis and Nocardia asteroids were
Sabouraud. Sabouraud Dextrose Agar is recommended by the USP and IP in sensitive to penicillin and streptomycin. Hantshke used colistin, novobiocin and

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cycloheximide to isolate Candida albicans. Dolan used gentamycin, Escherichia coli (25922) Luxuriant* More than 70%
chloramphenicol and cycloheximide for the selective isolation of pathogenic Lactobacillus casei (9595) Luxuriant* More than 70%
Key:
fungi.
* = inhibited on media with low pH
Principle
For growth RGI should be more than 70%
Mixture of peptic digest of animal tissue and pancreatic digest of casein provide For Inhibition RGI should be 0%
nitrogenous compounds, carbon and other growth factors. Dextrose is the RGI- Relative Growth Index
carbohydrate source. The low pH of approximately 5.6 is favourable for the Procedure
growth of fungi, especially dermatophytes and is slightly inhibitory to 1. Allow the agar surface to dry before inoculating.
contaminating bacteria. Various antibiotics can be added to this medium for 2. Inoculate and streak the specimen as soon as possible after collection. If the
bacterial inhibition as well as to make it selective for the isolation of pathogenic specimen to be cultured is on a swab, roll the swab over a small area of the
fungi from material containing large number of other fungi or bacteria. agar surface.
Sabouraud Dextrose Agar may also be used as the basis of Pagano- Levin 3. Streak for isolation with a sterile loop.
medium for the isolation of Candida albicans. 0.1 gm of filter sterilized
4. Incubate plates in an inverted position.
triphenyltetrazolium chloride is added to each liter of autoclaved molten medium
cooled to 550C. After incubation at 250C for 3 days, Candida albicans colonies are 5. Once inoculated, the medium should be protected from light and incubated
unpigmented or pale pink while other Candida species and other fungi form deep aerobically at 20-250C with increased humidity for four weeks or longer.
For Quantitative test
pink or red colonies. Other tests should be performed for identification of Candida
albicans. 1. Prepare decimal dilutions of the sample in a sterile diluent to obtain 30-300
Formula* colony forming units per plate.
Ingredients in grams per liter 2. Inoculate using the pour plate or streak plate technique.
Mixture of peptic digest of animal tissue 10.0
and pancreatic digest of casein(1:1) 3. Incubate plates aerobically for 7 days at 20-250C .
0
Dextrose 40.0 Note: After autoclaving, do not heat the medium longer than 3 hours at 45-50 C.
Agar 15.0 Sterile solidified medium can be remelted only once.
Final pH (at 250C) 5.6 ± 0.2 Interpretation of Results
* Formula adjusted to suit performance parameters
1. Identification of fungi is done by observing colony morphology,
Directions
characteristic microscopic structures, rate of growth, etc. Yeasts are identified
1. Suspend 65 gms of the powder in 1000 ml distilled water and mix
by various biochemical tests. Pour plate and spread plate method
thoroughly.
2. Count the number of colonies and express as colony forming units (CFU) per
2. Boil with frequent agitation to dissolve the powder completely. Avoid
gram or ml of sample, taking into account the applicable dilution factor.
overheating the agar as it could cause a softer medium. Precautions / Limitations
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. 1. Some of the pathogenic fungi may produce infective spores, which can be
Quality Control easily dispersed in the laboratory. Examine such organisms only within a
Dehydrated Appearance
protective cabinet.
Light yellow coloured, homogeneous, free flowing powder.
Prepared Appearance 2. When used for selective isolation, antimicrobials like chloramphenicol and
Light amber coloured, clear to slightly opalescent gel. cycloheximide may inhibit some pathogenic fungi. However, the mycelial
Cultural Response phase of Histoplama capsulatum, Paracoccidioides brasiliensis, Sporothrix
Cultural characteristics after 48-72 hours at 20-250C for fungi and 24-48 schoenckii and Blastomyces dermatidis is not inhibited by these antibiotics
hours at 35-370C for bacteria. when incubated at 20-250C .
Organisms (ATCC) Growth RGI
AAspergillus niger (16404) Luxuriant More than 70%
3. A non-selective and selective medium should be inoculated for isolation of
Candida albicans (10231) Luxuriant More than 70% fungi from potentially contaminated specimens.
Saccharomyces cerevisiae (9763) Luxuriant More than 70%

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Storage Shelf Life
Store at 22-300C and prepared medium at 2-80C. Use before expiry date as mentioned on the label.

Sabouraud Dextrose Agar AM1087/AM5087

Sabouraud Dextrose Broth AM1088/AM5088
Use approximately 5.6 is favourable for the growth of fungi, especially dermatophytes
Sabouraud Dextrose Agar and Sabouraud Dextrose Broth are general-purpose and is slightly inhibitory to contaminating bacteria. Various antibiotics can be
media used for the cultivation of yeasts, moulds and aciduric bacteria. added to this medium for bacterial inhibition as well as to make it selective for the
Summary isolation of pathogenic fungi from material containing large number of other fungi
Sabouraud Dextrose Agar and Sabouraud Dextrose Broth are Carliers (12) or bacteria.
modification of the formulation described by Sabouraud (99) for the cultivation of Sabouraud Dextrose Agar may also be used as the basis of Pagano-Levin medium
fungi, particularly those associated with skin infections. It is used in qualitative for the isolation of Candida albicans. 0.1 gm of filter sterilized triphenyltetrazolium
procedures for cultivation of pathogenic and non-pathogenic fungi, particularly chloride is added to each litre of autoclaved molten medium cooled to 550C. After
dermatophytes. Carlier showed that this medium gives reliable results with incubation at 250C for 3 days, Candida albicans colonies are unpigmented or pale
Microsporum audouini, M.canis, Trichophyton mentagrophytes, T.flavum, pink while other Candida species and other fungi form deep pink or red colonies.
T.rubrum and Candida albicans. The fungi maintain their typical cultural Other tests should be performed for identification of Candida albicans.
appearance and thus may be readily identified according to the standard Formula*
macroscopic characters described by Sabouraud. Sabouraud Dextrose Agar is Ingredients in grams Sabouraud Sabouraud
recommended by the USP (114) and IP (46) in Microbial Limit Tests for per liter Dextrose Agar Dextrose Broth
performing total yeast and mould count and is included in the Bacteriological Peptone 5.0 -
Analytical Manual for food testing (113). It is also recommended by APHA for the Tryptone 5.0 10.0
Dextrose 40.0 20.0
examination of foods (20).
Agar 15.0 -
Sabouraud Dextrose Broth is recommended in the Bacteriological Analytical Final pH (at 250C) 5.6 ± 0.2 5.6 ± 0.2
Manual for cosmetics testing (113). Sabouraud Dextrose Agar and Sabouraud * Formula adjusted to suit performance parameters
Dextrose Broth can be made inhibitory to most pathogenic fungi and bacteria by Directions
the addition of antibiotics. Gentamycin is an amino glycoside that inhibits the 1. Suspend the powder in 1000 ml distilled water and mix thoroughly.
growth of gram-negative bacteria. Chloramphenicol is inhibitory to a wide range Sabouraud Dextrose Agar - 65 gms
of gram-positive and gram-negative bacteria, cycloheximide is an antifungal
Sabouraud Dextrose Broth - 30 gms
agent that inhibits saprophytic fungi while allowing the growth of yeasts or
dermatophytes. George et al aseptically added 0.5 gm cycloheximide, 20000 2. Boil with frequent agitation to dissolve the powder completely. Avoid
units penicillin and 40000 units streptomycin to each litre of autoclaved, cooled overheating the agar as it could cause a softer medium.
medium. Cryptococcus neoformans, Aspergillus fumigatus and Allescheria boydii 3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
were found to be sensitive to cycloheximide; Actinomyces bovis and Nocardia Quality Control
asteroids were sensitive to penicillin and streptomycin. Hantshke used colistin, Dehydrated Appearance
Light yellow coloured, homogeneous, free flowing powder.
novobiocin and cycloheximide to isolate Candida albicans. Dolan used
Prepared Appearance
gentamycin, chloramphenicol and cycloheximide for the selective isolation of
Sabouraud Dextrose Agar - Light amber coloured, clear to slightly opalescent
pathogenic fungi. gel.
Principle Sabouraud Dextrose Broth - Light amber coloured clear solution, without any
Tryptone and mycological peptone provide nitrogenous compounds, carbon and precipitate.
other growth factors. Dextrose is the carbohydrate source. The low pH of Cultural Response
Cultural characteristics after 48-72 hours at 300C.

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Organisms (ATCC) Growth on Sabouraud RGI For Sabouraud Dextrose Broth
Dextrose Agar and 1. Inoculate the medium as soon as the specimen has been collected.
in Sabouraud
Dextrose Broth
2. Incubate with caps loosened at 300C for 18-24 hours or up to 7 days.
Aspergillus niger (16404) Luxuriant More than 70% Interpretation of Results
Candida albicans (10231) Luxuriant More than 70% Sabouraud Dextrose Agar
Saccharomyces cerevisiae Luxuriant More than 70% 1. Identification of fungi is done by observing colony morphology,
(9763)
characteristic microscopic structures, rate of growth, etc. Yeasts are
Escherichia coli (25922) Luxuriant* More than 70%
identified by various biochemical tests.
Lactobacillus casei (9595) Luxuriant* More than 70%
For growth RGI should be more than 70% Pour plate and spread plate method
RGI- Relative Growth Index 1. Count the number of colonies and express as colony forming units (CFU) per
Key: gram or ml of sample, taking into account the applicable dilution factor.
* = inhibited on media with low pH
Procedure
Sabouraud Dextrose Broth
For Sabouraud Dextrose Agar 1. Growth in the broth is indicated by the presence of turbidity compared to an
uninoculated control.
1. Allow the agar surface to dry before inoculating.
Precautions / Limitations
2. Inoculate and streak the specimen as soon as possible after collection. If the
1. Some of the pathogenic fungi may produce infective spores, which can be
specimen to be cultured is on a swab, roll the swab over a small area of the
easily dispersed in the laboratory. Examine such organisms only within a
agar surface.
protective cabinet.
3. Streak for isolation with a sterile loop.
2. When used for selective isolation, antimicrobials like chloramphenicol and
4. Incubate plates in an inverted position. cycloheximide may inhibit some pathogenic fungi. However, the mycelial
5. Once inoculated, the medium should be protected from light and incubated phase of Histoplama capsulatum, Paracoccidioides brasiliensis, Sporothrix
aerobically at 25-300C with increased humidity for four weeks or longer. schoenckii and Blastomyces dermatidis is not inhibited by these antibiotics
For Quantitative test when incubated at 25-300C.
1. Prepare decimal dilutions of the sample in a sterile diluent to obtain 30-300 3. A non-selective and selective medium should be inoculated for isolation of
colony forming units per plate. fungi from potentially contaminated specimens.
2. Inoculate using the pour plate or streak plate technique. Storage
0
3. Incubate plates aerobically for 7 days at 25-30 C. Store below 300C and prepared medium at 2-80C.
Shelf Life
Note: After autoclaving, do not heat the medium longer than 3 hours at 45-
Use before expiry date as mentioned on the label.
500C. Sterile solidified medium can be remelted only once.

Sabouraud Dextrose Broth (Harmonized) AMH5088

Sabouraud Dextrose Broth USP AM50881
Sabouraud Dextrose Broth EP AM50882
Use described by Sabouraud for the cultivation of fungi, particularly those associated
Sabouraud Dextrose Broth is general-purpose media used for the cultivation of with skin infections. It is used in qualitative procedures for cultivation of
yeasts, moulds and aciduric bacteria. pathogenic and non-pathogenic fungi, particularly dermatophytes. Carlier
Summary showed that this medium gives reliable results with Microsporum audouini,
Sabouraud Dextrose Broth is Carliers (12) modification of the formulation M.canis, Trichophyton mentagrophytes, T.flavum, T.rubrum and Candida

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albicans. The fungi maintain their typical cultural appearance and thus may be Procedure
readily identified according to the standard macroscopic characters described by 1. Allow the agar surface to dry before inoculating.
Sabouraud. Sabouraud Dextrose Broth is recommended in the Bacteriological 2. Inoculate and streak the specimen as soon as possible after collection. If the
Analytical Manual for cosmetics testing. specimen to be cultured is on a swab, roll the swab over a small area of the
Principle agar surface.
Mixture of peptic digest of animal tissue & pancreatic digest of casein provide 3. Streak for isolation with a sterile loop.
nitrogenous compounds, carbon and other growth factors. Dextrose is the
4. Incubate plates in an inverted position.
carbohydrate source. The low pH of approximately 5.6 is favorable for the growth
5. Once inoculated, the medium should be protected from light and incubated
of fungi, especially dermatophytes and is slightly inhibitory to contaminating
aerobically at 25-300C with increased humidity for four weeks or longer.
bacteria. Various antibiotics can be added to this medium for bacterial inhibition
as well as to make it selective for the isolation of pathogenic fungi from material For Quantitative test
containing large number of other fungi or bacteria. 1. Prepare decimal dilutions of the sample in a sterile diluent to obtain 30-300
Formula* colony forming units per plate.
Ingredients in grams per liter 2. Inoculate using the pour plate or streak plate technique.
Mixture of peptic digest of animal tissue 10.0
& pancreatic digest of casein (1:1) 3. Incubate plates aerobically for 7 days at 25-300C.
0
Dextrose 20.0 Note: After autoclaving, do not heat the medium longer than 3 hours at 45-50 C.
Final pH (at 250C) 5.6 ± 0.2 Sterile solidified medium can be remelted only once.
* Formula adjusted to suit performance parameters
Interpretation of Results
Directions
1. Identification of fungi is done by observing colony morphology, characteristic
1. Suspend 30 gms of the powder in 1000 ml distilled water and mix
microscopic structures, rate of growth, etc. Yeasts are identified by various
thoroughly.
biochemical tests. Pour plate and spread plate method
2. Boil with frequent agitation to dissolve the powder completely. Avoid
2. Count the number of colonies and express as colony forming units (CFU) per
overheating the agar as it could cause a softer medium.
gram or ml of sample, taking into account the applicable dilution factor.
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. Precautions / Limitations
Quality Control
1. Some of the pathogenic fungi may produce infective spores, which can be
Dehydrated Appearance
Light yellow coloured, homogeneous, free flowing powder.
easily dispersed in the laboratory. Examine such organisms only within a
Prepared Appearance protective cabinet.
Light amber coloured, clear to slightly opalescent gel. 2. When used for selective isolation, antimicrobials like chloramphenicol and
Cultural Response cycloheximide may inhibit some pathogenic fungi. However, the mycelial
Cultural characteristics after 2-5 days at 20-250C for fungi and 18-24 hours at phase of Histoplama capsulatum, Paracoccidioides brasiliensis, Sporothrix
35-370C for bacteria.
schoenckii and Blastomyces dermatidis is not inhibited by these antibiotics
Organisms (ATCC) Growth RGI
Candida albicans (10231) Luxuriant More than 70%
when incubated at 25-300C.
Escherichia coli (25922) Luxuriant* More than 70% 3. A non-selective and selective medium should be inoculated for isolation of
Lactobacillus casei (9595) Luxuriant* More than 70% fungi from potentially contaminated specimens.
Saccharomyces cerevisiae (9763) Luxuriant More than 70% Storage
Aspergillus niger (16404) Luxuriant More than 70%
Store at 22-300C and prepared medium at 2-80C.
Key:
Shelf Life
* = inhibited on media with low pH
For growth RGI should be more than 70% Use before expiry date as mentioned on the label.
RGI- Relative Growth Index

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Sabouraud Glucose Agar AM108821/AM508821
Use Quality Control
Sabouraud Glucose Agar is used for selective cultivation of yeasts, moulds and Dehydrated Appearance
Light yellow coloured, homogeneous, free flowing powder.
aciduric bacteria.
Prepared Appearance
Summary
Light amber coloured, clear to slightly opalescent gel.
Sabouraud Glucose Agar is Carliers (12) modification of the formulation described Cultural Response
by Sabouraud (99) for the cultivation of fungi, particularly those associated with Cultural characteristics after 48-72 hours at 20-25°C for fungi and 35-370C for
skin infections. It is used in qualitative procedure for the cultivation of pathogenic bacteria.
and non pathogenic fungi particularly dermatophytes. Carlier showed that this Organisms (ATCC) Growth
medium gives reliable results with Microsporum audouini, M. canis, Trichophyton Aspergillus niger (16404) Luxuriant
Candida albicans (10231) Luxuriant
mentagrophytes, T. flavum, T. rubrum and Candida albicans . The fungi maintain
Saccharomyces cerevisiae (9763) Luxuriant
their typical cultural appearance and thus may be readily identified according to
Escherichia coli (25922) Luxuriant*
the standard macroscopic characters described by Sabouraud. Lactobacillus casei (9595) Luxuriant*
Principle Staphylococcus aureus (6538) Luxuriant
Tryptone and mycological peptone provides nitrogenous compounds, carbon and Pseudomonas aeruginosa (9027) Luxuriant
other growth factors. Glucose is the carbohydrate source. The low pH of Clostridium sporogenes (11437) Luxuriant
approximately 5.6 is favourable for the growth of fungi, especially dermatophytes Bacillus subtilis (6633) Luxuriant
and slightly inhibitory to contaminating bacteria. Various antibiotic can be added Key:
* = inhibited on media with low pH
to this medium for bacterial inhibition as well as to make it selective for the
For growth RGI should be more than 70%
isolation of pathogenic fungi from from material containing large number of other
RGI- Relative Growth Index
fungi or bacteria. Procedure
Sabouraud Glucose Agar may also be used as the basis of Pagano-Levin medium 1. Allow the agar surface to dry before inoculation.
for the isolation of Candida albicans. 0.1 gm of filter sterilized
2. Inoculate and streak the specimen as soon as possible after collection.
triphenyltetrazolium chloride is added to each litre of autoclaved molten medium
3. If the specimen to be cultured is on a swab, roll the swab over a small area of
cooled to 550C. After incubation at 250C for 3 days, Candida albicans colonies are
the agar surface.
unpigmented or pale pink while other Candida species and other fungi from deep
pink or red colonies. Other tests should be performed for identification of Candida 4. Once inoculated, the medium should be protected from light and incubated
albicans. aerobically at 25-30°C with increased humidity for four weeks or longer.
Formula* For Quantitative test
Ingredients in grams per liter 1. Prepare decimal dilutions of the sample in a sterile diluent to obtain 30-300
Peptones 5.0
colony-forming units per plate.
Tryptone 5.0
Glucose 40.0 2. Isolate using pour plate or streak plate technique.
Agar 15.0 3. Incubate plates aerobically for 7 days at 25-300C.
Final pH (at 25°C) 5.6 ± 0.2 0
Note: After autoclaving, do not heat medium longer than 3 hours at 45-50 C.
* Formula adjusted to suit performance parameters
Sterile solid medium can be remelted only once.
Directions
Interpretation of Results
1. Suspend 65 gms of the powder in 1000 ml distilled water.
1. Identification of fungi is done by observation of colony morphology,
2. Mix thoroughly. characteristic microscopic structures, rate of growth, etc.
3. Boil with frequent agitation to dissolve the powder completely. 2. Yeasts are identified by various biochemical tests.
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes. For spread plate and pour plate method

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1. Count the number of colonies and express as colony forming units (CFU) per Shelf Life

gram or ml of sample, taking into account the applicable dilution factor. Use before expiry date as mentioned on the label.
Storage
Store at 22-300C and prepared medium at 2-80C.

Sabouraud Glucose Agar with Antibiotics IP AM50883

Use Organisms (ATCC) Growth RGI
Sabouraud Glucose Agar with antibiotics IP is used for selective cultivation of Candida albicans (10231) Luxuriant More than 70%
Escherichia coli (25922) Inhibited 0%
yeasts and moulds in compliance with IP.
Aspergillus niger (16404) Luxuriant More than 70%
Summary
Saccharomyces cerevisiae (9763) Luxuriant More than 70%
Sabouraud Glucose Agar with antibiotics is Carliers (12) modification of the For growth RGI should be more than 70%
formulation described by Sabouraud (99) with the addition of chloramphenicol, For Inhibition RGI should be 0%
for the cultivation of fungi, particularly those associated with skin infections. It is RGI- Relative Growth Index
used for the isolation of pathogenic fungi from materials containing large number Procedure
of saprophytic fungi or bacteria. 1. Allow the agar surface to dry before inoculation.
Principle 2. Inoculate and streak the specimen as soon as possible after collection.
Peptone(meat and casein) provides nitrogenous compounds, carbon and other 3. If the specimen to be cultured is on a swab, roll the swab over a small area of
growth factors. Glucose is the carbohydrate source. The low pH of approximately the agar surface.
5.6 is favourable for the growth of fungi, especially dermatophytes and slightly
4. Once inoculated, the medium should be protected from light and incubated
inhibitory to contaminating bacteria. Chloramphenicol inhibits a wide range of
aerobically at 25-30°C with increased humidity for four weeks or longer.
gram-positive and gram-negative bacteria making the medium selective for
fungi. For Quantitative test
Formula* 1. Prepare decimal dilutions of the sample in a sterile diluent to obtain 30300
Ingredients in grams per liter colony-forming units per plate.
Peptones (meat and casein) 10.0
2. Isolate using pour plate or streak plate technique.
Glucose monohydrate 40.0
Chloramphenicol 0.05 3. Incubate plates aerobically for 7 days at 25-300C.
0
Agar 15.0 Note: After autoclaving, do not heat medium longer than 3 hours at 45-50 C.
Final pH (at 25°C) 5.6 ± 0.2 Sterile solid medium can be remelted only once.
* Formula adjusted to suit performance parameters Interpretation of Results
Directions
1. Identification of fungi is done by observation of colony morphology,
1. Suspend 65.05 gms of the powder in 1000 ml distilled water. characteristic microscopic structures, rate of growth, etc.
2. Mix thoroughly. 2. Yeasts are identified by various biochemical tests.
3. Boil with frequent agitation to dissolve the powder completely. For spread plate and pour plate method
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes. 1. Count the number of colonies and express as colony forming units (CFU) per
Quality Control
gram or ml of sample, taking into account the applicable dilution factor.
Dehydrated Appearance
Storage
Light yellow coloured, homogeneous, free flowing powder.
Prepared Appearance Store at 22-300C and prepared medium at 2-80C.
Light amber coloured, clear to slightly opalescent gel. Shelf Life
Cultural Response Use before expiry date as mentioned on the label.
Cultural characteristics after 48-72 hours at20-25°C for fungi and 24-48 hours
at 35-370C for bacteria.

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Sabouraud Glucose Agar with Antibiotics AM50884
(Agar Medium C) EP
Sabouraud Glucose Agar with Antibiotics (Agar Medium C) BP AM50885
Use Organisms (ATCC) Growth RGI
Sabouraud Glucose Agar with antibiotics (Agar Medium C) EP/BP is used for Candida albicans (10231) Luxuriant More than 70%
Escherichia coli (25922) Inhibited 0%
selective cultivation of yeasts and moulds in compliance with EP/BP.
Lactobacillus casei (9595) lnhibited 0%
Summary
Saccharomyces cerevisiae (9763) Luxuriant More than 70%
Sabouraud Glucose Agar with antibiotics is Carliers (12) modification of the Aspergillus niger (16404) Luxuriant More than 70%
formulation described by Sabouraud (99) with the addition of chloramphenicol, For growth RGI should be more than 70%
for the cultivation of fungi, particularly those associated with skin infections. It is For Inhibition RGI should be 0%
used for the isolation of pathogenic fungi from materials containing large number RGI- Relative Growth Index
of saprophytic fungi or bacteria. It is also recommended by the EP/BP in Procedure
Microbial Limit Tests to perform total yeast and mould count. 1. Allow the agar surface to dry before inoculation.
Principle 2. Inoculate and streak the specimen as soon as possible after collection.
Peptone(meat and casein) provides nitrogenous compounds, carbon and other 3. If the specimen to be cultured is on a swab, roll the swab over a small area of
growth factors. Glucose is the carbohydrate source. The low pH of approximately the agar surface.
5.6 is favourable for the growth of fungi, especially dermatophytes and slightly
4. Once inoculated, the medium should be protected from light and incubated
inhibitory to contaminating bacteria. Chloramphenicol inhibits a wide range of
aerobically at 20-25°C with increased humidity for four weeks or longer.
gram-positive and gram-negative bacteria making the medium selective for
For Quantitative test
fungi.
Formula* 1. Prepare decimal dilutions of the sample in a sterile diluent to obtain 30-300
Ingredients in grams per liter colony-forming units per plate.
Peptones (meat and casein) 10.0 2. Isolate using pour plate or streak plate technique.
D-Glucose monohydrate 40.0
Chloramphenicol 0.05
3. Incubate plates aerobically for 7 days at 20-250C.
0
Agar 15.0 Note: After autoclaving, do not heat medium longer than 3 hours at 45-50 C.
Final pH (at 25°C) 5.6 ± 0.2 Sterile solid medium can be remelted only once.
* Formula adjusted to suit performance parameters Interpretation of Results
Directions
1. Identification of fungi is done by observation of colony morphology,
1. Suspend 65.05 gms of the powder in 1000 ml distilled water. characteristic microscopic structures, rate of growth, etc.
2. Mix thoroughly. 2. Yeasts are identified by various biochemical tests.
3. Boil with frequent agitation to dissolve the powder completely. For spread plate and pour plate method
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes. 1. Count the number of colonies and express as colony forming units (CFU) per
Quality Control gram or ml of sample, taking into account the applicable dilution factor.
Dehydrated Appearance
Storage
Light yellow coloured, homogeneous, free flowing powder.
Prepared Appearance
Store at 22-300C and prepared medium at 2-80C.
Light amber coloured, clear to slightly opalescent gel. Shelf Life
Cultural Response Use before expiry date as mentioned on the label.
Cultural characteristics after 48-72 hours at 20-25°C for fungi and 24-48 hours
at 35-370C for bacteria..

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Salt Meat Broth AM50886

Use 2. Warm slightly with frequent agitation to dissolve the powder completely. DO
Salt Meat Broth is used as an enrichment medium for the isolation of NOT OVERHEAT.
Staphylococci from grossly contaminated specimens. 3. Dispense in tubes or adequate containers and sterilize by autoclaving at
Summary 15lbs pressure (121ºC) for 15 minutes.
Staphylococci are tolerant of concentrations of sodium chloride that inhibit most Quality Control
other bacteria. Salt Meat Broth is a selective growth medium for staphylococci. It Dehydrated Appearance
is used for the preliminary enrichment for the isolation of small numbers of the Light yellow coloured, homogeneous, free flowing powder.
cocci from heavily contaminated materials (77.2). This medium can detect Prepared Appearance
halophilic Staphylococci from contaminated samples such as faeces especially Yellow coloured, clear solution without any precipitate.
Cultural Response
in case of food poisoning (77.3). The medium is also an excellent substrate for
Culture characteristics after 18-48hours at 35-370C.
the cultivation of some of the halophilic micrococci associated with hides and raw
Organisms (ATCC) Growth
salt supplies. Staphylococcus aureus (25923) Luxuriant
Principle Escherichia coli (25922) Inhibited
Salt Meat Broth is a selective medium for staphylococci due to the extra Proteus vulgaris (13315) Inhibited
concentration of sodium chloride. Peptic digest of animal tissue, beef extract and Procedure
ox heart tissue supply the essential nutrients and support the growth of the 1. For the isolation of staphylococci from samples of food, emulsify the specimen
bacteria. in peptone water (AM1079/5079) and inoculate a tube of Salt Meat Broth.
Formula* 2. After 24-48 hours incubation at 35-370C, subculture on Mannitol Salt Agar
Ingredients in grams per liter
(AM 1069/5069).
Peptic digest of animal tissue 10.0
Interpretation of Results
Beef Extract 10.0
Neutral Ox-heart tissue 30.0 Examine the colonies on Mannitol Salt Agar and do further biochemical tests for
Sodium chloride 100.0 the identification.
Final pH (at 250C) 7.6 ± 0.2 Storage
* Formula adjusted to suit performance parameters Store at 22-300C and prepared medium at 2-80C.
Directions Shelf Life
1. Suspend 15 gms in 100 ml distilled water. Soak for 5 minutes. Use before expiry date as mentioned on the label.

Salmonella Differential Agar (Twin Pack) AM50887

(Equivalent to RajhansTM Medium )
Use identification and differentiation of salmonella species (22.2) are based on
Salmonella Differential Agar medium is recommended for identification and lactose fermentation and hydrogen sulphide production.
differentiation of Salmonella species from members of Enterobacteriaceae, Principle
especially Proteus species. Peptone special and yeast extract supports the luxuriant growth of bacteria while
Summary sodium deoxycholate inhibits gram-positive organisms rendering the medium
Salmonella Differential Agar medium is a slight modification of original selective for enteric microorganisms. The BC indicator turns pink in presence of
formulation of Rambach (89.9) used for differentiation of Salmonella species acid produced from propylene glycol. Lactose fermentating ability is determined
from Proteus species and other enteric bacteria. Production of acid from by using an indicator which can detect the presence of enzyme ß-galactosidase.
propylene glycol is a novel characteristic of Salmonella species and is utilised in Lactose fermenting (ß-galactosidase producing) bacteria yield blue violet
this medium. Many of the media such as SS Agar, XLD Agar recommended for the coloured colony. Salmonellae produce acid from propylene glycol and combine

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with the pH indicator to give typical pink red colonies. Other enteric gram-negative Part B: Colourless viscous solution
Prepared Appearance
bacteria form colourless colonies. Salmonellae Typhimurium and Salmonellae
Light orange coloured, clear to slightly opalescent gel forms in Petri plates
Enteritidis produce pink to red colonies.
Cultural Response
Formula*
Cultural characteristics after 24-48 days at 35-370C.
Ingredients in grams per liter
Organisms Growth Colour of RGI
Part A -
colony
Peptone, special 8.0
Escherichia coli (25922) Luxuriant Blue-green More than 70%
Yeast extract 2.0
Klebsiella pneumoniae (13883) Luxuriant Blue-violet More than 70%
Sodium deoxycholate 1.0
Proteus mirabilis (25933) Luxuriant Colourless More than 70%
B. C. Indicator 2.0
Salmonella Typhimurium (14028) Luxuriant Pink-red More than 70%
Agar 12.0
Salmonella Enteritidis (13076) Luxuriant Pink-red More than 70%
Part B -
Salmonella Typhi (6539) Luxuriant Colourless More than 70%
Propylene glycol 10.0
Shigella flexneri (12022) Luxuriant Colourless More than 70%
Final pH ( at 25°C) 7.3±0.2
Staphylococcus aureus (25923) Inhibited - 0%
* Formula adjusted to suit performance parameters
For growth RGI should be more than 70%
Directions
For Inhibition RGI should be 0%
1. Suspend 10 grams of fluid Part B in 1000 ml distilled water. RGI- Relative Growth Index
2. Add 25 grams of Part A. Mix well and boil to dissolve the medium Storage
completely. DO NOT AUTOCLAVE. Store at 22-300C and prepared medium at 2-80C.
3. Cool to 45 - 50°C. Mix well before pouring into sterile Petri plates. Shelf Life
Quality Control Use before expiry date as mentioned on the label.
Dehydrated Appearance
Part A : Light yellow to light pink homogeneous free flowing powder

Selenite F Broth AM1089/AM5089

Use maintains the pH in the medium as selenite is reduced by bacterial growth and
Selenite F Broth is used as an enrichment medium for the isolation of Salmonella alkali is produced. An increase in pH lessens the toxicity of selenite and results in
species from faeces, urine, water, foods and other materials of sanitary the overgrowth of other bacteria. The acid produced by bacteria due to lactose
importance. fermentation helps to maintain a neutral pH. Disodium phosphate buffers the
Summary medium to maintain the pH and also lessens the toxicity of selenite, thus
Selenite F Broth is based on the formulation devised by Leifson (65), who showed increasing the capacity of the medium. Sodium selenite inhibits gram-positive
that selenite was beneficial in the isolation of Salmonella species while inhibiting bacteria and suppresses the growth of most gram-negative bacteria and
coliforms and certain other microbial species like faecal streptococci, present in enterococci other than Salmonella.
faecal specimens. An enrichment medium is routinely employed to detect Formula*
pathogens in faecal specimens since the pathogens are generally present in a very Ingredients in grams per liter
Tryptone 5.0
small number compared to the intestinal flora. This medium is useful in detecting
Lactose 4.0
Salmonella in the non-acute stages of illness when the organisms occur in faeces
Disodium Phosphate Dodecahydrate 10.0
in low numbers and for epidemiological studies to enhance the detection of low Sodium Hydrogen Selenite 4.0
numbers of organisms from asymptomatic or convalescent patients. Selenite F Final pH (at 250C) 7.0 ± 0.2
broth is used in the recovery of Salmonella with subcultures being made after 12- * Formula adjusted to suit performance parameters
18 hours of incubation. Directions
Principle 1. Suspend 23 gms of the powder in 1000 ml distilled water.
Tryptone provides nitrogenous substances and other amino acids. Lactose 2. Mix thoroughly.

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3. Boil with frequent agitation to dissolve the powder completely. Sterilize in 4. Incubate for 12-24 hours at 35-37oC.
boiling water bath or free flowing steam for 10 minutes. AVOID
5. Make sub-cultures after 12-18 hours of incubation.
OVERHEATING. DO NOT AUTOCLAVE. Excessive heating is detrimental. Interpretation of Results
4. Discard the prepared medium if a large amount of selenite is reduced, which 1. After incubation, there must be an increase in the number of pathogens that
is indicated by a red precipitate at the bottom of the tube/bottle. the medium is designed to select for and enrich.
Warning: Sodium Hydrogen Selenite is very toxic, corrosive and causes
teratogenicity. Handle with care. On contact with skin wash immediately 2. Subculture onto any combination of greater and lesser inhibitory, selective
with plenty of water. and differential media for Enterobacteriaceae. e.g. MacConkey Agar, XL
Quality Control Agar, etc to isolate pathogens for identification.
Dehydrated Appearance
Precautions / Limitations
Cream coloured, homogeneous, free flowing powder.
Prepared Appearance
1. Discard the prepared medium if large amounts of reduced selenite can be
Light yellow coloured, clear to very slightly opalescent solution, may have a seen as a red precipitate at the bottom of the tube.
slight precipitate. 2. Do not incubate for longer than 24 hours because the inhibitory effect of
Cultural Response selenite is reduced after 6-12 hours incubation and coliforms may overgrow
Cultural characteristics after 18-24 hours at 35-370C when subcultured on
the pathogens.
MacConkey Agar (AM10590/ AM50590).
Organisms (ATCC) Recovery Colour of 3. Take subcultures from the upper third of the broth column which should be at
Colony least 5 cm in depth
Escherichia coli (25922) Little to none Pink with bile
4. Enrichment broths should not be used as the sole isolation medium.
precipitate
Salmonella serotype Choleraesuis Good to excellent Colourless 5. Use in conjunction with selective and non-selective plating media to
(12011) increase the chances of isolating pathogens, particularly when they may be
Salmonella serotype Typhimurium Good to excellent Colourless present in small numbers.
(14028) Storage
Procedure
Store below 300C and prepared medium at 2-80C.
1. For faeces and other solid materials, suspend 1-2 gms of the specimen in Shelf Life
the broth (approximately 10-15% by volume) and emulsify if necessary. Use before expiry date as mentioned on the label.
2. Solid material is added to the normal strength broth.
3. Liquid samples are mixed with double strength medium in the ratio of 1:1.

Selenite F Broth IP (Twin Pack) AM10891/AM50891

Use or convalescent patients. Selenite F broth is used in the recovery of Salmonella
Selenite F Broth is used as an enrichment medium for the isolation of Salmonella with subcultures being made after 12-18 hours of incubation.
species, in accordance with IP. Principle
Summary Peptone provides nitrogenous substances and other amino acids. Lactose
Selenite F Broth is based on the formulation devised by Leifson (68), who showed maintains the pH in the medium as selenite is reduced by bacterial growth and
that selenite was beneficial in the isolation of Salmonella species while alkali is produced. An increase in pH lessens the toxicity of selenite and results in
inhibiting coliforms and certain other microbial species like faecal streptococci, the overgrowth of other bacteria. The acid produced by bacteria due to lactose
present in faecal specimens. An enrichment medium is routinely employed to fermentation helps to maintain a neutral pH. Disodium Hydrogen phosphate
detect pathogens in faecal specimens since the pathogens are generally present buffers the medium to maintain the pH and also lessens the toxicity of selenite,
in a very small number compared to the intestinal flora. This medium is useful in thus increasing the capacity of the medium. Sodium selenite inhibits gram-
detecting Salmonella in the non-acute stages of illness when the organisms positive bacteria and suppresses the growth of most gram-negative bacteria and
occur in faeces in low numbers and for epidemiological enterococci other than Salmonella.
studies to enhance the detection of low numbers of organisms from asymptomatic

288 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
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Formula* 2. Solid material is added to the normal strength broth.
Ingredients in grams per liter Part A Part B
3. Liquid samples are mixed with double strength medium in the ratio of 1:1.
Peptone 5.0 -
Lactose 4.0 - 4. Incubate for 12-24 hours at 35-37oC.
Disodium Hydrogen Phosphate 10.0 - 5. Make sub-cultures after 12-18 hours of incubation.
Sodium Hydrogen Selenite - 4.0 Interpretation of Results
Final pH (at 250C) 7.0 ± 0.2
1. After incubation, there must be an increase in the number of pathogens that
* Formula adjusted to suit performance parameters
the medium is designed to select for and enrich.
Directions
1. Suspend 19 gms of the Part A & 4 gm of the Part B powder in 1000 ml 2. Subculture onto any combination of greater and lesser inhibitory, selective and
distilled water. differential media for Enterobacteriaceae. e.g. MacConkey Agar, XLD Agar, etc
to isolate pathogens for identification.
2. Mix thoroughly.
Precautions / Limitations
3. Boil with frequent agitation to dissolve the powder completely. Sterilize by
1. Discard the prepared medium if large amounts of reduced selenite can be seen
maintaining at 1000C for 30 minutes. AVOID OVERHEATING. DO NOT
as a red precipitate at the bottom of the tube.
AUTOCLAVE. Excessive heating is detrimental.
2. Do not incubate for longer than 24 hours because the inhibitory effect of
4. Discard the prepared medium if a large amount of selenite is reduced, which
selenite is reduced after 6-12 hours incubation and coliforms may overgrow
is indicated by a red precipitate at the bottom of the tube/bottle.
the pathogens.
Quality Control
Dehydrated Appearance 3. Take subcultures from the upper third of the broth column which should be at
Cream coloured, homogeneous, free flowing powder. least 5 cm in depth.
Prepared Appearance Warning: Sodium Hydrogen Selenite is very toxic, corrosive and causes
Light yellow coloured, clear to very slightly opalescent solution, may have a teratogenicity. Handle with care. On contact with skin wash immediately
slight precipitate. with plenty of water.
Cultural Response 4. Enrichment broths should not be used as the sole isolation medium.
0
Cultural characteristics after 18-24 hours at 35-37 C when subcultured on 5. Use in conjunction with selective and non-selective plating media to increase
MacConkey Agar (AM10590/ AM50590).
the chances of isolating pathogens, particularly when they may be present in
Organisms (ATCC) Recovery Colour of Colony
Escherichia coli (25922) Little to none Pink with bile precipitate
small numbers.
Salmonella serotype Good to excellent Colourless Storage
Choleraesuis (12011) Store at 22-300C and prepared medium at 2-80C.
Salmonella serotype Good to excellent Colourless Shelf Life
Typhimurium (14028) Use before expiry date as mentioned on the label.
Procedure
1. For faeces and other solid materials, suspend 1-2 gms of the specimen in the
broth (approximately 10-15% by volume) and emulsify if necessary.

