UNIT – 5: INTrodUcTIoN aNd Types of

spoIlage-parT-1

Points to be covered in this topic

1. INTRODUCTION
2. TYPES OF SPOILAGE
✓ Enzymatic spoilage

✓ Physical spoilage

✓ Chemical spoilage

3. SPOILAGE OF FRUITS AND VEGETABLES

4. FACTORS AFFECTING THE MICROBIAL SPOILAGE OF
PHARMACEUTICAL PRODUCTS
5. SOURCES AND TYPES OF MICROBIAL CONTAMINANTS
6. PRESERVATION OF PHARMACEUTICAL PRODUCTS USING
ANTIMICROBIAL AGENTS
7. ASSESSMENT OF MICROBIAL CONTAMINATION AND
SPOILAGE
INTRODUCTION OF PHARMACEUTICAL SPOILAGE
• Spoilage is waste or scrap arising from the production
process. The term is most commonly applied to raw
materials that have a short life span, such as food used in
the hospitality industry.
• Normal spoilage is the standard amount of waste or scrap that is caused by
production, and which is difficult to avoid.
Abnormal spoilage exceeds the normal or expected rate of spoilage.
• For example, an overcooked meal cannot be served to a customer, and so is
instead classified as abnormal spoilage.
• Hence, spoilage is a complex reaction in which a combination of microbial
and biochemical activities are interact. Legally they are known as spoilage.
These substandard drugs when proceed further in order to be saleable as
goods units are known as defectives.
• Examples: Rancid meat, sour milk, moldy cheese, etc.
• The spoilage is mainly determined based on microbial colonization form
which depends on the characteristics of products, method of processing and
the storage condition.

TYPES OF SPOILAGE
• Microbial Spoilage Includes the contamination of pharmaceutical products
with the microbes which lead to spoilage of the product affecting drug safety
 and quality, and is not intended for use. Shortly, Microbial Spoilage is
defined as deterioration of pharmaceutical products by the contaminant
microbe.
Enzymatic spoilage:
• It is based on enzymatic reactions

occurred in foods and changes in

chemical nature and resulted rancidity.
• This rancidity occurred by two types of chemical reactions hydrolytic and

oxidative rancidity.
• For example: Triglyceride upon hydrolytic reaction in presence of lipase

enzyme forms glycerol and free fatty acid.

• Further, Linolenic acid with various oxidative reactions forms 3-(E)-hexenal

and 12-oxo-9 (Z) dodecenoic acid with the help of lipoxygenase and
hydroperoxide lyase.
ENZYMES FOODS SPOILAGE ACTION
Lipase Milk, oils Hydrolytic rancidity
Thiaminase Meat, fish Thiamine destruction
Peroxidases Fruits Browning
Proteases Egg, crab Reduction of shelf life
Lipoxygenases Vegetables Destruction of vitamin A

Physical spoilage:
• It occurs due to temperature, light, relative humidity and results in

mechanical damage of the food components.

 • For example: Oxidation of food occurs due to light and changes colour,
flavour and chemical nature, like greening of potatoes, sunlight flavour in
milk, loss of vitamin D, E etc.

Chemical spoilage:
• It is based on non-enzymatic chemical reaction occurred within the foods

and resulted change in flavor.

• Chemical reactions in food are responsible for changes in the colour and

flavour of foods during processing and storage.

• Others spoilage: It occurs due to insects, rodents, birds, and other animals

and results in changes in colour, odour, and chemical nature.

• Based on rate of spoilage, they are classified into three types like:

(i) High perishable: Meat, fish, poultry, eggs, milk, fruits and vegetables
(ii) Semi-perishable: Potatoes, some apple varieties, nutmeats
(iii) Stable or non-perishable: Sugar, flour, dry beans

Spoilage of fruits and vegetables

• Fruits and vegetables are rich source of energy, body-building
nutrients, vitamins and minerals. Protected mechanically by
the pectins which constitute a protective gum between the
cells and gives firmness.
• Spoilage in fruits and vegetables starts with the hydrolysis of the pectin.
• Once the pectinases have damaged the structure of the fruit/vegetable, other
organisms start to contribute to the soft rot.
 FACTORS AFFECTING THE MICROBIAL SPOILAGE OF
PHARMACEUTICAL PRODUCTS
• There are so many factors which affect Microbial spoilage. Some of these
factors reduce rate of spoilage whereas some factors increase the rate of
spoilage.
• These factors are related to nutritional requirement of micro-organisms,
environment and nature of micro-organism.
• These factors must be studied to minimize the impact of spoilage.
Following factors affect the microbial spoilage of Pharmaceutical products.
➤ pH
• Extremes of pH prevent microbial attack. They grow at neutral pH, therefore
acidic or alkaline formulations are less susceptible to spoilage.
 • Around neutrality bacterial spoilage is more likely, with reports of
pseudomonads and related Gram-negative bacteria growing in antacid
mixtures, flavoured mouth washes and in distilled or demineralized water.
• Above pH-8, the spoilage is rare for soap-based emulsions.
• Products with low pH levels such as the fruit juice-flavoured syrups are
attacked by mould or yeast.
• Yeasts are metabolizers of organic acids and raise the pH to levels where
secondary bacterial growth occurs.
In food industry, low pH adjustment is made to preserve foodstuffs only.

