MEVL-013

Environment Science Lab

Course - 3

School of Interdisciplinary and Tran-disciplinary Studies

Indira Gandhi National Open University
PROGRAMME DESIGN COMMITTEE
Dr. Himanshu Pathak, Director, Prof. P.A. Azeez, Prof I.S. Thakur,
ICAR-National Rice Research Director, Salim Ali Centre for School of Environmental
Institute Cuttack, Odisha Ornithology and Natural Sciences, JNU, New Delhi
History, Coimbatore
Prof Uma Melkania, Dean, Prof. Nidhi Rai, University Prof. Jitendra Pandey, Centre
College of Basic Sciences and College of Science, M.L. of Advanced Study in Botany,
Humanities, GBPUAT,Pantnagar Sukhadia University, Udaipur BHU
Prof. R. Baskar, Department of Dr.Tanushree Bhattacharya, Prof. Shachi Shah, School of
Environmental Science & Department of Civil and Interdisciplinary and Trans-
Engineering, Guru Jambheshwar Environmental Engineering, disciplinary Studies, IGNOU
University of Science & Birla Institute of Technology,
Technology, Hisar, Haryana Mesra, Ranchi
Prof. P.K. Biswas Prof. Jaswant Sokhi Prof. Neera Kapoor
STRIDE, IGNOU School of Science, IGNOU School of Science, IGNOU
Prof. S.K.Yadav, School of Prof. Vijay Kumar Baraik, Dr. Surendra Singh Suthar,
Agriculture, IGNOU SOS, IGNOU School of Environment &
Natural Resources, Doon
University, Dehradun
Prof. B. Rupini, School of Prof. Nandini Sinha Kapur, Dr. Pulak Das, School of
Interdisciplinary and Trans- School of Interdisciplinary and Human Ecology, Ambedkar
disciplinary Studies, IGNOU Trans-disciplinary Studies, University, Delhi
IGNOU
Prof. Shubhangi Vaidya, School Dr. Sadananda Sahoo, School Dr. Sushmitha Baskar, School
of Interdisciplinary and Trans- of Interdisciplinary and Trans- of Interdisciplinary and Trans-
disciplinary Studies, IGNOU disciplinary Studies, IGNOU disciplinary Studies, IGNOU
Dr. V. Venkat Ramanan Dr. Deeksha Dave Dr. Y.S.C. Khuman
SOITS, IGNOU, New Delhi SOITS, IGNOU, New Delhi SOITS, IGNOU, New Delhi

PROGRAMME COORDINATORS
Prof. Shachi Shah Dr. V. Venkat Ramanan Dr. Deeksha Dave
School of Interdisciplinary and School of Interdisciplinary and School of Interdisciplinary and
Transdisciplinary Studies, Transdisciplinary Studies, Transdisciplinary Studies,
IGNOU IGNOU IGNOU
COURSE COORDINATOR
Prof. Shachi Shah, Professor Environmental Studies, SOITS, IGNOU
COURSE PREPARATION TEAM
Course Contributor
Dr. Yogita Kharayat, Senior Scientist, Instrumentation Lab, Center Pollution Control Board, New Delhi
CONTENT EDITORS
Prof. Bhawana Pathak, Dean, Prof. Shachi Shah
School of Environment and Sustainable Professor Environmental Studies
Development, Gujrat Central University SOITS, IGNOU
FORMAT EDITOR AND LANGUAGE EDITOR
Prof. Shachi Shah, Professor Environmental Studies, SOITS, IGNOU, New Delhi
PRINT PRODUCTION
Mr. Rajiv Girdhar Mr. Heman Kumar Parida
Assistant Registrar, MPDD, IGNOU Section Officer, MPDD, IGNOU
June, 2022
Indira Gandhi National Open University, June 2022
ISBN:
All rights reserved. No part of this work may be reproduced in any form, by mimeograph or any other
means, without permission in writing from the Indira Gandhi National Open University.
Further Information on Indira Gandhi National Open University courses may be obtained from the
University’s office at Maidan Garhi, New Delhi-110068 or visit University website
http://www.ignou.ac.in.
Printed and published on behalf of the Indira Gandhi National Open University, New Delhi by
Director, School of Social Sciences.
Laser Composed by : Tessa Media & Computers, C-206, Shaheen Bagh, New Delhi-25
 CONTENTS
EXPERIMENTS 1 9
Sterilization, Media Preparation and Isolation of Pure Culture
EXPERIMENTS 2 16
Determination of Gram Staining of Bacterial Sample
EXPERIMENTS 3 22
Isolation and Characterization of Bacteria from Soil Samples
EXPERIMENTS 4 29
Isolation and Characterization of Actinomycetes from Soil Samples
EXPERIMENTS 5 36
Isolation of Pure Culture by Streak Plate Method, Pour Plate Method and
Spread Plate Method
EXPERIMENTS 6 46
Isolation and Characterization of Fungi from Soil Samples
EXPERIMENTS 7 54
Determination of Metabolic Activities of Bacteria through Biochemical Tests
EXPERIMENTS 8 66
Determination of Biological Activity of Soil by Dehydrogenase Assay
EXPERIMENTS 9 73
Isolation of Bio-Surfactant Producing Bacteria from Environmental
Samples
EXPERIMENTS 10 81
Isolation and Enumeration of Air Borne Microorganisms by All Glass
Impinger Technique
EXPERIMENTS 11 88
Detection of Coliform in Water Samples by Membrane Filter Technique
EXPERIMENTS 12 96
Determination of Effect of Industrial Effluents on Seed Germination and
Seedling Growth of Rice and Wheat
EXPERIMENTS 13 101
Composting of Biodegradable Waste and determination of compost quality
EXPERIMENTS 14 108
Determination of Pesticides in Environmental Samples Using Gas
Chromatography
EXPERIMENTS 15 119
Determination of Biogas Production from Organic Waste by Liquid
Displacement Method
EXPERIMENTS 16 126
Determination of Trace Metals in Environmental Samples using Inductively
Coupled Plasma Optical Emission Spectroscopy (ICP-OES)
EXPERIMENTS 17 133
Determination of Alcoholic fermentation from Grape Juice by
Saccharomyces cerevisiae
EXPERIMENTS 18 138
Separation of Amino Acids by Thin Liquid Chromatography
EXPERIMENTS 19 144
Determination of Sodium and Potassium in Water Using Flame Photometer
EXPERIMENTS 20 151
Determination of Suspended Particulate Matter (SPM) in Ambient Air
Using High Air Sampler (HVS)
COURSE INTRODUCTION
Rapid industrialization and population explosion has resulted in the
generation and dumping of various contaminants into the environment.
Environmental monitoring is essential for systematically sampling air, water,
soil, and biota in order to observe and study the environment, as well as to
derive knowledge from this process. Microorganisms can survive in a
divergent environment and produce metabolites that can degrade and
transform pollutants and are able to revive contaminated sites
naturally. Microorganisms, such as fungi, yeast, and bacteria have been
considered to be outstanding organisms in important ecological processes,
such as organic matter putrefaction, soil structure formation, recycling of
important chemical elements, detoxification of pollutants, biodegradation of
waste etc. Knowledge regarding microbial dynamics and their interactions
with biotic and abiotic factors is an indispensable tool in the fields of
environmental Microbiology, Environmental Biotechnology, bioremediation,
and energy production processes.

In the first two Environment Science Lab courses MEVL-011 and MEVL-
012 you have performed exercises based on habitat and biodiversity indices,
exercises on abiotic and biotic components and productivity, exercises on
weather and climate analysis, exercises based on rocks and soil analysis and
exercises based on water and waste water analysis. The Environment
Science Lab courses course 3 MEVL-013 is designed to make you familiar
with various microbiological analysis tool for the examination of soil, air and
water samples. There are a total of twenty experiments in this course.

The lab covers the experiment based on water and waste water analysis. As
we know that water is essential for the life, but many people lack the
accessibility to clean and healthy drinking water and die as a consequence of
water-borne infections. The analysis of wastewater for trace and heavy metal
contamination is very important in ensuring human and environmental effect.
Water is susceptible to contamination with microorganisms and organic
matter among other pollutants regardless of the source. Significantly,
microbial contaminants such as coliforms, E.coli, Cryptosporidium parvum,
and Giardia lamblia compromise the safety of the water. Among the
coliform, Escherichia coli is the indicator of fecal contamination. Similarly
there has been growing concern over diverse effects of heavy metals on
human and aquatic system A variety of techniques are used to measure trace
elements in water including Inductive couple plasma optical emission
spectrometry which is also covered in the course.

The course also includes the experiments based on air analysis by

determining the Suspended particulate matter (SPM) in ambient air Like wise
biological contaminants occur in the air as aerosols and may include bacteria,
fungi, viruses, and pollens. Aerosols are characterized as solid or liquid
particles suspended in air. Microbiologic air sampling is used as needed to
determine the numbers and types of microorganisms, or particulates, in
indoor air.

Similarly waste management issues are becoming increasingly pressing as

million tons of waste are produced each year. Waste poses numerous
challenges to human health, water and air quality. Biogas and anaerobic
systems provide an opportunity to create economic activity while
revolutionizing waste management. Anaerobic digestion offers solutions to
these pressing issues. Anaerobic digestion is the process of converting
organic materials, typically viewed as wastes, into usable products, including
biogas, renewable natural gas (RNG), as well as valuable organic fertilizer
and compost. The course also covers the experiments for biogas production
and composting which provide tremendous opportunities for rural and urban
communities alike to foster healthy communities and healthier environment.

The emphasis of the lab Course is on providing the necessary skills to use
and exploit different microbiological and instrumental techniques in different
ways. This lab course is designed to emphasizes the scientific method and
aims to develop laboratory skills, through experiments related to microbial
analysis of soil, air and water. The manual also stresses quantitative skills,
with included step-by-step instructions for the more difficult calculations.

Objectives

After studying the contents of the course and performing the experiment you
should be able to:

1. Define aseptic technique in the area of microbiology;

2. Understand media preparation and sterilization process;
3. Explain the difference between simple staining and differential staining
techniques;
4. Identify an unknown bacterium utilizing various staining techniques;
5. Determine the morphological, physiologic and biochemical
characteristics of an unknown bacterium
6. Isolate and identify fungi and actinomycetes from soil;
7. Determine the biological activity of soil by dehydrogenase assay.
8. Isolate Bio-Surfactant Producing bacteria.
9. Identify the main types of organisms found in the air;
10. Estimation of total coliform and fecal coliform in water samples;
11. Determine the effect of treated and untreated effluent on seed
germination.
12. Determine the compost quality
13. Explain the principle of gas chromatography;
14. Determine the concentration of pesticides in an environmental sample;
15. Explain the set up an experiment for biogas production from organic
waste;
16. Determination of the composition of biogas produced.
17. Explain the principle of Inductively Coupled Plasma Optical Emission
Spectroscopy (ICP-OES);
18. Determine the concentration of trace metals in an environmental sample;
19. Determine the trace metal through Inductively Coupled Plasma Optical
Emission Spectroscopy (ICP-OES);
20. Describe alcoholic fermentation;
21. Describe the process involved in alcoholic fermentation;
22. Determine the parameters to be analyzed in alcohol production
23. Explain the basic principle of TLC;
24. Perform the Separation of amino acids.
25. Determine the concentration of sodium and potassium in water sample;
26. Perform a flame photometric determination;
27. Determine the concentration of sodium and potassium ions in a given
sample.
28. Determine the particulate matter (PM) in the ambient air.