SIM Medium AM50892

Use Enterobacter and Serratia (6.2).
SIM Medium is used for differentiation of enteric bacteria on the basis of sulphide In SIM medium these two tests have been combined with sulphide-production
production, indole production and motility. test. The production of hydrogen sulphide is a useful diagnostic test in the
Summary identification of enteric bacteria and is helpful in the differentiation between
Tests for indole production and motility are very commonly used in the Salmonella and Shigella. Since these organisms are encountered very often in
identification of microorganisms in the diagnostic microbiology laboratory. A clinical material, the use of a there in one test can result in a substantial saving of
motility-indole medium has been found to be helpful in the identification of the materials and time.
Enterobacteriaceae, and especially in the differentiation of Klebsiella from

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Principle Prepared Appearance
Peptic digest of animal tissue serves as a source of carbon, nitrogen, vitamins and Semisolid, Medium amber coloured, Slightly opalescent gel forms in tubes.
Cultural Response
minerals. sodium thiosulphate and peptonized iron are indicators of hydrogen
Cultural characteristics after 18-24 hours at 35-370C.
sulfide production. hydrogen sulphide reacts with peptonized iron to form black
Organisms (ATCC) Growth Motility H2S INDOLE
precipitate of ferrous sulphide. The use of only 0.30% agar in the medium
Escherichia coli (25922) Luxuriant + _ +
results in the production of a semi-solid medium, ideal for the examination of S. serotype Typhimurium (14028) Luxuriant + + _
motility. Non-motile organisms will grow along the line of inoculation only, Shigella flexneri (12022) Luxuriant _ _ _
whereas motile species will grow away from it. Key: + = Positive reaction
H2S + = Blackening of the medium
Peptic digest of animal tissue is rich in tryptophan, which is attacked by certain
Procedure
microorganisms resulting in the production of indole, which is detected by the
addition of Kovac's reagents following the incubation period. 1. Charge straight wire inoculation loop with test organism from a pure culture.
Formula* 2. Inoculated once by inserting a straight wire to about one third of the depth of
Ingredients in grams per liter the medium in the center of the tube.
PPeptic digest of animal tissue 30.0
3. Incubate tubes with loosened caps for 18-24 hours at 35 ± 2°C in an
Beef extract 3.0
aerobic atmosphere.
Peptonized Iron 0.20
Sodium thiosulphate 0.025 Interpretation of Results
Agar 3.0 Observe for motility (diffuse growth outward from the stab line or turbidity
Final pH (at 250C) 7.3 ± 0.2 throughout the medium) and for H2S production (blackening along the stab line).
* Formula adjusted to suit performance parameters
Directions
For indole test add 0.2 ml of Kovac's Reagent to the tube and allow to stand for 10
minutes. A dark red colour in the reagent constitutes a positive indole test. No
1. Suspend the 36.23 gms of powder in 1000 ml distilled water.
change in the original colour of the reagent constitutes a negative test.
2. Mix thoroughly. Storage
3. Warm slightly with frequent agitation to dissolve the powder completely. Store at 22-300C and prepared medium at 2-80C.
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes. Shelf Life
Quality Control Use before expiry date as mentioned on the label.
Dehydrated Appearance
Yellow coloured, homogeneous, free flowing powder.

Simmons Citrate Agar BIS AM109011/AM509011

Use Bacteriological Analytical Manual for food and cosmetics analysis (113).
Simmons Citrate Agar is used for the differentiation of gram-negative bacteria on Principle
the basis of citrate utilization in compliance with BIS. Ammonium dihydrogen phosphate and sodium citrate serve as the sole nitrogen
Summary and carbon source respectively while bromothymol blue is the pH indicator.
Simmons Citrate Agar is recommended by APHA (20,39) and is used for the Organisms able to utilize the above compounds as sole source of nitrogen and
differentiation of Enterobacteriaceae and members of the aerogenes group on carbon, grow on this medium and produce an alkaline reaction as indicated by
the basis of citrate utilization. Koser (61) developed a liquid medium containing the change in colour of bromothymol blue indicator from green (neutral) to blue
ammonium salt as the only source of nitrogen and citrate as the only source of (alkaline).
carbon to differentiate between Escherichia coli and Enterobacter aerogenes Formula*
based on the IMVIC reactions. Simmons (103) later on, modified this medium Ingredients in grams per liter
with the addition of agar and bromothymol blue. Organisms capable of utilizing Sodium chloride 5.0
citrate grow well on this medium. Simmons Citrate Agar is included in the Sodium citrate 2.0

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Dipotassium phosphate 1.0 Procedure
Ammonium dihydrogen phosphate 1.0 1. The medium may be used as slants or as a plate medium in petri plates.
Magnesium sulphate 0.2
2. Inoculate with growth from a pure culture using a light inoculum.
Bromothymol blue 0.08
Agar 15.0
3. Incubate for 48 hours at 35-370C in an aerobic atmosphere.
Interpretation of Results
Final pH (at 250C) 6.8 ± 0.1
* Formula adjusted to suit performance parameters 1. A positive reaction (citrate utilization) is indicated by growth with an intense
Directions blue colour in the slant. A negative reaction is evidenced by no growth to
1. Suspend 24.28 gms of the powder in 1000 ml distilled water. slight growth with no change in colour of the medium.
2. Mix thoroughly. 2. E.coli including different serotypes from epidemic infantile enteritis, as well
as Shigella, Yersinia and Edwardsiella species do not grow on this medium.
3. Boil with frequent agitation to dissolve the powder completely.
Serratia and the majority of Enterobacter, Citrobacter, Klebsiella, Proteus
4. Dispense in tubes or as desired. and Providencia species, except Morganella morgani and Klebsiella
5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. rhinoscleromatis utilize citrate and produce the characteristic blue
6. Cool in slanted position for use as slants. colouration.
A Differential Dehydrated Culture Medium 3. This medium may also be able to differentiate citrate positive Salmonella
Quality Control enteritidis and members of Salmonella subgenus from the citrate negative
Dehydrated Appearance S.typhi, S.paratyphi A, S.pulloram and S.gallinarum.
Yellow coloured, homogeneous, free flowing powder. Precautions / Limitations
Prepared Appearance 1. Do not carry over any nutrients into the medium as it may lead to
Medium to dark green, clear to slightly opalescent gel. falsepositive results. Dilute the inoculum before inoculating the
Cultural Response medium to avoid a carry over of other carbon sources. Use a light inoculum
Cultural characteristics after 18-24 hours at 35-370C.
while streaking.
Organisms (ATCC) Growth Colour of Citrate
Storage
Medium Utilization
Enterobacter aerogenes (13048) Good to luxuriant Blue + Store at 22-300C and prepared medium at 2-80C.
Escherichia coli (25922) Inhibited Green - Shelf Life
Salmonella serotype Good to luxuriant Blue +
Use before expiry date as mentioned on the label.
Enteritidis (13076)
Salmonella serotype Good to luxuriant Blue +
Typhimurium (14028)
Shigella dysenteriae (13313) Inhibited Green -
Klebsiella Pneumoneae (33495) Good to luxuriant Green to -
Light blue

Simmons Citrate Agar AM1090/AM5090

Use with the addition of agar and bromothymol blue. Organisms capable of utilizing
Simmons Citrate Agar is used for the differentiation of gram-negative bacteria on citrate grow well on this medium. Simmons Citrate Agar is included in the
the basis of citrate utilization. Bacteriological Analytical Manual for food and cosmetics analysis (113).
Summary Principle
Simmons Citrate Agar is recommended by APHA (20, 39) and is used for the Ammonium dihydrogen phosphate and sodium citrate serve as the sole nitrogen
differentiation of Enterobacteriaceae and members of the aerogenes group on and carbon source respectively while bromothymol blue is the pH indicator.
the basis of citrate utilization. Koser (61) developed a liquid medium containing Organisms able to utilize the above compounds as sole source of nitrogen and
ammonium salt as the only source of nitrogen and citrate as the only source of carbon, grow on this medium and produce an alkaline reaction as indicated by the
carbon to differentiate between Escherichia coli and Enterobacter aerogenes change in colour of bromothymol blue indicator from green (neutral) to blue
based on the IMViC reactions. Simmons (103) later on, modified this medium (alkaline).

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Formula* (14028)
Ingredients in grams per liter Shigella dysenteriae (13313) Inhibited Green -
Sodium Chloride 5.0 Klebsiella Pneumoneae (33495) Good to luxuriant Green to
Sodium Citrate 2.0 light blue
Dipotassium Phosphate 1.0 Procedure
Ammonium Dihydrogen Phosphate 1.0 1. The medium may be used as slants or as a plate medium in petri plates.
Magnesium Sulphate 0.2
2. Inoculate with growth from a pure culture using a light inoculum.
Bromothymol Blue 0.08
Agar 15.0 3. Incubate for 48 hours at 35-370C in an aerobic atmosphere.
Final pH (at 250C) 6.8 ± 0.2 Interpretation of Results
* Formula adjusted to suit performance parameters 1. A positive reaction (citrate utilization) is indicated by growth with an intense
Directions blue colour in the slant. A negative reaction is evidenced by no growth to
1. Suspend 24.28 gms of the powder in 1000 ml distilled water. slight growth with no change in colour of the medium.
2. Mix thoroughly. 2. E.coli including different serotypes from epidemic infantile enteritis, as well
3. Boil with frequent agitation to dissolve the powder completely. as Shigella, Yersinia and Edwardsiella species do not grow on this medium.
Serratia and the majority of Enterobacter, Citrobacter, Klebsiella, Proteus
4. Dispense in tubes or as desired.
and Providencia species, except Morganella morgani and Klebsiella
5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
rhinoscleromatis utilize citrate and produce the characteristic blue
6. Cool in slanted position for use as slants. colouration.
Quality Control
3. This medium may also be able to differentiate citrate positive Salmonella
Dehydrated Appearance
enteritidis and members of Salmonella subgenus 2, 3 and 5 from the
Yellow coloured, homogeneous, free flowing powder.
Prepared Appearance citrate negative S.typhi, S.paratyphi A, S.pulloram and S.gallinarum.
Medium to dark green, clear to slightly opalescent gel. Precautions / Limitations
Cultural Response 1. Do not carry over any nutrients into the medium as it may lead to false
Cultural characteristics after 18-24 hours at 35-370C. positive results. Dilute the inoculum before inoculating the medium to avoid
Organisms (ATCC) Growth Colour of Citrate a carry over of other carbon sources. Use a light inoculum while streaking.
Medium Utilization
Storage
Enterobacter aerogenes (13048) Good to luxuriant Blue +
Escherichia coli (25922) Inhibited Green - Store below 300C and prepared medium at 2-80C.
Salmonella serotype Enteritidis Good to luxuriant Blue + Shelf Life
(13076) Use before expiry date as mentioned on the label.
Salmonella serotype Typhimurium Good to luxuriant Blue +

Skim Milk Agar AM10901/AM50901

Use elements to the organisms. Dextrose is the fermentable carbohydrate. Agar is the
Skim Milk Agar is used for the cultivation and enumeration of microorganisms in solidification agent.
milk and dairy products. Formula*
Summary Ingredients in grams per liter
Skim Milk Agar is used for detecting proteolytic microorganisms that hydrolyse Skim Milk Powder 28.0
Tryptone 5.0
casein (milk protein) and cause coagulation (clot formation) in dairy products
Yeast Extract 2.5
(105). Proteolytic microorganisms hydrolyse casein to form soluble nitrogenous
Dextrose 1.0
indicated as clear zone surrounding the colonies (20, 39). Agar 15.0
Principle Final pH (at 250 C) 7.0 ± 0.2
Skim milk powder is a source of casein. Tryptone and yeast extract provide the * Formula adjusted to suit performance parameters
essential nutrients of nitrogen, carbon, sulphur, vitamin B complex and trace

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Directions Proteus mirabilis (25933) Luxuriant + More than 70%
1. Suspend 51.5 grams of the powder in 1000 ml distilled water. Enterococcus faecalis Luxuriant - More than 70%
cescens (8100) Luxuriant + More than 70%
2. Boil with frequent agitation to dissolve the powder completely. For growth RGI should be more than 70%
3. Sterilize by autoclaving at 1210 C (15 lbs pressure) for 15 minutes. RGI- Relative Growth Index
Quality Control Procedure
Dehydrated Appearance 1. Standard operating techniques like streak plate method may be used for
Light yellow coloured, homogeneous, free flowing powder. isolating organisms.
Prepared Appearance
Interpretation of Results
Off-white coloured opaque gel.
Cultural Response
1. Proteolyitc bacteria produce a clear zone surrounding the colonies.
Storage
Cultural response after 18 - 24 hours at 35 - 370 C.
Organisms Growth Proteolyitc RGI Store below 300C and prepared medium at 2-80C.
(ATCC) Activity Shelf Life
Bacillus subtilis (6633) Luxuriant + More than 70% Use before expiry date as mentioned on the label.
Escherichia coli (25922) Luxuriant _ More than 70%
Pseudomonas aeruginosa Luxuriant + More than 70%
(27853)

Soyabean Casein Digest Agar (Tryptone Soya Agar) AM1091/AM5091

Soyabean Casein Digest Broth (Tryptone Soya Broth) AM1092/AM5092
Use the detection of contamination with low incidence fungi and aerobic bacteria and
Soyabean Casein Digest Agar and Soyabean Casein Digest Broth are general- in the performance of microbial limit test. It is used in the coliphage detection
purpose media used for the isolation and cultivation of a wide variety of fastidious procedure, a Methodology in Standard Methods for the Examination of Water and
and non-fastidious microorganisms. Wastewater.
Summary Soyabean Casein Digest Agar and Broth are included in the Bacteriological
Soyabean Casein Digest Agar (SCDA) is used for total aerobic microbial count and Analytical Manual for food and cosmetics testing (113), in the Compendia of
antimicrobial preservative-effective test. It is also used for testing bacterial Methods for the examination of milk (39), water and wastewater (36) and foods
contaminants in cosmetics and for a multitude of purpose including maintenance (20) and is also specified in the USP (113) and IP (46).
of stock cultures, plate counts, phage typing, colicin typing and as a base for Principle
media containing blood. Since this medium does not contain the X and V growth The combination of tryptone and soya peptone makes the medium highly
factors, it can be used for determining the requirements of these growth factors by nutritious by supplying organic nitrogen, particularly amino acids and long chain
isolates of Haemophilus. peptides. Sodium chloride maintains the osmotic balance. Soyabean Casein
Gunn et al used this medium for the study of haemolytic reactions after addition of Digest Agar and Soyabean Casein Digest Broth may be supplemented with blood
5% v/v blood. When Chocolate Agar is prepared, this medium supports good to provide a more nutritious medium for fastidious organisms, or with
growth of Neisseria species and Haemophilus influenzae. antimicrobials to provide a selective medium for specific organisms out of a mixed
Soyabean Casein Digest Broth (SCDB) is widely used for the cultivation of flora sample. Since Soyabean Casein Digest Agar contains no added
microorganisms from environmental sources, supporting the growth of a wide carbohydrate, it may be used with added blood to determine haemolysis.
variety of microorganisms including common aerobic, facultative and anaerobic When Soyabean Casein Digest Agar is supplemented with 0.7 gms lecithin and 5
bacteria and fungi. It is also used for preparing dilutions of organisms for colony gms polysorbate (Tween 80) per liter of medium, it can be used as microbial
counts and preparation of standard inocula for disc diffusion and dilution content test agar for testing quaternary ammonium compounds (collection of
antimicrobial susceptibility testing as standardized by the National Committee for samples from identical areas before and after treatment with disinfectant yields
Clinical Laboratory Standards (NCCLS). This medium is used in sterility testing for data useful in evaluating cleaning procedures in environmental sanitation).

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Formula* 2. If the specimen to be cultured is on a swab, roll the swab over a small area of
Ingredients in grams Soyabean Casein Soyabean Casein the agar surface.
per liter Digest Agar Digest Broth
Tryptone 15.0 17.0
3. Streak for isolation with a sterile loop.
Soya Peptone 5.0 3.0 4. Incubate plates aerobically at 35-370C for 18-24 hours.
Sodium Chloride 5.0 5.0 5. Since many pathogens require CO2 on primary isolation, plates may be
Dextrose - 2.5
incubated in an atmosphere containing 3-10% CO2.
Dipotassium Phosphate - 2.5
Agar 15.0 - For Quantitative test
Final pH (at 250C) 7.3 ± 0.2 7.3 ± 0.2 1. Prepare decimal dilutions of the sample in a sterile diluent to obtain 30-300
* Formula adjusted to suit performance parameters colony forming units per plate.
Directions
2. Inoculate using the pour plate or streak plate technique.
1. Suspend the powder in 1000 ml distilled water and mix thoroughly.
3. Incubate plates aerobically for 48 hours at 350C.
Soyabean Casein Digest Agar - 40.0 gms
Note: After autoclaving, do not heat the medium longer than 3 hours at 45-
Soyabean Casein Digest Broth - 30.0 gms
500C. Sterile solidified medium can be remelted only once.
2. Boil with frequent agitation to dissolve the powder completely. DO NOT
For Soyabean Casein Digest Broth
OVERHEAT.
Aerobic and anaerobic cultivation
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
1. Swab specimens may be inserted into the medium after inoculation of
4. To prepare Blood Agar plates, add 5-10% sterile, defibrinated blood to the appropriate plated media.
sterile agar cooled to 45-500C.
2. For liquid specimens, use a sterile inoculating loop to transfer a loopful of the
Quality Control
specimen to the broth medium.
Dehydrated Appearance
Light yellow coloured, homogeneous, free flowing powder. 3. Specimens known or suspected to contain obligate anaerobes should be
Prepared Appearance inoculated near the bottom of the tube.
Soyabean Casein Digest Agar - Basal Medium - Light yellow coloured, clear to 4. Incubate the containers with loosened caps at 35-370C aerobically with or
slightly opalescent gel.
without supplementation with CO2.
With the addition of blood - Cherry red coloured gel.
Soyabean Casein Digest Broth - Light yellow coloured, clear solution. 5. Containers intended for cultivation of anaerobes must be incubated under
Cultural Response anaerobic conditions.
Cultural characteristics after 18-48 hours at 35-370C. 6. Examine the growth after 18-24 hours and 42-48 hours of incubation.
Organisms (ATCC) Growth without Growth Haemolysis
Blood culture
blood on SCDA with blood on SCDA
and in SCDM on SCDA 1. The superior growth promoting properties of Soyabean Casein Digest Broth
Candida albicans (10231) Luxuriant Luxuriant None makes it especially useful for the isolation of organisms from blood or other
Escherichia coli (25922) Luxuriant Luxuriant Beta body fluids.
Neisseria meningitidis Luxuriant Luxuriant None 2. Anticoagulants such as 'liquoid' (Sodium polyanethyl sulphonate) or sodium
(13090) citrate may be added to the broth prior to sterilization.
Staphylococcus aureus Luxuriant Luxuriant Beta
3. 5-10 ml of blood may be added to 50 ml of medium.
(25923)
Streptococcus pyogenes Good to luxuriant Luxuriant Beta Selective culture media
(19615) 1. Soyabean Casein Digest Broth is used in food bacteriology as the basal
Procedure medium to which a variety of selective agents are added for selective
For Soyabean Casein Digest Agar enrichment of Staphylococcus aureus and Escherichia coli 0157.
1. Allow the agar surface to dry before inoculating. Inoculate and streak the Interpretation of Results
specimen as soon as possible after collection. Soyabean Casein Digest Agar

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1. Count the number of colonies and express as colony forming units (CFU) per Precautions / Limitations
gram or ml of sample, taking into account the applicable dilution factor. 1. Haemolytic reactions of streptococci on this medium can vary according to
2. Subculture colonies of interest so that positive identification can be made the origin of the blood.
by means of other tests. 2. The medium designed for sheep blood shows significant differences when
Soyabean Casein Digest Broth used with horse blood and vice versa.
Storage
1. Growth in broth medium is indicated by the presence of turbidity compared
to an un-inoculated control. Store below 300C and prepared medium at 2-80C.
Shelf Life
2. Broth cultures should be held at least for a week before discarding as
negative. Use before expiry date as mentioned on the label.

Soyabean Casein Digest Agar (Harmonized) AMH5091

Soyabean Casein Digest Agar AM10911/AM50911
(Casein Soyabean Digest Agar) IP
Soyabean Casein Digest Agar Medium USP AM10912/AM50912
Soyabean Casein Digest Agar AM10913/AM50913
(Agar Medium B)(Casein Soyabean Digest Agar) EP
Soyabean Casein Digest Agar (Agar Medium B) AM10914/AM50914
(Casein Soyabean Digest Agar) BP
Use meal makes the medium highly nutritious by supplying organic nitrogen,
Soyabean Casein Digest Agar is general-purpose media used for the isolation particularly amino acids and long chain peptides. Sodium chloride maintains the
and cultivation of a wide osmotic balance. In Soyabean Casein Digest Medium Dextrose is an energy source
variety of fastidious and non-fastidious microorganisms. and Dipotassium phosphate acts as a buffer to control pH. Soyabean Casein
Summary Digest Agar may be supplemented with blood to provide a more nutritious
Soyabean Casein Digest Agar (SCDA) is used for total aerobic microbial count and medium for fastidious organisms, or with antimicrobials to provide a selective
antimicrobial preservative-effective test. It is also used for testing bacterial medium for specific organisms out of a mixed flora sample. Since Soyabean
contaminants in cosmetics and for a multitude of purpose including maintenance Casein Digest Agar contains no added carbohydrate, it may be used with added
of stock cultures, plate counts, phage typing, colicin typing and as a base for blood to determine haemolysis. When Soyabean Casein Digest Agar is
media containing blood. Since this medium does not contain the X and V growth supplemented with 0.7 gms lecithin and 5 gms polysorbate (Tween 80) per liter
factors, it can be used for determining the requirements of these growth factors of medium, it can be used as microbial content test agar for testing quaternary
by isolates of Haemophilus. Gunn et al., used this medium for the study of ammonium compounds (collection of samples from identical areas before and
haemolytic reactions after addition of 5% v/v blood. When Chocolate Agar is after treatment with disinfectant yields data useful in evaluating cleaning
prepared, this medium supports good growth of Neisseria species and procedures in environmental sanitation).
Haemophilus influenzae. Soyabean Casein Digest Agar is included in the Formula*
Bacteriological Analytical Manual for food and cosmetics testing (113), in the Ingredients in grams per liter
Pancreatic digest of casein 15.0
Compendia of Methods for the examination of milk (39), water and wastewater
Papic digest of soyabean meal 5.0
(36) and foods (20).
Sodium chloride 5.0
Principle
Agar 15.0
The combination of Pancreatic Digest of casein and Papic Digest of soyabean Final pH (at 250C) 7.3 ± 0.2

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* Formula adjusted to suit performance parameters For growth RGI should be more than 70%
Directions RGI- Relative Growth Index
1. Suspend 40.0 gms of the powder in 1000 ml distilled water and mix Procedure
thoroughly.
1. Allow the agar surface to dry before inoculating. Inoculate and streak the
2. Boil with frequent agitation to dissolve the powder completely. DO
NOT OVERHEAT. specimen as soon as possible after collection.
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. 2. If the specimen to be cultured is on a swab, roll the swab over a small area of
4. To prepare Blood Agar plates, add 5-10% sterile, defibrinated blood to the agar surface.
the sterile agar cooled to 45-500C.
3. Streak for isolation with a sterile loop.
Quality Control
Dehydrated Appearance 4. Incubate plates aerobically at 35-370C for 18-24 hours.
Light yellow coloured, homogeneous, free flowing powder. 5. Since many pathogens require CO2 on primary isolation, plates may 2 be
Prepared Appearance
incubated in an atmosphere containing 3-10% CO2 .
Basal Medium - Light yellow coloured, clear to slightly opalescent gel.
Cultural Response For Quantitative test
0
Cultural characteristics after 18-48 hours at 35-37 C for bacteria and for fungi 1. Prepare decimal dilutions of the sample in a sterile diluent to obtain 30-300
2-5 days at 20-250C. colony forming units per plate.
Organisms(ATCC) Growth RGI
Candida albicans (10231) Luxuriant More than 70%
2. Inoculate using the pour plate or streak plate technique.
Escherichia coli (25922) Luxuriant More than 70% 3. Incubate plates aerobically for 48 hours at 350C.
Staphylococcus aureus (6538) Luxuriant More than 70% Note: After autoclaving, do not heat the medium longer than 3 hours at 45-50 C.
0

Streptococcus pyogenes (19615) Good to luxuriant More than 70%

Sterile solidified medium can be remelted only once.
Clostridium sporogenes (11437) Luxuriant More than 70%
Storage
Bacillus subtilis (6633) Luxuriant More than 70%
Aspergillus niger (16404) Luxuriant More than 70% Store at 22-300C and prepared medium at 2-80C.
Streptococcus pneumonia (6303) Luxuriant More than 70% Shelf Life
Pseudomonas aeruginosa (9027) Luxuriant More than 70% Use before expiry date as mentioned on the label.

Soyabean Casein Digest Broth (Harmonized) AMH5092

Soyabean Casein Digest Medium IP AM10921/AM50921
Soyabean Casein Digest Medium EP AM10923/AM50923
Soyabean Casein Digest Medium BP AM10924/AM50924
Soyabean Casein Digest Medium USP AM10922/AM50922
Use antimicrobial susceptibility testing as standardized by the National Committee
Soyabean Casein Digest Medium is general-purpose media used for the isolation for Clinical Laboratory Standards (NCCLS). This medium is used in sterility testing
and cultivation of a wide variety of fastidious and non-fastidious for the detection of contamination with low incidence fungi and aerobic bacteria
microorganisms. and in the performance of microbial limit test. It is used in the coliphage
Summary detection procedure, a Methodology in Standard Methods for the Examination of
Soyabean Casein Digest Medium (SCDM) is widely used for the cultivation of Water and Wastewater. Soyabean Casein Digest Agar and Medium are included
microorganisms from environmental sources, supporting the growth of a wide in the Bacteriological Analytical Manual for food and cosmetics testing, (113), in
variety of microorganisms including common aerobic, facultative and anaerobic the Compendia of Methods for the examination of milk (39), water and
bacteria and fungi. It is also used for preparing dilutions of organisms for colony wastewater (36) and foods (20).
counts and preparation of standard inocula for disc diffusion and dilution

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Principle Organisms(ATCC) Growth
The combination of Pancreatic Digest of casein and Papic Digest of soyabean meal Candida albicans (10231) Luxuriant
makes the medium highly nutritious by supplying organic nitrogen, particularly Escherichia coli (25922) Luxuriant
Neisseria meningitidis (13090) Luxuriant
amino acids and long chain peptides. Sodium chloride maintains the osmotic
Staphylococcus aureus (6538) Luxuriant
balance. In Soyabean Casein Digest Medium Dextrose is an energy source and
Streptococcus pyogenes (19615) Good to luxuriant
Dipotassium phosphate acts as a buffer to control pH. Soyabean Casein Digest Pseudomonas aeruginosa (9027) Luxuriant
Agar and Soyabean Casein Digest Medium may be supplemented with blood to Bacillus subtilis (6633) Luxuriant
provide a more nutritious medium for fastidious organisms, or with antimicrobials Bacteroides vulgatus (8482) Luxuriant
to provide a selective medium for specific organisms out of a mixed flora sample. Clostridium sporogenes (11437) Luxuriant
Since Soyabean Casein Digest Agar contains no added carbohydrate, it may be Aspergillus niger (16404) Luxuriant
used with added blood to determine haemolysis. When Soyabean Casein Digest Mucococcus luteus (9341) Luxuriant
Procedure
Agar is supplemented with 0.7 gms lecithin and 5 gms polysorbate (Tween 80)
per liter of medium, it can be used as microbial content test agar for testing Aerobic and anaerobic cultivation
quaternary ammonium compounds (collection of samples from identical areas 1. Swab specimens may be inserted into the medium after inoculation of
before and after treatment with disinfectant yields data useful in evaluating appropriate plated media.
cleaning procedures in environmental sanitation). 2. For liquid specimens, use a sterile inoculating loop to transfer a loopful of the
Formula* specimen to the broth medium.
Ingredients in grams per liter
3. Specimens known or suspected to contain obligate anaerobes should be
SCDM SCDM SCDM SCDM Harmonized
IP USP EP BP
inoculated near the bottom of the tube.
Pancreatic digest of casein 17.0 17.0 17.0 17.0 17.0 4. Incubate the containers with loosened caps at 35-370C aerobically with or
Papic digest of soyabean Meal 3.0 3.0 3.0 3.0 3.0 without supplementation with CO2 .
Sodium chloride 5.0 5.0 5.0 5.0 5.0
5. Containers intended for cultivation of anaerobes must be incubated under
Dibasic potassium phosphate – 2.5 – – –
Dipotassium hydrogen 2.5 – 2.5 2.5 2.5
anaerobic conditions.
phosphate 6. Examine the growth after 18-24 hours and 42-48 hours of incubation.
Dextrose – – – – – Blood culture
Dextrose monohydrate 2.5 2.5 – – –
1. The superior growth promoting properties of Soyabean Casein Digest Medium
Glucose monohydrate – – 2.5 2.5 2.5
Final pH (at 250C) 7.3 ± 0.2 makes it especially useful for the isolation of organisms from blood or other
* Formula adjusted to suit performance parameters body fluids.
Directions 2. Anticoagulants such as 'liquoid' (Sodium polyanethyl sulphonate) or sodium
1. Suspend 30.0 gms of the powder in 1000 ml distilled water and mix citrate may be added to the broth prior to sterilization.3. 5-10 ml of blood
thoroughly.
may be added to 50 ml of medium.
2. Boil with frequent agitation to dissolve the powder completely. DO NOT
OVERHEAT. Selective culture media
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. 1. Soyabean Casein Digest Medium is used in food bacteriology as the basal
4. To prepare Blood Agar plates, add 5-10% sterile, defibrinated blood to the medium to which a variety of selective agents are added for selective
sterile agar cooled to 45-500C.
enrichment of Staphylococcus aureus and Escherichia coli 0157.
Quality Control
Interpretation of Results
Dehydrated Appearance
Light yellow coloured, homogeneous, free flowing powder. 1. Growth in broth medium is indicated by the presence of turbidity compared to
Prepared Appearance an un-inoculated control.
Light yellow coloured, clear solution. 2. Broth cultures should be held at least for a week before discarding as
Cultural Response
negative.
Cultural characteristics after 48-72 hours at 30-350C for bacteria and 2-5 days
at 20-250C for fungi.

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Precautions / Limitations Storage
1. Haemolytic reactions of streptococci on this medium can vary according to Store at 22-300C and prepared medium at 2-80C.
the origin of the blood. Shelf Life
2. The medium designed for sheep blood shows significant differences when Use before expiry date as mentioned on the label.
used with horse blood and vice versa.

Soyabean Casein Digest Medium Sterile Powder AM50925/AM50925-5K

(girradiated)
Use Dextrose 2.5
Soyabean Casein Digest Medium, sterile powder is used for the evaluation of Dipotassium phosphate 2.5
Final pH (at 250C) 7.3 + 0.2
sterility in manufacturing process.
* Formula adjusted to suit performance parameters
Summary
Directions
Routine sampling for sterility testing is not sensitive enough to detect any low
1. For sterile liquid medium aseptically suspend the 30.0 gms medium in
level contamination in sterile pharmaceutical formulations. Sample numbers are
1000 ml sterile distilled water.
too small and only gross contamination is likely to be detected. Pharmaceutical
manufactures therefore need other means of guaranteeing the quality of their 2. Boil with frequent agitation to dissolve the powder completely.
product. This is why process stimulations (Media fill run) supported by 3. DO NOT AUTOCLAAVE OR OVERHEAT
environmental monitoring is must in pharmaceutical industry. 4. Dispense aseptically in desired sterile containers.
The new FDA guidelines pay particular attention to this aspect of aseptic 5. Carry out all operations in aseptic condition.
processing and it is becoming an area requiring more work and focus to satisfy the Quality Control
regulators. The FDA guidelines have recommended using Soyabean Casein Dehydrated Appearance
Digest Medium – a highly nutritious general-purpose medium that is ideal for Light yellow coloured, homogeneous, free flowing powder.
microbiological media fill. It also recommended that in order to more closely Prepared Appearance
Light yellow coloured, clear solution.
mimic the process, the culture medium should be filtered into the process, just as
Cultural Response
would occur to liquid pharmaceutical product. Regular dehydrated culture media
Cultural characteristics after 48-72 hours at 30-350C for bacteria and 2-5 days
is usually supplied in non-sterile form, which carries a high bioburden and should at 20-250C for fungi.
not taken directly into a controlled area. Therefore g irradiated, sterile SCDM Organisms(ATCC) Growth
powder is use for media fills. Irradiation of media also assures the sterile medium Candida albicans (10231) Luxuriant
is free from Mycoplasma. Escherichia coli (25922) Luxuriant
Principle Neisseria meningitides (13090) Luxuriant
Staphylococcus aureus (25923) Luxuriant
The combination of Tryptone and Soya peptone makes the medium highly
Streptococcus pyogenes (19615) Good to luxuriant
nutritious by supplying organic nitrogen, particularly amino acids and long chain Pseudomonas aeruginosa (9027) Luxuriant
peptides. Sodium Chloride maintains osmotic balance. This medium, which has Aspergillus niger (16404) Luxuriant
sterilized by gamma irradiation, can be directly used for media-fill runs as Storage
recommended by FDA guidelines. Store at 22-300C and prepared medium at 2-80C.
Formula*
Shelf Life
Ingredients in grams per liter
Tryptone 17.0
Use before expiry date as mentioned on the label.
Soya peptone 3.0
Sodium chloride 5.0

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Soyabean Casein Digest Medium with Lecithin & Tween 80 AM50927
Use Dextrose 2.50
Soyabean Casein Digest Medium with Lecithin & Tween 80 (Tryptose Soya Broth, Dipotassium phosphate 2.50
Lecithin 0.70
Antibiotic Assay Medium No. 37) is used for the isolation and cultivation of a
Tween 80 5.00
wide variety of microorganisms.
Final pH (at 250C) 7.3 ± 0.2
Summary
* Formula adjusted to suit performance parameters
Soyabean Casein Digest Medium with Lecithin & Tween 80 (Tryptose Soya Agar) Directions
is a nutritious base and a variety of supplements are added to enhance the 1. Suspend 35.7 gms of the powder in 1000 ml distilled water and mix
medium, including Lecithin and Tween 80. The Lecithin and Tween 80 inactivate thoroughly.
some preservatives that may inhibit bacterial growth, reducing “Preservative 2. Boil with frequent agitation to dissolve the powder completely. DO NOT
OVERHEAT.
carryover”.
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Principle
Quality Control
The combination of Tryptone and Soya peptone makes the medium highly Dehydrated Appearance
nutritious by supplying organic nitrogen, particularly amino acids and long chain Light yellow coloured, homogeneous, free flowing powder.
peptides. Sodium Chloride maintains osmotic balance. Dextrose is the energy Prepared Appearance:
source. Dipotassium phosphate act as a buffering system to control pH. Tween Light yellow coloured, clear solution.
80 and lecithin act as neutralizers to inactivate the residual disinfectants where Cultural Response
Cultural characteristics after 24-48 hours at 300C.
the samples are collected. Lecithin inactivates quaternary ammonium
Organisms(ATCC) Growth
compounds whereas tween 80 neutralizes formalin, phenolic disinfectants,
Listeria monocytogenes (19111) Good to luxuriant
hexachlorophene etc. Agar is the solidifying agent. Listeria monocytogenes (19118) Good to luxuriant
Formula* Storage
Ingredients in grams per liter
Store at 22-300C and prepared medium at 2-80C.
Tryptone 17.00
Shelf Life
Soya peptone 3.00
Sodium chloride 5.00 Use before expiry date as mentioned on the label.