➤ Storage Temperature
• The actual storage temperature determines the spoilage by particular types of
microorganisms. Spoilage of pharmaceuticals occurs potentially over the
range of about 20°C to 60°C.
• Storage in a deep freeze at –20°C or lower is used for
long-term storage of foodstuffs and some
pharmaceutical raw materials, and dispensed total
parenteral nutrition feeds are stored in hospitals for short
periods at –20°C to even further minimize the risk of
spoilage.

➤ Nutritional Factors
• Many spoilage microorganisms have simple nutritional requirements and
metabolic adaptability which enables them to utilize many formulation
components as substrates for biosynthesis, growth and also trace materials
contained in them.
 • The use of animal products and crude vegetable materials in a formulation
provides an additionally nutritious environment.
• Demineralized water prepared by ion-exchange methods, contains sufficient
nutrients to allow significant growth of many water-borne Gram-negative
bacteria like Pseudomonas spp.

➤ Water
• It is the most important cause of the survival and growth of micro-organisms.
• Some solute-rich medicines such as syrups appear to be ‘wet’, microbial
growth in them may be difficult since the microbes have to compete for
water molecules with the large numbers of sugar and other molecules of the
formulation which also interact with water via hydrogen bonding.
• An estimate of the proportion of the non-complexed water in a formulation
available to equilibrate with any microbial contaminants and facilitate
growth can be obtained by measuring its water activity (Aw).
Vapour pressure of formulation
𝐴𝑤 =
Vapour pressure of water under similar condition

➤ Redox Potential
• The ability of microbes to grow in an environment is influenced by its
oxidation-reduction balance since they require compatible terminal electron
acceptors to permit function of their respiratory pathways.
• The redox potential in viscous emulsion is high due to the high solubility of
oxygen in most fats and oils.
• It has a major influence on microbial stability of some formulations in
controlling the access of contaminants during both storage and use.
 • The most important dosage form such as parenteral drugs is protected
because of the high risk of infection by this route.
• Self-sealing rubber closures are used to prevent microbial entry into multi-
dose injection containers following withdrawals with a hypodermic needle.
• Wide-mouthed cream jars are replaced with narrow nozzle and flexible
screw capped tubes to remove the likelihood of operator-introduced
contamination during use.
• Other factors affecting microbial spoilage of pharmaceutical products
include:

SOURCES AND TYPES OF MICROBIAL CONTAMINANTS

• Source and types of microbial contaminants Microbes are very important part
of our environment.
• They are present almost and anywhere.
 • So, contamination may occur to pharmaceutical products in large scale
manufacturing, in small scale hospital manufacturing or during use by the
patient.
• Following are the different source of microbial contamination in
pharmaceutical products.
• In large scale manufacturing: - In large scale manufacturing as well as
medium and small scale manufacturing contamination may occur from
following sources.
Water
• Water is a major source of contamination. Common water-borne
microorganisms like Pseudomonas, Achromobacter and other low demand
gram-negative groups are present in portable water as well as in purified
water.
• Ion-exchange column may be contaminated by water
source and micro-organism may multiply there to
contaminate purified water.

Raw materials
• Pharmaceutical products are prepared from varieties of raw materials.

Clays and earth materials like Bentonite, Kaolin etc. may contain anaerobia
spores like Clostridium sp. Starch may contain coliform bacteria like E. coli.
Gums may contain actinomycetes.
• Animal products may contain a variety of bacterial like E. coli, Salmonella

sp etc.
Equipments
• Equipments of manufacturing may contain microbes if it is not sterilized

[Link], blender, filter etc. may contain non-specific and local

communities of micro-organisms.

Containers
• Containers may cause contamination if it is not sterile.

In hospital manufacturing – In hospital manufacturing water and

environment are the major source of contaminants.
• In hospitals, water is stored in storage tank which may develop fungus,

bacteria and algae type of microbes.

• Hospital air may be contaminated with pathogenic microorganisms due to

the presence of infected patients and numerous visitors.

Herman source
• Pharmaceutical products may be contaminated during use. Patient may self-

contaminate his medicine.

• Contaminants may travel to other patients through doctors, nurses, etc.

PRESERVATION OF PHARMACEUTICAL PRODUCTS USING

ANTIMICROBIAL AGENTS
• Antimicrobial agents are those substances which can kill or inhibit growth of
[Link] antimicrobial agents are included in formulation in
order to minimize levels of contaminated [Link]
antimicrobial agents are called [Link] preservatives can
generally prevent or kill low levels of contamination.
 • Preservatives are not used in those formulations which have low risk of
contamination and subsequent microbial growth.
• Formulations which contain high level of acid, alkali, sugar etc. may not
require preservatives.
• Formulations of antibiotics and other anti-microbial substances may not
require preservative.
➤ Air of the manufacturing area
• Air is filled with billions of suspending particles and microbes. Fungus
spores, like Penicillium, Mucor, Aspergillus etc.
• Bacterial spores like Bacillus sp. are also present. These spores and
microorganisms may contaminate pharmaceutical products.
• This type of contamination is minimized by practice of manufacturing in
clean room and in aseptic room under continuous flow of sterile air through
HEPA filter.