STUDY GUIDE

The course is worth 4 credits which as per IGNOU norms amount to 120
hours of engagement. This includes reading the contents, performing the
experiments, participating in the interaction with the peers and the counsellor,
preparing the laboratory notebook, etc. It is strongly advised that you read the
details of the experiments before performing them. This experimental work
will be conducted at the study centre allocated to you for the purpose. This
practical course involves 14 days of laboratory work. Each day of work
consists of 8 hours of tutor guided practicals. You are required to complete
all the 20 exercises described in this practical course. The first session is
meant for the introduction where your counsellor will give you an overview
of the entire experimental work and any useful instructions to be followed in
the course of the laboratory work. The last session is meant for the
evaluation. In the rest of the sessions, you would be performing the
experiments, detailed in the course, under the guidance of your counsellor.
These experiments are termed as 'Guided Experiments' and would be
evaluated on a continuous basis. These guided experiments carry a weightage
of 70%. You are required to maintain a laboratory notebook wherein you
would record the experiments performed and get them corrected by the
counsellor regularly. In the last session you would be required to perform one
of the experiments, allocated by your counsellor, on your own. This is termed
as 'Unguided Experiment' and has a weightage of 30%. It is necessary for you
to get pass marks (at least 35%) in both of these components of evaluation.

Please maintain a complete and upto date record of laboratory work. You
need to prepare and bring a laboratory practical note-book for writing and
porting the work you have carried out in each practical session. The note-
book should contain alternate pages of ruled and untitled pages. Buy a 100
page practical note-book for this purpose. You are advised to prepare the
page (such as principle, requirements, procedure, observations and
calculations, result etc.) for recording the laboratory exercise. For each
exercise, you should write down title of the exercise, date and time of the
exercise along with results obtained and your observations. What you need
to submit and write in the note-book is given at the end of each exercise.

Submission of Your Practical Record for Evaluation

You need to submit the practical note-book and other required items to the
counsellor for evaluation and grading. Marks will be allocated based on the
successful experiment and for recording the results and observations in the
note-book in a proper manner as asked.

We hope that you would find the contents of the course and your laboratory
experience stimulating and useful. In case you find difficulty in
understanding any concept, you may get it clarified in the counselling
sessions or feel free to mail your queries to us at: [email protected].
 Sterilization, Media
EXPT 1 STERILIZATION, MEDIA Preparation and
Isolation of Pure
PREPARATION AND ISOLATION Culture

OF PURE CULTURE

Structure

1.0 Introduction
1.1 Expected Learning Outcomes
1.2 Principle
1.3 Requirements
1.5 Procedure
1.5 Observations and Calculations
1.6 Result
1.7 Precautions

1.0 INTRODUCTION
Aseptic technique or sterile technique is very important and used to avoid
contamination of sterile media and equipment during microbiological
experiments in the laboratory and also to maintain germ free environment.
When dealing with live cell cultures and reagents/media, sterile method
should always be employed. This approach involves the use of flame to
destroy contaminating organisms as well as a general manner of operation
that minimizes contamination of sterile media and equipment to
contaminants. The Bunsen burner is the sterile technician's greatest
companion when it comes to destroying bacteria with heat. As heat is an
excellent way of killing microorganisms. In the microbiology lab, aseptic
technique helps to prevent contamination related to working with the specific
microorganism and secure room hygiene.

1.1 EXPECTED LEARNING OUTCOMES

After studying and performing this experiment you should be able to:

• Define aseptic technique in the area of microbiology;

• Describe how to prevent contamination of cultures and media;
• Understand media preparation and sterilization process;
• Understand the basics of an autoclave and hot air oven.

1.2 PRINCIPLE
In the realm of environmental microbiology, aseptic technique is a basic and
vital laboratory skill and the basics of good laboratory practices. In a
9
 Environment environmental microbiology lab, aseptic technique is used for different
Science Lab
Course - 3 procedures i.e., transferring cultures, inoculating medium, isolating pure
cultures, and conducting microbiological tests. The use of proper aseptic
techniques protects cultures against contamination by germs found in the
environment. Airborne microorganisms (including fungus), microbes taken
up from the researcher's body, the lab bench-top or other surfaces, germs
found in dust, and microbes found on unsterilized glassware and equipment,
may contaminate cultures which interfere the lab result. The use of proper
aseptic techniques may significantly reduce, if not completely eliminate, the
danger of infection may reduce the risk of contamination. Furthermore,
maintaining pure stock cultures while transferring cultures to fresh medium
necessitates the use of aseptic method. In order to isolate a single species of
bacterium from a mixed culture and get a pure culture, aseptic method is also
required. Appropriate aseptic procedure prevents microbes used in the
laboratory from accidentally leaking into the environment or infecting
laboratory workers. This is particularly important when dealing with
pathogens. Two following terms being used during performance in any
microbiological laboratory:

1) Sterilization: It is the process of removing all microorganisms from a

product, surface, or medium, whether they are vegetative or spores.

2) Disinfection: It is the process of removing all harmful pathogenic

microorganism (that is organisms that are capable of giving rise to
infection) from an object, surface, or medium.

There are many sources of contamination, such as the atmosphere, breath,

hands, clothing, hair, working surface, equipment etc., which may affect the
work performance.

1.3 REQUIREMENTS
1) Instruments/equipment

i) Laminar flow bench/UV radiation

ii) Hot air oven
iii) Autoclave

2) Media/Reagents

i) Dehydrated selective media

ii) Distilled water/reagent water
iii) 48-72-hour nutrient broth cultures of E. coli and Staphylococcus
aureus
iv) Articles to be sterilized (nutrient broth, nutrient agar etc.)
iv) Nutrient agar plates/slants (numbers as per requirement)

10
 Sterilization, Media
3) Glassware/plasticware Preparation and
Isolation of Pure
i) Sample bottles borosilicate glass. Culture

ii) Graduated measuring cylinders, volumetric flask

iii) Petri dishes
iv) Inoculating loops either of platinum-iridium or disposable loops of
0.2-0.3 cm dia.

4) Personal protective equipment

i) Gloves
ii) Apron
iii) Mask
iv) Shoe cover
v) Head cover

1.4 PROCEDURE OF ASEPTIC TECHNIQUES

There are various aseptic techniques/methods being used in the
microbiology/biotechnology laboratory. The detailed procedure for these
techniques are as below:

A-Physical methods

1) Heat: Glassware are sterilized either by dry heat or by Moist heat

(steam). Use a hot-air oven set at 170°C for 2 hours or more to sanitize
glassware using dry heat. Alternatively, autoclave glassware at 121°C for
at least 30 minutes to sanitize it. Before autoclaving, loosen the tops on
all bottles. After autoclaving, dry any remaining moisture in empty
sterile containers in a drying oven if required. Sterilize glass pipets in
metal containers for at least 2 hours in a hot-air oven at 170°C.

i) Moist heat (steam under high pressure):

Autoclave- culture media

ii) Dry heat:

Hot air oven- glassware

Flaming- inoculation loop &needle

Procedure for the setting up of autoclave:

• Pack the media, reagents, and plastic wares in appropriate autoclavable

resistant polypropylene or borosilicate glassware.
• If using closed containers or plastics, screw the lid of the tube and leave
one thread unfastened.

11
 Environment • For each run in any of the item to be autoclaved, stick at random
Science Lab
Course - 3 autoclavable indicators.
• Inspect the water level in the autoclave machine.
• Do not overcrowd the autoclave machine.
• Turn on the machine.
• Keep the machine's lid securely closed and one valve open until it
reaches boiling.
• Allow warm air to escape via the valve for a few minutes.
• Close the valve completely and wait for the temperature to reach 1210C
at 15 lbs pressure.
• Allow 15 minutes for the sterilization cycle to complete.
• When the sterilization cycle is complete, turn off the heat and allow the
machine to reach up to 65°C.
• After sterilization, lift the lid and take out the items back.

Procedure for the setting up of hot air oven

• Wrap the glassware that will be sterilized by hot air oven sterilization,
such as pipettes with pipette cans, glass Petri dishes, sample dishes, test
tubes, pestle and mortar, and mineral oil, with appropriate wrapping.
• Preheat the hot air oven to 160 °C.
• Keep the temperature at that level for an hour.
• Turn off the hot air oven's heating and open the door after the
temperature has dropped below 65°C.

Hot Air Oven Autoclave

12
 Sterilization, Media
Preparation and
Isolation of Pure
Culture
Platinum wire

Hand

An inoculating loop Laminar air Flow

2) Radiation: radiation is used for enclosed areas, such as inoculation

rooms and viral laboratories. Under some circumstances, Ultraviolet
(UV) radiation may be an effective sterilant. Bacteria, yeasts, molds, and
viruses are all easily inactivated if the cells are exposed to enough UV
light.

B- Chemical methods: Chemical methods being used for the work surface
and equipment sterilization.

1) Alcohols - surfaces, skin e.g., ethyl alcohol 70% conc.

2) Aldehydes: e.g., Formaldehyde- equipment such as centrifuge.
3) Phenolics e.g., Phenols(chloroxylenol) -floors, walls and benches.

Follow the following steps for transferring bacteria (24 -48hrs grown) from
one capped test tube/ Petri plate to another test tube/ Petri plate:

• Prepare the agar media (strength as per the instruction) by dissolving

dehydrated media in distilled water.
• Sterilize the media in autoclave as per the procedure stated above.
• Sterilize all glassware/plasticware being used in the exercise in hot air
oven as per the procedure stated above.
• Switch on UV-light of the laminar flow before 15 minutes, and after
switch off the UV-light, on the air flow button and clean the work
surface of the laminar flow bench by 70% alcohol properly.
• After surface sterilization, dispense the sterilized media in the sterilized
petri plates in laminar flow bench.
• Flame the inoculating loop.
• Flame the mouth of the test tube/ Petri plates while holding the lid.
• Remove some bacteria with the loop.
• Flame the mouth of the test tube/petri plates and replace the lid.
• Flame the mouth of the test tube/petri plates to which the bacteria are
being transferred.
13
 Environment • Inoculate the test tube/petri plates with bacteria from the loop.
Science Lab
Course - 3 • Flame the lid of the test tube/petri plates and replace the lid.
• Flame the inoculating loop.
• Close the test tube/petri plate.
• Incubate the inoculated test tubes/petri plates at 350C for 24 hrs.
• Always prepare the controls/blanks without cultures and incubate test
tubes/petri plates at 350C for 24 hrs.

Fig 1.1 Aseptic Transfer Technique

Fig 1.2 Colony Counter Fig 1.3 E Coli colonies on the plate

1.5 OBSERVATIONS AND CALCULATIONS

Observations

Examine all the inoculated plates/slants for the amount of growth of the test
organisms in the sterilized conditions by using the sterilized media.

14
 Sterilization, Media
Media used Unsterilized media Sterilized media Preparation and
(unautoclaved) (autoclaved) Isolation of Pure
Culture

E. coli
Staphylococcus aureus
Control (without
microbes)

Record the number of colonies of microorganisms observed in the control

(unautoclaved) and (autoclaved media.

1.6 RESULTS
The results of the performing the aseptic techniques followed during for
transferring bacteria (24 - 48hrs grown) from one capped test tube/ Petri plate
to another test tube/ Petri plate

…………………………………………………………………………………
…………………………………………………………………………………
…………………………………………………………………………………

1.7 PRECAUTIONS
No eating, drinking or smoking at any time. Do not bring food or drink items
into the lab. Avoid all finger/hand-to-mouth contact.