SS Agar (Salmonella Shigella Agar) AM1093/AM5093

Use a few lactose fermenting normal intestinal flora, acid is produced, which is
SS Agar is a differential and selective medium used for the isolation of Salmonella indicated by change in colour from yellow to red by the pH indicator neutral red
and some Shigella species from clinical and non-clinical specimens. and these organisms grow as red-pigmented colonies. Non-lactose fermenters
Summary grow as translucent colourless colonies. Sodium thiosulphate and ferric citrate
SS Agar is a modification of Deoxycholate Citrate Agar described by Leifson and is enable the detection of H2S production. Sodium thiosulphate is reduced by certain
an example of media used in the plating of samples for the detection of enteric species to sulphide and H2S gas in the presence of the enzyme thiosulphate
pathogens that contain bile salt mixtures. SS Agar is used in the performance of reductase. Production of H2S gas is detected as an insoluble black precipitate of
microbial limit tests and is recommended by APHA for the examination of foods ferrous sulphide, formed upon the reaction of H2S with ferric citrate, indicated as a
(20). black dot in the center of the colony. The high selectivity of SS Agar allows the use
Principle of large inocula directly from faeces, rectal swabs or other material suspected of
Peptone and beef extract provide sources of carbon, nitrogen and other growth containing the enteric pathogens.
factors. Brilliant green, bile salt mixture, thiosulphate and citrates selectively Formula*
inhibit gram-positive organisms and coliforms. Lactose is the fermentable Ingredients in grams per liter
carbohydrate and differentiation of enteric organisms is achieved based on Lactose 10.0
Sodium Citrate 10.0
lactose fermentation in the presence of neutral red. On fermentation of lactose by

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Bile Salts Mixture 8.5 RGI- Relative Growth Index

Sodium Thiosulphate 8.5 Procedure
Peptone 5.0
1. Use standard procedures like the streak plate method to obtain isolated
Beef Extract 5.0
Ferric Citrate 1.0
colonies.
Neutral Red 0.025 2. If the specimen to be cultured is on a swab, roll the swab on a small area of
Brilliant Green 0.00033 the agar surface and streak for isolation with a sterile loop.
Agar 15.0
3. Incubate plates aerobically, protected from light, at 35-370C for 18-24
Final pH (at 250C) 7.0 ± 0.2
* Formula adjusted to suit performance parameters hours.
Directions 4. If negative, incubate for an additional 24 hours.
1. Suspend 63 gms of the powder in 1000 ml distilled water. 5. Examine colony morphology.
2. Mix thoroughly. 6. Note: A non-selective medium should also be streaked to increase the
3. Boil with frequent agitation to dissolve the powder completely. chances of recovery when the population of gram-negative organisms is low
4. AVOID OVERHEATING. DO NOT AUTOCLAVE. and to provide an indication of other organisms present in the specimen.
5. Cool the medium to approximately 45-500C, pour into sterile petri plates. 7. In parallel with the SS Agar plate, inoculate a tube of Selenite Broth
enrichment medium, incubate for 12 hours at 350C, and subculture onto
6. Allow the plates to dry for about 2 hours with the covers partially removed
another SS Agar plate.
under aseptic conditions.
Interpretation of Results
Quality Control
Dehydrated Appearance Typical colonial morphology on SS Agar
Pinkish yellow coloured, homogeneous, free flowing powder. E.coli-------------------------- Slight growth, pink or red
Prepared Appearance Enterobacter/ Klebsiella------- Slight growth, pink
Reddish orange coloured, clear to slightly opalescent gel.
Cultural Response
Proteus------------------------ Slight growth, colourless, may have black center
Cultural characteristics after 18-24 hours at 35-370C. Salmonella-------------------- Good growth, colourless with black centers
Organisms (ATCC) Growth Colour of RGI Shigella----------------------- Slight or good growth, colourless
Colony
Pseudomonas----------------- Irregular, slight growth
Enterobacter aerogenes Poor to Cream pink 0%
(13048) good Gram-positive bacteria-------- No growth
Enterococcus None to Colourless 0% Precautions / Limitations
faecalis (29212) poor 1. This medium is highly selective; some strains of Shigella may not grow on it
Escherichia coli (25922) Poor to Pink with bile 0% and therefore must not be used for primary isolation of shigellae.
good precipitate
Proteus mirabilis (25933) Poor to Colourless may 0% 2. Inoculate plates of less inhibitory media like Deoxycholate Citrate Agar or XLD
good have black centre Agar for better isolation of Shigella species.
Salmonella serotype Good Colourless with More than 70% Storage
Enteritidis (13076) black centre Store below 300C and prepared medium at 2-80C.
Salmonella serotype Good Colourless with More than 70% Shelf Life
Typhimurium (14028) black centre
Use before expiry date as mentioned on the label.
Shigella flexneri (12022) Good Colourless More than 70%
without
black centre
For growth RGI should be more than 70%
For Inhibition RGI should be 0%

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SS Agar, Modified AM50931

Use Directions
SS Agar is a differential and selective medium used for the isolation of Salmonella 1. Suspend 57.0 gms of the powder in 1000 ml distilled water.
and some Shigella species from clinical and non-clinical specimens. 2. Mix thoroughly.
Summary
3. Boil with frequent agitation to dissolve the powder completely.
SS Agar is a modification of Deoxycholate Citrate Agar described by Leifson (68)
4. AVOID OVERHEATING. DO NOT AUTOCLAVE.
and is an example of media used in the plating of samples for the detection of
enteric pathogens that contain bile salt mixtures. SS Agar, Modified is especially 5. Cool the medium to approximately 45-500C, pour into sterile petri plates.
useful in isolating Shigella, because it is many times inhibited in SS Agar. By 6. Allow the plates to dry for about 2 hours with the covers partially removed
reducing the bile salt quantity and increasing the pH SS Agar has been modified under aseptic conditions.
such a way that easily it helps to isolate Shigella without interfering the growth of Quality Control
commensal organisms. Dehydrated Appearance
Pinkish yellow coloured, homogeneous, free flowing powder.
Principle
Prepared Appearance
Peptone and beef extract provide sources of carbon, nitrogen and other growth Reddish orange coloured, clear to slightly opalescent gel.
factors. Brilliant green, bile salt mixture, thiosulphate and citrates selectively Cultural Response
inhibit gram-positive organisms and coliforms. Lactose is the fermentable Cultural characteristics after 18-24 hours at 35-37ºC.
carbohydrate and differentiation of enteric organisms is achieved based on Organisms (ATCC) Growth Colour of RGI
Colony
lactose fermentation in the presence of neutral red. On fermentation of lactose by
Enterobacter aerogenes (13048) Poor to good Cream pink More than 70%
a few lactose fermenting normal intestinal flora, acid is produced, which is Enterococcus faecalis (29212) none to poor Colourless 0% or
indicated by change in colour from yellow to red by the pH indicator neutral red More than 70%
and these organisms grow as red-pigmented colonies. Non-lactose fermenters Escherichia coli (25922) Poor to good Pink with More than 70%
bile precipitate
grow as translucent colourless colonies. Sodium thiosulphate and ferric citrate
Proteus mirabilis (25933) Poor to good Colourless may More than 70%
enable the detection of H2S production. Sodium thiosulphate is reduced by certain have black centre
species to sulphide and H2S gas in the presence of the enzyme thiosulphate Salmonella serotype Enteritidis Good Colourless with More than 70%
(13076) black centre
reductase. Production of H2S gas is detected as an insoluble black precipitate of
Salmonella serotype Good Colourless with More than 70%
ferrous sulphide, formed upon the reaction of H2S with ferric citrate, indicated as a Typhimurium (14028) black centre
black dot in the center of the colony. The high selectivity of SS Agar allows the use Shigella flexneri (12022) Good Colourless More than 70%
without black centre
of large inocula directly from faeces, rectal swabs or other material suspected of Salmonella typhi (6538) Good Colourless More than 70%
containing the enteric pathogens. without black centre
Formula* For growth RGI should be more than 70%
Ingredients in grams per liter For Inhibition RGI should be 0%
Peptic digest of animal tissue 5.0 RGI- Relative Growth Index
Beef extract 5.0 Procedure
Lactose 10.0
1. Use standard procedures like the streak plate method to obtain isolated
Bile salts mixture 5.50
Sodium citrate 10.0 colonies.
Sodium thiosulfate 8.5 2. If the specimen to be cultured is on a swab, roll the swab on a small area of
Ferric citrate 1.0 the agar surface and streak for isolation with a sterile loop.
Brilliant green 0.00033
Neutral red 0.025 3. Incubate plates aerobically, protected from light, at 35-37ºC for 18-24
Agar 12.0 hours.
Final pH (at 25ºC) 7.2 ± 0.2 4. If negative incubate for an additional 24 hours.
* Formula adjusted to suit performance parameters
5. Examine colony morphology.

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6. Note: A non-selective medium should also be streaked to increase the Shigella--- Slight or good growth, Colourless
chances of recovery when the population of Gram negative organisms is low Pseudomonas--- Irregular, slight growth
and to provide an indication of other organisms present in the specimen. Gram-positive bacteria--- No growth
7. In parallel with the SS Agar plate, inoculate a tube of Selenite Broth Precautions / Limitations
enrichment medium, incubate for 12 hours at 35ºC, and subculture onto 1. This medium is highly selective; some strains of Shigella may not grow on it
another SS Agar plate. and therefore must not be used for primary isolation of shigellae.
Interpretation of Results
2. Inoculate plates of less inhibitory media like Deoxycholate Citrate Agar or XLD
Typical colonial morphology on SS Agar Agar for better isolation of Shigella species.
Escherichia coli --- Slight growth, Pink or red Storage
Enterobacter/ Klebsiella --- Slight growth, Pink Store below 300C and prepared medium at 2-80C.
Proteus--- Slight growth, Colourless, Shelf Life

may have black center Use before expiry date as mentioned on the label.
Salmonella--- Good growth, Colourless
with black centers

Standard Nutrient Agar AM509311

Use 3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
Standard Nutrient Agar is used as a general utility medium for cultivation and Directions

enumeration of not particularly fastidious microorganisms. 1. Suspend 45 gms of the powder in 1000 ml distilled water.
Summary 2. Boil with frequent agitation to dissolve the powder completely.
Standard Nutrient Agar is formulated as per the recommendation APHA as a 3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.Quality
general purpose medium for the cultivation of nonfastidious organisms from Control
water and wastewater, dairy and food products. Dehydrated Appearance
Principle Yellowish brown coloured, homogeneous, free flowing powder.
Prepared Appearance
Peptic digest of lean meat provides the amino acids and large chain peptides.
Light amber coloured, slightly opalescent gel.
Beef extract (meat infusion) provides water soluble substances like
Cultural Response
carbohydrates, vitamins, organic nitrogen compounds and salts. Sodium chloride Cultural characteristics after 18-24 hours at 35-37ºC.
maintains osmotic equilibrium. Organisms(ATCC) Growth RGI
Formula* Escherichia coli (25922) Good to luxuriant More than 70%
Ingredients in grams per liter Streptococcus pneumoniae (6303) Good to luxuriant More than 70%
Peptic digest of lean meat from 500.0 Staphylococcus aureus (25923) Good to luxuriant More than 70%
Beef extract 10.0 For growth RGI should be more than 70%
Sodium Chloride 5.0 RGI- Relative Growth Index
Agar 20.0 Storage
Final pH (at 250C) 7.6 ± 0.2
Store at 22-300C and prepared medium at 2-80C.
* Formula adjusted to suit performance parameters
Shelf Life
Directions
1. Suspend 25 grams in 1000 ml distilled water. Use before expiry date as mentioned on the label.
2. Heat if necessary to dissolve the completely.

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Standard Nutrient Broth (H.S. Vaccine Medium) AM509313
Use 2. Heat if necessary to dissolve the completely.
A highly nutritive medium recommended for the large scale cultivation of bacteria 3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
for production of vaccine preparations Quality Control
Principle Dehydrated Appearance
Peptic digest of lean meat is the principal source of organic nitrogen while meat Cream to light yellow coloured, homogeneous, free flowing powder.
infusion solids provides carbohydrates, vitamins, organic nitrogen compounds Prepared Appearance
and salts. Sodium chloride maintains osmolality of the medium. Amber coloured, clear solution without any precipitate.
Cultural Response
Formula*
Cultural characteristics observed after an incubation at 35 – 37°C for 18-48
Ingredients in grams per liter
hours.
Peptic digest of lean meat infusion, solids 10.00
Organisms(ATCC) Growth
Meat infusion, solids 10.00
Enterobacter aerogenes (13048) Good-luxuriant
Sodium chloride 5.00
Escherichia coli (25922) Good-luxuriant
Final pH (at 25°C) 7.6 ± 0.2
Salmonella typhi (6539) Good-luxuriant
* Formula adjusted to suit performance parameters
Staphylococcus aureus (25923) Good-luxuriant
Directions
Staphylococcus Epiderimidis (12228) Good-luxuriant
1. Suspend 25 grams in 1000 ml distilled water.
Staphylococcus pyogenes (19615) Good-luxuriant
2. Heat if necessary to dissolve the completely.
Storage
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
Directions Store at 22-300C and prepared medium at 2-80C.
Shelf Life
1. Suspend 25 grams in 1000 ml distilled water.
Use before expiry date as mentioned on the label.

Staphylococcus Agar No. 110 (Gelatin Mannitol Salt Agar) AM10932/AM50932

Use Formula*
Staphylococcus Agar No. 110 is used as a selective medium for the isolation and Ingredients in grams per liter
testing of pathogenic Staphylococci. Casein enzymic hydrolysate 10.00
Yeast extarct 2.50
Summary
Gelatin 30.00
Staphylococcus Agar No. 110 is formulated as described by chapman(16, 14.1, Lactose 2.00
16.5) for selective isolation and enumeration of Staphylococci from clinical as D-Mannitol 10.00
well as nonclinical specimens. This media is recommended by APHA (103.4). Sodium Chloride 75.00
Addition of blood in the medium enables to study haemolytic reaction and with Dipotassium phosphate 5.00
egg yolk enables to study lecithinase production by Staphylococcus aureus Agar 15.00
(12.2). Media is selective due to high concentration of salts and differential on the Final pH (at 250C) 7.0± 0.2
basis of ability of organisms to ferment the mannitol, pigment production and * Formula adjusted to suit performance parameters
Directions
gelatin liquefaction.
Principle 1. Suspend 149.5 gms of the powder in 1000 ml distilled water. Mix
Staphylococcus Agar No. 110 is very nutritive media as they contain casein Thoroughly.
enzymic hydrolysate, yeast extract which provide essential growth factors like 2. Heat to boiling to dissolve the medium completely.
vitamins, nitrogen, carbon compounds, sulphur and trace nutrient etc. to the 3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
organisms. High concentration of the sodium chloride inhibits many bacterial 4. Resuspend the precipitately gentle agitation to avoid bubbles and pour
species expect staphylococci. the plates while the medium to 45-500C and add blood or egg yolk if

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desired. faecalis (29212) More than 70%
Escherichia coli Inhibited – – – 0%
5. Medium also can be used without sterilization; it should be boiled for 5 (25922)
minutes and used at once Key: + = Positive reaction
Quality Control – = Negative reaction
Dehydrated Appearance v = Variable reaction
Light yellow coloured, homogeneous, free flowing powder. + = Slight reaction
Prepared Appearance For growth RGI should be more than 70%
Light amber coloured, opalescent gel with precipitate forms in petri plates.. For Inhibition RGI should be 0%
Cultural Response RGI- Relative Growth Index
Cultural characteristics after 48 hours at 35-37ºC. Storage
Organisms Growth Pigment Gelatinase Mannitol RGI Store at 22-300C and prepared medium at 2-80C.
(ATCC) production production fermentation
Shelf Life
Staphylococcus Luxuriant + + + More than 70%
aureus (25923) Use before expiry date as mentioned on the label.
Staphylococcus Luxuriant – + v More than 70%
epidermidis
(12228)
Enterococcus None-poor – v + 0% or

Streptococcus Selection Agar AM50933

Use Sodium sulphite 0.20
Streptococcus Selection Agar is recommended for selective isolation and L-cystine 0.20
Sodium azide 0.20
enumeration of all types of Streptococci, including group A beta haemolytic
Crystal violet 0.0002
strains.
Agar 15.00
Summary
Final pH (at 250C) 7.4± 0.2
Streptococcus Selection Agar is based on the suggestion of Pike (88.1), for the * Formula adjusted to suit performance parameters
selective isolation of Streptococci from various materials, specially those which Directions
are heavily contaminated with accompanying microbial flora (28.2). Welch et al., 1. Suspend 45.6 gms of the powder in 1000 ml distilled water. Mix
(118.1) also reported the ability of these media to recover group A â-haemolytic Thoroughly.
Streptococci.
2. Heat to boiling to dissolve the medium completely.
Principle
3. Autoclaving is not required if medium is used on the same day.
Casein enzymic hydrolysate, papaic digest of soyabean meal, dextrose and salts
provide nutrients essential for the growth of streptococci. Sodium azide, sodium 4. If storage is desired, Sterilize by autoclaving at 1180C (12 lbs pressure) for
sulphite inhibits gram-negative rods and the crystal violet suppresses 15 minutes.
Staphylococci. However, Streptococci are not affected by these inhibitors at these 5. Avoid overheating.
concentrations. Due to this reason, this media is useful in studies of streptococcal Caution: Sodium azide has a tendency to form explosive metal-azide with
plumbing material. It is advisable to use enough water to flush off the
flora from nutritional, dental and epidemiological research. disposable.
Formula* Quality Control
Ingredients in grams per liter Dehydrated Appearance
Casein enzymic hydrolysate 15.00 Light yellow coloured, homogeneous, free flowing powder.
Papaic digest of soyabean meal 5.00 Prepared Appearance
Dextrose 5.00 Light to medium amber coloured, clear to slightly opalescent gel forms in petri
Sodium Chloride 4.00 plates.
Sodium Citrate 1.00 Cultural Response
Cultural characteristics after 18-24 hours at 35-37ºC.

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Organisms(ATCC) Growth RGI For Inhibition RGI should be 0%
Streptococcus pyrogenes (19615) Luxuriant More than 70% RGI- Relative Growth Index
Enterococcus faecalis (29212) Luxuriant More than 70% Storage
Staphylococcus aureus (25923) None-poor 0% or more than 70% Store at 22-300C and prepared medium at 2-80C.
Escherichia coli (25922) None-poor 0% or more than 70% Shelf Life
Bacillus subtilis (6633) Inhibited 0%
Pseudomonas aeruginosa (27853) Inhibited 0%
Use before expiry date as mentioned on the label.
For growth RGI should be more than 70%

Streptococcus Selection Broth AM50934

Use Directions
Streptococcus Selection Broth is recommended for selective isolation and 1. Suspend 30.6 gms of the powder in 1000 ml distilled water. Mix
enumeration of all types of Streptococci, including group A beta haemolytic strains Thoroughly.
Summary 2. Heat to boiling to dissolve the medium completely.
Streptococcus Selection Broth is based on the suggestion of Pike (88.1), for the 3. Autoclaving is not required if medium is used on the same day.
selective isolation of Streptococci from various materials, specially those which
4. If storage is desired, Sterilize by autoclaving at 1180C (12 lbs pressure) for
are heavily contaminated with accompanying microbial flora (28.2). Welch et al.,
15 minutes.
(118.1) also reported the ability of these media to recover group A â-haemolytic
5. Avoid overheating.
Streptococci.
Caution: Sodium azide has a tendency to form explosive metal-azide with
Principle
plumbing material. It is advisable to use enough water to flush off the
Casein enzymic hydrolysate, papaic digest of soyabean meal, dextrose and salts disposable.
provide nutrients essential for the growth of streptococci. Sodium azide, sodium Quality Control
sulphite inhibits gram-negative rods and the crystal violet suppresses Dehydrated Appearance
Staphylococci. However, Streptococci are not affected by these inhibitors at these Light yellow coloured, homogeneous, free flowing powder.
Prepared Appearance
concentrations. Due to this reason, this media is useful in studies of streptococcal
Light to medium amber coloured, clear to slightly opalescent solution forms in
flora from nutritional, dental and epidemiological research. tubes.
Formula* Cultural Response
Ingredients in grams per liter Cultural characteristics after 18-24 hours at 35-37ºC.
Casein enzymic hydrolysate 15.00 Organisms(ATCC) Growth
Papaic digest of soyabean meal 5.00 Streptococcus pyrogenes (19615) Luxuriant
Dextrose 5.00 Enterococcus faecalis (29212) Luxuriant
Sodium chloride 4.00 Staphylococcus aureus (25923) None-poor
Escherichia coli (25922) None-poor
Sodium citrate 1.00
Bacillus subtilis (6633) Inhibited
Sodium sulphite 0.20
Pseudomonas aeruginosa (27853) Inhibited
L-cystine 0.20
Storage
Sodium azide 0.20
Crystal violet 0.0002
Store at 22-300C and prepared medium at 2-80C.
Final pH (at 250C) 7.4± 0.2 Shelf Life
* Formula adjusted to suit performance parameters Use before expiry date as mentioned on the label.

Stuart Transport Medium AM1094/AM5094

Use organisms.
Stuart Transport Medium is used for collecting, transporting and preserving Summary
microbiological specimens, particularly Neisseria species and other fastidious Stuart (104) originally designed Stuart Transport Medium when studying

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gonococci. Stuart et al modified this medium for the transportation of gonococcal Streptococcus pneumoniae (6303) Good Soyabean Casein
specimens for culturing. Transport media are basically chemically defined, Digest Agar with 5%
sheep blood
semisolid, non-nutritive, phosphate buffered media that provide a reduced
Key:
environment and are designed to maintain the viability of organisms without
* = Incubation in CO2 atmosphere.
much increase in growth. Ringertz (92) included thioglycollate in the Stuart Procedure
Medium and omitted charcoal. This medium is currently recommended for throat,
1. Obtain the specimen with a sterile swab and insert into the upper third of the
vaginal and wound samples.
medium in the transport container.
Principle
2. Break off the protruding portion of the swab stick and tightly screw the lid of
This medium by virtue of its composition prevents microbial proliferation but
the container.
ensures that the organisms present survive for sufficiently long period of time.
Calcium chloride provides essential ions that help maintain osmotic balance 3. Submit to laboratory within 24 hours for culture and analysis.
while controlling permeability of bacterial cells. Sodium thioglycollate is a Interpretation of Results

reducing agent providing anaerobiosis, which can be monitored by means of the 1. The survival of organisms depends on many factors including the type and
redox indicator; methylene blue. Sodium glycerophosphate is a buffer for use with concentration of bacteria in the specimen, the temperature and duration of
calcium chloride. transport and inoculation to appropriate culture media within 24 hours.
Formula* 2. Optimal growth and typical morphology can only be expected following direct
Ingredients in grams per liter inoculation and cultivation under appropriate conditions.
Sodium Glycerophosphate 10.0
Precautions / Limitations
Sodium Thioglycollate 0.9
Calcium Chloride 0.1 1. Prepared sterile media may undergo a slight degree of oxidation at the upper
Methylene Blue 0.002 periphery of the medium, however, if the vials or tubes show a distinct blue
Agar 3.0 colour throughout the medium, it must be discarded.
Final pH (at 250C) 7.4 ± 0.2 2. Fill the vials or tubes with the medium almost to capacity. Leave only enough
* Formula adjusted to suit performance parameters
space to allow insertion of the small swab.
Directions
3. For transportation of specimens that may contain N.gonnorhoeae, a selective
1. Suspend 14 gms of the powder in 1000 ml distilled water.
medium may be used.
2. Mix thoroughly.
4. Viability of cells will diminish over time and contaminants may overgrow
3. Boil with frequent agitation to dissolve the powder completely. during prolonged periods of transit. This is particularly true of faecal
4. Dispense in small screw cap bottles or vials, filling them almost to the top. specimens that contain high numbers of coliform organisms.
5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. 5. Specimens taken from transport media will not exhibit the optimal or
6. Tighten caps immediately and cool the tubes in an upright position. comparative growth as expected from direct inoculation and cultivation.
Note: The water used should be free from chlorine. However, these media provide an adequate degree of preservation for those
Quality Control specimens, which cannot be immediately forwarded to the laboratory.
Dehydrated Appearance 6. The condition of the specimen received by the laboratory for culture is an
White, homogeneous, free flowing powder. important variable in recovery and final identification of the suspect
Prepared Appearance
pathogen.
Colourless to whitish coloured, slightly opalescent butt with upper 10% or less
becoming blue on standing. 7. A specimen overgrown by contaminants or having the number of suspect
Cultural Response pathogens greatly diminished may lead to wrong or inconclusive results.
Cultural characteristics after 72 hours at 350C when subcultured onto a Storage
suitable medium.
Store below 300C and prepared medium at 2-80C.
Organisms (ATCC) Growth Subculture Medium
Shelf Life
Haemophilus influenzae (35056) Good *Chocolate Agar
Neisseria gonorrhoeae (19424) Good *Chocolate Agar Use before expiry date as mentioned on the label.

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Super Broth AM50935
Use Directions
Super Broth is used for the mass cultivation of Escherichia coli . 1. Suspend 60 gms of the powder in 1000 ml distilled water. Mix
Summary Thoroughly.
Escherichia coli is a bacterium that is commonly found in the gut of humans and 2. Heat if necessary to dissolve the medium completely.
warm-blooded animals. Most strains of E. coli are harmless. Some strains 3. Dispense as desired and sterilize by autoclaving at 15 lbs pressure
however, such as Enterohaemorrhagic E. coli (EHEC) can cause severe foodborne (121°C) for 15 minutes.
disease. Super Broth has a formulation slightly different from that described by Quality Control
Atlas (1.1.1) and it is used for the mass cultivation of E. coli . Dehydrated Appearance
Principle Cream to yellow homogeneous free flowing powder
Casein enzymic hydrolysate and yeast extract provide nitrogenous compounds, Prepared Appearance
vitamin B complex and other essential growth nutrients. Sodium chloride Light yellow coloured clear solution without any precipitate
Cultural Response
maintains osmotic equilibrium. Super Broth is nutritionally rich hence other
Cultural characteristics observed after an incubation at 35-37°C for 18-24
organisms can also grow in it easily. hours.
Formula* Organisms (ATCC) Growth
Ingredients in grams per literIngredients Gms/Liter Escherichia coli (23724) Good - luxuriant
Casein enzymic hydrolysate 35.00 Escherichia coli (25922) Good - luxuriant
Yeast extract 20.00 Staphylococcus aureus (25923) Good - luxuriant
Sodium chloride 5.00 Storage
Final pH ( at 25°C) 7.0±0 Store at 22-300C and prepared medium at 2-80C.
* Formula adjusted to suit performance parameters Shelf Life
Use before expiry date as mentioned on the label.

TB Broth Base AM50941

Use for the mycobacterial metabolism. Polysorbate 80, an oleic acid ester provides
TB Broth Base is used as a liquid medium for cultivation of Mycobacterium essential fatty acids for the replication of Mycobacteria.
tuberculosis. Formula*
Summary Ingredients in Gms/Liter
TB Broth media are based on the medium formulated by Dubos and Davis (21.1) Proteose peptone 4.00
Yeast extract 2.00
and are used as liquid media for the cultivation of Mycobacterium tuberculosis.
Disodium phosphate 2.50
Media provides dispersed growth of tubercle bacilli which is free of excessive
Monopotassium phosphate 1.00
clumps and so it can be used to prepare a uniform suspension of Mycobacteria. Sodium citrate 1.50
The media can be used without the additives and supplements however sterile Magnesium sulphate 0.60
dextrose and sterile serum can be added for the enrichment. Glycerol addition Polysorbate 80 0.50
helps in the cultivation of Mycobacterium tuberculosis though some bovine Final pH (at 250C) 7.0± 0.2
strains are inhibited by it. * Formula adjusted to suit performance parameters
Principle Directions

Proteose peptone and yeast extract provide nitrogenous nutrients like amino acids 1. Suspend 12.1 gms of the powder in 1000 ml distilled water. Which if desired
and peptides, vitamin B complex and other essential nutrients. The media are contains 5 ml glycerol (tested to be non-inhibitory to typical cultures).
well buffered by phosphates. The salts present in the media supply ions required 2. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.

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3. Cool to 450C and enrich with dextrose to a final concentration of 0.5% and Organisms (ATCC) Growth
eiter bovine albumin fraction V or serum as desired. Mycobacterium tuberculosis H37 RV (25618 Luxuriant
Quality Control Mycobaterium kansasi (12478) Luxuriant
Dehydrated Appearance Mycobacterium smegmatis (14468) Luxuriant
Light yellow coloured, homogeneous, free flowing powder. Storage
Prepared Appearance Store at 22-300C and prepared medium at 2-80C.
Yellow coloured, clear solution without any precipitate. Shelf Life
Cultural Response
Use before expiry date as mentioned on the label.
Cultural characteristics after 2-4 weeks at 35-37ºC.

TAT Broth Base AM509411

Use * Formula adjusted to suit performance parameters
T.A.T. Broth Base with added polysorbate 20 is used for cultivating Directions

microorganisms from highly viscous or gelatinous materials such as salves, 1. Suspend 25 grams in 960 ml distilled water and add 40 ml of polysorbate
ointments. 20.
Summary 2. Heat if necessary to dissolve the medium completely.
T.A.T. Broth is prepared according to the formula recommended by United States 3. Dispense as desired and sterilize by autoclaving at 15 lbs pressure
Food and Drug Administration (31.1.1) for enrichment and further isolation and (121°C) for 15 minutes.
cultivation of gram-negative bacteria in cosmetics, tropical drugs and in the
sterility testing of viscous or gelatinous substances. It is especially adapted for the Quality Control
testing of cosmetics. Cosmetics and pharmaceutical products are subject to Dehydrated Appearance
contamination during manufacturing and subsequent use by consumers Off-white to yellow homogeneous free flowing powder
(85.1.1). Preservatives are used in aqueous products to make them self- Prepared Appearance
Light amber coloured clear to slightly opalescent solution
sterilizing for vegetative bacteria, yeasts and moulds, and bacteriostatic or
Cultural Response
bactericidal for spores (85.1.1).
Cultural characteristics observed after an incubation at 35-37°C for 24-48
Principle hours.
Pancreatic digest of casein provides the nitrogen, vitamins, amino acids and Organisms (ATCC) Growth
carbon in T.A.T. Broth Base. Soy lecithin and polysorbate 20 neutralize Bacillus subtilis ATCC 6633 Good- luxuriant
preservatives in the cosmetics or pharmaceutical products, allowing bacteria to Candida albicans ATCC 10231 Fair- luxuriant
Pseudomonas aeruginosa ATCC 27853 Good-luxuriant
grow. Prepare decimal dilutions of the sample to be tested from 10-1 to 10-6.
Salmonella Typhi ATCC 6539 Good- luxuriant
Inoculate 1 gram (1 ml) sample and 1 ml of each dilution into 40 ml of T.A.T.
Staphylococcus aureus ATCC 25923 Good- luxuriant
Broth. After incubation, subculture the growth on MacConkey Agar and TSI Agar. Storage
Formula*
Ingredients in grams per literIngredients Gms/Liter
Store at 22-300C and prepared medium at 2-80C.
Pancreatic digest of casein 20.00 Shelf Life
Soy lecithin 5.00 Use before expiry date as mentioned on the label.
Final pH ( at 25°C) 7.2±0.2

Tartoff-Hobbs (Terrific) Broth AM509412

Use Summary
Tartoff-Hobbs (Terrific) Broth is recommended for the cultivation of recombinant Tartoff Hobbs developed the Terrific Broth Medium for cultivation of recombinant
strains of Escherichia coli. Escherichia coli strains. These strains have extended growth phase when
cultivated in this medium (107.1). Tartoff-Hobbs Medium supports high cellular

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density and mass and maintains the growth in the logarithmic phase for a long Directions
time. Due to this fact, it provides greater yields of recombinant proteins and 1. Dissolve 47.6 g of the powder in 1 L of purified water.
plasmid DNA. Often, Tartoff-Hobbs (Terrific) Broth substitutes Luria Bertani Broth 2. Add 4 ml of glycerol to the medium.
(AM50574), to get enhanced yields of plasmid DNA and recombinant proteins. 3. Autoclave at 121°C for 15 minutes.
The procedures for inoculation, incubation and generation of recombinant strains
4. Test samples of the finished product for performance using stable, typical
are detailed by Sambrook et al (102.1).
control cultures.
Principle
Quality Control
Casein enzymic hydrolysate and yeast extract supply the necessary nutrients and Dehydrated Appearance
cofactors for the excellent growth of recombinant strains of E. coli. The addition of Light yellow to beige homogeneous free flowing powder
extra peptone and yeast extract in the medium allows higher plasmid yield per Prepared Appearance
volume. Two phosphates provide good buffering action to the medium. Glycerol is Light to medium amber coloured clear solution without any precipitate.
used as the carbohydrate source. Unlike glucose, glycerol is not fermented to Cultural Response
acetic acid. Cultural characteristics observed after an incubation at 35-37°C for 18-24
hours.
Formula*
Organisms (ATCC) Growth
Ingredients in Gms/Liter
Escherichia coli (23724) Good
Casein enzymic hydrolysate 12.00
Escherichia coli (39403) Good
Yeast extract 24.00
Escherichia coli (47014) Good
Monopotassium phosphate 9.4
Escherichia coli (53868) Good
Dipotassium phosphate 2.2
Storage
Final pH ( at 25°C) 7.2±0.2
* Formula adjusted to suit performance parameters Store at 22-300C and prepared medium at 2-80C.
Shelf Life
Use before expiry date as mentioned on the label.

TCBS Agar AM1095/AM5095

Use the medium enhances the recovery of V.cholerae. Thymol blue and bromothymol
TCBS Agar is a selective medium used for isolating and cultivating vibrios causing blue are included as indicators of pH change. Sodium chloride maintains the
cholera and food poisoning from clinical and non-clinical specimens. osmotic balance.
Summary Formula*
Thiosulphate Citrate Bile Salts Sucrose (TCBS) Agar is a primary plating medium Ingredients in grams per liter
for the selective isolation of vibrios that cause cholera, diarrhoea and food Sucrose 20.0
Proteose Peptone 10.0
poisoning. It was developed by Nakanishi et al (83) and was modified by
Sodium Citrate 10.0
Kobayashi (59). The combination of alkaline peptone water and TCBS Agar is
Sodium Thiosulphate 10.0
used in many procedures for the isolation of V.cholerae and other Vibrio species Sodium Chloride 10.0
from foods (20), water (36) and faeces. This medium is included in the Oxgall 8.0
Bacteriological Analytical Manual for food testing (113). Yeast Extract 5.0
Principle Ferric Citrate 1.0
Proteose peptone and yeast extract provide nitrogenous compounds, vitamin B Thymol Blue 0.04
Bromothymol Blue 0.04
complex and other essential growth nutrients. Oxgall and sodium citrate inhibits
Agar 15.0
gram-positive bacteria. Sodium thiosulphate serves as the sulphur source and in
Final pH (at 250C) 8.6 ± 0.2
combination with ferric citrate detects hydrogen sulphide production. Sucrose is
* Formula adjusted to suit performance parameters
the fermentable carbohydrate for the metabolism of vibrios. The alkaline pH of

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Directions Interpretation of Results
1. Suspend 89 gms of the powder in 1000 ml distilled water. Colony morphology on TCBS Agar is as follows:
2. Mix thoroughly. V.cholerae ----------------------------------------------- Large yellow colonies
3. Boil with frequent agitation to dissolve the powder completely. V.parahaemolyticus -------------------------------------- Colonies with blue to
4. DO NOT AUTOCLAVE. green centers
5. Cool to 45-500C and pour into sterile petri plates, use immediately. V.alginolyticus ------------------------------------------- Large yellow colonies.
Quality Control Proteus/Enterococcus ------------------------------------ Partial inhibition. If
Dehydrated Appearance growth occurs, colonies are small, yellow and translucent.
Yellow coloured with tan cast, homogeneous, free flowing powder.
Pseudomonas/ Aeromonas ------------------------------ Partial inhibition. If
Prepared Appearance
growth occurs, colonies are blue.
Bluish green coloured, clear to slightly opalescent gel.
Precautions / Limitations
Cultural Response
0
Cultural characteristics after 18-24 hours at 35 C. 1. Cultures grown on TCBS Agar should be examined immediately after removal
Organisms (ATCC) Growth Colour of RGI from the incubator as yellow colonies of vibrios, e.g. V.cholerae may revert to
Colonies a green colour when left at room temperature.
Escherichia coli (25922) Inhibited - 0%
2. The identification of the various Vibrio species on TCBS Agar is presumptive
Proteus vulgaris (13315) Inhibited - 0%
and further tests are required for confirmation.
Shigella flexneri (12022) Inhibited - 0%
Streptococcus faecalis Inhibited - 0% 3. Yellow colonies on TCBS Agar will give unsatisfactory oxidase reactions.
(29212) 4. Initial isolation of V.parahaemolyticus may be confused with Aeromonas
Vibrio cholerae Good to Yellow More than 70% hydrophila, Plesiomonas shigelloids and Pseudomonas species.
(15748) luxuriant
Vibrio parahaemolyticus Good to Blue More than 70%
5. Sucrose fermenting Proteus species produce yellow colonies, which may
(17802) luxuriant resemble those of Vibrio.
For growth RGI should be more than 70% 6. A few strains of V.cholerae may appear green or colourless on TCBS Agar due
For Inhibition RGI should be 0% to delayed sucrose fermentation.
RGI- Relative Growth Index
7. Colonies taken from TCBS Agar are 'sticky' and react poorly in slide
Procedure
agglutination tests. Subculture to Nutrient Agar before slide agglutination
1. Use standard procedures to obtain isolated colonies from specimens.
tests are carried out.
2. Incubate the plates, protected from light in an inverted position at 350C for Storage
24-48 hours. Store below 300C and prepared medium at 2-80C.
Shelf Life
Use before expiry date as mentioned on the label.

Teepol Broth (Twin Pack ) AM509511

Use
lower temperature resuscitation is not required. Non-chlorinated organisms
Teepol Broth (Twin Pack) is used for selective isolation and identification of enteric benefit from 4 hour incubation at 300C but chlorinated organisms require 6 hours
lactose fermenting bacteria. incubation at 250C.
Summary Principle
Teepol Broth is formulated as described by Burman, where he substituted teepol The coliform and Escherichia coli count are made on separate volumes of water.
in place of bile salts in the formulation of Membrane Enrichment Teepol Broth. The water samples are filtered through membrane filter and this filter is placed
The use of Teepol in place of bile salts was previously recommended by Jameeson face upwards on an absorbent pad saturated with Teepol Borth. The yellow
and Emberley. Burman showed that if a preliminary incubation is carried out at a

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colonies formed are further identified. Presumptive coliform organisms: Yellow 3. Dispense in tubes containing inverted Durham's tubes.
colonies from membranes incubated at 350C, when subcultured in Lactose 4. Sterilize by autoclaving at 15 lbs pressure (1210C) for 15 minutes.
Peptone Water produce gas at 350C after 43 hours. Presumptive Escherichia coli Quality Control
Yellow colonies from membrane at 440C produce gas and indole after 24 hours. Dehydrated Appearance
Formula* Part A: Pink coloured, homogeneous, free flowing powder.
Ingredients in grams per liter Part B: Colourless viscous solution.
Part A : Peptic digest of animal tissue 20.00 Prepared Appearance
Lactose 10.00 Red coloured, clear to slightly opalescent solution.
Sodium chloride 5.00 Cultural Response
Phenol red 0.02 Cultural characteristics after 24-48 hours at …........
Part B: Teepol 1.00 Organisms (ATCC) Growth at 350C Growth at 440C
0
Final pH (at 25 C) 7.6 ± 0.2 Escherichia coli (25922) + +
* Formula adjusted to suit performance parameters Enterobacter aerogenes (13048) + –
Directions Storage
1. Suspend 35 grams of Part A in 1000 ml distilled water containing 1 grams Store at 22-300C and prepared medium at 2-80C.
of Part B. Shelf Life

2. Heat if necessary to dissolve the medium completely. Use before expiry date as mentioned on the label.

Tergitol-7 Agar Base AM10951/AM50951

Tergitol-7 Agar Base BIS AM10952/AM50952
Use fermenting organisms form blue colonies. Agar is the solidifying agent. When TTC
Tergitol-7 Agar is used for enumeration, differentiation and selective isolation of is added to the medium, it is rapidly reduced to insoluble red formazan by most
coliform bacteria. organisms. Non-lactose fermenters appear red due to uptake and reduction of TTC
Summary while lactose fermenting organisms continue to form yellow to greenish-yellow
Tergitol-7(Sodium heptadecyl sulphate) Agar or T-7 Agar is formulated as colonies.
described by Chapman (16.2). Tergitol-7 (sodium heptadecyl sulfate) inhibits Formula*
the growth of gram positive bacteria, gram negative sporeformers and swarming Ingredients in grams per liter
Proteose peptone 5.00
of Proteus while allowing the growth of coliform bacteria. Chapman modified his
Yeast extract 3.00
original T-7 agar by adding 40mg of triphenyl tetrazolium chloride (TTC). On
Lactose 10.00
modified media he found that surface colonies of Escherichia coli produce Tergitol-7 (sodium heptadecyl sulfate) 0.10
greenish yellow colonies surrounded by yellow halo while other coliforms Bromthymol blue 0.025
produce dark red colonies (16.3). Tergitol-7 Agar with TTC is used for routine Agar 15.00
analysis of water and food (63.1, 32.1). Final pH: 6.9 ± 0.2 at 25°C
Principle * Formula adjusted to suit performance parametersDirections
Proteose Peptone and Yeast extract serve as a source of nitrogen and vitamins. 1. Suspend 33.13 gms of the powder in 1000 ml distilled water
Tergitol-7 (sodium heptadecyl sulfate) as a selective agent inhibits the growth of 2. Mix thoroughly.
gram-positive bacteria, gram-negative sporeformers and swarming of Proteus 3. Heat with frequent agitation and boil for 1 minute to dissolve the powder
spp. Lactose is the fermentable sugar. Lactose fermentation is indicated by a completely.
colour change of the pH indicator, Bromothymol blue. Lactose fermenting
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
organisms form yellow colored colonies surrounded with yellow zones while
Klebsiella and Enterobacter form greenish yellow colonies. Non-Lactose 5. If desired, cool Tergitol-7 Agar to 500C. Add 4.0ml of a filter sterilized 1%
solution of TTC (AS0271).