➤ Personnel
• Manufacturing staff may also contaminate pharmaceutical
products.
• Personnel may be infected with various types of infections like coliform
bacteria, staphylococci, streptococci, Actinobacteria, Candida.
• This type of contamination may be minimized by proper health check-up,
vaccination and hygiene of the personnel.
Protective gear and proper training of the personnel may also minimize the
contamination.

➤ TYPES OF MICROBIAL CONTAMINANT

 • Microbial contamination is broadly classified into direct contamination and
cross contamination (Flowchart 2).
(a) Direct contamination: Contamination occurred by microbial components and
poorly maintained heating, ventilation and air conditioning system.
(b) Cross contamination: It is the process by which microbes are spread indirectly
from one to another through improper and unsterilized equipment.

➤ DIRECT CONTAMINATION IS CLASSIFIED AS FOLLOWS

(a) Direct physical contamination: Examples: Particles, fibres, metal parts, etc.
(b) Direct chemical contamination: Examples: Moisture, gases, vapors, etc.
(c) Direct biological contamination: Examples: Microorganisms like bacteria,
viruses, molds, fungi, etc.

➤ CROSS CONTAMINATION IS CLASSIFIED AS FOLLOWS

(a) Physical cross contamination: Example: Leakage of oil seal from the reactor.
(b) Chemical cross contamination: Example: Moisture content is increased when a
product is exposed to high relative humidity.
(c) Biological cross contamination: Example: Improper cleaning of equipment;
unclean equipment used for manufacturing process.
Pharmaceutical Products Contaminants
Plague vaccine Clostridium tetani
Serum vaccine Staphylococcus aureus
Thyroid tablets Salmonella muenchen
Antibiotic eye ointments Pseudomonas aeruginosa
Talcum powder Clostridium tetani
Saline solution Serratia marcescens
Antiseptic mouth wash Coliforms
Surgical dressings Clostridium species
Hand cream Klebsiella pneumoniae

➤ ASSESSMENT OF MICROBIAL CONTAMINATION AND SPOILAGE

• The evaluation of a medicinal product’s microbiological composition is

critical.
A sterile product should be completely devoid of microorganisms, as
determined by a sterility test.
• Non-sterile items, on the other hand, may include microorganisms.

These microorganisms have the potential to be both pathogenic and non-

pathogenic.
• Microorganisms like these can cause deterioration, which can pose health

risks.
 The overall number of microorganisms present in a product must be minimal
and below the allowable limit.
• To detect the existence of certain bacteria, the types and characteristics of the
microbes should also be examined.
• The evaluation of a medicinal product’s microbiological composition is
critical.
A sterile product should be completely devoid of microorganisms, as
determined by a sterility test.
• Microorganisms like these can cause deterioration, which can pose health
risks.
• The overall number of microorganisms present in a product must be minimal
and below the allowable limit.
• To detect the existence of certain bacteria, the types and characteristics of the
microbes should also be examined.
• Non-sterile items, on the other hand, may include microorganisms.
These microorganisms have the potential to be both pathogenic and non-
pathogenic.
• Tests to determine the total amount of microorganisms and types of bacteria
are called microbial limit tests.
• The following tests are carried out to evaluate microbial contamination and
consequent deterioration.
• Test of Sterility Some pharmaceutical items should be tested for sterility
according to the Indian Pharmacopoeia, the British Pharmacopoeia, and the
United States Pharmacopoeia. The procedures are comparable with minor
differences.
• The tests prescribed by the Indian Pharmacopoeia are briefly reviewed.
There are two methods: Direct inoculation and Membrane filtering.
1. Direct Inoculation
• In this procedure, a little amount of material is immediately introduced to the

pharmacopoeia-specified culture medium. This inoculation medium is then

incubated for a certain amount of time.
• The existence of growth implies the presence of a microorganism derived

from the sample. As a result, it is possible to determine that the sample is not
sterile. In the lack of any growth, a sample is said to be sterile.
2. Membrane Filtration
• The material is filtered through a membrane filter and rinsed with diluting

solution in this procedure.

• If microbes are present, they will be near the top of the filter paper.

This filter paper is now injected into the appropriate culture medium.
• If growth is detected, it shows that the product is not sterile.

• In both the above-mentioned methods, a positive and negative control test

must be performed.
• Microbial limit test - The European Pharmacopoeia suggests assessing

microorganisms both qualitatively and quantitatively. The microbiological

• Limit test is recommended by the United States Pharmacopoeia. It is divided

into two sections: .

1. Total Aerobic Microbial Count
2. Test for Specific Microorganisms.

i) Total Aerobic Microbial Count

• In this approach, a defined amount of test sample (10gm) is combined with a

specific amount of peptone water (90ml).