1) A lab coat must be worn at all times in the microbiology laboratory.

2) Before using the media, always use the autoclaved media.
3) Always use the sterile glassware.
4) Carefully prepare the dilution series when using the wastewater samples
or polluted samples
5) Before processing the samples in laminar flow bench, sterile the surface
to remove the contamination.
6) Always on the UV light before and after processing the samples in
laminar flow bench.
7) Were mask and gloves before start the analysis.
8) Always use the 70% alcohol for disinfect the working surface.

15
 Environment
Science Lab EXPT 2 DETERMINATION OF GRAM
Course - 3
STAINING OF BACTERIAL
SAMPLE

Structure

2.0 Introduction
2.1 Expected Learning Outcomes
2.2 Principle
2.3 Requirements
2.4 Procedure
2.5 Observations and Calculations
1.6 Result
2.7 Precautions

2.0 INTRODUCTION
The staining is a remarkable concept to evaluate the cell visually and
differentiate it from other cell types. Since most kinds of cells have no
natural pigment, it is difficult to spot under a light microscope unless they are
stained. To make bacterial cells more visible, a variety of strains are utilized.
Additionally, particular staining methods may be employed to determine the
cells' biochemical or structural features such as cell wall type and presence or
absence of endospores. This sort of data may aid in the identification and
classification of microorganisms.

Gram's stain is the most extensively used staining procedure in bacteriology

is a complex and differential staining procedure. The cell wall composition of
organisms in the Domain Bacteria is distinguished using a series of staining
and decolorization methods. The simple stain, in which just one stain is
employed and all forms of bacteria show as the colour of that stain when seen
under a microscope, is one sort of staining process that may be performed.
Simple stains may be used to assess the morphology and arrangement of
bacterial species, but they provide no more information. Differential
staining is often used by scientists since it helps them to learn more about the
germs they are dealing with. Multiple stains are used in differential stains,
and the appearance of cells varies depending on their chemical or structural
features. The Gram stain, acid-fast stain, and endospore stain are examples of
differential stains.

2.1 Expected Learning Outcomes

After studying and performing this experiment you should be able to

16
 Determination of
1) Explain the difference between simple staining and differential staining Gram Staining of
techniques; Bacterial Sample

2) Describe the differences between Gram-positive and Gram-negative

bacteria;
3) Perform the Gram stain procedure.

2.2 PRINCIPLE
Hans Christian Gram, a Danish bacteriologist, invented the commonly used
staining process in 1882 while dealing with tissue samples from the lungs of
patients who had died from pneumonia. Later this called as Gram’s Method.
Since then, microbiologists have employed the Gram stain process to gain
crucial information about the bacterial species they are dealing with all over
the world as fast, simple and effective approach. This technique is based on
the staining of bacterial cell wall’s which depends on the cell wall
composition of lipids and thickness. Some bacteria are able to preserve the
primary stain after being stained with Crystal Violet and fixed with the
mordant, whereas others are decolorized by alcohol. Gram-positive bacteria
have a thick covering of protein-sugar compounds termed peptidoglycan on
their cell walls, and their lipid content is minimal. The findings of the Gram
stain reveal changes in cell wall composition. Gram positive bacteria contain
extensive coatings of peptidoglycan (a carbohydrate) in their cell walls, while
Gram negative bacteria have none. Teichoic acids are present in Gram
positive bacteria, but not in Gram negative bacteria. (Figure 2.1).

Figure 2.1: The major differences between the Gram-positive and Gram-negative cell
walls.

17
 Environment
Science Lab
Course - 3

Figure 2.2: Gram-positive and Gram-negative bacteria and their common shapes

Gram negative cells have an outside membrane that mimics the cell
membrane's phospholipid bilayer. Lipopolysaccharides (LPS) are found in
the outer membrane of Gram-negative cells and are released as endotoxins
when they die. This is something to be concerned about if you have a gram-
negative infection. Fresh cultures are preferred for Gram stains since older
cells may have broken cell walls and so fail to generate the correct Gram
response. Due to the fact that certain species are Gram-variable, both Gram-
positive and Gram-negative responses may be evident on the slide. Gram-
positive bacteria have extensive coatings of peptidoglycan in their cell walls
(90 percent of cell wall). Gram-negative bacteria with thin peptidoglycan
coatings (10 percent of the wall) and high lipid content dye purple. These
leave a pink stain (Figure 2.2). Applying a primary stain (crystal violet) to a
heat-fixed smear, followed by the addition of a mordant (Gram's Iodine),
quick decolorization with alcohol, acetone, or a combination of alcohol and
acetone, and finally counterstaining with safranin are the four fundamental
processes of the Gram Stain.

2.3 REQUIREMENTS
a) 24 hrs. old culture of Escherichia coli and Staphylococcus aureus
b) Clean microscope slides (2-4)
c) Staining trays and blotting paper
d) Water bottle (for rinsing)
e) Bunsen burner/sprit lamp
f) Inoculating loop

g) Primary Stain: Reagent for Crystal Violet Staining. Crystal violet

coloring reagent Solution A 2g Ethanol, 95 percent (vol/vol), 20 ml
Crystal violet (confirmed % dye content) Crystal violet staining reagent
Solution B 0.8 g ammonium oxalate, 80 ml distilled water to make
18
 Determination of
crystal violet staining reagent, combine A and B. Before using, store for Gram Staining of
24 hours and filter through paper. Bacterial Sample

h) Mordant: Iodine, 1.0 g potassium iodide, 2.0 g distilled water, 300 ml

Gram's Iodine, 1.0 g potassium iodide, 2.0 g distilled water In a mortar,
pound the iodine and potassium iodide together, then gently add water
while continuing to ground until the iodine is dissolved. Amber bottles
are ideal for storing.

i) Decolorizing Agent Ethanol, 95% (vol/vol)

j) Counterstain:
k) Safranin Stock solution: 2.5g Safranin O
l) 100 ml 95% Ethanol
m) Working Solution:
n) 10 ml Stock Solution
o) 90 ml Distilled water
Personal protective equipment
i) Gloves
ii) Apron
iii) Mask
iv) Shoe cover
v) Head cover

2.4 PROCEDURE
Prepare a bacterial smear:

a) Label a clean glass slide.

b) Add a small drop of saline to the slide (you will usually put two bacteria
on one microscope slide). This can be done by placing a drop of saline
onto your inoculation loop and then transferring it to the slide. If you use
the saline dropper directly on the slide, do not release a full drop.

c) With an inoculation loop or needle, pick up a small amount of bacteria.

Mix it well with the saline and spread the mixture over a wider area of
the slide.

• Be careful not to have the two smears run into each other.

d) Air dry the bacterial specimen on the slide (slide warmers may also be
used).

e) When slides are completely air-dry, heat fix the bacterial specimen by
passing the slide slowly over the flame twice.

• Heat fixing kills cells, and adheres them to the slide.

19
 Environment • Cells will be rinsed off the slides if they are not heat fixed properly.
Science Lab
Course - 3 • Be careful not to overheat the slides in this procedure

After heat-fixing (Figure 2.3) is complete, you are ready to gram stain your
slide.

Figure 2.3: Heat fixation

Gram Staining

a) Flood air-dried, heat-fixed smear of cells for 1 minute with crystal violet
staining reagent.

(Please note that the quality of the smear (too heavy or too light cell
concentration) will affect the Gram Stain results).

b) Wash slide in a gentle and indirect stream of tap water for 2 seconds.

c) Flood slide with the mordant: Gram's iodine. Wait 1 minute.

d) Wash slide in a gentle and indirect stream of tap water for 2 seconds.

e) Flood slide with decolorizing agent. Wait 15 seconds or add drop by

drop to slide until decolorizing agent running from the slide runs clear.

f) Flood slide with counterstain, safranin. Wait 30 seconds to 1 minute.

g) Wash slide in a gentile and indirect stream of tap water until no color
appears in the effluent and then blot dry with absorbent paper.

h) Observe the results of the staining procedure under oil immersion using a
Brightfield microscope. At the completion of the Gram Stain, gram-
negative bacteria will stain pink/red and gram-positive bacteria will stain
blue/purple.

2.5 OBSERVATIONS AND CALCULATIONS

Observations

Observe your slide under the microscope. For each organism, determine
morphology, arrangement and Gram reaction.

20
 Determination of
Gram Staining of
Bacterial Sample

Figure 2.4: Gram positive Vs Gram negative

(blue/purple rods - gram-positive; pink/red rods- gram-negative)

In a smear that has been stained using the Gram Stain protocol, the shape,
arrangement and gram reaction of a bacterial culture will be revealed.

Draw sketches for each type of bacteria that you observe. Identify its
morphology, arrangement, and Gram reaction.

Bacteria Morphology Arrangements Gram reaction

1
2
3
4

2.6 RESULTS
The results of gram staining:

Bacterial strain 1----------------------------------

Bacterial strain 2----------------------------------

2.7 PRECAUTIONS
1) Avoid pressing the loop or needle too firmly against the agar surface as
this will damage it.

2) Before processing the samples in laminar flow bench, sterile the surface
to remove the contamination.

3) Always on the UV light before and after processing the samples in

laminar flow bench.

4) Always were the lab coat, mask and gloves before start the analysis.

5) Always use the 70% alcohol for disinfect the working surface and after
complete dry the surface turn on the burner.

21
 Environment
Science Lab EXPT 3 ISOLATION AND
Course - 3
CHARACTERIZATION OF
BACTERIA FROM SOIL SAMPLES

Structure

3.0 Introduction
3.1 Expected Learning Outcomes
3.2 Principle
3.3 Requirements
3.4 Sample preparation
3.5 Procedure
3.6 Observations
3.7 Result
3.8 Precautions

3.0 INTRODUCTION
In our environment, microorganisms are plentiful and omnipresent.
Microorganisms are the master of the universe and they may be found in the
air, soil, water, food, and sewage, among other places. Soil bacteria are the
most common organisms found in surface soils. The presence of these
organism is diverse and all of these creatures are prokaryotic, but the bacteria
may sustain their life in aerobic, anaerobic, or facultatively anaerobic
situations. According to their food needs they may also be autotrophic and
heterotrophic. Actinomycetes are filamentous microorganisms that belong to
prokaryotic category. Bacteria and actinomycetes play a significant role in
nutrient cycling and organic pollutant breakdown. They also interact with
plants as "rhizosphere" populations that live around plant roots. Soil
microorganisms may cause disease in both plants and people (e.g.,
Agrobacterium tumefaciens) (Clostridium perfringens and Bacillus
anthracis). Bacteria, algae, protozoans, yeasts, molds, and minute worms are
all present in soil. Soil is the excellent source for undiscovered
microorganisms.