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6. Cool the medium to approximately 45-500C, pour in to sterile petriplates. Procedure
Quality Control 1. Use standard procedures like the streak plate method to obtain isolated
Dehydrated Appearance colonies.
Yellow coloured w/ green tinge, homogeneous, free flowing powder. 2. Incubate plates aerobically, protected from light, at 35-370C for 18-48
hours.
Prepared Appearance
3. Examine colony morphology.
Green coloured, slightly opalescent gel.
Interpretation of Results
Cultural Response
Tergitol-7 Agar without TTC
Cultural characteristics after 18-48 hours at 35-370C.
E.coli produces yellow colonies with yellow halos; other coliforms produce
Organisms (ATCC) Growth Colour of RGI
yellow to yellow-green colonies. Non-lactose fermenters produce blue
colony colonies.
Escherichia coli (25922) Luxuriant Yyellow More than 70% Tergitol-7 Agar with TTC
Enterobacter aerogenes Luxuriant Yellow More than 70% E.coli produces yellow colonies; other coliforms produce yellow-green
(13048) colonies while Non-lactose fermenters produce red colonies.
S. serotype Typhimurium Luxuriant Blue More than 70% Limitations
(14028) 1. It is preferable that biochemical and / or serological tests be performed
Shigella flexneri (12022) Luxuriant Blue More than 70% on colonies from pure culture for complete identification.
Staphylococcus aureus Lnhibited - 0% 2. Pour plates do not give satisfactory results.
(25923) Storage
For growth RGI should be more than 70% Store at 22-300C and prepared medium at 2-80C.
For Inhibition RGI should be 0% Shelf Life
RGI- Relative Growth Index
Use before expiry date as mentioned on the label.

Tergitol-7 Broth AM50953

Use Klebsiella and Enterobacter form greenish yellow colonies. Non-Lactose
Tergitol-7 Broth is used for enumeration, differentiation and selective isolation of fermenting organisms form blue colonies.
coliform bacteria. Formula*
Summary Ingredients in grams per liter
Tergitol-7(Sodium heptadecyl sulphate) Broth or T-7 Broth is formulated as Proteose peptone 5.00
Yeast extract 3.00
described by Chapman (16.2). Tergitol-7 (sodium heptadecyl sulfate) inhibits
Lactose 10.00
the growth of gram positive bacteria, gram negative sporeformers and swarming
Tergitol-7 (sodium heptadecyl sulfate) 0.10
of Proteus while allowing the growth of coliform bacteria. Chapman modified his Bromthymol blue 0.025
original T-7 agar by adding 40mg of triphenyl tetrazolium chloride (TTC). On Final pH: 6.9 ± 0.2 at 25°C
modified media he found that surface colonies of Escherichia coli produce * Formula adjusted to suit performance parameters
greenish yellow colonies surrounded by yellow halo while other coliforms produce Directions
dark red colonies. Tergitol-7 Agar with TTC is used for routine analysis of water 1. Suspend 18.13 gms of the powder in 1000 ml distilled water
and food. 2. Mix thoroughly.
Principle
3. Heat with frequent agitation and boil for 1 minute to dissolve the powder
Proteose Peptone and Yeast extract serve as a source of nitrogen and vitamins.
completely.
Tergitol-7 (sodium heptadecyl sulfate) as a selective agent inhibits the growth of
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
gram-positive bacteria, gram-negative sporeformers and swarming of Proteus
spp. Lactose is the fermentable sugar. Lactose fermentation is indicated by a 5. Cool to 45-500C aseptically add 3 ml of 1% 2,3,5, Triphenyl Tetrazolium
colour change of the pH indicator, Bromothymol blue. Lactose fermenting chloride (TTC) solution (AS0271) if desired.
organisms form yellow colored colonies surrounded with yellow zones while

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Quality Control Enterobacter aerogenes (13048) Luxuriant Yellow
Dehydrated Appearance S. serotype Typhimurium (14028) Luxuriant Blue
Yellow coloured w/ green tinge, homogeneous, free flowing powder. Shigella flexneri (12022) Luxuriant Blue
Prepared Appearance Staphylococcus aureus (25923) Inhibited -
Green coloured, slightly opalescent solution forms in tubes. Storage
Cultural Response Store at 22-300C and prepared medium at 2-80C.
0
Cultural characteristics after 18-48 hours at 35-37 C. Shelf Life
Organisms (ATCC) Growth Colour of medium
Use before expiry date as mentioned on the label.
Escherichia coli (25922) Luxuriant Yellow

Tetrathionate Brilliant Green Bile Broth AM50954

Tetrathionate Brilliant Green Bile Broth IP AM50955
Tetrathionate Brilliant Green Bile Broth (Broth Medium I) EP AM50956
Tetrathionate Brilliant Green Bile Broth (Broth Medium I) BP AM50957
Use Directions
Tetrathionate Brilliant Green Bile Broth is used for the selective enrichment and 1. Suspend the 63 gms of powder in 1000 ml distilled water.
isolation of Salmonella for the examination of pharmaceutical products in raw 2. Mix thoroughly.
materials, foodstuffs, meat etc. 3. Heat with frequent agitation to dissolve the powder completely. Do not boil.
Summary
DO NOT AUTOCLAVE OR REHEAT.
Tetrathionate Brilliant Green Bile Broth is a selective enrichment broth used for
4. Pour into adequate containers homogenizing the medium well enough to
the isolation of Salmonella. Tetrathionate Brilliant Green Bile Broth is
distribute the calcium carbonate.
recommended in Indian Pharmacopoeia for isolation of Salmonella (46).
Quality Control
Principle
Dehydrated Appearance
Peptic Digest of Animal Tissue supply nutrients, nitrogen compounds and amino Greenish yellow coloured, homogeneous, free flowing powder.
acids. Ox bile supports the growth of enteric bacteria and inhibits other bacteria, Prepared Appearance
which do not normally live in the intestine. Brilliant-green specifically inhibits the Bluish green coloured, opalescent solution with white precipitate.
Gram-positive accompanying flora. Potassium tetrathionate inhibits normal flora Cultural Response
of faecal specimens. Sodium chloride provides sodium ions for the membrane Cultural characteristics after 18-24 hours at 35-370C.
Organisms (ATCC) Recovery Colour of
transport and maintains osmotic equilibrium of the medium. The Calcium on MacConkey Agar Colony
carbonate is a neutralizer that will absorb any toxic metabolites. AM1059 / AM5059
Formula* S. serotype Luxuriant Colourless
Ingredients in grams/ liter Microxpress IP EP BP Typhimurium (14028)
Peptone - 8.6 8.6 8.6 S. serotype Enteritidis (13076) Luxuriant Colourless
Peptic digest of animal tissue 8.6 - - - Escherichia coli (25922) Fair Pink with bile
Potassium tetrathionate 20.0 20.0 20.0 20.0 precipitate
Brilliant green 0.07 0.07 0.07 0.07 Staphylococcus aureus (25923) Inhibited -
Ox bile, dried 8.0 - 8.0 8.0 Procedure
Dehydrated ox bile - 8.0 - - 1. Add 1-2 ml enriched sample to Tetrathionate Brilliant Green Bile Broth and
Sodium chloride 6.4 6.4 6.4 6.4 incubate at 37°C for 18-24 hours.
Calcium carbonate 20.0 20.0 20.0 20.0
2. Sub-culture to at least 2 of the following media for confirmation of
Final pH (at 250) 7.0 ± 0.2
* Formula adjusted to suit performance parameters
Salmonellae spp:

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a. Deoxycholate Citrate Agar (Cat. AM1031/5031); Brilliant Green Agar small, transparent, colorless or pink or opaque-white
b. XLD Agar (Cat. AM1112/5112); or Brilliant Green Agar (Cat. colonies, often surrounded by a pink or red zone.
AM1018/5018). Storage
Interpretation of Results Storeat 22-300C and prepared medium at 2-80C.
Deoxycholate Citrate Agar well-developed, colorless colonies Shelf Life

XLD Agar well-developed, red colonies, with black centers Use before expiry date as mentioned on the label.

Tetrathionate Broth Base, Hajna AM1096/AM5096

Use Yeast Extract 2.0
Tetrathionate Broth Base, Hajna is a medium used for selective enrichment of Dextrose 0.5
Sodium Deoxycholate 0.5
Salmonella, particularly in food and dairy products prior to isolation.
Brilliant Green 0.01
Summary
Final pH (at 250C) 7.6 ± 0.2
Tetrathionate Broth Base, with added iodine-iodide solution is used as a selective * Formula adjusted to suit performance parameters
enrichment medium for the isolation of Salmonella from faeces, urine, foods and Directions
other materials of sanitary importance. Salmonellae can be injured in food- 1. Suspend 91.5 gms of the powder in 1000 ml distilled water.
processing procedures like exposure to low temperatures, sub-marginal heat,
2. Mix thoroughly.
drying, radiation, preservation and sanitizers. Although injured cells may not
form colonies on selective media, they can cause disease if ingested; causing 3. Heat to dissolve the powder completely or place in flowing steam for 30
many types of infections from mild self-limiting gastroenteritis to life threatening minutes.
typhoid fever. The most common type of Salmonella disease is self-limiting 4. DO NOT AUTOCLAVE.
gastroenteritis with fever lasting less than 2 days and diarrhoea lasting less than 5. Cool to 450C.
7 days. Tetrathionate Broth Base, Hajna conforms to the formulation of Hajna 6. Mix and add 40 ml of iodine solution (8 gms potassium iodide and 5 gms of
and Damon (41). The medium is a modification of the enrichment described by iodine per 40 ml).
Kauffmann (51) and Knox (58). It is specified in the Standard Methods (20, 36,
7. Mix and dispense 10 ml aliquots in tubes.
39), in the USP (114) and IP (46) as well as in the Bacteriological Analytical
8. Do not heat after the addition of iodine.
Manual for food testing (113).
Quality Control
Principle
Dehydrated Appearance
Peptone Special, provides nitrogen and amino acids, while yeast extract supplies Cream coloured, homogeneous, free flowing powder.
growth factors and vitamins. Dextrose and mannitol are fermentable Prepared Appearance
carbohydrates. Sodium thiosulphate and tetrathionate suppress coliforms. Light green coloured opalescent solution with heavy white precipitate.
Tetrathionate is formed in the medium by the addition of a solution containing Cultural Response
iodine and potassium iodide. Organisms containing the enzyme tetrathionate Cultural characteristics after18-24 hours at 35-370C.
reductase will proliferate in this medium. Sodium deoxycholate and brilliant Organisms (ATCC) Recovery Colour of Colony
on MacConkey Agar
green are selective agents that inhibit gram-positive organisms. Sodium chloride AM1059 / AM5059
maintains the osmotic balance while calcium carbonate is a neutralizer that Salmonella serotype Good to luxuriant Colourless
absorbs toxic metabolites. Typhimurium (14028)
Formula* Shigella dysenteriae(13313) Good to luxuriant Colourless
Ingredients in grams per liter Escherichia coli (25922) Fair to good Pink with
Sodium Thiosulphate 38.0 bile precipitate
Calcium Carbonate 25.0 Procedure
Peptone Special 18.0 1. After preparation, add 1-3 gms of fecal specimen to each tube (heavy
Sodium Chloride 5.0 inoculum).
D-Mannitol 2.5

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2. Incubate tubes for 18-24 hours at 35-370C in an aerobic atmosphere. Storage
Interpretation of Results Store at 22-300C and prepared medium at 2-80C.
1. Growth is indicated by turbidity in the medium. Shelf Life

2. Subculture to selective and differential enteric plating media for further Use before expiry date as mentioned on the label.
investigations.

Tetrathionate Broth Base IP AM10961/AM50961

Use Uulture Media, Bases, Supplements 2. Mix thoroughly.
Tetrathionate Broth Base is a medium used for selective enrichment of 3. Heat to dissolve the powder completely or place in flowing steam for 30
Salmonella, in Accordance with I P. minutes.
Summary 4. DO NOT AUTOCLAVE.
Tetrathionate Broth Base, with added iodine-iodide solution is used as a selective 5. Cool to 450C.
enrichment medium for the isolation of Salmonella from foods, faeces, urine, and
6. Mix and add a solution prepared by dissolving 5 g of potassium iodide and 6
other materials. Salmonellae can be injured in food-processing procedures like
g of iodine in 20 ml of water.
exposure to low temperatures, sub-marginal heat, drying, radiation, preservation
and sanitizers. Although injured cells may not form colonies on selective media, 7. Mix and dispense in tubes.
they can cause disease if ingested; causing many types of infections from mild 8. Do not heat after the addition of iodine.
self-limiting gastroenteritis to life threatening typhoid fever. The most common Quality Control
type of Salmonella disease is self-limiting gastroenteritis with fever lasting less Dehydrated Appearance
Cream coloured, homogeneous, free flowing powder.
than 2 days and diarrhea lasting less than 7 days. The medium is a modification
Prepared Appearance
of the enrichment described by Kauffmann and Knox. It is specified in the
Light green coloured opalescent solution with heavy white precipitate.
Standard Methods in the IP. Cultural Response
Principle Cultural characteristics after18-24 hours at 35-370C.
Peptone, provides nitrogen and amino acids, while yeast extract and beef extract Organisms (ATCC) Recovery Colour of Colony
supplies growth factors and vitamins. Sodium thiosulphate and tetrathionate on MacConkey Agar
AM1059 / AM5059
suppress coliforms. Tetrathionate is formed in the medium by the addition of a Salmonella serotype Good to luxuriant Colourless
solution containing iodine and potassium iodide. Organisms containing the Typhimurium (14028)
enzyme tetrathionate reductase will proliferate in this medium. Sodium chloride Shigella dysenteriae(13313) Poor to good Colourless
maintains the osmotic balance while calcium carbonate is a neutralizer that Escherichia coli (25922) none to poor Pink with
absorbs toxic metabolites. bile precipitate
Formula* Procedure
Ingredients in grams per liter 1. After preparation, add specimen to each tube.
Beef extract 0.9 2. Incubate tubes for 18-24 hours at 35-370C in an aerobic atmosphere.
Peptone 4.5
Interpretation of Results
Yeast extract 1.8
Sodium chloride 4.5 1. Growth is indicated by turbidity in the medium.
Calcium carbonate 25.0 2. Subculture to selective and differential enteric plating media for further
Sodium thiosulphate 40.7 investigations.
Final pH (at 250C) 7.6 ± 0.2 Storage
* Formula adjusted to suit performance parameters
Store at 22-300C and prepared medium at 2-80C.
Directions
Shelf Life
1. Suspend 77.40 gms of the powder in 1000 ml distilled water.
Use before expiry date as mentioned on the label.

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Tetrathionate Broth Base Medium USP AM10962/AM50962
Use Uulture Media, Bases, Supplements 2. Mix thoroughly.
Tetrathionate Broth Base is a medium used for selective enrichment of 3. Heat to boiling. Cool to below 600C.
Salmonella, in compliance with USP.
4. Add 20 ml of iodine solution (6.0 g of iodine crystals and 5.0 g of potassium
Summary
iodide in 20.0 ml of water).
Tetrathionate Broth Base, with added iodine-iodide solution is used as a selective
5. Then add 10 ml of a solution of Brilliant Green (1 in 1000) and mix.
enrichment medium for the isolation of Salmonella from faeces, urine, foods and
other materials of sanitary importance. Salmonellae can be injured in food- 6. DO NOT REHEAT MEDIUM AFTER ADDING BRILLIANT GREEN.
processing procedures like exposure to low temperatures, sub-marginal heat, 7. DO NOT AUTOCLAVE.
drying, radiation, preservation and sanitizers. Although injured cells may not 8. Mix and dispense 10 ml aliquots in tubes.
form colonies on selective media, they can cause disease if ingested; causing 9. Use immediately.
many types of infections from mild self-limiting gastroenteritis to life threatening Quality Control
typhoid fever. The most common type of Salmonella disease is self-limiting Dehydrated Appearance
gastroenteritis with fever lasting less than 2 days and diarrhoea lasting less than Cream coloured, homogeneous, free flowing powder.
7 days. Tetrathionate Broth Base, Hajna conforms to the formulation of Hajna Prepared Appearance
and Damon. The medium is a modification of the enrichment described by White to off white coloured opalescent solution with heavy white precipitate.
Kauffmann and Knox. It is specified in the Standard Methods, in the USP and IP as Cultural Response
Cultural characteristics after18-24 hours at 35-370C.
well as in the Bacteriological Analytical Manual for food testing .
Organisms (ATCC) Recovery Colour of Colony
Principle on MacConkey Agar
Pancreatic digest of casein & peptic digest of animal tissue provides nitrogen, AM1059 / AM5059
amino acids and vitamins. Sodium thiosulphate and tetrathionate suppress Salmonella serotype Good to luxuriant Colourless
Typhimurium (14028)
coliforms. Tetrathionate is formed in the medium by the addition of a solution
Shigella dysenteriae(13313) Poor to good Colourless
containing iodine and potassium iodide. Organisms containing the enzyme
Escherichia coli (25922) None to poor Pink with
tetrathionate reductase will proliferate in this medium. Sodium deoxycholate and bile precipitate
brilliant green are selective agents that inhibit gram-positive organisms. Sodium Procedure
chloride maintains the osmotic balance while calcium carbonate is a neutralizer 1. After preparation, add specimen to each tube.
that absorbs toxic metabolites.
2. Incubate tubes for 18-24 hours at 35-370C in an aerobic atmosphere.
Formula*
Interpretation of Results
Ingredients in grams per liter
Bile salts 1.0 1. Growth is indicated by turbidity in the medium.
Calcium carbonate 10.0 2. Subculture to selective and differential enteric plating media for further
Pancreatic digest of casein 2.5 investigations.
Peptic digest of animal tissue 2.5 Storage
Sodium thiosulphate 30.0
Store at 22-300C and prepared medium at 2-80C.
Final pH (at 250C) 8.4 ± 0.2
Shelf Life
* Formula adjusted to suit performance parameters
Directions Use before expiry date as mentioned on the label.
1. Suspend 46.0 gms of the powder in 1000 ml distilled water.

Tinsdale Agar Base AM50963

Use Summary
Tinsdale Agar Base with supplement is used for selective isolation and Tinsdale Agar was originally formulated by Tinsdale (110.2) and further modified
differentiation of Corynebacterium diphtheriae. by Billings (77.1) for selective isolation and differentiation of Corynebacterium

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diptheriae. 3. Heat to boiling to dissolve the medium completely.
Principle 4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Peptic digest of animal tissue provides nitrogenous compounds. L-cystein and 5. Cool to 500C and aseptically and Diphtheria Virulence Supplement (Part A &
sodium thiosulphate form H2S indicator system. Potassium tellurite from the B, AS0091)
supplement inhibits all gram-negative bacteria and most of the upper respiratory
6. Mix well and pour into sterile petri plates.
tract normal flora. Quality Control
Corynebacterium diptheriae forms greyish black colonies by a dark brown halo Dehydrated Appearance
while diphtheroids commonly found in the upper respiratory tract do not form Light yellow coloured, homogeneous, free flowing powder.
such colonies. Dark brown halo around the colony is due to H2S production from Prepared Appearance
cystein combining with the tellurite salt. Moore and Parsons (80.3) found Light amber coloured opalescent gel.
Cultural Response
Tinsdale medium as an ideal medium for the routien cultivation and isolation of
Cultural characteristics after 40-48 hours at 35-370C.
Corynebacterium diptheriae. They also confirmed the stability of halo formation
Organisms (ATCC) Growth Colour of RGI
on clear medium and its specificity for Corynebacterium diptheriae and Colony
Corynebacterium ulcerans. Corynebacterium ulcerans found in nasopharynx form
colonies same as Corynebacterium diptheriae and require further biochemical Corynebacterium diptheriae
testing. type gravis Good-luxuriant Brown to More than 70%
black with halo
Do not incubate the plate in 5-10 % CO2 as it retards the development of
type intermedius Good-luxuriant Brown to More than 70%
characteristic halos (69). black with halo
Formula* type mitis Good-luxuriant Brown to More than 70%
Ingredients in grams per liter black with halo
Peptic digest of animal tissue 20.0 Streptococcus Good Black pin point, More than 70%
Sodium chloride 5.00 pyogenes (19615) without halo
L-Cystine 0.24 Klebsiella Inhibited – 0%
Sodium thiosulphate 0.34 pneumoniae (13883)
Agar 15.00 For growth RGI should be more than 70%
Final pH (at 250C) 7.4 ± 0.2 For Inhibition RGI should be 0%
* Formula adjusted to suit performance parameters RGI- Relative Growth Index
Directions Storage
1. Suspend 40.7 gms of the powder in 1000 ml distilled water. Store at 22-300C and prepared medium at 2-80C.
2. Mix thoroughly. Shelf Life
Use before expiry date as mentioned on the label.

Todd Hewitt Broth AM10964/AM50964

Use and beef heart infusion. Dextrose stimulates haemolysin production. This
Todd Hewitt Broth is recommended for the Cultivation of group A haemolytic medium is well buffered by sodium phosphate and sodium carbonate to
Streptococci used for serological studies. neutralize the acid produced during dextrose fermentation. This restricts
Summary destruction of antigenic streptococcal haemolysin. It is also found that sodium
Todd hewitt Broth was originally formulated by Todd and Hewitt (112.1) for the phosphate have a stimulating effect on the pneumococcal growth. Todd and
haemolysin production by Streptococci. Updyke and Nickle (112.2) modified it Hewitt broth can be employed as an alternative to serum broth or horse flesh
for the cultivation of beta-haemolytic Streptococci used for fluorescent antibody digest broth for the cultivation of streptococci prior to serological typing.
procedures (80.4, 49.4) and serotyping based on M protein production (77.1). Formula*
Principle Ingredients Gms/Liter
Beef heart, infusion form 500.00
The medium is very nutritious due to the presence of peptic digest of animal tissue

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Peptic digest of animal tissue 20.00 Prepared Appearance:
Dextrose 2.00 Medium amber coloured, clear solution without any precipitate.
Sodium chloride 2.00 Cultural Response
Disodium phosphate 0.40 Cultural characteristics after 18-48 hours at 35-370C.
Sodium carbonate 2.50 Organisms (ATCC) Growth
Final pH (at 250C) 7.8 ± 0.2 Neisseria meningitidis (13090) Good to luxuriant
* Formula adjusted to suit performance parameters Streptococcus pneumoniae (6303) Good to luxuriant
Directions Streptococcus pyogenes (19615) Good to luxuriant
1. Suspend 37.0 gms of the powder in 1000 ml distilled water. Streptococcus mitis (9895) Good to luxuriant
Storage
2. Mix well and dispense as desired.
Store at 22-300C and prepared medium at 2-80C.
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Shelf Life
Quality Control
Dehydrated Appearance
Use before expiry date as mentioned on the label.
Yellow coloured, homogeneous, free flowing powder.

Tomato Juice Agar AM1097/AM5097

Tomato Juice Agar, Special AM1098/AM5098
Use Formula*
Tomato Juice Agar and Tomato Juice Agar, Special are used for the cultivation and Ingredients in grams per liter Tomato Juice Agar Tomato Juice
Agar Special
enumeration of Lactobacillus species. Tomato Juice Agar Special is also used for
Tomato Juice (from 400 ml) 20.0 20.0
the cultivation and enumeration of acidophilic microorganisms from clinical and Peptonized Milk 10.0 10.0
non-clinical specimens. Peptone 10.0 10.0
Summary Agar 11.0 20.0
Mickle and Breed (80) first reported the use of tomato juice in culture media for Final pH (at 250C) 6.1 ± 0.2 5.0 ± 0.2
lactobacilli. Kulp (64) investigated the use of tomato juice on bacterial * Formula adjusted to suit performance parameters
development and found that the growth of L.acidophilus was enhanced. Kulp Directions

and White (63) formulated Tomato Juice Agar, a modification of the original 1. Suspend the powder in 1000 ml distilled water.
formula, which yields high counts of lactobacilli from foodstuffs and clinical Tomato Juice Agar - 51 gms
specimens. Tomato Juice Agar, Special - 60 gms
Tomato Juice Agar Special is a formulation of Jay (47) and is recommended for 2. Mix thoroughly.
the direct plate counts of lactobacilli from saliva and acidophilic organisms in 3. Boil with frequent agitation to dissolve the powder completely.
foodstuffs. The acidic pH of Tomato Juice Agar Special enhances growth of 4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
lactobacilli while inhibiting growth of accompanying bacteria. The number of
5. Avoid overheating Tomato Juice Agar, Special as it could cause a softer
lactobacilli in saliva is an index of a predisposition to dental caries as described by
medium.
Jay. Many dentists use the direct count of lactobacilli for the diagnosis of caries.
Quality Control
This medium is more selective for lactobacilli than Tomato Juice Agar. Dehydrated Appearance
Principle Yellow coloured, free-flowing, homogeneous powder.
Peptone provides nitrogen, amino acids and carbon. Tomato juice provides Prepared Appearance
carbon, proteins and other nutrients. Peptonized milk contains lactose, which is Medium to dark amber coloured, very slightly opalescent gel.
the energy source. Cultural Response
Cultural characteristics after 40-48 hours at 35 ± 2 0C

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Organisms (ATCC) Growth on Growth on Storage
Tomato Juice Tomato Juice
Agar Agar, Special Store at 22-300C and prepared medium at 2-80C.
Lactobacillus casei (9595) Luxuriant Luxuriant Shelf Life
Lactobacillus acidophilus (4356) Luxuriant Luxuriant Use before expiry date as mentioned on the label.

Triple Sugar Iron Agar AM1099/AM5099

Use sucrose. A red colour in the slant and butt indicates that the organism being tested
Triple Sugar Iron Agar is used for the identification of gram-negative enteric is a non-fermenter.
bacilli on the basis of dextrose, lactose and sucrose fermentation and hydrogen Hydrogen sulphide results in a black precipitate in the butt of the tube because
sulphide production. reduction of thiosulphate proceeds in an acid environment. Some members of the
Summary Enterobacteriaceae and H2S producing Salmonella may not be H2S positive on
Sulkin and Willett originally developed Triple Sugar Iron Agar, which was later Triple Sugar Iron Agar (may be H2S positive on Kligler Iron Agar) because
modified by Hajna by adding sucrose to the double sugar (dextrose and lactose) utilization of sucrose in TSI Agar suppresses the enzyme pathway that results in
formulation of Kligler Iron Agar. The addition of sucrose increased the sensitivity H2S production. Splitting and cracking of the medium indicates gas production.
of the medium by facilitating the detection of sucrose fermenting bacilli as well as Formula*
lactose and/or dextrose fermenters. Acid and gas production is an indication of Ingredients in grams per liter
carbohydrate fermentation, which gives a visible colour change from red to yellow Lactose 10.0
due to change in the phenol red indicator. The production of hydrogen sulphide is Sucrose 10.0
indicated by the presence of a precipitate that blackens the medium in the butt of Peptone 10.0
Tryptone 10.0
the tube.
Sodium Chloride 5.0
The medium complies with the recommendations of APHA for examination of food Yeast Extract 3.0
(20), dairy (39), water and wastewater (36) and for microbial limit test (46, Beef Extract 3.0
114) in confirming the presence of Salmonella and in the identification of gram- Dextrose 1.0
negative bacilli. Triple Sugar Iron Agar is also included in the Bacteriological Sodium Thiosulphate 0.3
Ferrous Sulphate 0.2
Analytical Manual for food and cosmetics testing (113).
Phenol Red 0.024
Principle
Agar 12.0
Tryptone, peptone, yeast extract and beef extract provides nitrogenous 0
Final pH (at 25 C) 7.4 ± 0.2
compounds, sulphur, trace elements, vitamin B complex, etc. while sodium * Formula adjusted to suit performance parameters
chloride maintains the osmotic equilibrium. Lactose, sucrose and dextrose are the Directions
fermentable carbohydrates. Sodium thiosulphate and ferrous sulphate make the 1. Suspend 64.52 gms of the powder in 1000 ml distilled water.
H2S indicator system. Phenol red is the pH indicator. 2. Mix thoroughly.
Carbohydrate fermentation is indicated by the production of gas and a change in 3. Boil with frequent agitation to dissolve the powder completely.
the colour of the pH indicator from red to yellow. More amounts of acids are 4. Dispense in desired containers as per requirements.
liberated in the butt (fermentation) than in the slant (respiration). Growing
5. Sterilize by autoclaving at 1150C (10 lbs pressure) for 15 minutes.
bacteria also form alkaline products from the oxidative decarboxylation of
peptone and these alkaline products neutralize the large amount of acid present 6. Allow the medium to set in sloped form with a butt about 1 inch long.
Quality Control
in the butt, therefore, if the medium in the butt of the tube becomes yellow
Dehydrated Appearance
(acidic) while the medium in the slant becomes red (alkaline) the organism being
Light pink coloured, homogeneous, free flowing powder.
tested only ferments dextrose (glucose). A yellow colour in the slant and butt Prepared Appearance
indicates that the organism being tested ferments dextrose, lactose and/or Pinkish red coloured, clear to slightly opalescent gel.

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 319
 Exploring... Accumix
Cultural Response tested only ferments dextrose.
Cultural characteristics after 18-24 hours at 35-370C.
3. A yellow (acidic) colour in the slant and butt indicates that the organisms
Organisms (ATCC) Growth Slant Butt Gas H2S
being tested ferment dextrose, lactose and /or sucrose.
Enterobacter aerogenes (13048) Luxuriant A A + -
Escherichia coli (25922) Luxuriant A A + - 4. A red (alkaline) colour in the slant and butt indicates that the organism
Klebsiella pneumoniae (13883) Luxuriant A A + - being tested is a non-fermenter.
Proteus vulgaris (13315) Luxuriant K A - +
Salmonella serotype Typhimurium Luxuriant K A + +
5. Hydrogen sulphide production results in a black precipitate in the butt of the
(14028) tube.
Shigella flexneri (12022) Luxuriant K A - - 6. Splitting and cracking of the medium indicates gas production.
Key: Precautions / Limitations
A = acidic, yellow
1. It is important to stab the butt of the medium. Failure to stab the butt
K = alkaline, no change
+ = blackening (H2S) , positive reaction
invalidates this test. Do not use an inoculating loop to inoculate a tube of
- = no reaction
Triple Sugar Iron Agar because while stabbing the butt, mechanical splitting
Procedure of the medium occurs, causing a false positive result for gas production.
1. Touch only the center of an isolated colony on an enteric plated medium with Caps must be loosened during this test or erroneous results will occur.
a cool and sterile needle, stab into the medium and then streak back and 2. Triple Sugar Iron Agar must be read within the 18-24 hour stated incubation
forth along the surface of the slant. period. A false-positive reaction may be observed if read too early. A false-
2. Several colonies from each primary plate should be studied separately, since negative reaction may be observed if read later than 24 hours.
mixed infections may occur. 3. Hydrogen sulphide production may be evident on Kligler Iron Agar but
0
3. Incubate at 35 C with caps loosened and examine after 18-24 hours for negative on Triple Sugar Iron Agar. Studies by Bulmash and Fulton showed
carbohydrate fermentation, gas production and hydrogen sulphide that the utilization of sucrose could suppress the enzymatic mechanism
production. Any combination of these reactions may be observed. Do not responsible for H2S production. Not all H2S positive Salmonellae are positive
incubate longer than 24 hours because the acid reaction in the slant of on Triple Sugar Iron Agar.
lactose and sucrose fermenters may revert to an alkaline reaction. 4. Sucrose is added to Triple Sugar Iron Agar to eliminate some sucrose
Interpretation of Results fermenting, lactose non-fermenters such as Proteus species.
1. Compare reactions produced by the unknown isolate with those produced by Storage
the known control organisms. Store at 22-300C and prepared medium at 2-80C.
2. Carbohydrate fermentation is indicated by a yellow colouration of the Shelf Life

medium. If the medium in the butt of the tube becomes yellow (acidic), Use before expiry date as mentioned on the label.
while the medium in the slant becomes red (alkaline), the organism being

Triple Sugar Iron Agar IP AM10991/AM50991

Use indication of carbohydrate fermentation, which gives a visible colour change from
Triple Sugar Iron Agar is used for the identification of gram-negative enteric red to yellow due to change in the phenol red indicator. The production of
bacilli on the basis of dextrose, lactose and sucrose fermentation and hydrogen hydrogen sulphide is indicated by the presence of a precipitate that blackens the
sulphide production. medium in the butt of the tube. The medium complies with the recommendations
Summary of APHA for examination of food (115.1), dairy, water and wastewater and for
Sulkin and Willett (105.2) originally developed Triple Sugar Iron Agar, which was microbial limit test in confirming the presence of Salmonella (31.1, 36.1) and in
later modified by Hajna (40) by adding sucrose to the double sugar (dextrose the identification of gram-negative bacilli (77.1). Triple Sugar Iron Agar is also
and lactose) formulation of Kligler Iron Agar. The addition of sucrose increased the included in the Bacteriological Analytical Manual for food and cosmetics testing.
sensitivity of the medium by facilitating the detection of sucrose fermenting bacilli Principle
as well as lactose and/or dextrose fermenters. Acid and gas production is an Peptone, yeast extract and beef extract provides nitrogenous compounds,

320 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
 Exploring... Accumix
sulphur, trace elements, vitamin B complex, etc. while sodium chloride maintains Quality Control
the osmotic equilibrium. Lactose, sucrose and dextrose are the fermentable Dehydrated Appearance
Light pink coloured, homogeneous, free flowing powder.
carbohydrates. Sodium thiosulphate and ferrous sulphate make the H2S indicator
Prepared Appearance
system. Phenol red is the pH indicator. Carbohydrate fermentation is indicated by Pinkish red coloured, clear to slightly opalescent gel.
the production of gas and a change in the colour of the pH indicator from red to Cultural Response
yellow. More amounts of acids are liberated in the butt (fermentation) than in the Cultural characteristics after 18-24 hours at 35-370C.
slant (respiration). Growing bacteria also form alkaline products from the Organisms (ATCC) Growth Slant Butt Gas H2S
oxidative decarboxylation of peptone and these alkaline products neutralize the Enterobacter aerogenes (13048) Luxuriant A A + -
large amount of acid present in the butt, therefore, if the medium in the butt of the Escherichia coli (25922) Luxuriant A A + -
Klebsiella pneumoniae (13883) Luxuriant A A + -
tube becomes yellow (acidic) while the medium in the slant becomes red
Proteus vulgaris (13315) Luxuriant K A - +
(alkaline) the organism being tested only ferments dextrose (glucose). A yellow Salmonella serotype Typhimurium Luxuriant K A + +
colour in the slant and butt indicates that the organism being tested ferments (14028)
dextrose, lactose and/or sucrose. A red colour in the slant and butt indicates that Shigella flexneri (12022) Luxuriant K A - -
the organism being tested is a non-fermenter. Hydrogen sulphide results in a Key:
black precipitate in the butt of the tube because reduction of thiosulphate A = acidic, yellow
K = alkaline, no change
proceeds in an acid environment.
+ = blackening (H2S) , positive reaction
Some members of the Enterobacteriaceae and H2S producing Salmonella may - = no reaction
not be H2S positive on Triple Sugar Iron Agar (may be H2S positive on Kligler Iron Procedure
Agar) because utilization of sucrose in TSI 1. Touch only the center of an isolated colony on an enteric plated medium with
Agar suppresses the enzyme pathway that results in H2S production. Splitting and a cool and sterile needle, stab into the medium and then streak back and forth
cracking of the medium indicates gas production. along the surface of the slant.
Formula* 2. Several colonies from each primary plate should be studied separately, since
Ingredients in grams per liter
mixed infections may occur.
Lactose 10.0
Sucrose 10.0 3. Incubate at 350C with caps loosened and examine after 18-24 hours for
Peptone 20.0 carbohydrate fermentation, gas production and hydrogen sulphide
Sodium chloride 5.0 production. Any combination of these reactions may be observed. Do not
Yeast extract 3.0 incubate longer than 24 hours because the acid reaction in the slant of lactose
Beef extract 3.0
and sucrose fermenters may revert to an alkaline reaction.
Dextrose monohydrate 1.0
Interpretation of Results
Ferrous sulphate 0.2
Sodium thiosulphate 0.3 1. Compare reactions produced by the unknown isolate with those produced by
Phenol red 0.024 the known control organisms.
Agar 12.0
2. Carbohydrate fermentation is indicated by a yellow colouration of the
Final pH (at 250C) 7.0 ± 0.2
medium. If the medium in the butt of the tube becomes yellow (acidic), while
* Formula adjusted to suit performance parameters
Directions
the medium in the slant becomes red (alkaline), the organism being tested
only ferments dextrose.
1. Suspend 64.52 gms of the powder in 1000 ml distilled water.
3. A yellow (acidic) colour in the slant and butt indicates that the organisms being
2. Mix thoroughly.
tested ferment dextrose, lactose and /or sucrose.
3. Boil with frequent agitation to dissolve the powder completely.
4. A red (alkaline) colour in the slant and butt indicates that the organism being
4. Dispense in desired containers as per requirements.
tested is a non-fermenter.
5. Sterilize by autoclaving at 1150C (10 lbs pressure) for 15 minutes. 5. Hydrogen sulphide production results in a black precipitate in the butt of the
6. Allow the medium to set in sloped form with a butt about 1 inch long. tube.

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 321
 Exploring... Accumix
6. Splitting and cracking of the medium indicates gas production. 3. Hydrogen sulphide production may be evident on Kligler Iron Agar but negative
Precautions / Limitations on Triple Sugar Iron Agar. Studies by Bulmash and Fulton showed that the
1. It is important to stab the butt of the medium. Failure to stab the butt utilization of sucrose could suppress the enzymatic mechanism responsible for
invalidates this test. Do not use an inoculating loop to inoculate a tube of H2S production. Not all H2S positive Salmonellae are positive on Triple Sugar
Triple Sugar Iron Agar because while stabbing the butt, mechanical splitting of Iron Agar.
the medium occurs, causing a false positive result for gas production. Caps 4. Sucrose is added to Triple Sugar Iron Agar to eliminate some sucrose
must be loosened during this test or erroneous results will occur. fermenting, lactose non-fermenters such as Proteus species.
2. Triple Sugar Iron Agar must be read within the 18-24 hour stated incubation Storage
period. A false-positive reaction may be observed if read too early. A false- Store at 22-300C and prepared medium at 2-80C.
negative reaction may be observed if read later than 24 hours. Shelf Life
Use before expiry date as mentioned on the label.