 • This is called sample dilution. Dilutions of water-insoluble and fatty
compounds must follow precise procedures.
The total microbial count is determined using the following procedure:
• To begin with, the sample is filtered through a membrane filter by mixing 10
ml of distilled water with 90 ml of peptone water.
• After that, it is rinsed three times with sterile peptone water. One filter paper
is incubated for 5 days at 30–35°C in a Petri dish with Soyabean Casein
Digest Agar medium. Colonies are counted to ascertain bacterial count.
• Another filter paper is immersed in
Sabouraud Dextrose Agar Media and
incubated at 20–25°C for 5 days. The
fungal count is then calculated.
• Second, the sample plate count
technique is examined. In this approach, created dilution is directly
transferred to four Petri dishes.
• There are two for bacteria and two for fungus. 15 ml of Soyabean Casein
Digest Agar medium is added to the first two Petri dishes. After 5 days of
incubation at 30–35°C, colonies are counted.
• 15 ml of Sabouraud Dextrose Agar Media was transferred to the remaining
two Petri dishes and incubated at 20–25°C. Colonies are tallied.

ii) Test for Specific Microorganisms

• Escherichia coli, Salmonella, Pseudomonas aeruginosa, and

Staphylococcus aureus are identified using specific assays.

• Mac-Conkey agar medium is used to culture E. coli. Colonies

are distinguished by their metallic gleam.

• Salmonella colonies are detected as black or green on

bismuth sulphite agar medium.

 • Pseudomonas aeruginosa is recognized by growing it on cetrimide agar
medium. Colonies gradually turn a greenish colour.

Good Pharmaceutical Manufacturing Practice (GPMP)

• Quality Control (QC) is that part of GPMP dealing with specification,

documentation, and assessing conformance to specification.

• A high assurance of overall product quality is raised only from a detailed

specification, control, and monitoring of all the stages that contribute to the
manufacturing process.
• Parametric release is accepted as an operational

alternative to routine sterility testing for batch

release of some finished sterile products where
the manufacturer can provide assurance that the
product is of the stipulated quality, based on the
evidence of successful validation of the
manufacturing process and review of the
documentation on process monitoring carried out
during manufacturing.

Quality Assurance (QA)

• It is a combined scheme of management which embraces all the procedures

necessary to provide a high probability that a medicine will conform

consistently to a specified description of quality.
• It includes formulation design and development (R&D), Good

Pharmaceutical Manufacturing Practice (GPMP), Quality Control (QC), and

post-marketing surveillance.
• The risk of microbial infection and spoilage are raised from microbial

contamination during manufacture and storage and hence preservatives are

 recommended further protection against environmental microbial
contaminants but it is relatively non-specific in their reactivity.
• Laboratory tests are devised to challenge the product by used 'preservative
challenge tests' where relatively large inocula of various laboratory cultures
are added to aliquots of the product and determine their rate of inactivation
by viable counting methods (single challenge tests).

Post-market Surveillance
• It is most important stage to follow up a medicine that is smooth floating in

the market without any complain by the customers.

• A proper quality assurance system is included for monitoring in-use

performance and for responding to customer complaints.

• These are constantly followed up in great detail in order to decide carefully

constructed and implemented schemes for product safety.

 UNIT – 5: preserVaTIoN of
pHarMaceUTIcal prodUcTs UsINg
aNTIMIcroBIal ageNTs-parT-2

Points to be covered in this topic

1. INTRODUCTION
2. IDEAL PROPERTIES OF PRESERVATIVES
3. CLASSIFICATION OF PRESERVATIVES
o Classification of Preservatives based on Mechanism of Action

o Classification based on Source

4. METHODS OF PRESERVATION
5. MICROBIAL STABILITY TEST
6. GROWTH OF ANIMAL CELLS IN CULTURE
7. TYPES OF ANIMAL CELL CULTURE
8. GENERAL PROCEDURE FOR CELL CULTURE
9. APPLICATION OF CELL CULTURES IN PHARMACEUTICAL
INDUSTRY AND RESEARCH

INTRODUCTION
• Preservatives are the chemical substances used to improve or amplify shelf
life of drugs by decreasing or lowering the oxidation of active ingredients
and excipients by reducing microbial production.
 • Preservatives are substances added to various pharmaceutical dosage forms
and cosmetic preparations to prevent or inhibit microbial growth.
• An ideal preservative would be effective at low concentrations against all
possible micro-organisms, be nontoxic and compatible with other
constituents of the preparation, and be stable for the shelf life of the
preparation.

IDEAL PROPERTIES OF PRESERVATIVES

✓ It should not be irritant.
✓ It should not be toxic.
✓ It should be physically and chemically stable.
✓ It should be compatible with other ingredients used in formulation.
✓ It should act as a good antimicrobial agent and should exert a wide spectrum of
activity.
✓ It should act in small concentration, i.e., it must be potent.
✓ It should maintain activity throughout product manufacturing, shelf life, and
usage.
✓ It must decrease the percentage of microbes and prevent any regrowth.
They can be:
• Microbiostatic and Microbiocidal in nature.

Some preservatives are ineffective with certain microbial strains and should be
combined with others to be effective. Such as:
Benzalkonium chloride, Organo-mercurial, Cetrimide, Chlorhexidine, and 3-cresol
(combined).
CLASSIFICATION OF PRESERVATIVES

I. Cationic Detergents: Examples: Benzalkonium chloride, alkyl trimethyl

ammonium chloride.
II. Alcohols: Examples: Chlorbutanol, bronopol, phenyl, and phenoxy ethanol.
III. Phenolic Compounds: Examples: Chlorinated and isopropyl derivatives of meta
cresol.
IV. Organic Acids: Examples: Salicylic acid, benzoic acid, acetic acid, lactic acid,
hydroxyl benzoic acid.