3.1 EXPECTED LEARNING OUTCOMES:

After studying and performing this experiment you should be able to:

a) Describe the isolation of unknown bacterium from soil;

b) Identify an unknown bacterium utilizing various staining techniques;
c) Determine the motility of an unknown bacterium;
d) Describe the physiologic characteristics of an unknown bacterium
22
 Isolation and
3.2 PRINCIPLE Characterization of
Bacteria From Soil
Samples
Microorganisms are found in every aspect of the biosphere, including soil,
hot springs, and deep subterranean rocks at least nineteen kilometers deep.
Soil microorganisms serve a crucial part in sustaining the biological balance
of planet's existence. Bacteria, fungi, and viruses are found in various
concentrations in all soils, depending on soil conditions. The relative
abundance of bacteria is also determined by the allowed degree of acidity and
the kinds of wastes added. The quantity of bacteria in the soil determines its
fertility and the amount of organic matter it may accumulate in a short period
of time. Soil microorganisms play a critical part in biogeochemical cycles
and the biosphere's long-term sustainability. Soil microorganisms create and
consume two or three important naturally occurring greenhouse gases that
have a significant impact on agriculture. In many microbiology
investigations, isolating bacteria from the soil is a crucial initial step. once the
bacteria are isolated, bacteria may be further studied for the identification of
species and their role in the soil environment. Because even a little quantity
of soil might contain millions of bacteria, diluting a soil sample before
extracting bacteria from the sample is important. There are a variety of
methods for isolating and counting of microorganisms from the soil. The
serial dilution agar plating method and viable plate count method is one of
the most often used methods for the isolation and enumeration of
microorganisms from the soil sample.

3.3 REQUIREMENTS
• 25 g fresh soil of each soil type
• one plastic cup for each soil type
• benchtop balance (±0.01 g)
• weighing dishes
• deionized water
• plastic wrap
• rubber bands
• marking pens
• dissecting probe
• incubated soils from Period 1
• 9 peptone-yeast agar plates per soil type
• 9 glycerol-casein agar plates amended with cycloheximide per soil type
• 1 sterile, 95 ml water blank for each soil type
• 4 sterile, 9 ml water blanks for each soil type
• 10 sterile, 1 ml pipettes for each soil type
• pipette bulb
23
 Environment • 1 test tube rack
Science Lab
Course - 3 • glass hockey stick spreader
• ethyl alcohol for flame sterilization
• vortex
• gas burner
• pre-prepared R2A agar plates
• pre-prepared glycerol-casein agar plates

Personal protective equipment

i) Gloves
ii) Apron
iii) Mask
iv) Shoe cover
v) Head cover

3.4 SAMPLE PREPARATION

1) Fill the sterile container halfway with 100 mL distilled water from the
graduated cylinder.

2) Weigh out 1 g of soil sample and place it in the distilled water bottle.
Close the container tightly and shake it vigorously to properly mix the
fluid.

3) Write the numbers 10-3, 10-4, 10-5, and 10-6 on the sterile test tubes. Using
one of the pipettes, add 9 mL of distilled water to each of the tubes.

4) Using a fresh pipette, transfer 1 mL of the solution from the bottle to the
tube labelled 10-3. Cap the tube and gently swirl it until the solution is
evenly distributed.

5) Using a fresh pipette, transfer 1 ml of the solution from the 10-3 test tube
to the 10-4 tube. Swirl the 10-4 tube to combine it. Transfer solution from
the 10-4 tube to the 10-5 tube, then from the 10-5 tube to the 10-6 tube,
using this approach.

6) From the "10-4, 10-5, and 10-6 tubes, plate three samples each. Transfer 1
mL of solution from the tube to a Petri plate using a fresh pipette. Pour
approximately 15 mL of nutritious agar onto the plate, then cover with
the lid and gently swirl until the agar covers the whole bottom of the late.

7) Using a fresh pipette, pour 1 ml of distilled water onto a Petri plate to

create a control plate. Pour in the agar, cover, and spin the plate.

24
 Isolation and
8) After the agar has set, keep the Petri plates upright. Then flip the plates Characterization of
and incubate them for as little as 24 hours or as long as five days, either Bacteria From Soil
Samples
in an incubator or at room temperature.

9) After the specified period of incubation time, remove the plates from the
incubator. Count the bacterial colonies on plates with 30 to 300 colonies
on them. To prevent counting the same colonies repeatedly, use a
permanent marker to indicate the colonies you've previously counted

10) For each plate, divide the number of colonies counted by the soil
solution dilution-10-4,10-5, or 10-6. By averaging the findings from each
countable plate, determine the number of cultivable bacteria in the initial
gramme of soil.

11) Re-weigh each of the soil samples including the plastic wrap covering, to
allow for soil moisture calculation at the time of plating.

12) Prepare a dilution series of each of the soils (see Figure 3.1).

13) For each soil, suspend 10 g to a 95 ml water blank. Shake the suspension
well

14) Before the soil settles in the bottle, remove 1ml of the suspension with a
sterile pipette and add it to a 9 ml water blank. Vortex well.

15) Repeat the previous step three times, each time with a fresh 9 ml water
blank and sterile pipette. Vortex well. This will result in dilutions of ca.
10-1, 10-2, 10-3, 10-4, and 10-5 g soil ml-1.

3.5 PROCEDURE
Isolation of bacteria from soil sample:

1) Prepare two or three spread plates for each dilution 10-3, 10-4, and 10-5, as
follows. After vortexing, place a 0.1 ml drop of each dilution (this will
increase your effective dilution by a factor of ten) to three separates,
labeled peptone-yeast agar plates.

2) Inoculate three plates before spreading, as standing will allow too much
liquid to be absorbed into the agar in one spot.

3) Take the glass hockey stick spreader, dip it in ethanol, and flame the
spreader in a Bunsen burner just long enough to ignite the ethanol.

4) Moving the spreader out of the flame and holding it just above the first
of the inoculated plates allows all of the ethanol to burn off. Then
quickly open the plate, holding the lid nearby in one hand. Touch the
spreader to the agar away from the inoculum to cool it, and spread the
drop of inoculum around on the surface of the agar until all traces of free
liquid disappear (the surface will become somewhat tacky).

25
 Environment 5) Replace the lid, re-flame the spreader, and repeat with the next plate.
Science Lab
Course - 3 Work quickly so as not to contaminate the agar with air-borne
organisms.

6) Incubate the bacteria plates (inverted) at room temperature for one week.

Figure 3.1: preparation of dilution series

Identification of bacteria:

Identification and characterization of the isolated bacterial stains will be done

by performing the gram staining of the isolated single colony.

1) Examine your bacterial streak plates after one week. Observe the
colonies for uniformity of shape and size. Note the presence of any
contaminant and note the observation.

2) Transfer a small drop of tap water to a slide with a wire inoculating loop.
Flame the wire loop and remove a small amount of culture. Mix the
26
 Isolation and
bacteria in the drop of water, spreading it over an area about the size of a Characterization of
dime. Bacteria From Soil
Samples
3) Let the smear air-dry and then fix the film by passing the slide through
the Bunsen burner flame 2 or 3 times.

4) Apply 5 drops of crystal violet to the smear, allowing the dye to remain
on the slide for 2 or 3 minutes.

5) Rinse the slide with water and then with iodine solution. Cover with
fresh iodine and let stand for two minutes. Rinse with water, using a
gentle stream.

6) Decolorize with decolorizer. Add the decolorizer drop by drop to the

smear with the slide held tilted. Continue decolorization until no more
stain is seen to wash from the smear (usually 20 seconds is sufficient).
Rinse immediately in water.

7) Counterstain for 10 seconds with safranin and rinse the slide with water.

8) Carefully blot the slide to hasten drying. Examine the dry preparation
under oil using the oil immersion objective and observe the details in the
microscope.

3.6 OBSERVATIONS AND CALCULATIONS

Counting Bacteria (after 1 week incubation)

1) Examine all of the bacteria plates carefully. Note differences in colony

size and shape.

2) Count the total number of bacterial colonies (CFUs) for each plate.
Average the totals for each dilution. Count only those plate of a dilution
that are countable (30-200 colonies per plate).

3) Calculate the sample mean of CFUs per gram of dry soil for each of your
soils. The calculation is similar to that used in the soil fungi experiment,
except that the effective dilution is increased by one order of magnitude
since only 0.1 ml of inoculum was used for the spread plate as opposed
to 1 ml in the pour plates. Also, calculate for each soil the sample
standard deviation and the coefficient of variation.

Observations

The number of colonies appearing on dilution plates are counted, averaged

and multiplied by the dilution factor to find the number of cells/spores per
gram (milliliter) of the sample:

number of cells per gram dry soil = number of colonies X dilution

factor dry weight of soil (gm)

27
 Environment Where,
Science Lab
Course - 3
Number of colonies = average of the triplicate microbial counts,

dilution factor = Reciprocal of the dilution (e. g. 10-3 = 103).

Table: Colony characteristics and morphology of unknown bacteria by using

the microscope.

Colony Colony surface surface Prokaryote/ Shape

Colour Texture edge Elevation Appearance Eukaryote

1
2
3

3.7 RESULTS
The number of bacteria enumerate in a given soil sample is ………………
cfu/g

3.8 PRECAUTIONS
1) Isolation of microorganisms should be done from a composite sample
collected randomly from a field.
2) Soil should be in a powdered form.
3) Each dilution must be thoroughly shaken before removing an aliquot for
subsequent dilution.
4) Use separate sterile pipettes for each dilution.
5) Immediately after the addition of the medium to a plate, inoculum is to
be mixed with circular and side to side movements of the Petri plates.
6) Always use sterile glassware.
7) Apply dilution factor before reporting the results
8) Before processing the samples in a laminar flow bench, sterile the
surface to remove the contamination.
9) Always switch on the UV light before and after processing the samples
in laminar flow bench.
10) Always were the lab coat, mask and gloves before start the analysis.
11) Always use the 70% alcohol for disinfect the working surface and after
complete dry the surface turn on the burner.

28
 Isolation and
EXPT 4 ISOLATION AND Characterization of
Actinomycetes
CHARACTERIZATION OF From Soil Samples

ACTINOMYCETES FROM SOIL

SAMPLES

Structure

4.0 Introduction
4.1 Expected Learning Outcomes
4.2 Principle
4.3 Requirements
4.4 Sample Preparation
4.5 Procedure
4.6 Observations and Calculations
4.7 Result
4.8 Precautions

4.1 INTRODUCTION
Soil microorganism - Actinomycetes are Gram-positive, unicellular bacteria
that belong to the Order Actinomycetales. Members of this family are
extensively spread in nature and may be found in a broad range of
environments across the biosphere. The actinomycetes are varied and
encompass a range of subdivisions, as well as yet-unclassified isolates,
because certain genera are difficult to categorize due to a highly niche-
dependent phenotype. For example, Nocardia comprises multiple phenotypes
that were formerly thought to be separate species until it was discovered that
their differences were shown to be totally based on their growth condition.
Actinomycetales are gram-positive, anaerobic, and contain mycelium that
grows in a filamentous, branching manner. Some actinobacteria have the
shape of rods or coccoids, while others produce spores on aerial hyphae.

In most soils, actinomycetes are present as a large part of the microbial

community and produces extracellular enzymes that can breakdown a variety
of compounds. Enzymes of actinomycetes are more appealing than enzymes
from other sources because of their excellent stability and exceptional
substrate specificity. Actinomycetes found in extreme habitats, develop novel
enzymes with enormous economic value. Actinomycetales are found mostly
in soil and decomposing organic debris, as well as in living organism (i.e.,
humans and animals). They create symbiotic nitrogen-fixing relationships
with plant species and also act as growth promoters or biocontrol agents, as
well as cause disease in certain plant species. In the form of Helicobacter,
Actinomycetales may be found in the human urogenital tract as well as the
29
 Environment digestive system, including the mouth, throat, and gastrointestinal tract,
Science Lab
Course - 3 without producing illness in the host. They are used as a foundation of many
antibiotics and insecticides, and also have extensive medical and botanical
applications.