Triple Sugar Iron Agar USP AM10992/AM50992

Use bacteria also form alkaline products from the oxidative decarboxylation of
Triple Sugar Iron Agar is used for the identification of gram-negative enteric peptone and these alkaline products neutralize the large amount of acid present
bacilli on the basis of dextrose, lactose and sucrose fermentation and hydrogen in the butt, therefore, if the medium in the butt of the tube becomes yellow
sulphide production in complaince with USP. (acidic) while the medium in the slant becomes red (alkaline) the organism being
Summary tested only ferments dextrose (glucose). A yellow colour in the slant and butt
Sulkin and Willett (105.2) originally developed Triple Sugar Iron Agar, which indicates that the organism being tested ferments dextrose, lactose and/or
was later modified by Hajna (40) by adding sucrose to the double sugar sucrose. A red colour in the slant and butt indicates that the organism being tested
(dextrose and lactose) formulation of Kligler Iron Agar. The addition of sucrose is a non-fermenter.
increased the sensitivity of the medium by facilitating the detection of sucrose Hydrogen sulphide results in a black precipitate in the butt of the tube because
fermenting bacilli as well as lactose and/or dextrose fermenters. Acid and gas reduction of thiosulphate proceeds in an acid environment. Some members of the
production is an indication of carbohydrate fermentation, which gives a visible Enterobacteriaceae and H2S producing Salmonella may not be H2S positive on
colour change from red to yellow due to change in the phenol red indicator. The Triple Sugar Iron Agar (may be H2S positive on Kligler Iron Agar) because
production of hydrogen sulphide is indicated by the presence of a precipitate that
utilization of sucrose in TSI Agar suppresses the enzyme pathway that results in
blackens the medium in the butt of the tube. The medium complies with the
H2S production. Splitting and cracking of the medium indicates gas production.
recommendations of APHA for examination of food (115.1), dairy, water and
Formula*
wastewater and for microbial limit test in confirming the presence of Salmonella Ingredients in grams per liter
(31.1, 36.1) and in the identification of gram-negative bacilli (77.1). Triple Lactose 10.0
Sugar Iron Agar is also included in the Bacteriological Analytical Manual for food Sucrose 10.0
and cosmetics testing. Pancreatic digest of casein 10.0
Principle Pancreatic digest of animal tissue 10.0
Pancreatic Digest of Casein and Pancreatic Digest of animal tissue provides Sodium chloride 5.0
Dextrose 1.0
nitrogenous compounds, sulphur, trace elements, vitamin B complex, etc. while
Sodium thiosulphate 0.2
sodium chloride maintains the osmotic equilibrium. Lactose, sucrose and
Ferrous ammonium sulphate 0.2
dextrose are the fermentable carbohydrates. Sodium thiosulphate and ferrous Phenol red 0.025
sulphate make the H2S indicator system. Phenol red is the pH indicator. Agar 13.0
Carbohydrate fermentation is indicated by the production of gas and a change in Final pH (at 250C) 7.3 ± 0.2
the colour of the pH indicator from red to yellow. More amounts of acids are * Formula adjusted to suit performance parameters
liberated in the butt (fermentation) than in the slant (respiration). Growing Directions
1. Suspend 59.42 gms of the powder in 1000 ml distilled water.

322 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
 Exploring... Accumix
2. Mix thoroughly. Interpretation of Results

3. Boil with frequent agitation to dissolve the powder completely. 1. Compare reactions produced by the unknown isolate with those produced by
the known control organisms.
4. Dispense in desired containers as per requirements.
2. Carbohydrate fermentation is indicated by a yellow colouration of the
5. Sterilize by autoclaving at 1150C (10 lbs pressure) for 15 minutes.
medium. If the medium in the butt of the tube becomes yellow (acidic), while
6. Allow the medium to set in sloped form with a butt about 1 inch long.
the medium in the slant becomes red (alkaline), the organism being tested
Quality Control
only ferments dextrose.
Dehydrated Appearance
Light pink coloured, homogeneous, free flowing powder. 3. A yellow (acidic) colour in the slant and butt indicates that the organisms
Prepared Appearance being tested ferment dextrose, lactose and /or sucrose.
Pinkish red coloured, clear to slightly opalescent gel. 4. A red (alkaline) colour in the slant and butt indicates that the organism
Cultural Response being tested is a non-fermenter.
Cultural characteristics after 18-24 hours at 35-370C.
Organisms (ATCC) Growth Slant Butt Gas H2S
5. Hydrogen sulphide production results in a black precipitate in the butt of the
Enterobacter aerogenes(13048) Luxuriant A A + - tube.
Escherichia coli (25922) Luxuriant A A + - 6. Splitting and cracking of the medium indicates gas production.
Klebsiella pneumoniae(13883) Luxuriant A A + -
Precautions / Limitations
Proteus vulgaris (13315) Luxuriant K A - +
Salmonella serotype Luxuriant K A + + 1. It is important to stab the butt of the medium. Failure to stab the butt
Typhimurium (14028) invalidates this test. Do not use an inoculating loop to inoculate a tube of
Shigella flexneri (12022) Luxuriant K A - -
Triple Sugar Iron Agar because while stabbing the butt, mechanical splitting
Key:
of the medium occurs, causing a false positive result for gas production. Caps
A = acidic, yellow
K = alkaline, no change must be loosened during this test or erroneous results will occur.
+ = blackening (H2S) , positive reaction 2. Triple Sugar Iron Agar must be read within the 18-24 hour stated incubation
- = no reaction period. A false-positive reaction may be observed if read too early. A false-
For growth RGI should be more than 70% negative reaction may be observed if read later than 24 hours.
RGI- Relative Growth Index
3. Hydrogen sulphide production may be evident on Kligler Iron Agar but
Procedure
negative on Triple Sugar Iron Agar. Studies by Bulmash and Fulton showed
1. Touch only the center of an isolated colony on an enteric plated medium with
that the utilization of sucrose could suppress the enzymatic mechanism
a cool and sterile needle, stab into the medium and then streak back and
responsible for H2S production. Not all H2S positive Salmonellae are positive
forth along the surface of the slant.
on Triple Sugar Iron Agar.
2. Several colonies from each primary plate should be studied separately, since
4. Sucrose is added to Triple Sugar Iron Agar to eliminate some sucrose
mixed infections may occur.
fermenting, lactose non-fermenters such as Proteus species.
3. Incubate at 350C with caps loosened and examine after 18-24 hours for Storage
carbohydrate fermentation, gas production and hydrogen sulphide
Store at 22-300C and prepared medium at 2-80C.
production. Any combination of these reactions may be observed. Do not Shelf Life
incubate longer than 24 hours because the acid reaction in the slant of
Use before expiry date as mentioned on the label.
lactose and sucrose fermenters may revert to an alkaline reaction.

Triple Sugar Iron Agar EP AM10993/AM50993

Use Summary
Triple Sugar Iron Agar is used for the identification of gram-negative enteric Sulkin and Willett (105.2) originally developed Triple Sugar Iron Agar, which
bacilli on the basis of dextrose, lactose and sucrose fermentation and hydrogen was later modified by Hajna (40) by adding sucrose to the double sugar (dextrose
sulphide production in compliance with EP. and lactose) formulation of Kligler Iron Agar. The addition of sucrose increased

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 323
 Exploring... Accumix
the sensitivity of the medium by facilitating the detection of sucrose fermenting Sodium thiosulphate 0.3
bacilli as well as lactose and/or dextrose fermenters. Acid and gas production is Ferric ammonium citrate 0.3
Phenol red 0.025
an indication of carbohydrate fermentation, which gives a visible colour change
Agar 12.0
from red to yellow due to change in the phenol red indicator. The production of
Final pH (at 250C) 7.4 ± 0.2
hydrogen sulphide is indicated by the presence of a precipitate that blackens the * Formula adjusted to suit performance parameters
medium in the butt of the tube. The medium complies with the recommendations Directions
of APHA for examination of food (115.1), dairy, water and wastewater and for 1. Suspend 64.63 gms of the powder in 1000 ml distilled water.
microbial limit test in confirming the presence of Salmonella (31.1, 36.1) and in
2. Mix thoroughly.
the identification of gram-negative bacilli (77.1). Triple Sugar Iron Agar is also
included in the Bacteriological Analytical Manual for food and cosmetics testing . 3. Boil with frequent agitation to dissolve the powder completely.
Principle 4. Dispense in desired containers as per requirements.
Tryptone, peptone, yeast extract and beef extract provides nitrogenous 5. Sterilize by autoclaving at 1150C (10 lbs pressure) for 15 minutes.
compounds, sulphur, trace elements, vitamin B complex, etc. while sodium 6. Allow the medium to set in sloped form with a butt about 1 inch long.
chloride maintains the osmotic equilibrium. Lactose, Quality Control
sucrose and dextrose are the fermentable carbohydrates. Sodium thiosulphate Dehydrated Appearance
and ferrous sulphate make the H2S indicator system. Phenol red is the pH Light pink coloured, homogeneous, free flowing powder.
Prepared Appearance
indicator. Carbohydrate fermentation is indicated by the production of gas and a
Pinkish red coloured, clear to slightly opalescent gel.
change in the colour of the pH indicator from red to yellow. More amounts of acids
Cultural Response
are liberated in the butt (fermentation) than in the slant (respiration). Growing Cultural characteristics after 18-24 hours at 35-370C.
bacteria also form alkaline products from the oxidative decarboxylation of Organisms (ATCC) Growth Slant Butt Gas H2S RGI
peptone and these alkaline products neutralize the large amount of acid present Enterobacter Luxuriant A A + - More than 70%
in the butt, therefore, if the medium in the butt of the tube becomes yellow aerogenes (13048)
Escherichia Luxuriant A A + - More than 70%
(acidic) while the medium in the slant becomes red (alkaline) the organism being
coli (25922)
tested only ferments dextrose (glucose). A yellow colour in the slant and butt Klebsiella Luxuriant A A + - More than 70%
indicates that the organism being tested ferments dextrose, lactose and/or pneumoniae (13883)
sucrose. A red colour in the slant and butt indicates that the organism being tested Proteus vulgaris Luxuriant K A - + More than 70%
(13315)
is a non-fermenter. Hydrogen sulphide results in a black precipitate in the butt of Salmonella serotype Luxuriant K A + + More than 70%
the tube because reduction of thiosulphate proceeds in an acid environment. Typhimurium (14028)
Shigella flexneri (12022) Luxuriant K A - - More than 70%
Some members of the Enterobacteriaceae and H2S producing Salmonella may
Key:
not be H2S positive on Triple Sugar Iron Agar (may be H2S positive on Kligler Iron A = acidic, yellow
Agar) because utilization of sucrose in TSI K = alkaline, no change
+ = blackening (H2S) , positive reaction
Agar suppresses the enzyme pathway that results in H2S production. Splitting and - = no reaction
cracking of the medium indicates gas production. For growth RGI should be more than 70%
Formula* RGI- Relative Growth Index
Ingredients in grams per liter Procedure
Lactose monohydrate 10.0 1. Touch only the center of an isolated colony on an enteric plated medium with
Sucrose 10.0
a cool and sterile needle, stab into the medium and then streak back and
Peptone (casein & beef) 20.0
forth along the surface of the slant.
Sodium chloride 5.0
Yeast extract 3.0 2. Several colonies from each primary plate should be studied separately, since
Beef extract 3.0 mixed infections may occur.
Glucose monohydrate 1.0 3. Incubate at 350C with caps loosened and examine after 18-24 hours for

324 Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits
 Exploring... Accumix
carbohydrate fermentation, gas production and hydrogen sulphide Precautions / Limitations
production. Any combination of these reactions may be observed. Do not 1. It is important to stab the butt of the medium. Failure to stab the butt
incubate longer than 24 hours because the acid reaction in the slant of invalidates this test. Do not use an inoculating loop to inoculate a tube of
lactose and sucrose fermenters may revert to an alkaline reaction. Triple Sugar Iron Agar because while stabbing the butt, mechanical splitting of
Interpretation of Results the medium occurs, causing a false positive result for gas production. Caps
1. Compare reactions produced by the unknown isolate with those produced by must be loosened during this test or erroneous results will occur.
the known control 2. Triple Sugar Iron Agar must be read within the 18-24 hour stated incubation
organisms. period. A false-positive reaction may be observed if read too early. A false-
2. Carbohydrate fermentation is indicated by a yellow colouration of the negative reaction may be observed if read later than 24 hours.
medium. If the medium in the butt of the tube becomes yellow (acidic), 3. Hydrogen sulphide production may be evident on Kligler Iron Agar but
while the medium in the slant becomes red (alkaline), the organism being negative on Triple Sugar Iron Agar. Studies by Bulmash and Fulton showed
tested only ferments dextrose. that the utilization of sucrose could suppress the enzymatic mechanism
3. A yellow (acidic) colour in the slant and butt indicates that the organisms responsible for H2S production. Not all H2S positive Salmonellae are positive
being tested ferment dextrose, lactose and /or sucrose. on Triple Sugar Iron Agar.
4. A red (alkaline) colour in the slant and butt indicates that the organism being 4. Sucrose is added to Triple Sugar Iron Agar to eliminate some sucrose
tested is a non- fermenter. fermenting, lactose non-fermenters such as Proteus species.
Storage
5. Hydrogen sulphide production results in a black precipitate in the butt of the
tube. Store at 22-300C and prepared medium at 2-80C.
Shelf Life
6. Splitting and cracking of the medium indicates gas production.
Use before expiry date as mentioned on the label.

Triple Sugar Iron Agar BIS AM10994/AM50994

Triple Sugar Iron Agar ISO AM50995
Use Sugar Iron Agar is also included in the Bacteriological Analytical Manual for food
Triple Sugar Iron Agar is used for the identification of gram-negative enteric and cosmetics testing.
bacilli on the basis of dextrose, lactose and sucrose fermentation and hydrogen Principle
sulphide production. Peptone, yeast extract and beef extract provides nitrogenous compounds,
Summary sulphur, trace elements, vitamin B complex, etc. while sodium chloride maintains
Sulkin and Willett (105.2) originally developed Triple Sugar Iron Agar, which the osmotic equilibrium. Lactose, sucrose and glucose are the fermentable
was later modified by Hajna (40) by adding sucrose to the double sugar carbohydrates. Sodium thiosulphate and Iron (III) citrate make the H2S indicator
(dextrose and lactose) formulation of Kligler Iron Agar. The addition of sucrose system. Phenol red is the pH indicator. Carbohydrate fermentation is indicated by
increased the sensitivity of the medium by facilitating the detection of sucrose the production of gas and a change in the colour of the pH indicator from red to
fermenting bacilli as well as lactose and/or dextrose fermenters. Acid and gas yellow. More amounts of acids are liberated in the butt (fermentation) than in the
production is an indication of carbohydrate fermentation, which gives a visible slant (respiration). Growing bacteria also form alkaline products from the
colour change from red to yellow due to change in the phenol red indicator. The oxidative decarboxylation of peptone and these alkaline products neutralize the
production of hydrogen sulphide is indicated by the presence of a precipitate that large amount of acid present in the butt, therefore, if the medium in the butt of the
blackens the medium in the butt of the tube. The medium complies with the tube becomes yellow (acidic) while the medium in the slant becomes red
recommendations of APHA for examination of food (115.1), dairy, water and (alkaline) the organism being tested only ferments dextrose (glucose). A yellow
wastewater and for microbial limit test in confirming the presence of Salmonella colour in the slant and butt indicates that the organism being tested ferments
(31.1, 36.1) and in the identification of gram-negative bacilli (77.1). Triple dextrose, lactose and/or sucrose. A red colour in the slant and butt indicates that

Microxpress Dehydrated Culture Media, Bases, Supplements, Ready to use Media, Indicators & Stains, Test Kits 325
 Exploring... Accumix
the organism being tested is a non-fermenter. Hydrogen sulphide results in a Key:
A = acidic, yellow
black precipitate in the butt of the tube because reduction of thiosulphate K = alkaline, no change
proceeds in an acid environment. + = blackening (H2S) , positive reaction

Some members of the Enterobacteriaceae and H2S producing Salmonella may - = no reaction
For growth RGI should be more than 70%
not be H2S positive on Triple Sugar Iron Agar (may be H2S positive on Kligler Iron RGI- Relative Growth Index
Agar) because utilization of sucrose in TSI Procedure
Agar suppresses the enzyme pathway that results in H2S production. Splitting and 1. Touch only the center of an isolated colony on an enteric plated medium with
cracking of the medium indicates gas production. a cool and sterile needle, stab into the medium and then streak back and
Formula* forth along the surface of the slant.
Ingredients in grams per liter 2. Several colonies from each primary plate should be studied separately, since
Lactose 10.0
mixed infections may occur.
Sucrose 10.0
Peptone 20.0 3. Incubate at 350C with caps loosened and examine after 18-24 hours for
Sodium chloride 5.0 carbohydrate fermentation, gas production and hydrogen sulphide
Yeast extract 3.0 production. Any combination of these reactions may be observed. Do not
Meat extract 3.0 incubate longer than 24 hours because the acid reaction in the slant of
Glucose 1.0 lactose and sucrose fermenters may revert to an alkaline reaction.
Sodium thiosulphate 0.3 Interpretation of Results
Iron (III) citrate 0.3
1. Compare reactions produced by the unknown isolate with those produced by
Phenol red 0.024
Agar 12.0 the known control organisms.
Final pH (at 250C) 7.4 ± 0.2 2. Carbohydrate fermentation is indicated by a yellow colouration of the
* Formula adjusted to suit performance parameters medium. If the medium in the butt of the tube becomes yellow (acidic),
Directions while the medium in the slant becomes red (alkaline), the organism being
1. Suspend 64.52 gms of the powder in 1000 ml distilled water. tested only ferments dextrose.
2. Mix thoroughly. 3. A yellow (acidic) colour in the slant and butt indicates that the organisms
3. Boil with frequent agitation to dissolve the powder completely. being tested ferment dextrose, lactose and /or sucrose.
4. Dispense in desired containers as per requirements. 4. A red (alkaline) colour in the slant and butt indicates that the organism being
0
5. Sterilize by autoclaving at 115 C (10 lbs pressure) for 15 minutes. tested is a non-fermenter.
6. Allow the medium to set in sloped form with a butt about 1 inch long. 5. Hydrogen sulphide production results in a black precipitate in the butt of the
Quality Control tube.
Dehydrated Appearance 6. Splitting and cracking of the medium indicates gas production.
Light pink coloured, homogeneous, free flowing powder. Precautions / Limitations
Prepared Appearance 1. It is important to stab the butt of the medium. Failure to stab the butt
Pinkish red coloured, clear to slightly opalescent gel.
invalidates this test. Do not use an inoculating loop to inoculate a tube of
Cultural Response
Triple Sugar Iron Agar because while stabbing the butt, mechanical splitting
Cultural characteristics after 18-24 hours at 35-370C.
Organisms (ATCC) Growth Slant Butt Gas H2S
of the medium occurs, causing a false positive result for gas production. Caps
Enterobacter aerogenes (13048) Luxuriant A A + - must be loosened during this test or erroneous results will occur.
Escherichia coli (25922) Luxuriant A A + - 2. Triple Sugar Iron Agar must be read within the 18-24 hour stated incubation
Klebsiella pneumoniae(13883) Luxuriant A A + -
Proteus vulgaris (13315) Luxuriant K A - +
period. A false-positive reaction may be observed if read too early. A false-
Salmonella serotype Luxuriant K A + + negative reaction may be observed if read later than 24 hours.
Typhimurium (14028)
3. Hydrogen sulphide production may be evident on Kligler Iron Agar but
Shigella flexneri (12022) Luxuriant K A - -

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negative on Triple Sugar Iron Agar. Studies by Bulmash and Fulton showed Storage
that the utilization of sucrose could suppress the enzymatic mechanism Store at 22-300C and prepared medium at 2-80C.
responsible for H2S production. Not all H2S positive Salmonellae are positive Shelf Life
on Triple Sugar Iron Agar. Use before expiry date as mentioned on the label.
4. Sucrose is added to Triple Sugar Iron Agar to eliminate some sucrose
fermenting, lactose non-fermenters such as Proteus species.

Tryptone Salt Broth AM50996/AM50996-5K

Use Final pH ( at 25°C) 7.0±0.2
Tryptone Salt Broth is recommended for preparation of specimens, stock * Formula adjusted to suit performance parameters
Directions
suspensions and decimal dilutions for the purpose of microbiological tests of food
specimens. 1. Suspend 9.5 grams in 1000 ml distilled water.
Summary 2. Heat if necessary to dissolve the medium completely.
Tryptone Salt Broth is recommended by ISO Committee (46.6) for preparation of 3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
specimens, stock suspensions and decimal dilutions used in various 4. Mix well and dispense as desired.
microbiological tests of food specimens. Quality Control
Principle Dehydrated Appearance
Casein enzymic hydrolysate provides nitrogenous compounds and other essential Light yellow coloured, homogeneous, free flowing powder.
growth nutrients. Sodium chloride maintains the osmotic equilibrium. For ten- Prepared Appearance
fold serial dilutions, dispense the diluents in volume necessary for the preparation Light yellow coloured, clear to slightly opalescent gel.
Cultural Response
of the decimal dilutions into test tubes or flasks in quantities such that after
Cultural characteristics after 18-24 hours at 35-370C.
sterilization each tube or flask contains 9.0 ml. Transfer 1 ml of the initial
Organisms (ATCC) Growth
suspension by means of a pipette into a tube containing 9 ml of sterile diluent at Escherichia coli (25922) Luxuriant
the appropriate temperature. For optimal precision, avoid any contact between Salmonella Typhimurium (14028) Luxuriant
the pipette containing the inoculum and the sterile diluent. Mix thoroughly to Staphylococcus aureus (25923) Luxuriant
obtain dilutions until the appropriate number of microorganisms has been Storage
obtained. Store at 22-300C and prepared medium at 2-80C.
Formula* Shelf Life
Ingredients in grams per literIngredients Gms/Liter
Use before expiry date as mentioned on the label.
Casein enzymic hydrolysate 1.00
Sodium chloride 8.50

Tryptic Digest Broth AM1100/AM5100

Use Principle
Tryptic Digest Broth is used for the cultivation of fastidious microorganisms. Tryptic digest of beef heart provides carbon, nitrogen and other growth nutrients.
Summary Blood, serum or ascitic fluid provides additional growth factors. Sodium chloride
Tryptic Digest Broth was formulated by Field (31). It may be supplemented with maintains the osmotic equilibrium.
serum, blood or ascitic fluid to support the growth of fastidious organisms such as Formula*
Neisseria meningitidis, Haemophilus influenzae, and Streptococcus Ingredients in grams per liter
pneumoniae, etc. Shepard et al described its use for culturing Streptococcus Tryptic Digest of Beef Heart 10.0
Sodium Chloride 5.0
pneumoniae and Actinomycetes.

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Dextrose 1.0 With addition of blood - Cherry red coloured clear to slightly opalescent
Final pH (at 250C) 7.6 ± 0.2 solution.
* Formula adjusted to suit performance parameters With addition of serum or ascitic fluid - Dark yellow coloured clear to slightly
opalescent solution.
Directions
Cultural Response
1. Suspend 16 gms of the powder in 1000 ml distilled water. Cultural characteristics after 24-48 hours at 350C.
2. Mix thoroughly. Organisms (ATCC) Growth RGI
3. Heat if necessary to dissolve the powder completely. Haemophilus influenzae (35056) Luxuriant More than 70%
Neisseria meningitidis (13090) Luxuriant More than 70%
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. Staphylococcus aureus (25923) Luxuriant More than 70%
5. Cool below 500C and add sterile enrichment such as blood, serum or ascitic Streptococcus pneumoniae (6303) Luxuriant More than 70%
fluid as required. Streptococcus pyogenes (19615) Luxuriant More than 70%
Quality Control Storage
Dehydrated Appearance Store at 22-300C and prepared medium at 2-80C.
Yellow coloured, homogeneous, free flowing powder. Shelf Life
Prepared Appearance Use before expiry date as mentioned on the label.
Light yellow coloured clear solution.

Tryptone Agar AM11001/AM51001

Use Directions
Tryptone agar is a general-purpose medium for the growth of non-fastidious 1. Suspend 33.0 grams of the powder in 1000 ml distilled water.
microorganisms. 2. Boil with frequent agitation to dissolve the powder completely.
Summary
3. Sterilize by autoclaving at 1210 C (15 lbs pressure) for 15 minutes.
Tryptone Agar is a general-purpose agar medium, containing Tryptone, which Quality Control
will support the growth of a wide variety of microorganisms. It is suitable for the Dehydrated Appearance
cultivation of both aerobes and anaerobes. With the addition of 7% sterile blood Light yellow coloured, homogeneous, free flowing powder.
to the medium in molten state cooled to approximately 450 C, the medium serves Prepared Appearance
as a good blood agar base. The medium can also be used for the preparation of Yellow coloured, clear to slightly opalescent gel.
chocolate agar. Cultural Response

Principle Cultural characteristics after 18-24 hours at 35-370 C.

Organisms (ATCC) Growth RGI
Tryptone in the medium is a source of nitrogen and carbon. Sodium Chloride Escherichia coli (25922) Good to luxuriant More than 70%
maintains the osmotic equilibrium. Agar is the solidification agent. Pseudomonas aeruginosa (27853) Good to luxuriant More than 70%
Formula* Staphylococcus aureus (25923) Good to luxuriant More than 70%
Ingredients in grams per liter Storage
Tryptone 10.0
Store at 22-300C and prepared medium at 2-80C.
Sodium Chloride 8.0
Shelf Life
Agar 15.0
Use before expiry date as mentioned on the label.
Final pH (at 250 C) 7.0 ± 0.2
* Formula adjusted to suit performance parameters

Tryptone Glucose Beef Extract Agar AM11002/AM51002

Use Summary
Tryptone Glucose Beef Extract Agar (TGB) is used for the enumeration of bacteria Accurate methods are essential in the determination of the number of bacteria
in water, air, milk and dairy products. found in milk. The composition of medium is the most important factor that

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affects this accuracy. Bowers and Huckers (8) originally developed Tryptone Directions

Glucose Extract Agar, which was initially called Tryptone Glucose Skim Milk Agar. 1. Suspend the 24 gms of powder in 1000 ml distilled water and mix
It was later modified to the present composition for the cultivation and thoroughly.
enumeration of bacteria in air, water, milk and dairy products. Tryptone Glucose 2. Boil with frequent agitation to dissolve the powder completely.
Beef Extract Agar has been used for the study of various aspects like study of 3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
thermophilic bacteria in milk, influence of incubation temperature etc (122.1). Quality Control
Tryptone Glucose Beef Extract Agar used for the standard plate count of milk and Dehydrated Appearance
ice cream has been adopted by the committee on standard Methods for the Light yellow coloured, homogeneous, free flowing powder.
examination of dairy products (84.5). It is also recommended in Compendium of Prepared Appearance
Light yellow coloured, clear to slightly opalescent gel.
methods for the Microbiological Examination of Foods for performing the
Cultural Response
heterotrophic plate count procedure in testing bottled water (20).
Cultural characteristics after 18-24 hours at 35-370C.
Principle
Organisms (ATCC) Growth RGI
Tryptone, Beef extract and glucose supply nutrients, amino acids, carbon Bacillus subtilis (6633) Good – luxuriant More than 70%
compounds, carbohydrates, minerals and trace elements. Glucose is the energy Enterobacter aerogenes (13048) Good – luxuriant More than 70%
source. Escherichia coli (25922) Good – luxuriant More than 70%
Formula* Lactobacillus casei (9595) Good – luxuriant More than 70%
Ingredients in grams per liter Pseudomonas aeruginosa (27853) Good – luxuriant More than 70%
Tryptone 5.0 Staphylococcus aureus (25923) Good – luxuriant More than 70%
Beef extract 3.0 For growth RGI should be more than 70%
Glucose 1.0 RGI- Relative Growth Index
Agar 15.0 Storage
Final pH (at 250C) 7.0 ±0.2 Store at 22-300C and prepared medium at 2-80C.
Formula adjusted to suit performance parameters Shelf Life
Use before expiry date as mentioned on the label.

Tryptone Glucose Extract Agar AM1101/AM5101

Tryptone Glucose Extract Broth AM1102/AM5102
Use Principle
Tryptone Glucose Extract Agar is used for the enumeration of bacteria in water, air, Tryptone, yeast extract and glucose supply nutrients, amino acids, carbon
milk and dairy products while Tryptone Glucose Extract Broth is used as a general compounds, carbohydrates, minerals and trace elements. Glucose is the energy
purpose enrichment medium for a wide variety of microorganisms. source. Dipotassium phosphate is the buffer.
Summary Formula*
Bowers and Huckers (8) originally developed Tryptone Glucose Extract Agar, Ingredients in grams Tryptone Glucose Tryptone Glucose
per liter Extract Agar Extract Broth
which was initially called Tryptone Glucose Skim Milk Agar. It was later modified
Tryptone 5.0 10.0
to the present composition for the cultivation and enumeration of bacteria in air,
Yeast Extract 3.0 1.0
water, milk and dairy products. Tryptone Glucose Extract Agar has been used for Glucose 1.0 5.0
the study of various aspects like study of thermophilic bacteria in milk, influence Dipotassium Phosphate - 1.25
of incubation temperature etc. It is used as a standard medium for the Agar 15.0 -
bacteriological plate count of milk and dairy products. Final pH (at 250C) 7.0 ± 0.2 6.8 ± 0.2
Tryptone Glucose Extract Agar is recommended by APHA for the enumeration of * Formula adjusted to suit performance parameters
Directions
microorganisms in milk (39) during microbiological examination of food material
by MPN technique (20). 1. Suspend the powder in 1000 ml distilled water and mix thoroughly.

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Tryptone Glucose Extract Agar - 24 gms Organisms (ATCC) Growth on Tryptone RGI
Glucose Extract
Tryptone Glucose Extract Broth - 17.25 gms Agar and in Tryptone
2. Boil with frequent agitation to dissolve the powder completely. Glucose Extract Broth
Bacillus subtilis (6633) Luxuriant More than 70%
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Enterobacter aerogenes (13048) Luxuriant More than 70%
Quality Control
Enterococcus faecalis (29212) Luxuriant More than 70%
Dehydrated Appearance Escherichia coli (25922) Luxuriant More than 70%
Light yellow coloured, homogeneous, free flowing powder. Lactobacillus casei (9595) Luxuriant More than 70%
Prepared Appearance Pseudomonas aeruginosa (27853) Luxuriant More than 70%
Tryptone Glucose Extract Agar - Light yellow coloured, clear to slightly Staphylococcus aureus (25923) Luxuriant More than 70%
opalescent gel. For growth RGI should be more than 70%
Tryptone Glucose Extract Broth - Light yellow coloured clear solution without RGI- Relative Growth Index
any precipitate. Storage
Cultural Response
Store at 22-300C and prepared medium at 2-80C.
Cultural characteristics after 18-24 hours at 35-370C.
Shelf Life
Use before expiry date as mentioned on the label.

Tryptone Phosphate Broth AM1103/AM5103

Use 2. Mix thoroughly.
Tryptone Phosphate Broth is used for the enrichment of enteropathogenic E.coli. 3. Heat if necessary to dissolve the powder completely.
Summary 4. Dispense in 100 ml aliquots.
Tryptone Phosphate Broth is recommended by APHA (20) and is also included in 5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
the Bacteriological Analytical Manual for enriching pathogenic E.coli (113). Quality Control
Principle Dehydrated Appearance
Tryptone is the source of nitrogen. Polysorbate 80 provides the fatty acids required Yellow coloured, homogeneous, free flowing powder.
for bacterial metabolism. The inorganic phosphates serve as the buffer while Prepared Appearance
sodium chloride maintains the osmotic balance. Light amber coloured, clear solution without any precipitate.
Cultural Response
Formula*
Cultural characteristics after 18-24 hours at 35-370C.
Ingredients in grams/liter
Organisms (ATCC) Growth RGI
Tryptone 20.0
Escherichia coli (25922) Good to luxuriant More than 70%
Sodium Chloride 5.0
For growth RGI should be more than 70%
Monopotassium Phosphate 2.0
RGI- Relative Growth Index
Dipotassium Phosphate 2.0
Polysorbate 80 1.50 Storage

Final pH (at 250C) 7.0 ± 0.2 Store at 22-300C and prepared medium at 2-80C.
* Formula adjusted to suit performance parameters Shelf Life
Directions Use before expiry date as mentioned on the label.
1. Suspend 30.5 gms of the powder in 1000 ml distilled water.

Tryptone Soya Agar with Lecithin and Tween 80 AM11031/AM51031

Use
cleanliness on surface of containers, equipment surfaces and water miscible
Tryptone soya agar with lecithin and tween 80 is used for detection of cosmetics.
microorganisms on surfaces sanitized with quaternary ammonium compounds.
For the microbiological examination of surfaces RODAC (Replicate Organism
Summary
Detection and Counting) and surface plates are used (115.4). Microbiological
Tryptone soya agar with lecithin and tween 80 is recommended for validation of

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examination of surfaces before and after treatment with disinfectant provides For growth RGI should be more than 70%
data about cleanliness, which is used for validation of cleaning procedures in RGI- Relative Growth Index
To Check Neutralizing activity of the medium:
environmental sanitation (9.2).
Ø Prepare the medium as per label directions.
Principle
Ø Test the medium in parallel with Plate Count Agar (AM1081/AM5018), by
Casein enzymic hydrolysate and papaic digest of soyabean meal serves as a using the pour plate method.
source of nitrogen. Sodium chloride provides sodium ions for the membrane Ø Apply disks impregnated with varying dilutions of quaternary ammonium
transport and maintains osmotic equilibrium of the medium. Lecithin and tween compound to the medium surface.
Ø Incubate plates at 35-370C for 40-48 hours and inspect for zone of
80 inactivates disinfectant. Lecithin neutralizes quaternary ammonium inhibition.
compounds and tween 80 neutralizes substituted phenolic disinfectant. Agar is Organism (ATCC) Growth*
the solidifying agent. Escherichia coli (11229) Smaller zone of inhibition
Formula* of growth compare to Plate Count Agar
Ingredients in grams per liter Staphylococcus aureus (6538) Smaller zone of inhibition
TCasein enzymic hydrolysate 15.00 of growth compare to Plate Count Agar
Papaic digest of soyabean meal 5.00 * Interpretation- The smaller zone of inhibition indicate neutralization of the
Sodium chloride 5.00 quaternary ammonium compound by the medium.
Lecithin 0.70 Procedure
Polysorbate 80 (Tween 80) 5.00 1. Use standard procedures like the streak plate method to obtain isolated
Agar 15.00 colonies.
Final pH (at 250C) 7.3 ± 0.2 2. If the specimen to be cultured is on a swab, roll the swab on a small area of the
* Formula adjusted to suit performance parameters
agar surface and streak for isolation with a sterile loop.
Directions
3. Incubate plates aerobically, protected from light, at 35-370C for 18-24 hours
1. Suspend 45.7 gms of the powder in 1000 ml distilled water
or as required.
2. Mix thoroughly.
4. After incubation count the colonies.
3. Boil with frequent agitation to dissolve the powder completely. 5. Examine colony morphology and carry out biochemical testing for
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes. identification.
5. Cool the medium to approximately 45-500C, pour in to sterile petriplates. Interpretation of Results
Quality Control 1. Count all developing colonies.
Dehydrated Appearance 2. Interpretation of results are relative, each laboratory should establish its own
Light yellow coloured, homogeneous, free flowing powder. values for cleanness and compare the counts for results.
Prepared Appearance Limitations
Light to slightly amber, clear to slightly opalescent gel, may have a precipitate.
1. Neutralization of disinfectant depends on its concentration and type.
Cultural Response Storage
Cultural characteristics after 18-24 hours at 35-370C.
Store at 22-300C and prepared medium at 2-80C.
Organisms (ATCC) Growth Colour of colony RGI
Shelf Life
Staphylococcus aureus (6538) Luxuriant Yellow to gold More than 70%
Pseudomonas aeruginosa (27853) Luxuriant Yellow to green More than 70% Use before expiry date as mentioned on the label.

Tryptone Soya Yeast Extract Agar ISO AM51032

Use monocytogenes is the causative agent of listeriosis. Tryptone Soya Yeast Extract
Tryptone Soya Yeast Extract Agar is recommended for confirmation of Listeria in Agar is recommended by APHA (115.1) for the isolation and cultivation of Listeria
Henry's light, in compliance with ISO specifications ISO 10560: 1993. monocytogenes from foods. ISO Committee (46.2)has recommended these
Summary media for confirmation of Listeria species.
Listeriosis has been recognized as an important public health problem. Listeria

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Principle Quality Control
Casein enzymic hydrolysate, papaic digest of soyabean meal and yeast extract Dehydrated Appearance
Light yellow coloured, homogeneous, free flowing powder.
provide the carbon, nitrogen and other growth factors. Dextrose is the source of
Prepared Appearance
energy. Dipotassium hydrogen phosphate acts as a buffering agent.
Yellow coloured, clear gel forms in petri plates.
Formula*
Cultural Response
Ingredients in grams per liter
Cultural characteristics after after 24-48 hours at 300C.
Casein enzymic hydrolysate 15.00
Organisms (ATCC) Growth RGI
Papaic digest of soyabean meal 5.00
Listeria monocytogenes (19111) Good to luxuriant More than 70%
Sodium chloride 5.00
Listeria monocytogenes (19118) Good to luxuriant More than 70%
Lecithin 0.70
For growth RGI should be more than 70%
Polysorbate 80 (Tween 80) 5.00
RGI- Relative Growth Index
Agar 15.00
Procedure
Final pH (at 250C) 7.3 ± 0.2
* Formula adjusted to suit performance parameters Refer to appropriate references for specific procedures.
Directions Interpretation of Results

1. Suspend 51 grams of the powder in 1000 ml of Distilled water. Refer to appropriate references and procedures for results.
Storage
2. Boil to dissolve the medium completely.
Store at 22-300C and prepared medium at 2-80C.
3. Sterilize by autoclaving at 1210C (15 lbs) for 15 minutes
Shelf Life
Use before expiry date as mentioned on the label.