CLASSIFICATION OF PRESERVATIVES BASED ON MECHANISM OF

ACTION
1. Antioxidants:
Agents which prevent oxidation of active pharmaceutical ingredients that otherwise
undergo degradation due to oxidation (sensitive to oxygen).
Examples: Vitamin E, Vitamin C, Butylated-hydroxyanisole (BHA), Butylated-
hydroxytoluene (BHT).
2. Antimicrobial Agents:
Agents that are active against gram-positive and gram-negative microorganisms,
which cause degradation of pharmaceutical preparations, active in small inclusion
levels.
Examples: Benzoates, Sodium benzoate, Sorbates, etc.
3. Chelating Agents:
Agents that form complexes with pharmaceutical ingredients and prevent
degradation of pharmaceutical formulations.
Examples: Disodium ethylenediaminetetraacetic acid (EDTA), Polyphosphates,
Citric acid.

CLASSIFICATION BASED ON SOURCE

i. Natural Preservatives:
These preservatives are obtained from natural sources such as plants, minerals, and
[Link]: Neem oil, Salt (sodium chloride), Lemon, Honey.
ii. Artificial Preservatives:
These preservatives are man-made by chemical synthesis and active against various
microorganisms in small concentrations. Examples: Benzoates, Sodium benzoate,
Sorbates, Propionates, Nitrites.

Factors affecting the efficacy and availability of preservatives:

• Temperature.

• Chemical structure of preservatives.

• Capacity of preservatives.

• Inoculum size.

• Effect of pH.

• Effect of containers and packaging.

• Changes of concentration.

METHODS OF PRESERVATION
➤ Physical protection:
 • It is used for proper packaging of the pharmaceutical products under aseptic
condition or else there is a chance for microbial growth.
• Operating persons are also an important factor for proper processing of the
products under aseptic environment.

➤ Preservative coating:
• Aqueous raw materials used in the formulation of
paints and coatings create the perfect environment for
the growth of bacteria, fungi, and yeast. They can
destroy valuable pharmaceutical formulations.
• Controlling these microorganisms helps increase efficiency, helps deliver a
better end-use product, and helps employees and consumers avoid contact
with spoilage microorganisms.
• Biocides are necessary for protecting the integrity and functionality of water-
based paints and coatings from destruction by microbial contamination. This
results in a longer product shelf life and protection of dry films from algae,
mold, and mildew.

➤ Water proof protection:

• Packaging of the pharmaceutical products should be under water proof

protection because water favours the growth of microorganisms.

➤ Water vapour proof protection:

• This method is applicable for certain pharmaceutical products when they are

packed under proper care to minimize microbial activity.

 • For dry dosage forms with very low water activity (Aw), it provides
protection against microbial attack.
• The moisture vapour properties of packaging materials require careful
examination.

➤ Water vapour proof protection with desiccant:

• This method is also used for dry products that absorb moisture from the
environment and spoil due to growth of microorganisms.
• Packing should be proper with this method to minimize microbial growth
and spoilage of products.

➤ Mode of Action: Preservatives interfere with the growth, multiplication, and

metabolism of microorganisms by one or more of the following mechanisms:
(i) Modifying the membrane permeability.
(ii) Denaturation of enzymes and other cellular proteins.
(iii) Oxidation of cellular constituents.
(iv) Hydrolysis.
• For use in pharmaceutical products, the antimicrobial preservatives should

be selected from those recommended in pharmacopoeias and used in

minimum effective concentrations.

Commonly used preservatives:

Benzalkonium chloride Benzoic acid Benzyl alcohol
Butyl paraben Cetrimonium bromide Cetylpyridinium chloride
Chlorobutanol Chlorocresol Cresol
Benzalkonium chloride Benzoic acid Benzyl alcohol
Ethyl paraben Methyl paraben Phenol
Phenoxyethanol Phenylethyl alcohol Phenylmercuric acetate
Phenylmercuric nitrate Potassium benzoate Potassium sorbate
Propyl paraben Sodium benzoate Sodium propionate
Sorbic acid Thimerosal Thymol

Examples of antimicrobial preservatives commonly employed in manufacturing of

pharmaceutical products:
• Methyl, ethyl, propyl, and butyl parabens.

• Sorbic acid, Na, K, and Ca sorbates.

• Benzoic acid, Na, K, and Ca benzoate.

• Sodium metabisulfite.

• Propylene glycol.

• BHT (butylhydroxy toluene).

• BHA (butylhydroxyanisole).

• Benzaldehyde.

• Essential oils, phenol, and mercury compounds.

Parabens are among the most commonly used antimicrobial preservatives.

MICROBIAL STABILITY TEST:

 • Microbial contaminants usually originate from two different sources —
during production and filling, and during the use of the cosmetics by
consumers.
• It is necessary to carry out routine microbiological analysis of each batch of

the finished product.

• The main potential pathogens in cosmetics are Pseudomonas aeruginosa,

Candida albicans, and Staphylococcus aureus.

• These pathogens must not be detectable in more than 0.1 g or 0.1 ml of a

cosmetic product.
SOME TESTS ARE AS FOLLOWS:
1. Screening Test:
• Also known as plate count or dip slide method.

This method is used to detect aerobic bacteria in aqueous samples. The dip
slide is coated on both sides with a solid agar gel medium.
• A small quantity of TTC (2,3,5-triphenyl tetrazolium chloride) is added to

detect aerobic bacteria in the sample.