Figure 4.1: Actinomycetes

4.1 EXPECTED LEARNING OUTCOMES

After studying and performing this experiment you should be able to:

a) Isolate unknown actinomycetes from soil;

b) Identify actinomycetes utilizing various staining techniques;
c) Determine the motility of actinomycetes;
d) Determine the physiologic characteristics of actinomycetes.

4.2 PRINCIPLE
Actinomycetes are Gram-positive, soil born very high GC content microbes
having fungal morphological characteristics. They are marvelous source of
secondary metabolites and unique enzymes with a wide range of biological
activities. In most of soils, actinomycetes make up a considerable portion of
the microbial community. Actinomycetes counts of above 1 million per gram
are frequent, according to estimates. Over twenty genera have been identified
from soil, with streptomycetes accounting for 95% of the isolates. The kind
and number of actinomycetes in soil are influenced by environmental
conditions. In culture, most actinomycete isolates behave like neutrophils,
with a pH range of 5.0 to 9.0 and an optimal pH of approximately 7.0.

The distribution and activity of soil actinomycetes are influenced by pH as a

major environmental factor. Acidophilic and acidoduric streptomycetes are
abundant in acidic soils below pH 5.0, but neutrophilic and acidoduric
streptomycetes are rare. However, there are just a few cases of to 9.5 being
isolated from soil near Salt Lake. In the laboratory, most actinomycetes
behave as mesophiles, with optimal growth temperatures of 25 to 30°C. In
compost, several mesophilic actinomycetes are active.

30
 Isolation and
CLASSIFICATION OF ACTINOMYCETES Characterization of
Actinomycetes
From Soil Samples
Section Characteristics

Aerobic, acid-alcohol fast; rods, cocci, and

Nocardioform branching filaments or substrate and aerial
actinomycetes mycelium that fragment; wall chemotype IV;
mycolic acids.

Actinomycetes with
Mycelium splits in all planes, no aerial hyphae, wall
multilocular
chemotype III. Aerobic to facultatively anaerobic
sporangia

Aerobic sporoactinomycetes, nonmotile, having

spores that may be encapsulated in vesicles; no
Actinoplanetes
aerial mycelium; wall chemotype II; arabinose and
xylose in whole-organism hydrolysates.

Streptomycetes and Aerobic sporoactinomycetes; form an extensively

related genera branched substrate and aerial mycelium.

Aerobic spordactinomycetes produce a branching

Thermomonospora substrate and aerial mycelium, both of which may
and related genera contain single spore chains. either motile or non-
motile spores; chemotype III of the wall

Aerial growth is produced by the stable filaments.

On both aerial and vegetative filaments, single
spores (endospores) develop. Thermophilia is a trait
Thermoactinomycetes
shared by all organisms. Meso-DAP is found in the
cell wall, but no other amino acids or carbohydrates
are present.

They all generate spore-bearing aerial growth

Other genera
chains.

Actinomycetes are prokaryotic organisms that are considered as true bacteria.

Bacteria generate slimy colonies that vary in colour from colourless to
brilliantly coloured orange, yellow, or pink colonies when cultivated on agar.
Actinomycetes, on the other hand, have a filamentous growth habit that
allows them to be visually distinguished from bacteria. Colonies of
actinomycetes are chalky, hard, and leathery, and will shatter if pressed.
Bacterial colonies, on the other hand, will smear under pressure.
Actinomycetes, unlike bacteria, are drought tolerant and have an ecological
benefit in desert soils, which are often dry and alkaline. As a result, drying
the soil prior to doing culturable heterotrophic plate counts may significantly 31
 Environment minimize bacterial influence. To manage fungus, antifungal medicines such
Science Lab
Course - 3 as cycloheximide are used.

Fig 4.2. Actinomycetes on Starch Casein Agar

Actinomycetes are one of the most common and dominant groups of gram-
positive bacteria. Pharmaceuticals, neutraceuticals, enzymes, anticancer
drugs, enzyme inhibitors, and other products derived from actinomycetes
have all been commercially utilized.

4.3 REQUIREMENTS
• 25 g fresh soil of each soil type
• one plastic cup for each soil type
• benchtop balance (±0.01 g)
• weighing dishes
• deionized water
• plastic wrap
• rubber bands
• marking pens
• dissecting probe
• incubated soils from Period 1
• 9 peptone-yeast agar plates per soil type
• 9 glycerol-casein agar plates amended with cycloheximide per soil type
• 1 sterile, 95 ml water blank for each soil type
• 4 sterile, 9 ml water blanks for each soil type
• 10 sterile, 1 ml pipettes for each soil type
• pipette bulb
• 1 test tube rack
• glass hockey stick spreader
• ethyl alcohol for flame sterilization
• vortex
32
 Isolation and
• gas burner Characterization of
Actinomycetes
• pre-prepared R2A agar plates From Soil Samples
• pre-prepared glycerol-casein agar plates

Personal protective equipment

• Gloves
• Apron
• Mask
• Shoe cover
• Head cover

4.4 SAMPLE PREPARATION

1) Measure 100 ml. distilled water in the graduated cylinder and add it to
the sterile bottle

2) Weigh out 1 g of the soil sample and add it to the bottle of distilled
water. Tightly cap the bottle and shake it to thoroughly mix the solution

3) Label the sterile test tubes 10-3, 10-4, 10-5 and 10-6, Add 9 ml of distilled
water to each of the tubes, using one of the pipettes

4) Transfer 1 ml of the solution in the bottle to the tube labeled 10-3, using
a new pipette. Cap the tube and swirl it gently until the solution is well
mixed

5) Transfer 1 ml of the solution in the 10-3 test tube to the 10-4 tube with a
new pipette. Cap the 10-4 tube and swirl to mix. Repeat this method to
transfer solution from the 10-4 tube to the 10-5 tube and then from the
10-5 tube to the 10-6 tube.

6) Plate three samples each from the "10-4, 10-5 and 10-6 tubes. Use a new
pipette to transfer 1 ml of solution from the tube into a Petri plate. Add
about 15 ml of nutrient agar to the plate; then put the lid on the plate
and swirl gently so that the agar covers the bottom of the plate

7) Make a control plate by putting 1 ml of distilled water into a Petri plate,

using a new pipette. Add agar; put the lid on and swirl the plate.\

8) Leave the Petri plates upright until the agar has set. Then invert the
plates and incubate them either in an incubator or at room
temperature—for as little as 24 hours and up to five days.

9) Remove the plates from the incubator after the desired amount of
incubation time. Count the bacterial colonies on plates containing about
30 to 300 colonies. Use a permanent marker to mark the colonies you
have already counted in order to avoid counting the same colonies
twice.
33
 Environment 10) Divide the number of colonies counted by the dilution—10-4, 10-5 or
Science Lab
Course - 3 10-6 of the soil solution for each plate. Find the number of cultivatable
bacteria in the original gram of soil by averaging the results from each
countable plat

11) Re-weigh each of the soil samples including the plastic wrap covering, to
allow for soil moisture calculation at the time of plating.

12) Prepare a dilution series of each of the soils

13) For each soil, suspend 10 g to a 95 ml water blank. Shake the suspension
well.

14) Before the soil settles in the bottle, remove 1ml of the suspension with a
sterile pipette and add it to a 9 ml water blank. Vortex well

15) Repeat the previous step three times, each time with a fresh 9 ml water
blank and sterile pipette. Vortex well. This will result in dilutions of ca.
10-1, 10-2, 10-3, 10-4, and 10-5 g soil ml-1

4.5 PROCEDURE
Isolation of Actinomycetes from soil sample:

1) Use the dilutions 10-2, 10-3, and 10-4 from above. Spread plate 0.1 ml of
vortexed suspension on glycerol-casein plates, make three replicates for
each dilution.

2) Incubate the actinomycete plates (inverted) at room temperature for two

weeks.

Counting Actinomycetes (after two-week incubation)

1) Examine all of the actinomycete plates carefully. Note differences in

colony size and shape.

2) Count the total number of actinomycete colonies (CFUs) for each plate.
Average the totals for each dilution. Count only those plates of a dilution
that are countable.

3) Calculate the sample mean of actinomycete CFUs per gram of dry soil
for each of soil sample.

4.6 OBSERVATIONS AND CALCULATIONS

Observations

The number of colonies appearing on dilution plates are counted, averaged

and multiplied by the dilution factor to find the number of cells/spores per
gram (milliliter) of the sample:

34
 Isolation and
number of cells per gram dry soil = number of colonies X dilution Characterization of
factor dry weight of soil (gm) Actinomycetes
From Soil Samples
Where,

Number of colonies: average of the triplicate microbial counts,

dilution factor= Reciprocal of the dilution (e. g. 10-3 = 103).

Table: Colony characteristics and morphology of unknown

Actinomycetes by using the microscope.

Colony Colony surface surface Prokaryote/ Shape

Colour Texture edge Elevation Appearance Eukaryote

1
2
3

4.7 RESULTS
The number of actinomycetes in a given soil sample is ……………… cfu/g

4.8 PRECAUTIONS
1) Isolation of microorganisms should be done from a composite sample
collected randomly from a field.
2) Soil should be in a powdered form.
3) Each dilution must be thoroughly shaken before removing an aliquot for
subsequent dilution.
4) Use separate sterile pipettes for each dilution.
5) Immediately after the addition of the medium to a plate, inoculum is to
be mixed with circular and side to side movements of the Petri plates.
6) Always use the sterile glassware.
7) Apply dilution factor before report the results
8) Before processing the samples in laminar flow bench, sterile the surface
to remove the contamination.
9) Always on the UV light before and after processing the samples in
laminar flow bench.
10) Always were the lab coat, mask and gloves before start the analysis.
11) Always use the 70% alcohol for disinfect the working surface and after
complete dry the surface turn on the burner.

35
 Environment
Science Lab EXP 5 ISOLATION OF PURE CULTURE
Course - 3
BY STREAK PLATE METHOD,
POUR PLATE METHOD AND
SPREAD PLATE METHOD

Structure

5.0 Introduction
5.1 Expected Learning Outcomes
5.2 Principle
5.3 Requirements
5.4 Procedure
5.5 Observations and Calculations
5.6 Result
5.7 Precautions

5.0 INTRODUCTION
In nature, microorganisms are present in mixed populations. To obtain a pure
microbial culture from the mixed population in the lab is very important
technique. Pure culture, preparation means one in which all organisms are
offspring of the same organism. Various environmental parameters that are
optimal for the growth of specific culture and also able to eliminate other
growth is very significant in the field of microbiology and biotechnology.
Microorganisms can be isolated from various sources (soil, air, water,
sewage, decomposing waste, etc.) by growing under well-defined conditions.
Microbial culture contains millions or billions of individuals. To study
colony features, biochemical properties, morphology, staining responses, and
immunological reactions of a specific strain of bacteria, actinomycetes, or
fungus or to test sensitivity to antimicrobial agents, pure culture is required.
Identification of microorganisms is difficult solely based on microscopic
observations due to their small size, comparable morphological traits, and
staining responses. Culture the microorganisms on specific artificial medium
and observe the growth pattern and colonial properties is one of the methods
to study microorganisms. Cultural techniques are not effective only for
identifying microorganisms, but also for isolating and determining the kind,
behavior and load of microorganisms present in any environmental sample.