Tryptone Soya Yeast Extract Broth ISO AM51033

Use Directions

Tryptone Soya Yeast Extract Broth is recommended for confirmation of Listeria in 1. Suspend 36 grams of the powder in 1000 ml of Distilled water.
Henry's light, in compliance with ISO specifications ISO 10560: 1993. 2. Boil to dissolve the medium completely.
Summary 3. Sterilize by autoclaving at 1210C (15 lbs) for 15 minutes.
Listeriosis has been recognized as an important public health problem. Listeria Quality Control
monocytogenes is the causative agent of listeriosis. Tryptone Soya Yeast Extract Dehydrated Appearance
Broth is recommended by APHA (115.1) for the isolation and cultivation of Light yellow coloured, homogeneous, free flowing powder.
Listeria monocytogenes from foods. ISO (46.2) Committee has recommended Prepared Appearance
these media for confirmation of Listeria species. Yellow coloured, clear solutions forms in tubes.
Cultural Response
Principle
Cultural characteristics after after 24-48 hours at 300C.
Casein enzymic hydrolysate, papaic digest of soyabean meal and yeast extract Organisms (ATCC) Growth
provide the carbon, nitrogen and other growth factors. Dextrose is the source of Listeria monocytogenes (19111) Good to luxuriant
energy. Dipotassium hydrogen phosphate acts as a buffering agent. Listeria monocytogenes (19118) Good to luxuriant
Formula* Procedure
Ingredients in grams per liter Refer to appropriate references for specific procedures.
Casein enzymic hydrolysate 17.00
Interpretation of Results
Papaic digest of soyabean meal 3.00
Sodium chloride 5.00 Refer to appropriate references and procedures for results.
Dipotassium hydrogen phosphate 2.50 Storage
Dextrose 2.50 Store at 22-300C and prepared medium at 2-80C.
Yeast extract 6.00 Shelf Life
Final pH (at 250C) 7.3 ± 0.2 Use before expiry date as mentioned on the label.
* Formula adjusted to suit performance parameters

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Tryptone Water AM1104/AM5104
Use 4. Dispense in tubes as per requirements.
Tryptone Water is used for the detection of indole production especially by 5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
coliforms. Quality Control
Summary Dehydrated Appearance
Tryptone Water is based on the formula described in an ISO standard, where it is Light yellow coloured, homogenous, free flowing powder.
used with Brilliant Green Bile Broth 2% to determine the most probable number Prepared Appearance
(MPN) of E.coli present in meat and meat products. Growth and gas production in Yellow coloured clear solution, without any precipitate.
Cultural Response
Brilliant Green Bile Broth 2% and indole production in Tryptone Water followed
Cultural characteristics after 24 hours at 370C.
by incubation of both media at 44±10C is used as the basis for the presumptive
Organisms Growth Indole reaction
E.coli test. Tryptone Water is also used for the differentiation of other bacteria Escherichia coli (25922) Good to luxuriant +
based on indole production and is recommended by APHA (20, 36). It is also Enterobacter aerogenes (13048) Good to luxuriant -
included in the Bacteriological Analytical Manual for food testing (113). Pure Procedure
cultures are used for indole production in tryptophan-containing media to Indole determination using pure cultures.
differentiate bacteria and to identify E.coli isolated from food and water samples. 1. Inoculate Tryptone Water using a light inoculum of an 18-24 hour pure
Principle
culture.
Tryptone is suitable for detecting indole production by bacteria. Tryptophan is
2. Incubate the tubes at 370C with loosened caps for 24 hours.
hydrolyzed and deaminated to produce indole, pyruvic acid and ammonia. Indole
3. Add 0.5 ml of Indole Reagent (Kovac’s) directly to the tube and agitate.
can be detected by the addition of either Kovac's or Ehrlich's Reagent, which
contains an aldehyde group. The aldehyde group combines with indole to 4. Allow tubes to stand for 5-10 minutes.
produce a red colour in the alcohol layer. Sodium chloride is added to the medium Interpretation of Results

to provide a suitable osmotic environment. 1. Examine the tubes for the formation of a red ring at the top of the tube
Formula* indicating indole production.
Ingredients in grams per liter Precautions / Limitations
Tryptone 10.0 1. Detection of E.coli in meats using Tryptone Water is a presumptive test.
Sodium Chloride 5.0
2. Indole testing is recommended as an aid in the differentiation of
Final pH (at 250C) 7.5 ± 0.2
* Formula adjusted to suit performance parameters
microorganisms based on indole production. Other biochemical tests need
Directions to be performed for complete identification.
Storage
1. Suspend 15 gms of the powder in 1000 ml distilled water.
Store at 22-300C and prepared medium at 2-80C.
2. Mix thoroughly.
Shelf Life
3. Heat if necessary to dissolve the powder completely. Use before expiry date as mentioned on the label.

Tryptone Water without Sodium Chloride AM110411/AM510411

Use parahaemolyticus isolates food and water samples.
Tryptone Water without Sodium Chloride is used for the detection of Vibrio Principle
cholerae and Vibrio parahaemolyticus in compliance with BIS specification IS: Tryptone provides all essential growth nutrient.
5887 (Part 5) 1976 reaffirmed 1986. Formula*
Summary Ingredients in grams per liter
Tryptone Water without Sodium Chloride, is used for differentiation of Vibrio Tryptone 10.0
cholerae and Vibrio parahaemolyticus, Vibrio cholerae and Vibrio Final pH (at 250C) 7.0 ± 0.2
* Formula adjusted to suit performance parameters

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Directions Prepared Appearance
1. Suspend 10 gms of the powder in 1000 ml distilled water. Yellow coloured clear solution, without any precipitate.
Cultural Response
2. Mix thoroughly. Cultural characteristics after 24 hours at 35-370C.
3. Sodium chloride in requisite amount is to be added if desired. Organisms (ATCC) Growth
4. Dispense in 5 ml amount into sterilized tubes as per requirements. Vibrio cholerae Good to luxuriant
Vibrio parahaemolyticus Good to luxuriant
5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Storage
Quality Control
Dehydrated Appearance
Store at 22-300C and prepared medium at 2-80C.
Light yellow coloured, homogeneous, free flowing powder. Shelf Life
Use before expiry date as mentioned on the label.

Tryptose Agar AM11041/AM51041

Use 5. Dispense as desired.
Tryptose Agar is recommended for the cultivation of Brucella species and other Quality Control
fastidious microorganisms. Dehydrated Appearance
Summary Light yellow coloured, homogeneous, free flowing powder.
Prepared Appearance
Tryptose Agar is prepared with Tryptose, and recommended for the cultivation and
Yellow coloured, clear to slightly opalescent gel.
isolation of pathogenic and saprophytic bacteria. Historically, it was considered
Cultural Response
necessary to include meat extract or infusion as a nutritional supplement in Cultural characteristics after 48-72 hours at 35-370C under 10% CO2.
culture media. Tryptose was developed while studying growth requirements of Organisms (ATCC) Growth RGI
Brucella species. Huddleson found Tryptose media to be equal or superior to Brucella abortus (4315) Good-luxuriant More than 70%
meat infusion media, providing uniformity for the cultivation and differentiation Brucella melitensis (4309) Good-luxuriant More than 70%
of fastidious microorganisms. Tryptose Agar is particularly well suited for the Brucella suis (4314) Good-luxuriant More than 70%
isolation of Brucella from blood. Streptococcus pneumoniae (6303) Good-luxuriant More than 70%
Principle Streptococcus pyogenes (19615) Good-luxuriant More than70%
For growth RGI should be more than 70%
Tryptose in the medium serves as a nitrogen source. Dextrose is a source of energy.
RGI- Relative Growth Index
Sodium Chloride maintains the osmotic equilibrium. Agar is the solidification
Procedure
agent.
1. Refer to U.S.P. and other appropriate references for procedure and
Formula*
Ingredients in grams per liter
interpretation of results.
Tryptose 20.0 Precautions / Limitations
Dextrose 1.0 1. Tryptose Agar is a general-purpose, non-selective medium. Therefore, a
Sodium Chloride 5.0 number of non-pathogenic, bacteria will grow on this medium and must be
Agar 15.0 distinguished from the pathogenic bacterial strains by additional
Final pH (at 250 C) 7.2 ± 0.2 biochemical tests.
* Formula adjusted to suit performance parameters
2. When preparing blood agar, hemolytic reactions of some strains of Group D
Directions
Streptococci may be effected due to differences in animal blood.
1. Suspend 41.0 grams of the powder in 1000 ml distilled water.
3. The incubation atmosphere may affect the hemolytic reactions of some Beta
2. Boil with frequent agitation to dissolve the powder completely.
hemolytic Streptococci. For optimal performance, incubate the medium
3. Sterilize by autoclaving at 1210 C (15 lbs pressure) for 15 minutes. supplemented with blood under increased CO2 or anaerobic conditions.
4. To prepare blood agar, aseptically add 5% sterile defibrinated sheep, horse
4. Dextrose has been shown to inhibit hemolysin production by some
or rabbit blood.
microorganisms.

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Storage Shelf Life
Store at 22-300C and prepared medium at 2-80C. Use before expiry date as mentioned on the label.

Universal Beer Agar AM11042

Use Directions

Universal Beer agar is recommended for cultivation of microorganisms related to 1. Suspend 62.16 grams in 750 ml of distilled water.
brewing industry. 2. Heat to boiling to dissolve the medium completely.
Summary 3. Add 250 ml Beer, without degassing, to the hot medium and mix gently.
Universal Beer Agar is designed according to the formula developed by Kozulis 4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 10 minutes.
and Page (61.1) for culturing microorganisms which are significant in the
* If required, add 5 mcg/ml of Amphotercin B to sterile medium to dispensing.
brewing industry. They had suggested that the beer should be added to the
Quality Control
medium to stimulate the growth of beer spoilage organisms, thereby increasing Dehydrated Appearance
the selectivity of the medium. Beer contains hop constituents and ethyl alcohol Light yellow coloured, homogeneous, free flowing powder.
which eliminates many airborne contaminants and thus help in minimizing false Prepared Appearance:
positive results. Medium amber coloured, clear to slightly opalescent gel forms in petri plates.
Principle Cultural Response
Peptonized milk, yest extract, dextrose and salts provide all essential growth Cultural characteristics after after 40-48 hours at 35-370C.
Organisms (ATCC) Growth RGI
nutrient. Tomato juice gives acidic environment. The organisms which survive or
Acinetobacter calcoaceticus Good-luxuriant More than 70%
grow in wort and beer during the beer manufacturing can be recovered due to this
(19606)
particular composition of the medium (77.1). Lactobacillus fermentum Good-luxuriant More than 70%
Formula* (9338)
Ingredients in grams per liter Lactobacillus acidophilus Good-luxuriant More than 70%
Peptonized milk 15.00 (4356)
Yeast extract 6.10 Proteus vulgaris Fair-good More than 70%
Dextrose 16.10 (13315)
Tomato juice 12.20 For growth RGI should be more than 70%
Dipotassium phosphate 0.31 RGI- Relative Growth Index
Monopotassium phosphate 0.31 Procedure
Magnesium sulphate 0.12
Refer to appropriate references for specific procedures.
Sodium chloride 0.006
Interpretation of Results
Ferrous sulphate 0.006
Manganese sulphate 0.006 Refer to appropriate references and procedures for results.
Agar 12.00 Storage
Final pH (at 250C) 6.3 + 0.2 Store at 22-300C and prepared medium at 2-80C.
* Formula adjusted to suit performance parameters Shelf Life
Use before expiry date as mentioned on the label.

Urea Agar Base, Christensen AM1105/AM5105

Use differentiation of enteric bacilli. It differentiates between rapid urease-positive
Urea Agar Base with added urea is used for the detection of urease production, Proteeae organisms (Proteus species, Morganella morganii subspecies morganii,
particularly by the genus Proteus. Providencia rettgeri, and some Providencia stuartii ) and other urease-positive
Summary organisms: Citrobacter, Enterobacter and Klebsiella and bacteria other than
Christensen (17) devised Urea Agar Base for use as a solid medium for the Enterobacteriaceae, i.e., some Bordetella and Brucella species. Urea Agar Base is

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included in the Bacteriological Analytical Manual for food and cosmetics testing Salmonella serotype Luxuriant - More than 70%
(113), in IP (46) and is recommended by APHA for the examination of foods (20). Typhimurium
(14028)
Principle
Key:
Rustigian and Stuart (98) had originally formulated Urea Broth to differentiate + = positive, pink-red colour
Proteus species from other gram-negative enteric bacilli capable of utilizing urea; - = negative, no change
the latter were unable to do so because of limited nutrients and the high buffering Procedure
capacity of the Urea Broth. To provide a medium with greater use Christensen 1. Using a heavy inoculum of growth from an 18-24 hour pure culture,
devised Urea Agar Base with the addition of peptone, dextrose and reduced inoculate the agar by streaking back and forth over the entire surface of
content of buffer to promote rapid growth of many of the Enterobacteriaceae and the s l a n t .
permit a reduction in incubation time.
2. Do not stab the butt since it serves as a colour control.
Formula*
Ingredients in grams per liter 3. Incubate tubes with loosened caps at 350C in an incubator or water bath.
Sodium Chloride 5.0 4. Observe reactions after 2, 4, 6, 18, 24 and 48 hours.
Disodium Phosphate 1.2
5. Continue to check everyday for a total of 6 days; even longer incubation
Dextrose 1.0
periods may be necessary.
Peptone 1.0
Interpretation of Results
Monopotassium Phosphate 0.8
Phenol Red 0.012 1. When organisms utilize urea, ammonia is formed which makes the
Agar 15.0 medium alkaline, producing an intense pink-red colour on the slant.
Final pH (at 250C) 6.8 ± 0.2 2. The colour may penetrate into the agar butt; the extent of colour
* Formula adjusted to suit performance parameters indicates the rate of urea hydrolysis.
Directions
3. A negative reaction is no colour change. The agar medium remains pale
1. Suspend 24 gms of the powder in 950 ml distilled water.
yellow to buff.
2. Mix thoroughly. Precautions / Limitations
3. Boil with frequent agitation to dissolve the powder completely. DO NOT 1. Do not reheat the medium as urea decomposes very easily.
OVERHEAT. 2. The alkaline reaction produced in this medium after prolonged
4. Sterilize by autoclaving at 1150C (10 lbs pressure) for 20 minutes. incubation may not be caused by urease activity. False positive
5. Cool to 500C and aseptically add 50 ml of sterile 40% Urea (AS028) solution reactions may occur due to the utilization of peptones (P.aeruginosa for
and mix well. e.g.) or other proteins, which raises the pH due to protein hydrolysis, and
6. Dispense into sterile test tubes and allow to set in a slanting position. the release of excess amino acid residue. To eliminate possible protein
hydrolysis, perform a control test without urea.
7. Do not reheat the medium after the addition of Urea 40%, as urea
decomposes very easily. 3. Urea Agar rapidly detects urease activity of only the urease positive Proteus
Quality Control species. For results to be valid for the detection of Proteus, the results must
Dehydrated Appearance be read within first 2-6 hours after incubation.
Yellowish orange coloured, homogeneous, free flowing powder. 4. Urease positive Enterobacter, Citrobacter or Klebsiella in contrast, hydrolyze
Prepared Appearance urea much more slowly, showing only slight reaction into the butt of the
Yellow orange coloured, clear gel.
medium in 6 hours and requiring 3-5 days to change the reaction of the
Cultural Response
entire butt.
Cultural characteristics after 18-24 hours at 350C.
Storage
Organisms (ATCC) Growth Urease RGI
Enterobacter aerogenes (13048) Luxuriant - More than 70% Store at 22-300C and prepared medium at 2-80C
Escherichia coli (25922) Luxuriant - More than 70% Shelf Life
Proteus vulgaris (13315) Luxuriant + More than 70% Use before expiry date as mentioned on the label.

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Urea Agar Base, Christensen BIS AM11051/AM51051
Use 7. Do not reheat the medium after the addition of Urea 40%, as urea
Urea Agar Base with added urea is used for the detection of urease production, decomposes very easily.
particularly by the genus Proteus in compliance with BIS specification IS: 5887 Quality Control
(Part 1), 1976 and IS: 5887 (Part 3): 1999. Dehydrated Appearance
Summary Yellowish orange coloured, homogeneous, free flowing powder.
Prepared Appearance
Christensen (17, 77.1) devised Urea Agar Base for use as a solid medium for the
Yellow orange coloured, clear gel.
differentiation of enteric bacilli. It differentiates between rapid urease-positive
Cultural Response
Proteeae organisms (Proteus species, Morganella morganii subspecies morganii, Cultural characteristics after 18-24 hours at 35-37ºC.
Providencia rettgeri, and some Providencia stuartii) and other urease-positive Organisms (ATCC) Growth Urease
organisms: Citrobacter, Enterobacter and Klebsiella and bacteria other than Enterobacter aerogenes (13048) Luxuriant -
Enterobacteriaceae, i.e., some Bordetella and Brucella species. Urea Agar Base is Escherichia coli (25922) Luxuriant -
included in the Bacteriological Analytical Manual for food and cosmetics testing, Proteus vulgaris (13315) Luxuriant +
in IP and is recommended by APHA and BIS for the examination of foods. Salmonella serotype Typhimurium (14028) Luxuriant -
Principle Key:
+ = positive, pink-red colour
Rustigian and Stuart (98) had originally formulated Urea Broth to differentiate
- = negative, no change
Proteus species from other gram-negative enteric bacilli capable of utilizing urea; Procedure
the latter were unable to do so because of limited nutrients and the high buffering
1. Using a heavy inoculum of growth from an 18-24 hour pure culture, inoculate
capacity of the Urea Broth. To provide a medium with greater use Christensen
the agar by
devised Urea Agar Base with the addition of peptone, dextrose and reduced
streaking back and forth over the entire surface of the slant.
content of buffer to promote rapid growth of many of the Enterobacteriaceae and
permit a reduction in incubation time. 2. Do not stab the butt since it serves as a colour control.
Formula* 3. Incubate tubes with loosened caps at 35ºC in an incubator or water bath.
Ingredients Gms/Liter 4. Observe reactions after 2, 4, 6, 18, 24 and 48 hours.
Sodium chloride 5.0
Glucose 1.0
5. Continue to check everyday for a total of 6 days; even longer incubation
Peptone 1.0 periods may be necessary.
Potassium dihydrogen phosphate 2.0 Interpretation of Results
Phenol red 0.012 1. When organisms utilize urea, ammonia is formed which makes the medium
Agar 15.0 alkaline, producing an
Final pH (at 25ºC) 6.8 ± 0.2
intense pink-red colour on the slant.
* Formula adjusted to suit performance parameters
Directions 2. The colour may penetrate into the agar butt; the extent of colour indicates the
1. Suspend 24.012gms of the powder in 950 ml distilled water. rate of urea hydrolysis.
2. Mix thoroughly. 3. A negative reaction is no colour change. The agar medium remains pale
yellow to buff.
3. Boil with frequent agitation to dissolve the powder completely. DO NOT
Precautions / Limitations
OVERHEAT.
1. Do not reheat the medium as urea decomposes very easily.
4. Sterilize by autoclaving at 115ºC (10 lbs pressure) for 20 minutes.
2. The alkaline reaction produced in this medium after prolonged incubation
5. Cool to 50ºC and aseptically add 50 ml of sterile 40% Urea (AS028)
may not be caused by urease activity. False positive reactions may occur due
solution and mix well.
to the utilization of peptones (P. aeruginosa for e.g.) or other proteins, which
6. Dispense into sterile test tubes and allow to set in a slanting position. raises the pH due to protein hydrolysis, and the release of excess amino acid

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residue. To eliminate possible protein hydrolysis, perform a control test urea much more slowly, showing only slight reaction into the butt of the
without urea. medium in 6 hours and requiring 3-5 days to change the reaction of the
3. Urea Agar rapidly detects urease activity of only the urease positive Proteus entire butt.
species. For results to be valid for the detection of Proteus, the results must be Storage

read within first 2-6 hours after incubation. Store at 22-300C and prepared medium at 2-80C.
4. Urease positive Enterobacter, Citrobacter or Klebsiella in contrast, hydrolyze Shelf Life
Use before expiry date as mentioned on the label.

Urea Agar Base, Christensen ISO AM51052

Use 2. Mix thoroughly.
Urea Agar Base with added urea is used for the detection of urease production, 3. Boil with frequent agitation to dissolve the powder completely. DO NOT
particularly by the genus Proteus in compliance with ISO. OVERHEAT.
Summary
4. Sterilize by autoclaving at 1150C (10 lbs pressure) for 20 minutes.
Christensen (17, 77.1) devised Urea Agar Base for use as a solid medium for the
5. Cool to 500C and aseptically add 50 ml of sterile 40% Urea (AS028) solution
differentiation of enteric bacilli. It differentiates between rapid urease-positive
and mix well.
Proteeae organisms (Proteus species, Morganella morganii sub species
morganii, Providencia rettgeri, and some Providencia stuartii) and other urease- 6. Dispense into sterile test tubes and allow to set in a slanting position.
positive organisms: Citrobacter, Enterobacter and Klebsiella and bacteria other 7. Do not reheat the medium after the addition of Urea 40%, as urea
than Enterobacteriaceae, i.e., some Bordetella and Brucella species. Urea Agar decomposes very easily.
Base is included in the Bacteriological Analytical Manual for food and cosmetics Quality Control
testing, in IP and is recommended by APHA for the examination of foods. Dehydrated Appearance
Principle Yellowish orange coloured, homogeneous, free flowing powder.
Prepared Appearance
Rustigian and Stuart (98) had originally formulated Urea Broth to differentiate
Yellowish orange coloured, clear gel.
Proteus species from other gram-negative enteric bacilli capable of utilizing Cultural Response
urea; the latter were unable to do so because of limited nutrients and the high Cultural characteristics after 18-24 hours at 35-370C.
buffering capacity of the Urea Broth. To provide a medium with greater use Organisms (ATCC) Growth Urease
Christensen devised Urea Agar Base with the addition of peptone, dextrose and Enterobacter aerogenes (13048) Luxuriant -
reduced content of Escherichia coli (25922) Luxuriant -
Proteus vulgaris (13315) Luxuriant +
buffer to promote rapid growth of many of the Enterobacteriaceae and permit a
Salmonella serotype Typhimurium (14028) Luxuriant -
reduction in incubation time. Key:
Formula* + = positive, pink-red colour
Ingredients Gms/Liter - = negative, no change
Sodium chloride 5.0 Procedure
Glucose 1.0
1. Using a heavy inoculum of growth from an 18-24 hour pure culture,
Peptone 1.0
inoculate the agar by streaking back and forth over the entire surface of the
Potassium dihydrogen phosphate 2.0
Phenol red 0.012 slant.
Agar 15.0 2. Do not stab the butt since it serves as a colour control.
Final pH (at 25ºC) 6.8 ± 0.2
3. Incubate tubes with loosened caps at 350C in an incubator or water bath.
* Formula adjusted to suit performance parameters
Directions 4. Observe reactions after 2, 4, 6, 18, 24 and 48 hours.
1. S. Suspend 24.012 gms of the powder in 950 ml distilled water. 5. Continue to check everyday for a total of 6 days; even longer incubation
periods may be necessary.

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Interpretation of Results residue. To eliminate possible protein hydrolysis, perform a control test
1. When organisms utilize urea, ammonia is formed which makes the medium without urea.
alkaline, producing an intense pink-red colour on the slant. 3. Urea Agar rapidly detects urease activity of only the urease positive Proteus
2. The colour may penetrate into the agar butt; the extent of colour indicates the species. For results to be valid for the detection of Proteus, the results must be
rate of urea hydrolysis. read within first 2-6 hours after incubation.
3. A negative reaction is no colour change. The agar medium remains pale 4. Urease positive Enterobacter, Citrobacter or Klebsiella in contrast, hydrolyze
yellow to buff. urea much more slowly, showing only slight reaction into the butt of the
Precautions / Limitations medium in 6 hours and requiring 3-5 days to change the reaction of the
1. Do not reheat the medium as urea decomposes very easily. entire butt.
2. The alkaline reaction produced in this medium after prolonged incubation Storage

may not be caused by urease activity. False positive reactions may occur due Store at 22-300C and prepared medium at 2-80C.
to the utilization of peptones (P.aeruginosa for e.g.) or other proteins, which Shelf Life
raises the pH due to protein hydrolysis, and the release of excess amino acid Use before expiry date as mentioned on the label.

Urea Broth Base AM1106/AM5106

Use Phenol Red 0.01
Urea Broth Base with added urea is used for the detection of urease production, to Final pH (at 250C) 6.8 ± 0.2
* Formula adjusted to suit performance parameters
differentiate Proteus species from Salmonella and Shigella species.
Directions
Summary
Rustigian and Stuart (98) developed Urea Broth Base for the identification of 1. Suspend 18.7 gms of the powder in 950 ml distilled water.
bacteria on the basis of urea utilization and is particularly recommended for the 2. Mix thoroughly.
differentiation of the genus Proteus from those of Salmonella and Shigella in the 3. Boil with frequent agitation to dissolve the powder completely.
diagnosis of enteric infections. The broth is positive for Proteus, Morganella 4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
morganii subspecies morganii, Providencia rettgeri, and a few Providencia
5. Cool to 550C.
stuartii strains with the reclassification of the members of the genus Proteeae.
6. Aseptically add 50 ml of sterile 40% Urea (AS028) Solution and distribute
Urea Broth Base is included in the Bacteriological Analytical Manual for food and
10 ml aliquots into sterile tubes.
cosmetics testing (113), in IP (46) and is recommended by APHA in the
Quality Control
examination of milk (39) and foods (20).
Dehydrated Appearance
Principle
Light orange coloured, homogeneous, free flowing powder.
Yeast extract provides trace elements, vitamins and amino acids. Gram-negative Prepared Appearance
enteric bacilli are unable to utilize urea because of less nutrients and high Yellow orange coloured, clear solution without any precipitate.
buffering capacity of the medium. Urea Broth becomes alkaline as utilization of Cultural Response
urea by the organisms liberates ammonia during incubation, which is indicated Cultural characteristics after 18-48 hours at 35-370C.
by pink red colour. Since this test relies on the alkalinity formation, it is not Organisms (ATCC) Growth Urease RGI
Enterobacter aerogenes Luxuriant - More than 70%
specific for urease testing. The utilization of proteins may raise the pH to alkalinity
(13048)
due to protein hydrolysis and excess of amino acids results in false positive
Escherichia coli (25922) Luxuriant - More than 70%
reaction. Proteus vulgaris (13315) Luxuriant + More than 70%
Formula* Salmonella serotype Luxuriant - More than 70%
Ingredients in grams per liter Typhimurium (14028)
Yeast Extract 0.1 For growth RGI should be more than 70%
Dipotassium Phosphate 9.5 RGI- Relative Growth Index
Monopotassium Phosphate 9.1 Key:

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+ = Positive, pink-red colour 3. To rule out false-positives due to protein hydrolysis (as opposed to urea
- = Negative, no change hydrolysis) that may occur in the medium after prolonged incubation,
Procedure
perform a control test without urea.
1. Using a heavy inoculum of growth from an 18-24 hour pure culture,
4. The high buffering system in this medium masks urease activity in
inoculate the broth.
organisms that are delayed positive. This medium is therefore
2. Shake tubes gently to suspend the bacteria. recommended for the detection of urease activity in all Proteus species.
3. Incubate tubes with loosened caps at 35 -370C in an incubator or water bath. Providencia rettgeri and urease-positive Providencia stuartii, M.morganii
4. Observe reactions after 2, 4, 6, 18, 24 and 48 hours. slowly hydrolyze urea and may require approximately a 36-hour incubation
Interpretation of Results for strong urease positive reaction to occur. When in doubt as to a result,
1. The production of urease is indicated by an intense pink-red colour compare with an un-inoculated tube or incubate for an additional 24 hours.
throughout the broth. 5. Variations in the size of the inoculum can affect the time required to reach
2. A negative reaction is no colour change. The broth medium remains pale positive results.
yellow to buff. Storage
Precautions / Limitations Store at 22-300C and prepared medium at 2-80C.
1. It is preferable that the medium be used on the day of preparation. If not, Shelf Life

examine the tubes carefully to ensure sterility. Use before expiry date as mentioned on the label.
2. Do not reheat the medium after the addition of Urea 40% as urea
decomposes very easily.

Urea Broth IP AM51061

Use Yeast extract 0.10
Urea Broth is recommended for the identification of bacteria on the basis of urea Phenol red 0.01
Urea 20.00
utilization, specifically for the differentiation of Proteus species from Salmonella
Final pH (at 25ºC) 6.8 ± 0.2
and Shigella species in compliances with IP.
* Formula adjusted to suit performance parameters
Summary
Directions
Urea Broth was developed by Rustigian and Stuart (98). This medium is
1. Suspend 38.7 gms of the powder in1000 ml distilled water.
especially recommended for the differentiation of Proteus species from
2. Mix well and sterilize by filtration..
Salmonella and Shigella species in the enteric infection diagnosis, based on urea
utilization. 3. DO NOT AUTOCLAVE OR HEAT the medium.
Principle 4. Dispense into sterile test tubes.
Gram-negative enteric bacilli are unable to utilize urea because of less nutrients Quality Control
and high buffering capacity of the medium. Urea Broth becomes alkaline as the Dehydrated Appearance
Light orange coloured, homogeneous, free flowing powder.
utilization of urea by the organisms liberate ammonia during the incubation,
Prepared Appearance
indicated by pink colour. All urea test media rely on the alkalinity formation and
Yellow orange coloured, clear solution without any precipitate.
so they are not specific for urease testing. Cultural Response
The utilization of protein may raise the pH to the alkalinity due to protein Cultural characteristics after 18-24 hours at 35-370C.
hydrolysis and excess of amino acids results in false-positive reaction. Organisms (ATCC) Growth Urease
Formula* Enterobacter aerogenes (13048) Luxuriant -
Ingredients Gms/Liter Escherichia coli (25922) Luxuriant -
Potassium dihydrogen ortho phosphate 9.10 Klebsiella pneumoniae (13883) Luxuriant +
Anhydrous disodium hydrogen phosphate 9.50 Proteus vulgaris (13315) Luxuriant +
Salmonella serotype Typhimurium (14028) Luxuriant -

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Key: Storage
+ = Positive, cerise colour Store at 22-300C and prepared medium at 2-80C.
- = Negative, no change Shelf Life
Use before expiry date as mentioned on the label.

Violet Red Bile Agar AM1107/AM5107

Use Quality Control
Violet Red Bile Agar is a selective medium used for the detection and enumeration Dehydrated Appearance
Beige coloured homogeneous, free flowing powder.
of coliforms.
Prepared Appearance
Summary
Reddish purple coloured, clear to slightly opalescent gel.
Violet Red Bile Agar is recommended by APHA for the detection and enumeration Cultural Response
of coliforms in water (36), dairy (39) and other food products (20). Druce et al Cultural characteristics after 18-24 hours at 350C.
(21) found that this medium was as good an indicator of coli-aerogenes bacteria Organisms (ATCC) Growth Colour of RGI
in milk as MacConkey Broth. Colony
Principle Enterobacter aerogenes Luxuriant Pink More than 70%
Peptone provides nitrogen and carbon required for growth while yeast extract (13048)
Escherichia coli (25922) Luxuriant Pinkish red with More than 70%
supplies B complex vitamins. Bile salts mixture and crystal violet inhibit gram-
bile precipitate.
positive organisms especially staphylococci and makes the medium selective.
Salmonella serotype Luxuriant Colourless More than 70%
Sodium chloride maintains the osmotic equilibrium. Lactose is the carbohydrate Enteritidis (13076)
source and neutral red is the pH indicator. Organisms, which rapidly ferment Staphylococcus aureus Inhibited - 0%
lactose, produce red colonies surrounded by red-purple haloes. Lactose non- (25923)
fermenters and late lactose fermenters produce pale colonies. Other related For growth RGI should be more than 70%
gram-negative bacteria can be suppressed by incubation greater than 420C or by For Inhibition RGI should be 0%
anaerobic incubation. RGI- Relative Growth Index
Procedure
Formula*
Presumptive test for coliforms
Ingredients in grams per liter
Lactose 10.0 1. Transfer 1 ml aliquot of test sample to a petri plate.
Peptone 7.0 2. Add 10 ml of Violet Red Bile Agar cooled to 450C and swirl to mix.
Sodium Chloride 5.0
Yeast Extract 3.0
3. Allow medium to solidify before incubating at 350C for 18-24 hours. For
Bile Salts Mixture 1.5 dairy products, incubate at 320C.
Neutral Red 0.03 4. Do confirmatory testing of typical coliform colonies.
Crystal Violet 0.002
5. An overlay method is helpful to improve the specificity of the medium. A thin
Agar 15.0
layer of cooled molten medium is poured over the inoculated base layer and
Final pH (at 250C) 7.4 ± 0.2
allowed to set before incubation. Incubation can be carried out at greater
* Formula adjusted to suit performance parameters
Directions than 420C for 18 hours, 320C for 24-48 hours or 40C for 10 days, depending
on the temperature characteristics of the organism to be recovered. For
1. Suspend 41.53 gms of the powder in 1000 ml distilled water.
E.coli, a temperature of 440C is specifically recommended.
2. Mix thoroughly.
Interpretation of Results
3. Heat with frequent agitation to dissolve the powder completely. DO NOT 1. Lactose fermenters including coliforms form purple red colonies, with or
AUTOCLAVE. without a zone of precipitate around the colonies. (Generally surrounded by
4. Cool to 450C, and pour into sterile petri plates containing the inoculum. a reddish zone of precipitated bile)

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2. Non-lactose fermenters form colourless to transparent colonies. 4. This medium may not be completely inhibitory to gram-positive organisms.
3. Gram-positive cocci, if present, form colourless, pinpoint colonies. Enterococci may grow as pinpoint colonies. Perform gram stain and
Precautions / Limitations biochemical tests to identify isolates.
1. Boiling the medium for more than 2 minutes may decrease the ability to 5. Gram-negative bacilli other than Enterobacteriaceae may also grow.
support growth. Perform biochemical tests to identify isolates to genus and species.
Storage
2. Do not incubate inoculated plates for more than 24 hours because
microorganisms that are only slightly inhibited may grow after extended Store at 22-300C and prepared medium at 2-80C.
incubation. Shelf Life
Use before expiry date as mentioned on the label.
3. Prepare and use the medium within 24 hours for optimum performance.

Violet Red Bile Agar BIS AM11071/AM51071

Use * Formula adjusted to suit performance parameters
Violet Red Bile Agar is a selective medium used for the detection and enumeration Directions

of coliforms in compliance with BIS. 1. Suspend 41.53 gms of the powder in 1000 ml distilled water.
Summary 2. Mix thoroughly.
Violet Red Bile Agar is recommended by APHA for the detection and enumeration 3. Heat with frequent agitation to dissolve the powder completely. DO NOT
of coliforms in water (36), dairy (39) and other food products (20). Druce et al., AUTOCLAVE.
(21)found that this medium was as good an indicator of coli-aerogenes bacteria 4. Cool to 450C, and pour into sterile petri plates containing the inoculum.
in milk as MacConkey Broth. Recently the agar formulation is recommended by Quality Control
ISO committee for the enumeration of coliforms (5). Dehydrated Appearance
Principle Beige coloured homogeneous, free flowing powder.
Peptone provides nitrogen and carbon required for growth while yeast extract Prepared Appearance
supplies B complex vitamins. Bile salts mixture and crystal violet inhibit gram- Reddish purple coloured, clear to slightly opalescent gel.
positive organisms especially staphylococci and makes the medium selective. Cultural Response
Cultural characteristics after 18-24 hours at 35-370C.
Sodium chloride maintains the osmotic equilibrium. Lactose is the carbohydrate
Organisms (ATCC) Growth Colour of RGI
source and neutral red is the pH indicator. Organisms, which rapidly ferment
Colony
lactose, produce red colonies surrounded by red-purple haloes. Lactose non- Enterobacter aerogenes Luxuriant Pink More than 70%
fermenters and late lactose fermenters produce pale colonies. Other related (13048)
gram-negative bacteria can be suppressed by incubation greater than 420C or by Escherichia coli (25922) Luxuriant Pinkish red with More than 70%
anaerobic incubation. bile precipitate.
Formula* Salmonella serotype Luxuriant Colourless More than 70%
Ingredients in grams per liter Enteritidis (13076)
Lactose 10.0 Staphylococcus aureus Inhibited - 0%
Peptone 7.0 (25923)
Sodium chloride 5.0 For growth RGI should be more than 70%
Yeast extract 3.0 For Inhibition RGI should be 0%
Bile salts mixture 1.5 RGI- Relative Growth Index
Neutral red 0.03 Procedure
Crystal violet 0.002 Presumptive test for coliforms
Agar 15.0 1. Transfer 1 ml aliquot of test sample to a petri plate.
Final pH (at 250C) 7.4 ± 0.2
2. Add 10 ml of Violet Red Bile Agar cooled to 450C and swirl to mix.

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3. Allow medium to solidify before incubating at 35-370C for 18-24 hours. For Precautions / Limitations
dairy products, incubate at 320C. 1. Boiling the medium for more than 2 minutes may decrease the ability to
4. Do confirmatory testing of typical coliform colonies. support growth.
5. An overlay method is helpful to improve the specificity of the medium. A thin 2. Do not incubate inoculated plates for more than 24 hours because
layer of cooled molten medium is poured over the inoculated base layer and microorganisms that are only slightly inhibited may grow after extended
allowed to set before incubation. Incubation can be carried out at greater incubation.
than 420C for 18 hours, 320C for 24-48 hours or 40C for 10 days, depending 3. Prepare and use the medium within 24 hours for optimum performance.
on the temperature characteristics of the organism to be recovered. For 4. This medium may not be completely inhibitory to gram-positive organisms.
E.coli, a temperature of 440C is specifically recommended. Enterococci may grow as pinpoint colonies. Perform gram stain and
Interpretation of Results biochemical tests to identify isolates.
1. Lactose fermenters including coliforms form purple red colonies, with or 5. Gram-negative bacilli other than Enterobacteriaceae may also grow. Perform
without a zone of precipitate around the colonies. (Generally surrounded by biochemical tests to identify isolates to genus and species.
a reddish zone of precipitated bile) Storage

2. Non-lactose fermenters form colourless to transparent colonies. Store 22- 300C and prepared medium at 2-80C.
Shelf Life
3. Gram-positive cocci, if present, form colourless, pinpoint colonies.
Use before expiry date as mentioned on the label.