• The slide is dipped into the aqueous solution for 10 seconds, and excess

liquid is drained off from the slide.

Then it is incubated at 35–37°C for 18–48 hours.
• The color appearance is compared with a calibration chart.

Aerobic bacterial species grow on this medium and are detected by their
ability to reduce TTC dye to a red-colored Formosan dye.
Developing bacterial colonies alter the TTC dye and appear as red spots.
Quantitative test:
• Quantitative tests determine the actual count level of bacteria, molds and

yeasts in cosmetic products. This method is used for isolation of

microorganisms from cosmetic products and includes direct colony counts
and enrichment culturing.
 • Microbial stability studies are also carried out in pharmaceutical products.
Raw materials play a vital role in the product formulation. Pharmaceutical
raw material is defined as a substance that is used in the manufacturing of
pharmaceutical products.
• During the manufacture of pharmaceuticals and cosmetics,
untreated raw materials are contaminated with microorganisms
beyond acceptable limits.
• Hence, it is necessary to control microbiological contamination
from raw materials under GMP regulations in the
pharmaceutical industry.
• These microorganisms from plants (e.g. species of Erwinia, Pseudomonas,
Lactobacillus, Bacillus or Streptococcus) such as gum acacia, tragacanth,
agar, powdered rhubarb, and starches contain bacteria, which cause disease.
• Water plays a major role in product formulations because water is a good
medium for microbial growth.
• The water activity (Aw) of pharmaceutical and cosmetic products is the
measure of free water in the formulation.
The amount of water that is in free form is available to microorganisms for
survival.
• The measurement of Aw in pharmaceutical and cosmetic products predicts
the type and number of microorganisms responsible for product degradation
and helps maintain chemical stability.
• Water activity is determined by dew point method and using electric
hygrometer (measure relative humidity).

Antimicrobial Effectiveness Test

• This test is used for estimating preservative in a product.

It is mainly used during development of formulation and stability studies.

 • In this test, a product is inoculated with a controlled quantity of specific
microorganisms.
• The test then compares the level of microorganisms found on a control
sample versus the test sample over a period of 28 days.
• Test organisms are used for the purpose of challenging the preservative
system in a product.
The suitable media are Soyabean Casein Digest or Sabouraud Dextrose Agar
used for the test.
The bacteria, yeast, and mold are used for this test.
• A product is inoculated or contaminated with a number of organisms
between
1 × 105 (100,000) to 1 × 106 (1,000,000) colony-forming units (CFU) per ml
of product.
At various intervals, the product is tested to determine its ability to control,
reproduce, or destroy microorganisms.
• Test and standard are incubated at 32.5 ± 2.5°C and 22.5 ± 2.5°C for bacteria
and yeast respectively, and the concentration of the microorganisms in each
of the standardized inoculum is determined by the plate count method.

Pharmaceutical Products: For testing purposes, the USP has divided test articles
into four separate categories:
Category 1: Injections, other parenterals including emulsions, sterile nasal products
made with aqueous bases or vehicles.
Category 2: Topically used products made with aqueous bases or vehicles, non-
sterile nasal products, and emulsions, including those applied to mucous
membranes.
Category 3: Oral products other than candies made with aqueous bases or vehicles.
Category 4: Antacids made with an aqueous base.
Growth of Animal Cells in Culture
•Cell culture is a technique that involves the isolation of cells from
animal/plant body (i.e., from their natural environment in vivo) and
practicing to grow isolated cells in cell-specific media in plastic flasks or
Petri dishes in a controlled environment (in vitro).
• Cell culture means to keep cells alive and growing in vitro in a nutritive

media, which are widely used for research, diagnosis, and understanding of
the function and mechanism of operation of many cells.
• Animal cell cultures are initiated by the dispersion of a piece of tissue into a

suspension of its component cells, which is then added to a culture dish

containing nutrient media.
• In animal cells, fibroblasts and epithelial cells are selected because these

cells grow very fast.

• The plastic surface of dishes is used for cell culture.

In 1907, Harrison cultivated frog nerve cells in a lymph clot and observed
the growth of nerve fibres in vitro for several weeks.
Hence, he is recognized as the father of cell culture.
TYPES OE ANIMAL CELL CULTURE:
• Based on the number of cell divisions, cell culture is classifñed as primary

cell culture and cell lines. Cell lines can undergo finite or infinite cell
divisions.
Primary Cell Culture:
• This cell culture is obtained from the cells of a host tissue.

The cells dissociated from the parental tissue are grown on a suitable
container and the culture thus obtained is called primary cell culture.
• The culture is comprised of heterogeneous cells and most of the cells divide

only for a limited time.

Based on their origin, primary cells grow either as an adherent monolayer or
in a suspension.
Adherent Cells:
• These cells are propagated as a monolayer and are anchorage dependent.

Monolayer cultures are defined as when the bottom of the culture vessel is
covered with a continuous layer of cells of one need to be attached to a solid
or semi-solid substrate for proliferation.
• They adhere to the culture vessel with the use of an extracellular matrix,

which is derived from tissues of organs that are immobile and embedded as
connective tissue.
• For example: Fibroblasts and epithelial cells.