5.1 EXPECTED LEARNING OUTCOMES

After studying and performing this experiment you should be able to:

1) Identify the difference between pure cultures and mixed culture;

36
 Isolation of Pure
2) Describe a good aseptic technique in culture transfer or inoculation and Culture by Streak
handling sterile materials; Plate Method, Pour
Plate Method and
3) Explain isolation of organisms from a mixed culture using selective and Spread Plate
Method
differential media;
4) Isolate microorganisms from a wide variety of sources;
5) Describe the preparation of the dilution series.

5.2 PRINCIPLE
The microbial community in the environmental sample is very diverse. To
properly grasp each group of species' contributions to the ecology of the
mass, this mixed culture must first be dissected into separate colonies. Pure
culture contains just one species of microbe, whereas mixed culture contains
more than one species of microbes. Dilution techniques such as pour plate,
spread plate, and streak plate may be used to create pure cultures. In the
streak plate approach, a mixed sample is streaked multiple times over the
surface of the solid culture media using an inoculating loop. Spread and pour
plates are quantitative methods for determining the quantity of bacteria
present in a sample. In a spread plate, a known quantity of diluted sample is
distributed over the surface of the nutritional medium with the aid of a
spreader, while in a pour plate, a known amount of diluted sample is spread
over the surface of the nutrient medium with the use of a spreader. In sterile
Petri plates, diluted material is combined with melted nutrient media under
aseptic circumstances. Bacterial growth is observable as surface colonies (in
the case of the spread plate method) and surface/embedded colonies at the
conclusion of the incubation period (in case of pour plate technique).

Two major steps are involved in obtaining pure cultures from a mixed
population:

1) First, dilute the mixture until the individual microorganisms are

separated far enough away on an agar surface that following incubation,
they form visible colonies apart from other microbe colonies. An
isolation plate is the name for this kind of plate.

2) An isolated colony may then be "picked off" the isolation plate and
transferred to a fresh sterile medium in an aseptic manner. All organisms
in the new culture will be descendants of the same organism after
incubation, resulting in a pure culture.

5.3 REQUIREMENTS
a) Mixed culture of bacteria
b) Nutrient agar plates (3-4)
c) Inoculating loop
d) Empty sterile culture tubes with cotton plug
37
 Environment e) Sterile 9-ml water blanks
Science Lab
Course - 3 f) Sterile petri plates
g) Sterile 1-ml pipettes
h) Test tube rack
i) Water bath
j) Alcohol (95 percent)
k) Beaker (50 ml)
l) L-shaped bent glass rod spreader
m) Bunsen burner
n) Marker pen
Personal protective equipment
i) Gloves
ii) Apron
iii) Mask
iv) Shoe cover
v) Head cover

5.4 PROCEDURE
A. Streak Plate Method:

This approach is most typically used to isolate pure bacterium cultures. A

tiny quantity of mixed culture is streaked over the surface of the agar medium
using the tip of an inoculation loop/needle. The inoculum is suitably thinned
out by the consecutive streaks, and the microorganisms are isolated from one
another. Without re-inoculation, it's typically best to streak out a second plate
with the same loop/needle. These plates are incubated to enable colonies to
develop. The basic premise of this approach is that when bacterial cells are
deposited on the agar surface, streaking creates a dilution gradient across the
face of the Petri plate.

The streak plate is prepared using the loop. This entails gradually diluting of
a bacterium inoculum across the surface of solidified agar medium in a Petri
dish such that colonies develop well apart from one another.

1) Loosen the cap of the bottle containing the inoculum.

2) Hold the loop in the right hand.

3) Flame the loop and allow to cool.

4) Lift the bottle/test tube containing the inoculum with the left hand.

5) Remove the cap/cotton wool plug of the bottle/test tube with the little
finger of the right hand.
38
 Isolation of Pure
6) Flame the neck of the bottle/test tube. Culture by Streak
Plate Method, Pour
7) Insert the loop into the culture broth and withdraw. At all times, hold the Plate Method and
loop as still as possible. Spread Plate
Method

8) Flame neck of the bottle/test tube.

9) Replace the cap/cotton wool plug on the bottle/test tube using the little
finger. Place bottle/test tube on bench.

10) Partially lift the lid of the Petri dish containing the solid medium.

11) Hold the charged loop parallel with the surface of the agar; smear the
inoculum backwards and forwards across a small area of the medium
(see streaked area ‘1’ in figure 1 (A).

12) Remove the loop and close the Petri dish.

13) Flame the loop and allow it to cool. Turn the dish through 90°
anticlockwise.

14) With the cooled loop streak the plate from area ‘A’ across the surface of
the agar in three or four parallel lines (‘2’). Make sure that a small
amount of culture is carried over.

15) Remove the loop and close the Petri dish.

16) Flame the loop and allow to cool. Turn the dish through 90°
anticlockwise again and streak from ‘B’ across the surface of the agar in
three or four parallel lines (‘3’).

17) Remove the loop and close the Petri dish.

18) Flame the loop and allow to cool. Turn the dish through 90°
anticlockwise and streak loop across the surface of the agar from ‘3’ into
the center of the plate (‘4’).

19) Remove the loop and close the Petri dish. Flame the loop.

20) Seal and incubate the plate in an inverted position at 35 oC for 48-72
hours.

39
 Environment
Science Lab
Course - 3

B
Figure 5.1: Streak Plate Method

B. Pour Plate Method:

1) Label around the edge of the bottom (not the lid) of a sterile but empty
Petri dish with at least your name, date, type of growth medium, and the
type of organism to be added to the melted agar medium.
• Include the dilution factor if plating serial dilutions, or a series of
repeated dilutions, which results in a systematic reduction in the
concentration of cells in the sample. Preparing serial dilutions is
necessary if the number of cells in the sample exceeds the capacity
of the agar plate, in which the statistically significant range is 30 to
300 CFU. If there are more than 300 CFU on a plate, then the
colonies will be crowded and overlapping.
2) Obtain a tube containing 18 ml of melted agar medium.
• The agar medium should be dispensed into test tubes and pre-
sterilized in an autoclave. On the same day, it is needed for an
experiment, the agar should be melted in a steamer for 30 minutes
40
 then transferred to a 55 °C water bath. Only as much agar as is Isolation of Pure
Culture by Streak
needed for the experiment should be melted as it cannot be re-used. Plate Method, Pour
Plate Method and
• Ten minutes before pouring plates, the tubes of melted agar should Spread Plate
Method
be transferred from the 55 °C water bath to a heat block on the
laboratory bench set at 48 °C. Once the agar reaches this
temperature, it is ready to pour. If the agar is too hot, the bacteria in
the sample may be killed. If the agar is too cool, the medium may be
lumpy once solidified.
3) Obtain your sample, which should be either a broth culture or a
suspension of cells produced by mixing cells from a colony into buffer or
saline.
• The samples may be derived from a dilution series of a single sample.
• The sample volume to be plated should be between 0.1 and 1.0 ml.
4) Open the lid of the empty Petri dish, and dispense your sample into the
middle of the plate. Close the lid.
• Use aseptic technique throughout this procedure.
• Use either a serological pipette or micropipette to transfer your
sample to the plate. Control the flow of the sample so it does not
splash out of the plate.

5) Remove the cap from the tube of the melted agar, and pass the rim of the
open tube through the flame of the Bunsen burner.
6) Open the lid of the Petri dish containing your sample and pour the agar in
carefully. Close the lid then mix the sample with the agar by gently
swirling the plate.
7) Allow the agar to thoroughly solidify before inverting the plate for
incubation.

Figure 5.2: Pour Plate Method

41
 Environment
Science Lab
Course - 3

Pour plate method has certain disadvantages as follows:

i) The picking up of subsurface colonies needs digging them out of the agar
medium thus interfering with other colonies, and

ii) The microbes being isolated must be able to withstand temporary

exposure to the 42-45° temperature of the liquid agar medium; therefore,
this technique proves unsuitable for the isolation of psychrophilic
microorganisms.

However, the pour plate method, in addition to its use in isolating pure
cultures, is also used for determining the number of viable bacterial cells
present in a culture.

C. Spread Plate Method:

Figure 5.3: Spread Plate Method

a) Make a dilution series from a sample.

b) Pipette out 0.1 ml from the appropriate desired dilution series onto the
center of the surface of an agar plate.
c) Dip the L-shaped glass spreader into alcohol.
d) Flame the glass spreader (hockey stick) over a Bunsen burner.
e) Spread the sample evenly over the surface of agar using the sterile glass
spreader, carefully rotating the Petri dish underneath at the same time.
f) Incubate the plate at 35°C for 24 hours.

42
 Isolation of Pure
g) Calculate the CFU value of the sample. Once you count the colonies, Culture by Streak
multiply by the appropriate dilution factor to determine the number of Plate Method, Pour
Plate Method and
CFU/mL in the original sample. Spread Plate
Method
D. Procedure for Serial Dilution:

This method is commonly used to obtain pure cultures of those

microorganisms that have not yet been successfully cultivated on solid media
and grow only in liquid media.

A microorganism that predominates in a mixed culture can be isolated in pure

form by a series of dilutions. The inoculum is subjected to serial dilution in a
sterile liquid medium, and a large number of tubes of sterile liquid medium
are inoculated with aliquots of each successive dilution.

The aim of this dilution is to inoculate a series of tubes with a microbial

suspension so dilute that there are some tubes showing growth of only one
individual microbe. For convenience, suppose we have a culture containing
10 ml of liquid medium, containing 1,000 microorganisms i.e., 100
microorganisms/ml of the liquid medium.

If we take out 1 ml of this medium and mix it with 9 ml of fresh sterile liquid
medium, we would then have 100 microorganisms in 10 ml or 10
microorganisms/ml. If we add 1 ml of this suspension to another 9 ml. of
fresh sterile liquid medium, each ml would now contain a single
microorganism.

Figure 5.4: Preparation of serial dilution

43
 Environment
Science Lab 5.5 OBSERVATIONS AND CALCULATIONS
Course - 3
After incubation, examine each of the three plates for the growth of the
colonies.

Observations

Examine all the inoculated plates/slants or in the broth for the amount of
growth of the test organisms in the sterilized conditions by using the
sterilized media.

Sample Sample volume Colonies No. of

source taken observed after CFU/100ml
incubation
1
2
3
4

5.6 RESULTS
The number of bacterial colonies in Streak plate method = …………… cfu

The number of bacterial colonies in Pour plate method =………………… cfu

The number of bacterial colonies in Spread plate method = …………… cfu

5.7 PRECAUTIONS
Avoid pressing the loop or needle too firmly against the agar surface as this
will damage it.

1) Inoculating loop should be cooled by touching the agar surface away

from the set of streaks, before streaking of the inoculum.
2) Petri plate lid should never be lifted completely.
3) Plating of the medium should be done twenty-four hours in advance of
performing the exercise.
4) Use a fresh sterile pipette for each dilution.
5) The medium to be poured in the petri plates should have a temperature of
45oC.
6) While transferring a suspension from one tube to another it should be in
motion for the uniform distribution of cells.
7) The plate should be incubated in an inverted position to prevent
collection of condensation on the agar surface. Unless the surface is dry
it will difficult to obtain discrete surface colonies.
44
 Isolation of Pure
8) Always use the sterile glassware. Culture by Streak
Plate Method, Pour
9) Apply dilution factor before report the results Plate Method and
Spread Plate
10) Before processing the samples in laminar flow bench, sterile the surface Method
to remove the contamination.
11) Always on the UV light before and after processing the samples in
laminar flow bench.
12) Always were the lab coat, mask and gloves before start the analysis.
13) Always use the 70% alcohol for disinfect the working surface and after
complete dry the surface turn on the burner.