Violet Red Bile Agar (1.2%) ISO AM510711

Use Yeast extract 3.0
Violet Red Bile Agar (1.2%) ISO is a selective medium used for the detection and Bile salts mixture 1.5
Neutral red 0.03
enumeration of coliforms.
Crystal violet 0.002
Summary
Agar 12.0
Violet Red Bile Agar is recommended by APHA for the detection and enumeration Final pH (at 250C) 7.4 ± 0.2
of coliforms in water (36), dairy (39) and other food products (20). Druce et al., * Formula adjusted to suit performance parameters
(21)found that this medium was as good an indicator of coli-aerogenes bacteria Directions
in milk as MacConkey Broth. Recently the agar formulation is recommended by 1. Suspend 38.53 gms of the powder in 1000 ml distilled water.
ISO committee for the enumeration of coliforms (5).
2. Mix thoroughly.
Principle
3. Heat with frequent agitation to dissolve the powder completely. DO NOT
Peptone provides nitrogen and carbon required for growth while yeast extract
AUTOCLAVE.
supplies B complex vitamins. Bile salts mixture and crystal violet inhibit gram-
positive organisms especially staphylococci and makes the medium selective. 4. Cool to 450C, and pour into sterile petri plates containing the inoculum.
Sodium chloride maintains the osmotic equilibrium. Lactose is the carbohydrate Quality Control
Dehydrated Appearance
source and neutral red is the pH indicator. Organisms, which rapidly ferment
Beige coloured homogeneous, free flowing powder.
lactose, produce red colonies surrounded by red-purple haloes. Lactose non-
Prepared Appearance
fermenters and late lactose fermenters produce pale colonies. Other related Reddish purple coloured, clear to slightly opalescent gel.
gram-negative bacteria can be suppressed by incubation greater than 420C or by Cultural Response
anaerobic incubation. Cultural characteristics after 18-24 hours at 35-370C.
Formula* Organisms (ATCC) Growth Colour of RGI
Ingredients in grams per liter Colony
Lactose 10.0 Enterobacter aerogenes Luxuriant Pink More than 70%
Peptone 7.0 (13048)
Sodium chloride 5.0

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Escherichia coli (25922) Luxuriant Pinkish red with More than 70% Interpretation of Results
bile precipitate. 1. Lactose fermenters including coliforms form purple red colonies, with or
Salmonella serotype Luxuriant Colourless More than 70%
without a zone of precipitate around the colonies. (Generally surrounded by
Enteritidis (13076)
a reddish zone of precipitated bile)
Staphylococcus aureus Inhibited - 0%
(25923) 2. Non-lactose fermenters form colourless to transparent colonies.
For growth RGI should be more than 70% 3. Gram-positive cocci, if present, form colourless, pinpoint colonies.
For Inhibition RGI should be 0% Precautions / Limitations
RGI- Relative Growth Index
1. Boiling the medium for more than 2 minutes may decrease the ability to
Procedure
support growth.
Presumptive test for coliforms
1. Transfer 1 ml aliquot of test sample to a petri plate. 2. Do not incubate inoculated plates for more than 24 hours because
microorganisms that are only slightly inhibited may grow after extended
2. Add 10 ml of Violet Red Bile Agar cooled to 450C and swirl to mix.
incubation.
3. Allow medium to solidify before incubating at 35-370C for 18-24 hours. For
3. Prepare and use the medium within 24 hours for optimum performance.
dairy products, incubate at 320C.
4. This medium may not be completely inhibitory to gram-positive organisms.
4. Do confirmatory testing of typical coliform colonies.
Enterococci may grow as pinpoint colonies. Perform gram stain and
5. An overlay method is helpful to improve the specificity of the medium. A thin biochemical tests to identify isolates.
layer of cooled molten medium is poured over the inoculated base layer and
5. Gram-negative bacilli other than Enterobacteriaceae may also grow.
allowed to set before incubation. Incubation can be carried out at greater
Perform biochemical tests to identify isolates to genus and species.
than 420C for 18 hours, 320C for 24-48 hours or 40C for 10 days, depending
Storage
on the temperature characteristics of the organism to be recovered. For
Store 22- 300C and prepared medium at 2-80C.
E.coli, a temperature of 440C is specifically recommended.
Shelf Life
Use before expiry date as mentioned on the label.

Violet Red Bile Agar with MUG AM510712

Use E. coli produces the enzyme glucuronidase which hydrolyzes MUG to yield a
Violet Red Bile Agar with MUG is used for enumerating Escherichia coli and fluorogenic compound detectable with long wave UV light (366 nm). Typical
coliform bacteria in food and dairy products. strains of E. coli (red colonies surrounded by a bile precipitate) exhibit blue
Summary fluorescence. Non- E. coli coliform may produce red colonies with zone of
Violet Red Bile Agar is specified in standard methods procedures to enumerate precipitated bile but they are MUG negative.
coliforms in food and dairy products (17.3, 61.2, 43.1). In 1982, Feng and Formula*
Hartman (30.1) developed a rapid fluorogenic assay for Escherichia coli by Ingredients in grams per liter
incorporating 4-methylumbelliferyl-ß – D- glucuronide (MUG) into Lauryl Yeast extract 3.0
Peptone 7.0
Tryptose Broth. Incorporating MUG into Violet Red Bile Agar permits the detection
Bile salts No. 3 1.5
of E. coli among the coliform colonies.
Lactose 10.0
Principle
Sodium chloride 5.0
Peptone as a source of carbon, nitrogen, vitamin and minerals. Yeast extract Agar 15.0
supplies B-complex vitamins which stimulate bacterial growth. Bile salt and Neutral red 0.03
crystal violet inhibit gram-positive bacteria. Lactose is a carbohydrate source. Crystal violet 0.002
Neutral red is a pH indicator. MUG is a substrate used for detecting glucuronidase MUG (4-methylumbelliferyl-â– D- glucuronide ) 0.1
activity. Agar is the solidifying agent. Final pH (at 250C) 7.4 ± 0.2
* Formula adjusted to suit performance parameters

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Directions Organisms (ATCC) Growth Colour of Fluorescence RGI
Colony
1. Suspend 41.6 gms of the powder in 1000 ml distilled water.
Enterobacter Luxuriant Pink may have - More than 70%
2. Mix thoroughly. aerogenes (13048) bile precipitate.
(25922) Escherichia coli Luxuriant Deep red with bile + More than 70%
3. Heat with frequent agitation to dissolve the powder completely. DO NOT
Staphylococcus Inhibited - - 0%
AUTOCLAVE. aureus (25923)
4. Cool to 45-500C, and use immediately. For growth RGI should be more than 70%
Quality Control For Inhibition RGI should be 0%
Dehydrated Appearance RGI- Relative Growth Index
Reddish beige, homogeneous, free flowing powder. Storage
Prepared Appearance Store at 22- 300C and prepared medium at 2-80C.
Reddish purple coloured, clear to slightly opalescent gel. Shelf Life
Cultural Response
Use before expiry date as mentioned on the label.
Cultural characteristics after 18-24 hours at 35-370C.

Violet Red Bile Broth AM51072

Use 0
Final pH (at 25 C) 7.4 ± 0.2
Violet Red Bile Broth is selective media used for the detection and enumeration of * Formula adjusted to suit performance parameters
coliform organisms from water and food. Directions

Summary 1. Suspend 26.53 gms of the powder in 1000 ml distilled water.

Violet Red Bile Broth is recommended by APHA for the detection and enumeration 2. Mix thoroughly.
of coliform organisms in water, milk, dairy and other food products (103.2, 3. Heat with stirring to boiling to dissolve the medium completely. DO NOT
91.2). Druce et al.,(21) found this media equally good as the indicator of coli- AUTOCLAVE.
aerogenes in milk as MaConkey Broth. Recently, the agar formulation is 4. Cool to 450C,and pour into tubes containing the inoculum..
recommended by ISO committee for the enumeration of coliforms (46.5). Quality Control
Principle Dehydrated Appearance
The media is selective due to the presence of the inhibitors- bile salts and crystal Beige coloured, homogeneous, free flowing powder.
violet. Crystal violet inhibit gram-positive microorganisms especially Prepared Appearance
staphylococci. Organisms which rapidly ferment lactose produce red colonies Reddish purple coloured, clear to slightly opalescent.
Cultural Response
surrounded by red-purple halo (18.4). Lactose non-fermenters and late lactose
Cultural characteristics after 18-24 hours at 35-370C.
fermenters produce pale colonies.
Organisms (ATCC) Growth
Formula*
Enterobacter aerogenes (13048) Luxuriant
Ingredients in grams per liter Escherichia coli (25922) Luxuriant
Yeast extract 3.0 Salmonella serotype Enteritidis (13076) Luxuriant
Peptic digest of animal tissue 7.0 Staphylococcus aureus (25923) Inhibited
Bile salt mixture 1.5 For growth RGI should be more than 70%
Lactose 10.0 RGI- Relative Growth Index
Sodium chloride 5.0 Storage
Neutral red 0.03 Store at 22- 300C and prepared medium at 2-80C.
Crystal violet 0.002 Shelf Life
Use before expiry date as mentioned on the label.

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Violet Red Bile Glucose Agar (Harmonized ) AMH51073
Violet Red Bile Glucose Agar BP AM510731
Violet Red Bile Glucose Agar USP AM510732
Violet Red Bile Glucose Agar AM51073
Use Directions
Violet Red Bile Glucose Agar is used for detection and enumeration of 1. Suspend 41.53 gms of the powder in 1000 ml distilled water.
Enterobacteriaceae in food products. 2. Mix thoroughly.
Summary
3. Boil with frequent agitation to dissolve the powder completely. DO NOT
Mossel et al., (81.4)added glucose and excluded lactose from the media AUTOCLAVE.
observing improved detection of coliforms. Incubation can be carried out at
4. Cool to 45ºC, and pour into sterile petri plates containing the inoculum.
different temperature and incubation time depending upon the group of
Quality Control
Enterobacteriaceae to be recovered. Dehydrated Appearance
Principle Beige coloured, homogeneous, free flowing powder.
Pancreatic digest of gelatin and yeast extract provide nitrogenous compounds, Prepared Appearance
vitamin B complex and other nutrients essential for the bacterial metabolism Reddish purple coloured, clear to slightly opalescent gel
while glucose and neutral red helps to detect glucose fermentation. Bile salts Cultural Response
mixture and crystal violet select the growth of gram-negative intestinal bacteria Cultural characteristics after 18-24 hours at 35-370C.
Organisms (ATCC) Growth Colony of colour RG
inhibiting gram-positive bacteria.
Enterobacter aerogenes (13048) Luxuriant Pink More than 70%
Formula* Escherichia coli (25922) Luxuriant Pinkish red More than 70%
Ingredients in grams per liter with bile precipitate.
Yeast extract 3.0 Salmonella serotype Luxuriant Light pink with More than 70%
Pancreatic digest of gelatin 7.0 Enteritidis (13076) bile precipitate
Bile salts 1.5 Staphylococcus Inhibited - 0%
aureus (25923)
Sodium chloride 5.0
For growth RGI should be more than 70%
Glucose monohydrate 10.0
For Inhibition RGI should be 0%
Neutral red 0.03
RGI- Relative Growth Index
Crystal violet 0.002
Agar 15.0 Storage
Final pH (at 25ºC) 7.4 ± 0.2 Store at 22- 300C and prepared medium at 2-80C.
* Formula adjusted to suit performance parameters Shelf Life
Use before expiry date as mentioned on the label.

Violet Red Bile Glucose Agar without Lactose ISO AM51074

Use aerogenes in milk as MaConkey Broth. Recently, the agar formulation is
Violet Red Bile Glucose Agar without Lactose ISO is selective media used for the recommended by ISO committee for the enumeration of coliforms (5).
detection and enumeration of Enterobacteriaceae in food product. It is also Principle
recommended by ISO committee under the specifications ISO 7402:1993. The media is selective due to the presence of the inhibitors- bile salts and crystal
Summary violet. Crystal violet inhibit gram-positive microorganisms especially
Violet Red Bile Broth is recommended by APHA for the detection and enumeration staphylococci. Organisms which rapidly ferment glucose produce red colonies
of coliform organisms in water (36), milk, dairy (39) and other food products surrounded by red-purple halo. Glucose non-fermenters and late glucose
(20). Druce et al., (21) found this media equally good as the indicator of coli- fermenters produce pale colonies.

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Formula* Prepared Appearance
Ingredients in grams per liter Reddish purple coloured, clear to slightly opalescent gel.
Yeast extract 3.0 Cultural Response
Peptic digest of animal tissue 7.0 Cultural characteristics after 18-24 hours at 35-370C.
Bile salt mixture 1.5 Organisms (ATCC) Growth Colony colour RGI
Glucose 10.0 Enterobacter Good-luxuriant Pink-red More than 70%
Sodium chloride 5.0 aerogenes (13048)
Neutral red 0.03 Escherichia coli Good-luxuriant Pink-red with bile More than 70%
Crystal violet 0.002 (25922) precipitate
Agar 12.00 Salmonella serotype Good-luxuriant Light pink More than 70%
Final pH (at 250C) 7.4 ± 0.2 Enteritidis(13076)
* Formula adjusted to suit performance parameters Staphylococcus aureus Inhibited - 0%
Directions (25923)
For growth RGI should be more than 70%
1. Suspend 38.53 gms of the powder in 1000 ml distilled water.
For Inhibition RGI should be 0%
2. Mix thoroughly. RGI- Relative Growth Index
3. Heat with stirring to boiling to dissolve the medium completely. DO NOT Storage
AUTOCLAVE. Store at 22- 300C and prepared medium at 2-80C.
4. Cool to 450C,and pour into petri plates containing the inoculum.. Shelf Life
Quality Control Use before expiry date as mentioned on the label.
Dehydrated Appearance
Beige coloured, homogeneous, free flowing powder.

Violet Red Bile Glucose Agar with Lactose (Agar Medium F) EP AM51075
Violet Red Bile Glucose Agar with Lactose (Agar Medium F) BP AM51076
Use Bile salts 1.5
Violet Red Bile Glucose Agar (VRBGA) is used for the detection and enumeration Sodium chloride 5.0
Glucose monohydrate 10.0
of Enterobacteriaceae in foods and dairy products.
Lactose monohydrate 10.0
Summary
Crystal violet 0.002
Violet Red Bile Agar is recommended by APHA for the detection and enumeration Neutral red 0.03
of coliforms in water(36), milk, dairy (39) and other food products (20). Druce et Agar 15.0
al., (21) found that this medium was as good an indicator of coli-aerogenes Final pH (at 25ºC) 7.4 ± 0.2
bacteria in milk as MacConkey Broth. European Pharmacopoeia also * Formula adjusted to suit performance parameters
recommends this medium for microbiological examination of non-sterile Directions
products. 1. Suspend the 51.53 gms of powder in 1000 ml distilled water and mix
Principle thoroughly.
Pancreatic digest of gelatin and Yeast extract are the source of nutrients, amino 2. Boil with frequent agitation to dissolve the powder completely.
acids, carbon compounds, vitamin B -complex, minerals & trace elements. 3. DO NOT AUTOCLAVE.
Glucose is the energy source. Bile Salts and Crystal Violet inhibit gram-positive Quality Control
bacteria. Neutral red is pH indicator. Agar is the solidifying agent. Dehydrated Appearance
Formula* Pink- beige coloured, homogeneous, free flowing powder.
Ingredients in grams per liter Prepared Appearance
Pancreatic digest of gelatin 7.0 Reddish purple coloured, clear to slightly opalescent gel.
Yeast extract 3.0

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Cultural Response 2. Incubate plates aerobically, protected from light, at 35-37ºC for 18-24
Cultural characteristics after 18-24 hours at 35-37ºC hours or as required.
Organisms (ATCC) Growth Colony colour RGI
Enterobacter Good-luxuriant Pinkish purple More than 70%
3. After incubation count the colonies.
aerogenes (13048) 4. Examine colony morphology and carry out biochemical testing for
Escherichia coli Good-luxuriant Pink-red with bile More than 70% identification.
(25922) precipitate Interpretation of Results
Salmonella serotype Good-luxuriant Colourless More than 70%
1. Enterobacteriaceae ferment glucose and produce red-pink colonies.
Enteritidis(13076)
Staphylococcus Inhibited - 0% 2. Count all developing red-pink colonies.
aureus (25923) Storage
For growth RGI should be more than 70% Store at 22- 300C and prepared medium at 2-80C.
For Inhibition RGI should be 0% Shelf Life
RGI- Relative Growth Index
Use before expiry date as mentioned on the label.
Procedure
1. Use standard procedures like the spread or pour plate method.

Vogel Johnson Agar Base w/o Tellurite IP AM11081/AM51081

Vogel Johnson Agar Base w/o Tellurite USP AM11082/AM51082
Use present are easily distinguished by their inability to produce black colonies.
Vogel Johnson Agar Base with the addition of potassium tellurite is used for the Formula*
isolation of Staphylococcus aureus from clinical and nonclinical specimens. Ingredients in grams per liter
Summary Mannitol 10.0
Pancreatic digest of casein 10.0
Vogel Johnson (116) Agar is used for the early detection of coagulase positive
Glycine 10.0
and mannitol fermenting Staphylococcus aureus from heavily contaminated
Yeast extract 5.0
food and clinical specimens. Vogel Johnson modified the original Tellurite Glycine Lithium chloride 5.0
Agar formula of Zebovitz et al., (124) by increasing the mannitol concentration Dipotassium phosphate 5.0
and adding phenol red as the pH indicator. This medium is specified as a standard Phenol red 0.025
method medium for cosmetics, pharmaceutical articles and nutritional Agar 16.0
supplements and the formulation complies with recommendations by the USP Final pH (at 250C) 7.2 ± 0.2
and IP for microbial limit testing. It selects and differentiates the coagulase * Formula adjusted to suit performance parameters
positive staphylococci, which ferment mannitol and reduce tellurite. Directions

Principle 1. Suspend 61 gms of the powder in 1000 ml distilled water.

Pancreatic digest of casein is a source of carbon, nitrogen, minerals and other 2. Mix thoroughly.
growth factors. Yeast extract provides B complex vitamins. Mannitol is the 3. Boil with frequent agitation to dissolve the powder completely.
carbohydrate source while dipotassium phosphate is the buffer. Potassium 4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
tellurite, lithium chloride and the high glycine content inhibit non-staphylococcal
5. Cool to 45-500C and add 20 ml of sterile 1% Potassium Tellurite Solution
organisms. Staphylococcu s may also be slightly inhibited by the above
(AS022).
inhibitors; but this is compensated by the addition of mannitol and glycine.
Coagulase positive strains of S.aureus reduce tellurite to metallic free tellurium, 6. Mix well and pour into sterile petri plates.
Quality Control
producing characteristic black colonies. The fermentation of mannitol is detected
Dehydrated Appearance
by a change in colour of the phenol red indicator from red (alkaline) to yellow
Light pink coloured, homogeneous, free flowing powder.
(acid); and the colonies are surrounded by a yellow zone. Other microorganisms if

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Prepared Appearance 2. After 48 hours, many coagulase negative, mannitol fermenting or coagulase
Red coloured, slightly opalescent gel. negative, mannitol non-fermenting staphylococci will grow on the medium.
Cultural Response
3. The coagulase positive cocci form small, black colonies against the red
Cultural characteristics after 18-48 hours at 35-370C.
Organisms (ATCC) Growth Colony colour RGI
background.
Escherichia coli (25922) Inhibited - 0% 4. If mannitol is fermented, the colonies are surrounded by yellow zones due to
Proteus mirabilis (25933) Poor Black More than 70%
the colour change of the phenol red indicator in response to the acid
Staphylococcus aureus (25923) Luxuriant Black with More than 70%
yellow halo. formation.
Staphylococcus epidermidis Fair Translucent More than 70% Precautions / Limitations
(12228) to blackish.
1. Do not heat the medium after the addition of potassium tellurite.
For growth RGI should be more than 70%
For Inhibition RGI should be 0%
2. Prolonged incubation may result in the growth of black
RGI- Relative Growth Index coagulase negative colonies and if these organisms also ferment mannitol they
Procedure may be falsely identified from their appearence as S.aureus.
1. Use standard procedures like the streak plate method to obtain isolated 3. If tellurite is reduced but mannitol is not fermented, the medium surrounding
colonies from specimens. colonies may be a deeper red colour due to utilization of proteins in the
2. If material being cultured is directly from a swab, roll the swab over a small medium resulting in alkalinity.
area of the agar surface and streak for isolation. Warning: Lithium chloride is harmful, bodily contact or inhalation of
vapours must be avoided. On contact with skin, wash with plenty of water
3. The medium is highly selective and the inoculum may be applied heavily. immediately.
4. Incubate the plates aerobically at 35-370C for 18-48 hours. Storage
Interpretation of Results Store at 22- 300C and prepared medium at 2-80C.
1. During the first 24 hours of incubation, most organisms other than coagulase Shelf Life
positive staphylococci, are markedly or totally inhibited. Use before expiry date as mentioned on the label.

Vogel Johnson Agar Base w/o Tellurite AM1108/AM5108

Use high glycine content inhibit non-staphylococcal organisms. Staphylococcus may
Vogel Johnson Agar Base with the addition of potassium tellurite is used for the also be slightly inhibited by the above inhibitors; but this is compensated by the
isolation of Staphylococcus aureus from clinical and non-clinical specimens. addition of mannitol and glycine. Coagulase positive strains of S.aureus reduce
Summary tellurite to metallic free tellurium, producing characteristic black colonies. The
Vogel Johnson Agar is used for the early detection of coagulase positive and fermentation of mannitol is detected by a change in colour of the phenol red
mannitol fermenting Staphylococcus aureus from heavily contaminated food indicator from red (alkaline) to yellow (acid); and the colonies are surrounded by
and clinical specimens. Vogel Johnson (116) modified the original Tellurite a yellow zone. Other microorganisms if present are easily distinguished by their
Glycine Agar formula of Zebovitz et al (124) by increasing the mannitol inability to produce black colonies.
concentration and adding phenol red as the pH indicator. This medium is Formula*
specified as a standard method medium for cosmetics (113), pharmaceutical Ingredients in grams per liter
articles and nutritional supplements and the formulation complies with Mannitol 10.0
Tryptone 10.0
recommendations by the USP (114) and IP (46) for microbial limit testing. It
Glycine 10.0
selects and differentiates the coagulase positive staphylococci, which ferment
Yeast Extract 5.0
mannitol and reduce tellurite. Lithium Chloride 5.0
Principle Dipotassium Phosphate 5.0
Tryptone is a source of carbon, nitrogen, minerals and other growth factors. Yeast Phenol Red 0.025
extract provides B complex vitamins. Mannitol is the carbohydrate source while Agar 16.0
dipotassium phosphate is the buffer. Potassium tellurite, lithium chloride and the Final pH (at 250C) 7.2 ± 0.2

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* Formula adjusted to suit performance parameters colonies from specimens.
Directions
2. If material being cultured is directly from a swab, roll the swab over a small
1. Suspend 61 gms of the powder in 1000 ml distilled water. area of the agar surface and streak for isolation.
2. Mix thoroughly. 3. The medium is highly selective and the inoculum may be applied heavily.
3. Boil with frequent agitation to dissolve the powder completely. 4. Incubate the plates aerobically at 35-370C for 18-48 hours.
0
4. Sterilize by autoclaving at 121 C (15 lbs pressure) for 15 minutes. Interpretation of Results
0
5. Cool to 45-50 C and add 20 ml of sterile 1% Potassium Tellurite Solution 1. During the first 24 hours of incubation, most organisms other than coagulase
(AS022). positive staphylococci, are markedly or totally inhibited.
6. Mix well and pour into sterile petri plates. 2. After 48 hours, many coagulase negative, mannitol fermenting or coagulase
Warning: Lithium chloride is harmful, bodily contact or inhalation of negative, mannitol non-fermenting staphylococci will grow on the medium.
vapours must be avoided. On contact with skin, wash with plenty of 3. The coagulase positive cocci form small, black colonies against the red
water immediately.
background.
Quality Control
Dehydrated Appearance 4. If mannitol is fermented, the colonies are surrounded by yellow zones due to
Light pink coloured, homogeneous, free flowing powder. the colour change of the phenol red indicator in response to the acid
Prepared Appearance formation.
Red coloured, slightly opalescent gel. Precautions / Limitations
Cultural Response 1. Do not heat the medium after the addition of potassium tellurite.
Cultural characteristics after 18-48 hours at 35-370C.
2. Prolonged incubation may result in the growth of black coagulase negative
Organisms (ATCC) Growth Colour of RGI
Colony
colonies and if these organisms also ferment mannitol they may be falsely
Escherichia coli (25922) Inhibited - 0% identified from their appearence as S.aureus.
Proteus mirabilis (25933) Poor Black 0% 3. If tellurite is reduced but mannitol is not fermented, the medium surrounding
Staphylococcus Luxuriant Black with More than 70% colonies may be a deeper red colour due to utilization of proteins in the
aureus (25923) yellow halo. medium resulting in alkalinity.
Staphylococcus Fair Translucent 0% Storage
epidermidis (12228) to blackish.
Store at 22-300C and prepared medium at 2-80C.
For growth RGI should be more than 70%
Shelf Life
For Inhibition RGI should be 0%
Use before expiry date as mentioned on the label.
RGI- Relative Growth Index
Procedure
1. Use standard procedures like the streak plate method to obtain isolated

Wilson Blair Agar Base AM51083

Use Dextrose is the source of energy whereas sodium chloride maintains the osmotic
Wilson Blair Agar Base is used for isolation and differentiation of Salmonella balance. Agar is used as a solidifying agent.
serotype Typhi. Formula*
Summary Ingredients in grams per liter
Wilson Blair Agar Base is a valuable medium for the isolation of Typhi. Peptone, special 10
Dextrose 10
Salmonellae produce hydrogen sulfide that causes the colony to be surrounded
Beef extract 5
by a metallic sheen. Wilson Blair Agar Base is highly selective for Salmonellae,
Sodium chloride 5
being inhibitory to coliforms, Proteus and Shigellae (77.2). Agar 30
Principle Final pH (at 250C) 7.3 ± 0.2
Peptone and beef extract provide carbon, nitrogen and other growth factors. * Formula adjusted to suit performance parameters

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Directions Cultural Response
1. Suspend 60gms of powder in 1000ml-distilled water. Cultural characteristics after 24-48 hours at 35-37ºC.
Organisms (ATCC) Growth Colour of RGI
2. Boil to dissolve the medium completely.
Colony
3. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes. Escherichia coli (25922) Inhibited - 0%
4. To sterile melted base, add 4 ml of 1% Brilliant Green solution and 70 ml of Proteus mirabilis (25933) Luxuriant Green More than 70%
selected reagent. Salmonella serotype Luxuriant Black with More than 70%
Typhi (6539) Sheen
Selective Reagent: Salmonella serotype Luxuriant Black with More than 70%
Solution 1: 40 gm Sodium sulphite in 100mld/w Typhimurium sheen
For growth RGI should be more than 70%
Solution 2: 21 gm Dibasic Sodium phosphate in 100 ml d/w
For Inhibition RGI should be 0%
Solution 3: 12.5 gm Bismuth ammonium citrate in 100 ml d/w RGI- Relative Growth Index
Solution 4: 0.96 gm Ferrous sulphate in 20 ml d/w with 2 drops of Procedure
hydrochloric acid. Refer to appropriate references for specific procedures.
Prepare each solution separately and then combine. Interpretation of Results

Boil the combined solution until a slate grey colour develops. Refer to appropriate references and procedures for results.
Quality Control Storage
Dehydrated Appearance Store at 22- 300C and prepared medium at 2-80C.
Light yellow coloured, homogeneous free flowing powder. Shelf Life
Prepared Appearance Use before expiry date as mentioned on the label.
Light yellow coloured, clear to slightly opalescent gel.

WL Differential Agar AM1109/AM5109

Use Formula*
WL Differential Agar is used for the selective isolation and enumeration of Ingredients in grams per liter
Dextrose 50.0
bacteria encountered in breweries and industrial fermentation.
Tryptone 5.0
Summary
Yeast Extract 4.0
Green and Gray (35) developed WL (Wallerstein Laboratory) Differential Agar to Monopotassium Phosphate 0.55
study various fermentation processes. Development of this medium came about Potassium Chloride 0.425
due to an exhaustive study examining the methods of fermentation control Magnesium Sulphate 0.125
procedures in worts, beers, liquid yeasts and similar fermentation products. By Calcium Chloride 0.125
adjusting the pH to 6.5, the medium is made suitable for obtaining counts of Bromocresol Green 0.022
baker and distiller's yeast. Cycloheximide 0.004
Ferric Chloride 0.0025
Principle
Manganese Sulphate 0.0025
Yeast extract provides trace elements, vitamins and amino acids. Tryptone Agar 20.0
provides carbon, nitrogen and amino acids while dextrose is the source of Final pH (at 250C) 5.5 ± 0.2
carbohydrate. Monopotassium phosphate buffers the medium. Potassium * Formula adjusted to suit performance parameters
chloride, calcium chloride and ferric chloride are essential ions and help to Directions
maintain osmotic balance. Magnesium sulphate and manganese sulphate are 1. Suspend 80.26 gms of the powder in 1000 ml distilled water.
sources of divalent cations. Bromocresol green is the pH indicator. Cycloheximide 2. Mix thoroughly.
suppresses the growth of yeasts and moulds in brewing samples, permitting the
3. Boil with frequent agitation to dissolve the powder completely.
detection and enumeration of bacteria that may be present in small numbers.
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.

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5. If desired, use 1% solution of sodium bicarbonate to adjust the pH to 6.5. 2. For baker's yeast and alcohol fermentation mash analysis, incubate at 300C.
Quality Control 3. Incubate aerobically for the growth of acetic acid bacteria, Flavobacterium
Dehydrated Appearance
species, Proteus and thermophilic bacteria.
Greenish yellow coloured, homogeneous, free flowing powder.
Prepared Appearance 4. Incubate anaerobically for the growth of lactic acid bacteria and Pediococcus
Bluish green coloured, very slightly opalescent gel. species.
Cultural Response Precautions / Limitations
0
Cultural characteristics after 40-48 hours at 35 C. 1. While reconstituting vials containing cycloheximide ensure that the vial
Organisms (ATCC) Growth RGI solution does not touch the skin, also avoid the formation of aerosol and the
Saccharomyces cerevisiae (9763) Inhibited 0% inhalation of the compound.
Escherichia coli (25922) Luxuriant More than 70%
Proteus mirabilis (25933) Luxuriant More than 70%
2. Wear personal protective equipment.
For growth RGI should be more than 70% Storage
For Inhibition RGI should be 0% Store at 22-300C and prepared medium at 2-80C.
RGI- Relative Growth Index Shelf Life
Procedure Use before expiry date as mentioned on the label.
1. For brewing materials, incubate at 250C.

WL Differential Broth AM51091

Use Monopotassium phosphate 0.55
WL Differential Broth is used for the selective isolation and enumeration of Potassium chloride 0.425
Magnesium sulphate 0.125
bacteria encountered in breweries and industrial fermentation.
Calcium chloride 0.125
Summary
Bromocresol green 0.022
Green and Gray (35) developed WL (Wallerstein Laboratory) Differential Broth to Captan 0.004
study various fermentation processes. Development of this medium came about Ferric chloride 0.0025
due to an exhaustive study examining the methods of fermentation control Manganese sulphate 0.0025
procedures in worts, beers, liquid yeasts and similar fermentation products. By Final pH (at 250C) 5.5 ± 0.2
adjusting the pH to 6.5, the medium is made suitable for obtaining counts of * Formula adjusted to suit performance parameters
Directions
baker and distiller's yeast.
Principle 1. Suspend 60.26 gms of the powder in 1000 ml distilled water.
Yeast extract provides trace elements, vitamins and amino acids. Tryptone 2. Mix thoroughly.
provides carbon, nitrogen and amino acids while dextrose is the source of 3. Boil with frequent agitation to dissolve the powder completely.
carbohydrate. Monopotassium phosphate buffers the medium. Potassium 4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
chloride, calcium chloride and ferric chloride are essential ions and help to 5. If desired, use 1% solution of sodium bicarbonate to adjust the pH to 6.5.
maintain osmotic balance. Magnesium sulphate and manganese sulphate are Quality Control
sources of divalent cations. Bromocresol green is the pH indicator. Cycloheximide Dehydrated Appearance
suppresses the growth of yeasts and moulds in brewing samples, permitting the Greenish yellow coloured, homogeneous, free flowing powder.
detection and enumeration of bacteria that may be present in small numbers. Prepared Appearance
Formula* Bluish green coloured, very slightly opalescent solution form in tube.
Ingredients in grams per liter Cultural Response
Dextrose 50.0 Cultural characteristics after 40-48 hours at 35-370C.
Casein enzyme hydrolysate 5.0 Organisms(ATCC) Growth
Yeast extract 4.0 Saccharomyces cerevisiae (9763) Inhibited

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Escherichia coli (25922) Luxuriant 4. Incubate anaerobically for the growth of lactic acid bacteria and Pediococcus
Saccharomyces uvarum (9080) Inhibited species.
Lactobacillus fermentum (9338) Luxuriant
Precautions / Limitations
Proteus mirabilis (25933) Luxuriant
For growth RGI should be more than 70%
1. While reconstituting vials containing cycloheximide ensure that the vial
For Inhibition RGI should be 0% solution does not touch the skin, also avoid the formation of aerosol and the
RGI- Relative Growth Index inhalation of the compound.
Procedure 2. Wear personal protective equipment.
1. For brewing materials, incubate at 250C. Storage
2. For baker's yeast and alcohol fermentation mash analysis, incubate at 30 C.0
Store at 22- 300C and prepared medium at 2-80C.
3. Incubate aerobically for the growth of acetic acid bacteria, Flavobacterium Shelf Life

species, Proteus and thermophilic bacteria. Use before expiry date as mentioned on the label.

WL Nutrient Agar AM51092

Use Agar 20.0
WL Nutrient Agar is used for cultivating yeasts, moulds and bacteria encountered Final pH (at 250C) 5.5 ± 0.2
* Formula adjusted to suit performance parameters
in brewing and fermentation processes.
Directions
Summary
WL (Wallerstein Laboratory) Nutrient Agar is based on the formulation of Green 1. Suspend 80.25 gms of the powder in 1000ml distilled water.
and Gray and is used for the determination of the microbiological flora in brewing 2. Mix thoroughly.
and fermentation processes (35). 3. Boil with frequent agitation to dissolve the powder completely.
Principle 4. Dispense in desired containers as per requirements.
Yeast extract provides trace elements, vitamins and amino acids. Tryptone 5. Sterilize by autoclaving at 1210C (15lbs pressure) for 15 minutes.
provides carbon, nitrogen and amino acids while dextrose is the source of
6. If desired, use 1% solution of sodium bicarbonate to adjust the pH to 6.5.
carbohydrates. Monopotassium phosphate buffers the medium. Potassium
Quality Control
chloride, calcium chloride and ferric chloride are essential ions and help to Dehydrated Appearance
maintain osmotic balance. Magnesium sulphate and manganese sulphate are Greenish yellow coloured, homogeneous, free flowing powder.
sources of divalent cations. Bromocresol green is the pH indicator. Prepared Appearance
Adding 0.004 grams/litre of cycloheximide suppresses the growth of yeast and Bluish green coloured, very slightly opalescent gel forms in petri plate and
tube.
renders the medium selective for bacteria. Adjusting the pH to 6.5 facilitates the
Cultural Response
growth of bakers' and distillers' yeasts.
Cultural characteristics after 40-48 hours at 300C.
Formula*
Organisms (ATCC) Growth RGI
Ingredients in grams per liter
Saccharomyces cerevisiae (9763) Good to luxuriant More than 70%
Dextrose 50.0
Saccharomyces uvarum (9080) Good-luxuriant More than 70%
Tryptone 5.0
Escherichia coli (25922) Fair to good More than 70%
Yeast extract 4.0
Lactobacillus fermentum (9338) Fair to good More than 70%
Monopotassium phosphate 0.55
Proteus mirabilis (25933) Fair to good More than 70%
Potassium chloride 0.425
For growth RGI should be more than 70%
Magnesium sulphate 0.125
RGI- Relative Growth Index
Calcium chloride 0.125
Bromocresol green 0.022
Test Procedure
Manganese sulphate 0.0025 1. Prepare the medium and inoculate the medium.
Ferric chloride 0.0025 2. For brewing materials, incubate at 250C.

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3. For Baker's yeast and alcohol fermentation mash analysis, incubate at 300C. Storage

4. For bacteria incubate at 35 ± 2 C. 0 Store at 22- 300C and prepared medium at 2-80C.
Interpretation of Results Shelf Life

Refer to appropriate references and procedures for result. Use before expiry date as mentioned on the label.

WL Nutrient Broth AM1110/AM5110

Use Directions
WL Nutrient Broth is used for cultivating yeasts, moulds and bacteria encountered 1. Suspend 60.25 gms of the powder in 1000 ml distilled water.
in brewing and fermentation processes. 2. Mix thoroughly.
Summary
3. Boil with frequent agitation to dissolve the powder completely.
WL Nutrient Broth is based on the formulation of Green and Gray (35) and is used
4. Dispense in desired containers as per requirements.
where there are advantages for broth media e.g. using larger samples of liquid
products or for enrichment cultures with cycloheximide. 5. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Principle 6. If desired, use 1% solution of sodium bicarbonate to adjust the pH to 6.5.
Yeast extract provides trace elements, vitamins and amino acids. Tryptone 7. Adding 0.004 gms of cycloheximide per liter of broth will make it WL
provides carbon, nitrogen and amino acids while dextrose is the source of Differential Broth.
carbohydrates. Monopotassium phosphate buffers the medium. Potassium Quality Control
chloride, calcium chloride and ferric chloride are essential ions and help to Dehydrated Appearance
maintain osmotic balance. Magnesium sulphate and manganese sulphate are Greenish yellow coloured, homogeneous, free flowing powder.
Prepared Appearance
sources of divalent cations. Bromocresol green is the pH indicator.
Bluish green coloured, very slightly opalescent solution.
Adding 0.004 grams/litre of cycloheximide suppresses the growth of yeast and Cultural Response
renders the medium selective for bacteria. Adjusting the pH to 6.5 facilitates the Cultural characteristics after 40-48 hours at 300C.
growth of bakers' and distillers' yeasts; the medium at pH 5.5 is used for the Organisms (ATCC) Growth
growth of bakers' yeasts. Saccharomyces cerevisiae (9763) Good to luxuriant
Formula* Escherichia coli (25922) Fair to good
Ingredients in grams per liter Proteus mirabilis (25933) Fair to good
Dextrose 50.0 For growth RGI should be more than 70%
Tryptone 5.0 RGI- Relative Growth Index
Yeast Extract 4.0 Precautions / Limitations
Monopotassium Phosphate 0.55 1. While reconstituting vials containing cycloheximide ensure that the vial
Potassium Chloride 0.425 solution does not touch the skin, also avoid the formation of aerosol and the
Magnesium Sulphate 0.125 inhalation of the compound.
Calcium Chloride 0.125
Bromocresol Green 0.022
2. Wear personal protective equipment.
Manganese Sulphate 0.0025 Storage
Ferric Chloride 0.0025 Store at 22-300C and prepared medium at 2-80C.
0
Final pH (at 25 C) 5.5 ± 0.2 Shelf Life
* Formula adjusted to suit performance parameters Use before expiry date as mentioned on the label.