• Majority of continuous cell lines grow as monolayers, and such types of cells

are transferred directly to a cover slip for examination under a microscope.

Suspension Cells:
• These types of cells do not attach to the surface of the culture vessels.

Hence, they are also known as anchorage independent or non-adherent cells,

which are grown in liquid culture medium.
• Hematopoietic stem cells and tumor cells are grown in suspension much

faster, which do not require frequent replacement of the medium.

These cultures have a short lag period.
Secondary Cell Culture and Cell Line:
• Secondary culture is the culture when a primary culture is sub-cultured.

It is also known as a cell line or sub-clone.

• The process involves removing the growth media and disassociating the

adhered cells by enzymatic treatment.

• Sub-culturing of primary cells to different divisions leads to the generation

of cell lines.
During the growth period, cells with the highest growth capacity
predominate and result in genotypic and phenotypic uniformity in the
population.
• Based on the life span of the culture, cell lines are of two types, viz.

Finite cell lines and Continuous cell lines.

Finite Cell Lines
• It is defined as the cell line that undergoes a limited number of cell divisions

with a limited life span.

The cells passage several times and then lose their ability to proliferate,
which is a genetically determined event known as senescence.
• Cell lines derived from primary cultures of normal cells are finite cell lines.

Continuous Cell Lines

• It is defined as a finite cell line which undergoes transformation and acquires

the ability to divide indefinitely. This transformation occurs spontaneously

or is chemically or virally induced, or from the establishment of cell cultures
from malignant tissue.
• Prepared cell cultures are then sub-cultured and grown indefinitely as

permanent cell lines. These cells are less adherent and fast-growing.
As a result, the cell density becomes higher and different in phenotypes from
the original tissue. They grow more in suspension medium.
GENERAL PROCEDURE FOR CELL CULTURE
(a) Requirements:
• Vertical Laminar Air Flow

• Incubator

• Refrigerator

• Microscope

• Tissue Culture Ware

(b) Temperature:
• The temperature is set the same as the body temperature of the host from

which cells are procured.

• Most animal cells require 36–37°C.
(c) Substrate:
• Good compatible substrate is required for attachment and optimum growth.

Glass and specially treated plastics are commonly used as substrates.

• Thereafter, attachment factors such as collagen, gelatin, and laminin etc. are

used as substrate coatings to improve growth and function of normal cells

derived from brain, blood vessels, kidney, liver, skin, etc.

(d) Culture Medium:

• It is an important and complex factor for cell growth.

The culture medium is supplemented with various growth factors, pH and

osmolality regulators, and provides essential gases like oxygen and carbon
dioxide.
• The medium is also supplemented with amino acids, vitamins, minerals, and

carbohydrates, which are essential for the growth of cells and provide energy
for metabolism.
• Choice of media is used based on the cells being cultured.

Generally, media like:

o Dulbecco’s Modified Eagle’s Medium

(DMEM)
o Eagle’s Minimal Essential Medium (EMEM)

o Glasgow Minimum Essential Medium (GMEM)

are used for cell culture.

• Prepared media is filtered and incubated at 4°C.

(e) Media and Growth Requirement:

• Temperature should be maintained at 37°C and optimum pH = 7.2 to 7.5.
 • The humidity is required to be maintained properly in the media with proper
gas phase ratio (bicarbonate concentration and carbon dioxide in
equilibrium).
• For growth of cultured cells Light intensity also plays a vital role. Inside
environments, cells are cultured in dark because light induces the production
of toxic compounds.
• Commonly used antibiotics are penicillin, streptomycin, kanamycin, etc.
Trace elements like iron, zinc, selenium, sugar, amino acids, vitamins,
choline, inositol, etc.

(f) Selection of Organ:

• Different types of cells are grown in cultures including connective tissue
elements such as fibroblasts, skeletal tissue, cardiac, epithelial tissue (liver,
breast, skin, and kidney) and many different types of tumor cells.
• On the basis of morphology (shape and appearance) or on the functional
characteristics of cells, they are divided into three types:
• Epithelial like: attached to a substrate and appear flattened and polygonal in

shape.
• Lymphoblast like: cells do not attach; remain in suspension with a spherical

shape.
• Fibroblast like: cells attached to a substrate appear elongated and bipolar.

(g) Culturing of Cell:

• Cells are cultured as anchorage dependent or independent.
Cell lines derived from normal tissues are considered as anchorage-
dependent, which grows only on a suitable substrate e.g., tissue cells.
 • Suspension cells are anchorage-independent e.g., blood cells, whereas
transformed cell lines either grow as a monolayer or as a suspension.