45
 Environment
Science Lab EXPT 6 ISOLATION AND
Course - 3
CHARACTERIZATION OF FUNGI
FROM SOIL SAMPLES

Structure

6.0 Introduction
6.1 Expected Learning Outcomes
6.2 Principle
6.3 Requirements
6.4 Procedure
6.5 Observations and Calculations
6.6 Result
6.7 Precautions

6.0 INTRODUCTION
The soil serves as a reservoir for many microbial communities of plants and
herbs which produces CO2 and nitrogen cycle. The microorganisms play
major role in soil ecosystem. Through breakdown of organic matter, nutrient
recycling, and biological control, microbial composition and function affect
soil quality. Soil is an oligotrophic medium for the growth of fungi. Fungal
growth is extremely limited for most of the time. These are readily available
for short periods and their presence is in a limited zone. For most of the part,
fungi are either dormant or they metabolized by utilizing a range of organic
molecules and grow very slowly. A fungus (plural: fungi or fungus) is
member of the group of eukaryotic organisms that includes microorganisms
such as yeasts and molds, as well as the more familiar mushrooms. These
organisms belong to a different kingdom from the other eukaryotic life
kingdoms of plants and animals. Fungi are heterotrophic eukaryotic
organisms, and with the exception of yeasts, are aerobic. The fungi distribute
organic matter away from the roots. In general, the concentration of microbes
is greatest close to the surface of roots (rhizosphere) and hyphae of
arbuscular mycorrhizal fungi (mycorhizosphere), where exudates are
extraordinarily important source of organic energy entering from soils. In
general, the concentration of microbes is closer to the surface of roots
(rhizosphere) and hyphae of arbuscular mycorrhizal fungi (mycorhizosphere),
where exudates are important source of organic energy source from soils.
E.g., the degrading of aromas is known in white rot fungi (Phanerochaete
chryosporium).

6.1 Expected learning Outcomes

After studying and performing this experiment you should be able to:
46
 Isolation and
1) Perform isolation of fungi from soil; Characterization of
Fungi From Soil
2) Describe identification of fungus utilizing various staining techniques; Samples
3) Determine the motility of fungi;
4) Describe the physiologic characteristics of isolated fungi.

6.2 PRINCIPLE
Fungi are not only attractive, but they also serve an important part in the daily
life of human existence. Their use is also in industry, agriculture, medicine,
food industry, textiles, bioremediation, natural cycles, as bio fertilizers, and
many others. Fungal biotechnology has become an essential component of
human well-being. Because soils typically contain millions of fungi per
gramme. The soil is typically created by suspending a given amount of soil in
a dispersing solution (often deionized water) and transferring aliquots of the
suspensions to fresh solution until the suspension is diluted sufficiently to
allow individual discrete fungal colonies to grow on agar plates. The plates
are incubated after inoculation on several replicate plates and counted when
they form macroscopic fungal colonies at a suitable temperature (Figure:1).
Since one fungal colony is formed from an organism, it is ultimately used as
the term "colony forming units" (CFUs) with the results expressed as CFUs
per gramme of dry soil of oven. The results are given in CFUs per gramme of
oven dried soil. Counts of culturable fungal spores, hyphae, or hyphal
fragments per gramme of dry soil have been recorded to be about 106 fungal
"propagules" (spores, hyphae, or hyphal fragments).

Figure 6.1: Fungal pour plate, with macroscopic colonies

Based on classification fungi are grouped into four divisions:

the Chytridiomycota (chytrids), Zygomycota (bread
molds), Ascomycota (yeasts and sac fungi), and the Basidiomycota (club
fungi).

6.3 REQUIREMENTS
• fresh soil sample
47
 Environment • deionized water
Science Lab
Course - 3 • Pipette
• pipette bulb
• plastic vial
• rubber bands
• plastic wrap
• benchtop balance (±0.01 g)
• sterile water blank (95 mL) per soil type
• sterile, 9 ml water blanks per soil type
• 150-ml Rose Bengal agar
• filter-sterilized streptomycin solution to bring the agar to 30mg/ml
• sterile Petri dishes per soil sample
• sterile, 1 ml pipettes per soil sample
• deionized water
• test tube stand
• pan for collecting excess agar
• vinyl gloves
• marking pens
• vortex
• water bath at 45°C to keep agar molten prior to pouring
• lactophenol mounting fluid
• pressure or transparent tape
• dissecting probe forceps
• microscope slides
• immersion oil
• microscope
• fungal identification key

Personal protective equipment

i) Gloves
ii) Apron
iii) Mask
iv) Shoe cover
v) Head cover

48
 Isolation and
6.4 PROCEDURE Characterization of
Fungi From Soil
Samples
A- Sample Preparation

1) Fill the sterile bottle halfway with 100 mL distilled water from the
graduated cylinder.

2) Weigh out 1 g of the soil sample and add it to the bottle of distilled
water. Close the bottle tightly and shake it vigorously to properly mix the
fluid.

3) Label the sterile test tubes 10-3, 10-4, 10-5, and 10-6 with the numbers 10-3,
10-4, 10-5, and 10-6. Using one of the pipettes, add 9 mL of distilled water
to each of the tubes.

4) Using a fresh pipette, transfer 1 mL of the solution from the bottle to the
tube labelled 10-3. Cap the tube and gently swirl it until the solution is
evenly distributed.

5) With a fresh pipette, transfer 1 ml of the solution from the 10-3 test tube
to the 10-4 tube. Swirl the 10-4 tube to combine it. Transfer solution from
the 10-4 tube to the 10-5 tube, then from the 10-5 tube to the 10-6 tube,
using this approach.

B- Isolation of fungi from soil sample:

6) From the 10-4, 10-5, and 10-6 tubes, plate three samples each. Transfer 1
mL of solution from the tube to a Petri plate using a fresh pipette. Pour
about 15 mL of nutritious agar into the plate, then cover with the lid and
gently swirl until the agar covers the whole bottom of the plate.

7) Using a fresh pipette, pour 1 ml of distilled water into a Petri plate to

create a control plate. Pour in the agar, cover, and spin the plate.

8) After the agar has set, keep the Petri plates upright. Then invert the plates
and incubate them for as little as 24 hours or as long as five days, either
in an incubator or at room temperature.

9) After the specified period of incubation time, remove the plates from the
incubator. Count the bacterial colonies on plates with 30 to 300 colonies
on them. To prevent counting the same colonies repeatedly, use a
permanent marker to indicate the colonies you've previously counted.

10) Divide the number of colonies counted by the dilution—10-4, 10-5 or 10-6
of the soil solutions for each plate. Find the number of cultivatable
bacteria in the original gram of soil by averaging the results from each
countable plate.

49
 Environment
Science Lab
Course - 3

Figure 6.2: Process for culturable heterotrophic plate counts of filamentous fungus

Figure 6.3: macroscopic fungal colonies that result from Incubation following soil
dilution and plating of soil

C- Counting fungal stains (after one week incubation)

1) Make colony counts at one dilution of each soil. Countable colonies

should be present on the plates being counted. Plates that have become
overgrown should not be counted. Similarly, plates with Take a close
look at all of the fungal plates. Take note of the size and form of the
colonies. Subtract any bacteria from the total number of actinomycete
50
 Isolation and
colonies (CFUs) for each plate. For each dilution, average the totals. Characterization of
Only count the plates of a dilution that can be counted (as for the Fungi From Soil
Samples
bacteria). For each of soils, calculate the sample mean of actinomycete
CFUs per gramme of dry soil.

2) Use the following process to prepare pressure tape (transparent tape)

mounts on slides for comprehensive microscope study:

• Using a clean glass slide, place a drop of lactophenol mounting fluid

in the Centre.

• From the stock roll, cut a 3 cm long strip of transparent cellophane

tape. When handling the tape, use forceps to prevent contaminating
the adhesive surface. The tape will be easier to remove from the
forceps with the help of a dissecting needle.

• The tape's adhesive side is placed to a sporulating fungal colony's

surface. Excessive pressure on the tape will result in a thick clump
of hyphae and spores being gathered.

• Remove the tape off the fungal colony and stick it to the drop of
mounting solution on the glass slide, sticky side down. To release air
bubbles, gently rub the tape with a smooth, flat tool.

Figure 6.4: Illustrations of fungal fruiting bodies, frequently separated from soil

51
 Environment 3) Examine the fungus under a microscope with an oil immersion objective.
Science Lab
Course - 3
4) Examine the mycelium of "rapid spreading" fungus on the plate
immediately under the microscope's 10 objective.

5) Using the fungal identification key provided, identify three distinct

fungal genera (Figure 6.4). Using drawings, describe and show the
fruiting bodies of these fungus.

6.5 OBSERVATIONS AND CALCULATIONS

Observations

The number of cells/spores per gram (milliliters) of the sample is calculated

by counting, averaging, and multiplying the number of colonies showing on
dilution plates by the dilution factor:

number of cells per gram dry soil =number of colonies X dilution factor
dry weight of soil (gm)

Where,

Number of colonies: average of the triplicate microbial counts,

dilution factor= Reciprocal of the dilution (e. g. 10-3 = 103).

Tabulate all of the findings, including individual plate counts and mean
counts, as shown below. Calculate the total number of filamentous fungus per
gramme dry weight of soil (in colony forming units (CFUs).

Table.1 The colony morphology of different species isolated from soil

sample

Colony Morphology
Colour Nature of hyphae Conidia
shape
1
2
3
4
5
6
7
8

52
 Isolation and
6.6 RESULTS Characterization of
Fungi From Soil
Samples
The number of fungal strains isolated in a given soil sample is …………cfu/g

Fungal Isolates isolated from soil sample on the basis of fungal identification
key as per figure 6.4 are as below:

6.7 PRECAUTIONS
1) Isolation of fungus should be done from a composite sample collected
randomly from a field.
2) Soil should be in a powdered form.
3) Before withdrawing an aliquot for future dilution, each dilution must be
vigorously shaken.
4) For each dilution, use separate sterile pipettes.
5) Immediately after the addition of the medium to a plate, inoculum is to
be mixed with circular and side to side movements of the Petri plates.
6) Always use the sterile glassware.
7) Before processing the samples in laminar flow bench, sterile the surface
to remove the contamination.
8) Always on the UV light before and after processing the samples in
laminar flow bench.
9) Always were the lab coat, mask and gloves before start the analysis.
10) Always use the 70% alcohol for disinfect the working surface and after
complete dry the surface turn on the burner.
11) To assure a countable number of colonies, prepare plates from three
sequential dilutions.
12) When plating different dilutions, change pipettes as needed.
13) During plating, gently spin the media to spread the soil inoculum without
putting media on the Petri dish lid.
14) Before flipping the plates, ensure sure the agar has solidified.
15) Only count one type of soil dilution.
16) Streptomycin should always be added to the medium.
17) Never add media to the soil inoculum when the temperature is too high,
since this can kill bacteria.
18) Allowing the medium to cool too much can cause it to harden, so keep it
at a temperature that is pleasant to touch.
62) Don't let excess media harden in flasks since it'll be tough to clean up.
63) Avoid squeezing the tape mounts too hard, since this may kill the fungal
fruiting bodies.