Wort Agar AM1111/AM5111

Use Summary
Wort Agar is used for the cultivation and enumeration of yeasts. Parfitt (86) formulated Wort Agar for the cultivation of fungi especially yeasts in
syrups and butter. Culture media containing dextrose and maltose with an acidic

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pH enhance the growth of yeasts. 4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
Principle Quality Control
Malt extract and peptone provide nitrogenous compounds and other nutrients for Dehydrated Appearance
the growth of yeasts. Maltose and dextrin are fermentable carbohydrates. The Yellow coloured, homogeneous, free flowing powder.
Prepared Appearance
high acidic pH inhibits the growth of many bacteria.
Yellow coloured, opalescent gel.
Formula*
Cultural Response
Ingredients in grams per liter
Cultural characteristics after 40-48 hours at 300C.
Malt Extract 15.0
Organisms (ATCC) Growth RGI
Maltose 12.75
Aspergillus niger (16404) Luxuriant More than 70%
Peptone 0.78
Saccharomyces cerevisiae (9763) Luxuriant More than 70%
Dextrin 2.75
Candida albicans (10231) Luxuriant More than 70%
Ammonium Chloride 1.0
For growth RGI should be more than 70%
Dipotassium Phosphate 1.0
RGI- Relative Growth Index
Agar 15.0
Precautions / Limitations
Final pH (at 250C) 4.8 ± 0.2
* Formula adjusted to suit performance parameters 1. Do not reliquify agar medium as it may cause alteration of the medium with
Directions hydrolysis of agar at low pH and results in failure to gel when cooled.
1. Suspend 48.3 gms of the powder in 1000 ml distilled water containing 2.35 Storage

gms glycerol. Store at 22-300C and prepared medium at 2-80C.

Shelf Life
2. Mix thoroughly.
Use before expiry date as mentioned on the label.
3. Boil with frequent agitation to dissolve the powder completely.

Xylose Lysine Deoxycholate Agar (XLD Agar) AM1112/AM5112

Use nutrients while sodium deoxycholate inhibits gram-positive organisms. Xylose is
XLD Agar is a moderately selective medium used for the isolation and fermented practically by all enterics except shigellae, which enables the
differentiation of Salmonella and Shigella. differentiation of Shigella species. Incorporation of lysine enables the
Summary Salmonella group to be differentiated from the non-pathogens since, without
XLD Agar is a differential medium used for the isolation of salmonellae and lysine, Salmonella would rapidly ferment xylose and be indistinguishable from
shigellae from clinical and non-clinical specimens like faeces and foods. It was non-pathogenic species. After Salmonella exhausts the supply of xylose, lysine is
developed by Taylor (108) in order to increase the efficiency of isolation of the attacked, with reversion to an alkaline pH, which mimic the Shigella reaction.
enteric pathogens, particularly Shigella from faecal specimens. The pathogens However, to prevent this reaction by lysine positive coliforms, lactose and sucrose
are differentiated not only from the non-pathogenic lactose fermenters but also are added in excess to produce acid and hence non-pathogenic H2S producers do
from many non-pathogens, which do not ferment lactose or sucrose. Also, the not decarboxylate lysine. The acid reaction produced by them prevents the
medium was formulated to increase the frequency of growth of the more blackening of the colonies. Sodium thiosulphate and ferric ammonium citrate are
fastidious pathogens, which in other formulations have often failed to grow due to included for the visualization of hydrogen sulphide production, resulting in the
the inclusion of excessively toxic inhibitors. This medium is used in the microbial formation of colonies with black centers. Sodium chloride maintains the osmotic
limit test for screening specimens for the detection of Salmonella and is balance.
recommended by APHA for the examination of foods (20), dairy products (39) Formula*
and water (36). XLD Agar conforms to the specifications of the USP (114), IP (46) Ingredients in grams per liter
and is included in the Bacteriological Analytical Manual for food testing (113). Sucrose 7.5
Principle Lactose 7.5
Sodium Thiosulphate 6.8
XLD Agar is both, a selective and differential medium. Yeast extract provides
L-Lysine 5.0

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Sodium Chloride 5.0 5. Examine colonial morphology, characteristics and haemolytic reactions.
Xylose 3.5
6. It is recommended that selective enrichment broth, such as Selenite Cystine
Yeast Extract 3.0
Sodium Deoxycholate 2.5
Broth be used in conjunction with other selective plating media to maximize
Ferric Ammonium Citrate 0.8 the recovery of enteric pathogens.
Phenol Red 0.08 Interpretation of Results
Agar 15.0 1. Degradation of xylose, lactose and sucrose leads to formation of acid products,
Final pH (at 250C) 7.4 ± 0.2 causing a colour change in the medium from red to yellow.
* Formula adjusted to suit performance parameters
2. H2S production under alkaline conditions causes colonies to form black
Directions
centers. This reaction is inhibited by the acid conditions that accompany
1. Suspend 56.68 gms of the powder in 1000 ml distilled water.
carbohydrate fermentation.
2. Mix thoroughly.
3. Lysine decarboxylation in the absence of lactose and sucrose fermentation
3. Heat with frequent agitation until the medium just boils to dissolve the causes reversion to alkaline condition and the colour of the medium changes
powder completely. back to red.
4. DO NOT OVERHEAT OR AUTOCLAVE.Overheating causes precipitation. Typical colonial morphology and reactions on XLD Agar:
5. Cool immediately in a water bath at 45-500C and pour into sterile petri E.coli------------------------------------ Large, flat, yellow; some strains may be
plates. inhibited.
Quality Control Enterobacter/ Klebsiella------------ Mucoid, yellow.
Dehydrated Appearance Proteus--------------------------------- Red to yellow, most strains have black
Pink coloured, homogeneous, free flowing powder. centers.
Prepared Appearance Salmonella---------------------------- Red- yellow with black centers.
Red coloured, clear to very slightly opalescent gel. H2S negative Shigella,
Cultural Response Salmonella --------------------------- Red.
Cultural characteristics after 18-24 hours at 35-370C. Pseudomonas------------------------ Red.
Organisms (ATCC) Growth Colour of Colony Gram-positive bacteria------------- No growth or slight growth.
Enterobacter aerogenes (13048) Fair Yellow Precautions / Limitations
Escherichia coli (25922) Fair Yellow 1. It is advisable not to prepare large volumes, which will require prolonged
Proteus vulgaris (13315) Good to luxuriant Yellow heating.
Salmonella serotype Enteritidis Good to luxuriant Red with black
(13076) centers
2. Longer incubation may result in false positive results.
Salmonella serotype Typhimurium Good to luxuriant Red with black 3. Some species of Salmonella like S.paratyphi A, S.choleraesuis, S.gallinarum
(14028) centers and S.pullorum form red colonies without black centers, which resemble
Shigella dysenteriae (13313) Good to luxuriant Red Shigella colonies.
Staphylococcus aureus (25923) Inhibited -
4. Also, a few species of Shigella that ferment lactose, and Salmonella that fail
Procedure
to decarboxylate lysine would not be detected on this medium.
1. Use standard procedures like the streak plate method to obtain isolated
5. Red, false positive colonies may occur with some Proteus and Pseudomonas
colonies from specimens.
species. Some Proteus strains will give black centered colonies on XLD Agar.
2. If material being cultured is directly from a swab, roll the swab over a small Storage
area on the agar surface and streak for isolation.
0
Store at 22-300C and prepared medium at 2-80C.
3. Incubate the plates, protected from light at 35-37 C for 18-24 hours. Shelf Life
4. Colonies on XLD Agar may require 48 hours incubation for full colour Use before expiry date as mentioned on the label.
development.

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Xylose Lysine Deoxycholate Agar (XLD Agar) Harmonized AMH5112
Xylose Lysine Deoxycholate Agar (XLD Agar) IP AM51121
Xylose Lysine Deoxycholate Agar (XLD Agar)USP AM51122
Xylose Lysine Deoxycholate Agar (XLD Agar) EP AM51123
Xylose Lysine Deoxycholate Agar (XLD Agar) BP AM51124
Use Formula*
XLD Agar is a moderately selective medium used for the isolation and Ingredients in grams per liter
Sucrose 7.5
differentiation of Salmonella and Shigella.
Lactose 7.5
Summary
Sodium thiosulphate 6.8
XLD Agar is a differential medium used for the isolation of salmonellae and L-Lysine 5.0
shigellae from clinical and non-clinical specimens like faeces and foods. It was Sodium chloride 5.0
developed by Taylor (108) in order to increase the efficiency of isolation of the Xylose 3.5
enteric pathogens, particularly Shigella from faecal specimens. The pathogens Yeast extract 3.0
are differentiated not only from the non-pathogenic lactose fermenters but also Sodium deoxycholate 2.5
from many nonpathogens, which do not ferment lactose or sucrose. Also, the Ferric ammonium citrate 0.8
Phenol red 0.08
medium was formulated to increase the frequency of growth of the more
Agar 13.5
fastidious pathogens, which in other formulations have often failed to grow due
Final pH (at 250C) 7.4 ± 0.2
to the inclusion of excessively toxic inhibitors. This medium is used in the * Formula adjusted to suit performance parameters
microbial limit test for screening specimens for the detection of Salmonella Directions
(111.2) and is recommended by APHA for the examination of foods, dairy 1. Suspend 55.18 gms of the powder in 1000 ml distilled water.
products and water. XLD Agar conforms to the specifications of the USP, IP and is
2. Mix thoroughly.
included in the Bacteriological Analytical Manual for food testing.
Principle 3. Heat with frequent agitation until the medium just boils to dissolve the
XLD Agar is both, a selective and differential medium. Yeast extract provides powder completely.
nutrients while sodium deoxycholate inhibits gram-positive organisms. Xylose is 4. DO NOT OVERHEAT OR AUTOCLAVE. Overheating causes precipitation.
fermented practically by all enterics except 5. Cool immediately in a water bath at 45-500C and pour into sterile petri
shigellae, which enables the differentiation of Shigella species. Incorporation of plates.
lysine enables the Salmonella group to be differentiated from the non- Quality Control
pathogens since, without lysine, Salmonella would rapidly ferment xylose and Dehydrated Appearance
Pink coloured, homogeneous, free flowing powder.
be indistinguishable from non-pathogenic species. After Salmonella exhausts
Prepared Appearance
the supply of xylose, lysine is attacked, with reversion to an alkaline pH, which
Red coloured, clear to very slightly opalescent gel.
mimic the Shigella reaction. However, to prevent this reaction by lysine positive Cultural Response
coliforms, lactose and sucrose are added in excess to produce acid and hence non- Cultural characteristics after 18-24 hours at 35-370C.
pathogenic H2S producers do not decarboxylate lysine. The acid reaction produced Organisms (ATCC) Growth Colour RGI
by them prevents the blackening of the colonies. Sodium thiosulphate and ferric of Colony
ammonium citrate are included for the visualization of hydrogen sulphide Enterobacter Fair Yellow More than 70%
production, resulting in the formation of colonies with black centers. Sodium aerogenes (13048)
Escherichia coli (25922) Fair Yellow More than 70%
chloride maintains the osmotic balance.
Proteus vulgaris (13315) Good to Yellow More than 70%
Luxuriant

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Salmonella serotype Good to Red with black More than 70% 3. Lysine decarboxylation in the absence of lactose and sucrose fermentation
Enteritidis (13076) Luxuriant centers causes reversion to alkaline condition and the colour of the medium changes
Salmonella serotype Good to Red with black More than 70%
back to red.
Typhimurium (14028) Luxuriant centers
Shigella dysenteriae Good to Red More than 70% Typical colonial morphology and reactions on XLD Agar:
(13313) Luxuriant E.coli------------------------Large, flat, yellow; some strains may be inhibited.
Staphylococcus Inhibited - 0%
Enterobacter/ Klebsiella-----Mucoid, yellow.
aureus (25923)
For growth RGI should be more than 70% Proteus----------------------Red to yellow, most strains have black centers.
For Inhibition RGI should be 0% Salmonella------------------Red- yellow with black centers. H2S negative
RGI- Relative Growth Index Shigella,
Procedure
Salmonella ---------------- Red.
1. Use standard procedures like the streak plate method to obtain isolated
Pseudomonas ---------------Red.
colonies from specimens.
Gram-positive bacteria------ No growth or slight growth.
2. If material being cultured is directly from a swab, roll the swab over a small
Precautions / Limitations
area on the agar surface and streak for isolation.
1. It is advisable not to prepare large volumes, which will require prolonged
3. Incubate the plates, protected from light at 35-370C for 18-24 hours.
heating.
4. Colonies on XLD Agar may require 48 hours incubation for full colour
2. Longer incubation may result in false positive results.
development.
3. Some species of Salmonella like S.paratyphi A, S.choleraesuis,
5. Examine colonial morphology, characteristics and haemolytic reactions.
S.gallinarum and S.pullorum form red colonies without black centers,
6. It is recommended that selective enrichment broth, such as Selenite Cystine which resemble Shigella colonies.
Broth be used in conjunction with other selective plating media to maximize
4. Also, a few species of Shigella that ferment lactose, and Salmonella that
the recovery of enteric pathogens.
fail to decarboxylate lysine would not be detected on this medium.
Interpretation of Results
5. Red, false positive colonies may occur with some Proteus and Pseudomonas
1. Degradation of xylose, lactose and sucrose leads to formation of acid
species. Some Proteus strains will give black centered colonies on XLD Agar.
products, causing a colour change in the medium from red to yellow.
Storage
2. H2S production under alkaline conditions causes colonies to form black
Store at 22- 300C and prepared medium at 2-80C.
centers. This reaction is inhibited by the acid conditions that accompany Shelf Life
carbohydrate fermentation.
Use before expiry date as mentioned on the label.

XLD Agar, Modified ISO AM51125

Use medium was formulated to increase the frequency of growth of the more
XLD Agar, Modified is a moderately selective medium used for the isolation and fastidious pathogens, which in other formulations have often failed to grow due to
differentiation of Salmonella and Shigella in compliances with ISO specification the inclusion of excessively toxic inhibitors. This medium is used in the microbial
ISO 6579:2002.. limit test for screening specimens for the detection of Salmonella (111.2) and is
Summary recommended by APHA for the examination of foods, dairy products and water.
XLD Agar is a differential medium used for the isolation of salmonellae and XLD Agar conforms to the specifications of the ISO 6579:2002 and is included in
shigellae from clinical and non-clinical specimens like faeces and foods. It was the Bacteriological Analytical Manual for food testing.
developed by Taylor (108) in order to increase the efficiency of isolation of the Principle
enteric pathogens, particularly Shigella from faecal specimens. The pathogens XLD Agar is both, a selective and differential medium. Yeast extract provides
are differentiated not only from the non-pathogenic lactose fermenters but also nutrients while sodium deoxycholate inhibits gram-positive organisms. Xylose is
from many nonpathogens, which do not ferment lactose or sucrose. Also, the fermented practically by all enterics except

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shigellae, which enables the differentiation of Shigella species. Incorporation of Quality Control
lysine enables the Salmonella group to be differentiated from the non- Dehydrated Appearance
Pink coloured, homogeneous, free flowing powder.
pathogens since, without lysine, Salmonella would rapidly ferment xylose and
Prepared Appearance
be indistinguishable from non-pathogenic species. After Salmonella exhausts the
Red coloured, clear to very slightly opalescent gel.
supply of xylose, lysine is attacked, with reversion to an alkaline pH, which mimic Cultural Response
the Shigella reaction. However, to prevent this reaction by lysine positive Cultural characteristics after 18-24 hours at 35-370C.
coliforms, lactose and sucrose are added in excess to produce acid and hence non- Organisms (ATCC) Growth Colour RGI
pathogenic H2S producers do not decarboxylate lysine. The acid reaction produced of Colony
by them prevents the blackening of the colonies. Sodium thiosulphate and ferric Enterobacter Fair Yellow More than 70%
ammonium citrate are included for the visualization of hydrogen sulphide aerogenes (13048)
Escherichia coli (25922) Fair Yellow More than 70%
production, resulting in the formation of colonies with black centers. Sodium
Proteus vulgaris (13315) Good to Yellow More than 70%
chloride maintains the osmotic balance.
luxuriant
Formula*
Salmonella serotype Good to Red with More than 70%
Ingredients in grams per liter
Enteritidis (13076) luxuriant black centers
Sucrose 7.5
Salmonella serotype Good to Red with More than 70%
Lactose 7.5
Typhimurium (14028) luxuriant black centers
Sodium thiosulphate 6.8
Shigella dysenteriae Good to Red More than 70%
L-Lysine 5.0
(13313) luxuriant
Sodium chloride 5.0
Staphylococcus Inhibited - 0%
Xylose 3.75
aureus (25923)
Yeast extract 3.0
Proteus mirabilis Good to Yellow More than 70%
Sodium deoxycholate 1.0
(25933) luxuriant
Ferric ammonium citrate 0.8
Salmonella typhi Good to Red with More than 70%
Phenol red 0.08
(6539) luxuriant black center
Agar 15.0
Shigella sonnei Good to Red More than 70%
Final pH (at 250C) 7.4 ± 0.2
(25931) luxuriant
* Formula adjusted to suit performance parameters
For growth RGI should be more than 70%
Directions
For Inhibition RGI should be 0%
1. Suspend 55.43 gms of the powder in 1000 ml distilled water. RGI- Relative Growth Index
2. Mix thoroughly. Storage

3. Heat with frequent agitation until the medium just boils to dissolve the Store at 22- 300C and prepared medium at 2-80C.
powder completely. Shelf Life

4. DO NOT OVERHEAT OR AUTOCLAVE. Overheating causes precipitation. Use before expiry date as mentioned on the label.

5. Cool immediately in a water bath at 45-500C and pour into sterile petri
plates.

XLT4 Agar AM571251

Use Although most Salmonella cannot be distinguished by biochemical
XLT4 Agar Base medium is recommended for selective isolation of Salmonella characteristics, one serotype, namely S. Typhi produce only a trace amount of
species other than Salmonella Typhi. hydrogen sulphide and is less active biochemically than the more common
Summary serotypes (60.1). XLT4 Agar Base is formulated as described by Miller and Tate
Salmonella is a genus of gram-negative enterobacteria commonly implicated in (79.5) for isolating Salmonella from faecally contaminated farm samples, which
foodborne illness and is the causative agent of typhoid and paratyphoid fever. contains other bacteria as well. XLT4 Agar Base enhances the recovery of

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Salmonella species other than Salmonella Typhi (79.6, 79.7, 20.1 & 20.2). 2. Add 4.6 ml XLT4 Supplement (AS0281).
Principle 3. Heat to boiling to dissolve the medium completely.
Proteose peptone is a source of carbon, nitrogen and other essential amino acids 4. DO NOT AUTOCLAVE OR OVERHEAT.
and growth factors. Yeast extract supplies nitrogenous requirements and vitamins Quality Control
required for growth. The sugars namely lactose, saccharose and xylose are the Dehydrated Appearance
fermentable carbohydrates. Salmonella rapidly utilize xylose, producing acidity. Light yellow to pink homogeneous free flowing powder
Subsequently they decarboxylate lysine and revert to alkalinity. To add to the Prepared Appearance
differentiating ability of the formulation, an H2S indicator system, consisting of Red coloured clear to slightly opalescent gel forms in Petri plates.
Cultural Response
sodium thiosulphate and ferric ammonium citrate is included for the visualization
Cultural characteristics observed after an incubation at 35-37°C for 18-24
of the hydrogen sulphide produced, resulting in the formation of colonies with hours with added XLT4
black centers. The non-pathogenic H2S producers do not decarboxylate lysine; Organisms (ATCC) Growth Colour RGI
therefore, the acid reaction generated by them prevents the blackening of the of Colony
colonies. XLT4 Agar is both selective and differential. Salmonella Enterococcus Inhibited 0% -
faecalis (29212)
Formula*
Escherichia coli (25922) Fair-good Yellow More than 70%
Ingredients in grams per liter
Salmonella Enteritidis Good- Red with More than 70%
Proteose peptone 1.60
(13076) luxuriant black centers
Yeast extract 3.00
Salmonella Typhimurium Good- Red with More than 70%
L-Lysine 5.00
(14028) luxuriant black centers
Xylose 3.75
Staphylococcus aureus Inhibited 0% -
Lactose 7.50
( 25923)
Saccharose 7.50
For growth RGI should be more than 70%
Ferric ammonium citrate 0.80
For Inhibition RGI should be 0%
Sodium thiosulphate 6.80
RGI- Relative Growth Index
Sodium chloride 5.00
Storage
Phenol red 0.08
Agar 18.00 Store at 22- 300C and prepared medium at 2-80C.
Final pH ( at 25°C) 7.4±0.2 Shelf Life
* Formula adjusted to suit performance parameters Use before expiry date as mentioned on the label.
Directions
1. Suspend 59.03 grams in 1000 ml distilled water.

Yeast and Mould Broth AM51126

Use Formula*
Yeast and Mould Broth is used for isolation and cultivation of yeasts and moulds. Ingredients in grams per liter
Summary Yeast extract 3.0
Malt extract 3.0
Yeast and Mould Broth is formulated on the basis of the description given by
Peptone 5.0
Wickerham (119.1). The medium is recommended for the isolation and Dextrose 10.0
maintenance of yeasts and moulds. Final pH (at 250C) 6.2 ±0.2
Principle * Formula adjusted to suit performance parameters
Peptone, yeast extract and malt extract provide all the essential nutrients for Directions
growth. Dextrose is the source of energy. Acidic pH inhibits the bacterial growth 1. Suspend the 21.0gms of powder in 1000 ml distilled water.
and favours the growth of mould. 2. Mix thoroughly.

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3. Heat with frequent agitation to dissolve the powder completely. DO NOT Candida albicans (10231) Good
OVERHEAT. Saccharomyces cerevisiae (9763) Good
Procedure
4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Refer to appropriate references for specific procedures for the cultivation of yeast
Quality Control
Dehydrated Appearance
and mould.
Cream coloured, homogenous, free flowing powder. Interpretation of Results
Prepared Appearance Refer to appropriate references and procedures for results.
Light amber couloured, slightly opalescent solution forms in tubes. Storage
Cultural Response Store at 22- 300C and prepared medium at 2-80C.
0
Cultural characteristics after 48-72 hours at 20-25 C.
Shelf Life
Organisms (ATCC)
Aspergillus niger (16404) Good
Use before expiry date as mentioned on the label.

Yeast Dextrose Agar AM51127

Use 4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Yeast Dextrose Agar is used for the cultivation of a variety of heterotrophic Dehydrated Appearance
microorganisms. Light yellow coloured, homogeneous, free flowing powder.
Summary Prepared Appearance
Yellow coloured opalescent gel forms in petriplates.
Yeast Dextrose Agar is recommended for the cultivation of a variety of
Cultural Response
heterotrophic microorganisms. Cultural characteristics after 5 days at 20-25°C.
Principle Organisms (ATCC) Growth RGI
Yeast extract supplies carbon, nitrogen and other ingredients for the growth of Candida albicans (10231) Luxuriant More than 70%
microorganisms. Dextrose is the energy source. Agar is the solidifying agent. Saccharomyces cerevisiae (9763) Luxuriant More than 70%
Formula* Aspergillus niger (16404) Luxuriant More than 70%
Ingredients in grams per liter For growth RGI should be more than 70%
Yeast extract 10.0 RGI- Relative Growth Index
Dextrose 10.0 Procedure
Agar 15.0 Refer to appropriate references for specific procedures.
Final pH (at 250C) 7.0 ± 0.2 Interpretation of Results
* Formula adjusted to suit performance parameters
Refer to appropriate references and procedures for results.
Directions
Storage
1. Suspend the 35 gms of powder in 1000 ml distilled water.
Store at 22- 300C and prepared medium at 2-80C.
2. Mix thoroughly. Shelf Life
3. Heat gently with frequent agitation to dissolve the powder completely. Use before expiry date as mentioned on the label.

Yeast Extract Agar AM1113/AM5113

Use microorganisms in water and is also used for the cultivation of a wide variety of
Yeast Extract Agar is a highly nutritive medium used for cultivation of a wide bacteria. It is included in the Bacteriological Analytical Manual for food and
variety of bacteria. cosmetics testing (113).
Summary Principle
Windle Taylor (109) formulated Yeast Extract Agar for the plate count of Yeast extract and peptone provide nitrogenous compounds, vitamin B complex
and other nutrients. Agar is the solidifying agent.

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Formula* Organisms (ATCC) Growth RGI
Ingredients in grams per liter Escherichia coli (25922) Luxuriant More than 70%
Peptone 5.0 Staphylococcus aureus (25923) Luxuriant More than 70%
Yeast Extract 3.0 For growth RGI should be more than 70%
Agar 15.0 RGI- Relative Growth Index
Final pH (at 250C) 7.2 ± 0.2 Procedure
* Formula adjusted to suit performance parameters 1. Incubate plates at 35-370C for 18-24 hours.
Directions
2. Separate counts are also made of the organisms forming visible colonies
1. Suspend 23 gms of the powder in 1000 ml distilled water. after 24 hours at 350C and organisms forming visible colonies after 3 days at
2. Mix thoroughly. 20-220C.
3. Boil with frequent agitation to dissolve the powder completely. Interpretation of Results
0
4. Sterilize by autoclaving at 121 C (15 lbs pressure) for 15 minutes. 1. Select plates containing 20-300 colonies.
Quality Control 2. Record results as colony forming units (CFU) per volume of sample taking
Dehydrated Appearance into account the dilution factor.
Light yellow coloured, homogeneous, free flowing powder. Storage
Prepared Appearance
Store at 22-300C and prepared medium at 2-80C.
Yellow coloured, clear to very slightly opalescent gel.
Shelf Life
Cultural Response
0
Cultural characteristics after 18-24 hours at 35-37 C.
Use before expiry date as mentioned on the label.

Yeast Extract Chloramphenicol Dextrose Agar AM51131

Use Directions
Yeast Extract Chloramphenicol Dextrose Agar recommended for isolation and 1. Suspend 40.0 gms of the powder in 1000 ml distilled water.
enumeration of fungi-yeasts and moulds in milk and milk products 2. Heat to boiling to dissolve the medium completely.
Summary
3. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes.
The antibiotic method for enumerating yeasts and molds in dairy products has Dehydrated Appearance
become the method of choice, replacing the traditional acidified method. The use Light yellow colour, homogeneous, free flowing powder.
of antibiotics for supressing bacteria results in better recovery of injured fungal Prepared Appearance:
cells. Which are sensitive to an acid environment, and in less interference from Yellow coloured, clear to slightly opalescent gel forms in petriplates.
precipitated food particles during the counting. Yeast Extract Chloramphenicol Cultural Response
Dextrose Agar is a nutrient medium that inhibits the growth of organisms other Cultural characteristics after 2-5 days at 20-25°C.
Organisms (ATCC) Growth RGI
than yeasts and molds due to the presence of Chloramphenicol.
Aspergillus niger (16404) Good-luxuriant More than 70%
Principle
Candida albicans (10231) Good-luxuriant More than 70%
Yeast extract provides basic nutrient. Dextrose is a carbon energy source. Escherichia coli (25922) Inhibited 0%
Chloramphenicol inhibits bacterial growth. Agar is the solidifying agent. Saccharomyces cerevisiae (9763) Good-luxuriant More than 70%
Formula* For growth RGI should be more than 70%
Ingredients in grams per liter For Inhibition RGI should be 0%
Yeast extract 5.0 RGI- Relative Growth Index
Dextrose 20.0 Procedure
Chloramphenicol 0.10 Refer to appropriate references for specific procedures.
Agar 14.90
Interpretation of Results
Final pH (at 250C) 6.6 ± 0.2
* Formula adjusted to suit performance parameters
Refer to appropriate references and procedures for results.

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Storage Shelf Life
0 0
Store at 22- 30 C and prepared medium at 2-8 C. Use before expiry date as mentioned on the label.

Yeast Malt Agar AM1114/AM5114

Yeast Malt Broth AM1115/AM5115
Use 6. DO NOT REHEAT the media after the addition of antibiotics or acid.
Yeast Malt Agar and Yeast Malt Broth are used for the isolation and cultivation of Quality Control
yeasts, moulds and aciduric bacteria. Dehydrated Appearance
Summary Light yellow coloured, homogeneous, free flowing powder.
Prepared Appearance
Wickerham (119) formulated Yeast Malt Agar. Media selectivity can be enhanced
Yeast Malt Agar - Light amber coloured, slightly opalescent gel.
by adding selective agents or by acidifying the medium. Yeast Malt Agar and
Yeast Malt Broth - Light amber coloured, slightly opalescent solution.
Yeast Malt Broth should be sterilized without prior pH adjustment and sterile acid Cultural Response
should be added to sterile cooled molten agar. Antibiotics may be aseptically Cultural characteristics after 40-72 hours at 25-300C.
added to the medium. Fungistatic material like sodium propionate and diphenyl Organisms (ATCC) Growth at pH 3.0-4.0 Growth at pH 6.2
may be added to eliminate moulds thus permitting the enumeration of yeasts in a Aspergillus niger (16404) Good to luxuriant Good to luxuriant
mixed population. Candida albicans (10231) Good to luxuriant Good to luxuriant
Principle Saccharomyces cerevisiae (9763) Good to luxuriant Good to luxuriant
Escherichia coli (25922) Inhibited Good to luxuriant
Yeast extract provides trace elements, vitamins and amino acids. Malt extract
Procedure
provides carbon, protein and other nutrients. Peptone is an additional source of
carbon and provides amino acids and nitrogen. Dextrose provides carbon as 1. Inoculate the Yeast Malt Agar plates and Yeast Malt Broth tubes with
energy source. samples to evaluate the presence of yeasts, moulds or aciduric organisms.
Formula* 2. Incubate at 25-300C for 40-72 hours.
Ingredients in grams per liter Yeast Malt Agar Yeast Malt Broth 3. To favour isolation of fermentative species in Yeast Malt Broth, add a layer of
Dextrose 10.0 10.0 sterile paraffin oil 1 cm deep on the surface of the inoculated broth. Incubate
Peptone 5.0 5.0
the culture till growth appears then streak onto Yeast Malt Agar to obtain
Malt Extract 3.0 3.0
isolated yeast colonies.
Yeast Extract 3.0 3.0
Agar 20.0 - 4. To isolate oxidative or fermentative strains, place the acidified Yeast Malt
Final pH (at 250C) 6.2 ± 0.2 6.2 ± 0.2 Broth on a rotary shaker for 1-2 days. This favours yeast recovery while
* Formula adjusted to suit performance parameters preventing the sporulation of moulds.
Directions Interpretation of Results
1. Suspend the powder in 1000 ml distilled water. For Yeast Malt Agar
Yeast Malt Agar - 41gms 1. Examine the plates for growth.
Yeast Malt Broth - 21 gms 2. Record as colony forming units (CFU) per volume of the sample.
2. Mix thoroughly. For Yeast Malt Broth
3. Boil with frequent agitation to dissolve the powder completely. 1. Record result as growth or no growth on the basis of turbidity of the medium.
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. Storage

5. To make the medium selective: acidify the medium to pH 3.0 to 4.0 by Store at 22-300C and prepared medium at 2-80C.
Shelf Life
adding sterile acid (10% HCl, tartaric acid or 10% citric acid) or antibiotics.
Use before expiry date as mentioned on the label.

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Yeast Mannitol Agar with 1.5% Agar AM51151
Use 3. Heat with frequent agitation to dissolve the powder completely. DO NOT
Yeast Mannitol Agar with 1.5% agar is used for cultivation, isolation and OVERHEAT.
enumeration of soil microorganism like Rhizobium species. 4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Summary Dehydrated Appearance
Yeast Mannitol Agar with 1.5% agar is recommended for the cultivation of the Yellow coloured, homogenous, free flowing powder.
symbiotic nitrogen-fixing organisms like Rhizobium species (106.1). Prepared Appearance
Principle Whitish buff coloured, opalescent solution forms in petri plates.
Cultural Response
Yeast extract provides amino acids and vitamins while sodium chloride maintains
Cultural characteristics after 5 days at 20-250C.
the osmotic balance. Mannitol is the source of energy. Calcium and magnesium
Organisms (ATCC) Growth RGI
supports the growth of rhizobia. RRhizobium meliloti (9930) Luxuriant More than 70%
Formula* Rhizobium leguminosarum (10004) Luxuriant More than 70%
Ingredients in grams per liter For growth RGI should be more than 70%
Yeast extract 1.0 RGI- Relative Growth Index
Mannitol 10.0 Procedure
Dipotassium phosphate 0.50
Refer to appropriate references for specific procedures for the cultivation of
Magnesium sulphate 0.20
Sodium chloride 0.10 phosphate solubilizing soil microorganisms.
Calcium carbonate 1.00 Interpretation of Results
Agar 15.00 Refer to appropriate references and procedures for results.
Final pH (at 250C) 6.8 ± 0.2 Storage
* Formula adjusted to suit performance parameters Store at 22- 300C and prepared medium at 2-80C.
Directions
Shelf Life
1. Suspend the 27.8 gms of powder in 1000 ml distilled water. Use before expiry date as mentioned on the label.
2. Mix thoroughly.

Yeast Mannitol Agar with Congo Red AM51152

Use Magnesium sulphate 0.2
Yeast Mannitol Agar with Congo Red is used for the cultivation of soil Sodium chloride 0.1
Congo red 0.025
microorganisms like Rhizobium species.
Agar 20.0
Summary
Final pH (at 250C) 6.8 ± 0.2
Yeast Mannitol Agar with Congo Red is recommended for the cultivation of the * Formula adjusted to suit performance parameters
symbiotic nitrogen-fixing organisms like Rhizobium species (106.1). Directions
Principle
1. Suspend the 31.83gms of powder in 1000 ml distilled water.
Yeast extract provides amino acids and vitamins while sodium chloride maintains
2. Mix thoroughly.
the osmotic balance. Mannitol is the source of energy. Magnesium ion supports
3. Heat with frequent agitation to dissolve the powder completely. DO NOT
the growth of rhizobia.
OVERHEAT.
Formula*
Ingredients in grams per liter 4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Yeast extract 1.0 Dehydrated Appearance
Mannitol 10.0 Orangish red colour. homogeneous, free flowing powder.
Dipotassium phosphate 0.5 Prepared Appearance
Orangish red coloured slightly opalescent gel forms in petri plates.

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Cultural Response Interpretation of Results
Cultural characteristics after 5 days at 20-250C. Refer to appropriate references and procedures for results.
Organisms (ATCC) Growth RGI
Storage
Rhizobium meliloti (9930) Luxuriant More than 70%
Rhizobium leguminosarum (10004) Luxuriant More than 70%
Store at 22- 300C and prepared medium at 2-80C.
For growth RGI should be more than 70% Shelf Life
RGI- Relative Growth Index Use before expiry date as mentioned on the label.
Procedure
Refer to appropriate references for specific procedures for the cultivation of
Rhizobium species.

Yeast Mannitol Broth AM51153

Use 3. Heat with frequent agitation to dissolve the powder completely. DO NOT
Yeast Mannitol Broth is used for cultivation of Rhizobium species. OVERHEAT.
Summary 4. Sterilize by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
Yeast Mannitol Broth is recommended for the cultivation of the symbiotic Dehydrated Appearance
nitrogen-fixing organisms like Rhizobium species (106.1). Yellow coloured, homogenous, free flowing powder.
Principle Prepared Appearance
Yeast extract provides amino acids and vitamins while sodium chloride maintains Whitish buff coloured, opalescent solution forms in petri plates.
Cultural Response
the osmotic balance. Mannitol is the source of energy. Calcium and magnesium
Cultural characteristics after 5 days at 20-250C.
supports the growth of rhizobia.
Organisms (ATCC) Growth
Formula*
Rhizobium meliloti (9930) Luxuriant
Ingredients in grams per liter
Rhizobium leguminosarum (10004) Luxuriant
Yeast extract 1.0
Procedure
Mannitol 10.0
Dipotassium phosphate 0.50 Refer to appropriate references for specific procedures for the cultivation of
Magnesium sulphate 0.20 phosphate solubilizing soil microorganisms.
Sodium chloride 0.10 Interpretation of Results
Calcium carbonate 1.00 Refer to appropriate references and procedures for results.
Final pH (at 250C) 6.8 ± 0.2 Storage
* Formula adjusted to suit performance parameters
Store at 22- 300C and prepared medium at 2-80C.
Directions
Shelf Life
1. Suspend the 12.8gms of powder in 1000 ml distilled water.
Use before expiry date as mentioned on the label.
2. Mix thoroughly.

Yersinia Selective Agar Base AM1116/AM5116

Use alternative to MacConkey Agar and other commonly used media for isolation of
Yersinia Selective Agar Base with added supplements is recommended for Y.enterocolitica, a causative agent of gastroenteritis and was found to be far
isolation and enumeration of Yersinia enterocolitica from clinical and non- superior. The formula is based on CIN (Cefsulodin-Igrasan-Novobiocin) Agar of
clinical specimens. Schiemann and is recommended by the ISO committee and APHA (20).
Summary Principle
Yersinia Selective Agar Base was first described by Schiemann (102) as an The medium differentiates between mannitol fermenting and non-mannitol

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fermenting bacteria. Fermentation of mannitol in the presence of neutral red Quality Control
results in a characteristic “bull's-eye” colony, colourless with red center. Sodium Dehydrated Appearance
deoxycholate and crystal violet inhibit most gram-positive and a number of Beige coloured, homogeneous, free flowing powder.
Prepared Appearance
gram-negative bacteria. Addition of antibiotic supplement makes it highly
Orange red coloured, clear to slightly opalescent gel.
selective for Yersinia by inhibiting normal enteric pathogens. Organisms that do
Cultural Response
not metabolize mannitol to acid end products form colourless, translucent Cultural characteristics after 48 hours at 320C.
colonies. Organisms (ATCC) Growth Colour of RGI
Formula* Colony
Ingredients in grams per liter Enterococcus faecalis (29212) Inhibited - 0%
Mannitol 20.00 Escherichia coli (25922) Inhibited - 0%
Peptone, Special 20.00 Pseudomonas aeruginosa Inhibited - 0%
Sodium Pyruvate 2.00 (27853)
Yeast Extract 2.00 Yersinia enterocolitica Good to Translucent More than
Sodium Chloride 1.00 (27729) luxuriant with dark pink 70%
Sodium Deoxycholate 0.50 center and
Neutral Red 0.03 bile precipitate.
Magnesium Sulphate 0.01 For growth RGI should be more than 70%
Crystal Violet 0.001 RGI- Relative Growth Index
Agar 12.50 Interpretation of Results
Final pH at (250C) 7.4 ± 0.2
1. Typical Y.enterocolitica colonies will have deep red centers surrounded by a
* Formula adjusted to suit performance parameters
transparent border giving the appearance of a “bull's-eye”. Growth of other
Directions
organisms is markedly inhibited.
1. Suspend 29 gms of the powder in 500 ml distilled water.
Precautions / Limitations
2. Mix thoroughly. 1. Serratia liquefaciens, Citrobacter freundii and Enterobacter agglomerans
3. Boil with frequent agitation to dissolve the powder completely. may grow on this medium and resemble Y.enterocolitica, which needs to be
4. Sterilize by autoclaving at 1210C (15 lbs pressure) for 15 minutes. differentiated by biochemical tests.
5. Cool to 45-500C and add reconstituted contents of 1 vial of Yersinia Selective Storage

Supplement (AS029). Store at 22-300C and prepared medium at 2-8 0C.

Shelf Life
6. Mix well and pour into sterile petri plates.
Use before expiry date as mentioned on the label.

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