Steps:
1. Use sterile technique: Tissue part is harvested and processed using sterile
equipment, reagents, and techniques. Personal protective equipment is used to
avoid contamination. All enzymes and reagents are filtered under sterile conditions
using a 0.22 micron membrane.
2. Mince/Cut Tissue: Mince the tissue specimen into small pieces (usually 2 × 4
mm) with sterile scissors or scalpel, and then place the small pieces into selected
buffer, media, or salt solution.
3. Wash and Add Enzyme: Wash tissue two to three times to eliminate excess blood
proteins and then add enzyme(s) of choice — likely collagenase, protease, papain,
or trypsin. Usually about 0.5 to 1.5 mg/ml of selected enzyme is sufficient.
4. Incubation: Further, the tissue specimen is incubated at optimum temperature
37°C for 30 to 90 minutes, with periodic mixing.
5. Disperse and Wash Cells: Cells are dispersed by gentle pipetting and then the
cell suspension is filtered using a fine mesh. The cells become settled and excess
liquid containing enzymes is decanted. Further, cells are washed two to three times
with Fetal Bovine Serum (FBS), Bovine Serum Albumin (BSA), or other inhibitors
to halt enzyme digestion.
6. Resuspension and Measure Cells: Cells are resuspended in the correct medium or
buffer and then quantitatively determined for cell yield and viability. This is an
important step in the cell isolation process to evaluate results by dissociation
technique. Most researchers use a haemocytometer for determining cell yield and
trypan blue diazo dye to measure cell viability.
➤ PRIMARY CULTURE
• Cells, when surgically or enzymatically removed from the organism and
placed in a suitable culture environment and grown, are called primary
culture.
• They have a finite life span and contain a heterogeneous population of cells.
These cells upon subculture lead to generate cell lines which have limited
life span.
• Lineage of cells originated from the primary culture is known as cell strain.
Primary cultures are morphologically similar to the parent tissues.

ESTABLISHED CELL CULTURE

 • Primary cell culture, when first subcultured, is known as secondary cell
culture. Established or immortalized cell lines have the ability to proliferate
indefinitely by random mutation and artificial modification such as artificial
expression of the telomerase gene.
➤ Advantages:
• Many kinds of cell lines.
• Generally easy to grow and manipulate.

• Proliferate indefinitely.

• Contact inhibition.

Example: HeLa, Sf-9, Cervical cancer.

Transformed Cell Culture
• Transformation is a process of conversion of a normal cell into a cell having
some or many of the attributes of a different cell. When a cell is transformed,
it loses contact inhibition and becomes immortal.
• For exanmple: NIH 3T3 mouse cells are partially transformed and becarme

immortal but contact inhibited and grows in a mono layer.

• Transformed cells lack contact inhibition of movement due to change of cell

surface property and loss of many receptors.

• They continue to grow and pile up on top of one another as they proliferate.

• Transformed cells are also cultured through suspension medium where cells

do not attach to the surface of the culture vessels and are grown liquid
culture medium.
• Hematopoietic stem cells and tumor cells are grown in suspension much

faster which do not require the frequent replacement of the medium.

 APPLICATION OF CELL CULTURES IN PHARMACEUTICAL
INDUSTRY AND RESEARCH
• Cell culture is used in cellular and molecular biology, providing excellent
model systems for studying the normal physiology and biochemistry of cells
(for example: metabolic studies, aging), the effects of drugs and toxic
compounds on the cells, and mutagenesis and carcinogenesis.
✔ Model System:
• Cell culture is used as a model system to study basic cell biology and
biochemistry.
• It is also used to study the interaction between cell and disease-causing
agents like bacteria, viruses.
• It helps to study the effect of drugs and to study triggers for ageing.

✔ Genetic Counseling:
• Fetal cell culture extracted from pregnant women is used to
study or examine the abnormalities of chromosomes, genes
using karyotyping.
• These findings are used in early detection of fetal disorders.

✔ Toxicity Testing:
• Animal cell culture is used to study the effects of new
drugs, cosmetics, and chemicals and growth of multiple
cells, especially liver and kidney cells.
• This technique is also used to determine the maximum
permissible dosage of new drugs.
✔ Cancer Research:
• Cell culture is one of the most important tools in cancer
research.
• The basic difference between normal cell and cancer cell
can be studied using animal cell culture technique.
• Normal cells are induced into cancer cells by using radiation, chemicals, or
viruses and then cause of cancer is studied.

✔ Virology:
• Animal cell cultures are used to replicate the viruses instead of animals for
the production of vaccines.
• Cell culture can also be used to detect and isolate
viruses, and also to study the growth and development
cycle of viruses.

✔ Vaccine Production:
• Cultured animal cells are used for virus production,
and these viruses are used to produce vaccines.
• For example: Vaccines like polio, rabies,
chickenpox, measles, and hepatitis B are produced
using animal cell culture.

✔ Gene Therapy:
• Cultured animal cells are genetically altered and are
used in gene therapy techniques.
 • First, cells are removed from the patient lacking a functional gene or missing
a functional gene.
• These genes are replaced by functional genes and altered cells are cultured
and grown in laboratory conditions and then introduced into the patient.

✔ Drug Screening and Development:

• Animal cell cultures are used to study the cytotoxicity of new
drugs.
• This is also used to find out the effective and safe dosage of
new drugs.
• Cell-based assay plays an important role in the
pharmaceutical industry.

✔ Genetically Engineered Protein:

• Animal cell cultures are used to produce commercially important genetically
engineered proteins such as monoclonal antibodies, insulin, and hormones
etc.
• Proteins extracted from biological sources are important for substitution
therapy.
• Interferon was discovered in 1957 by cell culture method through viral
infection.

✔ Replacement Tissue or Organ:

• Animal cell culture is used as replacement tissue or organs.
• For example: Artificial skin is produced to treat patients with burns and
ulcers.
• Recently, artificial organ culture such as liver, kidney, and pancreas are
successfully carried out for transplantation.