53
 Environment
Science Lab EXPT 7 DETERMINATION OF
Course - 3
METABOLIC ACTIVITIES OF
BACTERIA THROUGH
BIOCHEMICAL TESTS

Structure

7.0 Introduction
7.1 Expected Learning Outcomes
7.2 Principle
7.3 Requirements
7.4 Procedure
7.5 Observations and Calculations
7.6 Result
7.7 Precautions

7.0 INTRODUCTION
The procedure for storage of bacteria at the species level are insufficient.
Many gram negative and Gram positive stains of E. coli, P. aeruginosa, P.
vulgaris, E. aerogenes, and many other bacteria are indistinguishable. As a
result, additional procedures for identifying particular species are necessary.
Even while various bacterial species may seem similar under the microscope,
they all have unique genetic composition (different DNA) and hence develop
unique enzymes that enable them to perform a specific set of biochemical
activities. A specific metabolism is used for distinguishing each species from
others. Based on the appearance and metabolism, unknown bacteria may
identify from the other bacteria under the microscope and their metabolic
features. Biochemical assays are performed to distinguish between bacterial
species based on the biochemical activity. Some of the factors contributing
for bacterial identification include the differences in protein and fat
metabolism, carbohydrate metabolism, enzymes production, chemical
utilization abilities etc.

7.1 EXPECTED LEARNING OUTCOMES:

After studying and performing this experiment you should be able to:

1) Explain the importance of biochemical tests;

2) Describe how to perform the biochemical tests in the laboratory.

7.2 PRINCIPLE

54 Biochemical tests:
 Determination of
Biochemical tests are based on the biochemical activity of bacteria for Metabolic
different biochemical compounds on various bacteria in order to identify Activities of
Bacteria Through
them. It is important to understand that the reactions in responses might vary Biochemical Tests
depending on the incubation period, incubation temperature, the number of
bacteria in the initial inoculum, and how the medium is inoculated, among
other factors. An inoculum having a high number of bacteria, for example,
may elicit a strong positive reaction, whereas one containing a small number
of bacteria may only cause a mildly positive, or even a negative reaction. It is
also important that not all bacterial species will produce unambiguous
positive or negative findings on these medium. Since it is not necessary that
every differential media provides relevant findings for all bacterial species.
Therefore, the media must be carefully selected when unknowns are
identified. End products that change the pH of the medium are generated in
many metabolic assays. pH indicators (chemicals that change colour
according to pH) are included in the medium in order to measure this pH
shift. Phenol red, bromocresol purple, and bromothymol blue are other
typical pH indicators. Each pH indicator has a pH range that it changes
colour across,

pH Indicator Observation
phenol red < pH 6.8 = pH 6.8 – 7.4 pH >7.4 =
yellow = red pink/magenta
bromocresol < pH 6.8 = > 6.8 pH =
purple yellow purple
bromothymol blue < pH 6.0 pH 6.1 – 7.5 pH >7.5 = blue
= yellow = green

A. Carbohydrate Fermentation

Fermentation is a metabolic process used by certain bacteria to break down

glucose in the absence of oxygen. Fermentation involves reaction of
glycolysis (the breakdown of a single molecule of glucose into two molecules
of pyruvate) as well as other events that result in a range of end products
(acids, alcohols, gases). The end products are characteristics of individual
bacterial species.

Although glucose is always the final product of fermentation, certain bacteria

employ extra chemical processes to convert other monosaccharides and
disaccharides to glucose. As a result, bacteria may be distinguished based on
their capacity to ferment a variety of carbohydrates as well as the end
products of the fermentation process. A nutrient broth that contains a
fermentable carbohydrate (typically a monosaccharide or disaccharide),
peptone (amino acids), and a pH indicator is used to assess carbohydrate
fermentation. A nutrient broth that contains a fermentable carbohydrate
(typically a monosaccharide or disaccharide), peptone (amino acids), and a
pH indicator is used to assess carbohydrate fermentation. When employing
phenol red, the pH of the medium is adjusted to around 7.5, giving it an
55
 Environment orange/red appearance. A colour change to yellow will occur if the
Science Lab
Course - 3 carbohydrate in the medium is fermented and acidic end products are
produced. Occasionally, bacteria will not ferment the glucose, instead break
down proteins producing ammonia (NH3) in the growth media. The medium
will become more alkaline in this instance.

Figure 7.1: Carbohydrate fermentation

Some bacteria produce gases when fermenting a carbohydrate. To detect

these gases, a Durham tube is used. This is a small inverted tube that is
placed within the larger glass tube containing the fermentation medium. If
gases (typically CO2) are produced during the fermentation process, a bubble
will form at the top of the Durham tube. The medium will be acidic If bubble
is observed in the Durham tube. Carbohydrate fermentation media are often
used to differentiate members of the family Enterobacteriaceae (e.g.,
Escherichia coli, Enterobacter aerogenes) from each other.

B. Gelatin Hydrolysis

Gelatin is a protein generated from collagen, which are found in bones, skin,
and tendons. Robert Koch first used gelatin as a solidifying agent for growth
media. It is switched to agar when it is observed that gelatin does not stay
solid at temperatures above 28oC. some bacteria have the ability to
chemically break down. Some species of bacteria secrete an extracellular
enzyme (exoenzyme) called gelatinase (a type of proteinase), that breaks
gelatin down into its constituent polypeptides and amino acids. The small
amino acids can then be brought into the bacterial cell. This is why agar is
used as the solidification agent in bacterial growth media. Since gelatinase
breaks down connective tissue, it is sometimes called “spreading factor” and
contributes to the virulence of a pathogen.
56
 Determination of
H2 O H2 O Metabolic
Activities of
Gelatin Polypeptides Amino acids Bacteria Through
Biochemical Tests
gelatinase gelatinase

The presence of gelatinase can be detected by using nutrient gelatin deeps.

This medium contains gelatin and nutrients for bacterial growth. A positive
result is indicated by the gelatin becoming liquefied due to the enzyme
activity. It is important to note that if the nutrient gelatin deeps are incubated
at 37oC, then the medium must be cooled to below 25oC before a result can
be recorded.

C. Starch Hydrolysis

Starch is a polymer of glucose that is too large to be transported into bacterial

cells. For bacteria to be able to use the glucose in starch as an energy source,
the macromolecule must be broken down extracellularly into smaller
monosaccharide and disaccharide subunits. Some bacteria secrete the
extracellular enzyme (exoenzyme) amylase that breaks down starch into
monosaccharides and disaccharides (glucose and maltose). These smaller
molecules can then be transported inside the bacterial cell.

Starch Glucose + Maltose

Amylase

Starch agar is used to determine if bacteria can produce the amylase enzyme.
Bacteria that secrete amylase, break down the starch in the media that
surrounds the colony. Iodine is poured on the plate after the plate is
inoculated and incubated. The mixture of iodine and starch produces a dark
blue-black colour. The colour of the iodine persist in areas of the agar where
no starch is present (i.e., where starch has been broken down by amylase)
(light yellow or gold).

D. Casein Hydrolysis

Some bacteria secrete enzymes called proteinases that break down proteins.
Milk contains large proteins called caseins. Some bacteria secrete caseinases
that break down casein outside of the bacterial cell so the smaller products
(e.g., amino acids) can be transported inside the cell and further metabolized.
Milk agar (which contains powdered milk) is used to detect the presence of
bacterial caseinases. This medium is cloudy because when milk is mixed with
agar, the casein forms a colloid through which light cannot pass. A clearing
in the agar around the bacterial growth suggests that the caseins have been
broken down into transparent end products (amino acids and peptides), which
are subsequently taken up by the cells, indicating the existence of caseinases.

57
 Environment E. Urea Hydrolysis
Science Lab
Course - 3
Urea is a waste product that is produced when proteins are broken down. The
existence of a urease enzyme generated by certain bacteria is tested using
urea agar slants. Urea may be broken down into ammonia and carbon dioxide
by bacteria that generate ureases. The ammonia produced by the
decomposition of urea leads the slant's medium to become more alkaline,
which is recognised by the phenol red, which turns a hot pink colour.
Organisms that don't break down urea can develop on the slant, but the slant
will either stay the same colour or get yellower (due to the production of
acids).
H2 O
NH2CONH2 2NH3 + CO2
Urease

F. Citrate Utilization

Citrate permease is an enzyme that transports citrate molecules into cells of

organisms and that can survive using citrate as the only source of carbon.
Subsequently citrate is transformed into pyruvate, which can be converted
into variety of different products. Simmon's citrate is a chemically defined
medium that contain sodium citrate as the only carbon source and
bromothymol blue as a pH indicator. Bacteria that can thrive on this medium
(i.e., bacteria that can survive only on citrate as a carbon source) create
alkaline byproducts that turn the medium blue (pH neutral) (alkaline pH).

G. Catalase Activity

Two hazardous byproducts of aerobic metabolism are superoxide free

radicals (2O2 -) and hydrogen peroxide (H2O2). Cells produce enzymes to
break down these substances in order to eliminate them. The enzyme
superoxide dismutase (SOD) converts superoxide free radicals to hydrogen
peroxide, whereas catalase breaks down hydrogen peroxide into water and
oxygen.
Superoxide dismutase
2H+ + O2 - H2O2 + O2
Superoxide
free radical
Catalase
2H2O2 2H2O + O2

A simple test to determine if bacteria produce catalase is to add hydrogen

peroxide to bacteria on an agar slant or to bacteria spread on a slide. If
catalase is present, the hydrogen peroxide will be broken down into water and
oxygen gas, resulting in the production of bubbles. This test does not require
any special type of medium, however it should never be performed on
organisms that have been grown on blood agar (a medium that contains
blood). This is because there is a catalase activity in blood that would
58
 Determination of
produce a false positive result. Most aerobic and facultatively anaerobic Metabolic
organisms produce SOD and catalase (note: some species use peroxidase Activities of
Bacteria Through
rather than catalase to break down hydrogen peroxide). Obligate anaerobes Biochemical Tests
lack these enzymes, which is why they cannot survive in an atmosphere
containing oxygen.

H. Tryptophan Hydrolysis:

The Indole Experiment Most proteins include tryptophan as an amino acid.

Tryptophanase is an enzyme that breaks down tryptophan into indole,
ammonia, and pyruvate in bacteria. The pyruvate and ammonia are
transformed to other molecules, but the indole accumulates in the media and
may be identified.

Tryptophanase Fermentation

Tryptophan Indole + NH3 +Pyruvate

Respiration

The presence of indole shows that the enzyme tryptophanase is produced by

the organism. Kovac's reagent is a chemical that may be used to detect
indole. To identify indole, BBL dry slide is used. A dry reagent slide
impregnated with Kovac's reagent is used in this disposable slide. To enable
multiple tests on one slide, slide is separated into four broad portions. If
indole is present, a pink colour develops on the dry slide; if no indole is
present, no color change occurs.

Note: The organism must be cultured on tryptophan-containing media in

order to perform the indole test.

Indole + Kovac’s reagent + HCl + Amyl Alcohol

Rosindole dye (pink-red)

7.3 REQUIREMENTS
a) For carbohydrate fermentation test: 1-1 tube of the Lactose + phenol red,
Sucrose + phenol red, Glucose + phenol red and bacterial culture of
Proteus vulgaris, Escherichia coli, Bacillis subtilis

b) Gelatin Hydrolysis: 1 nutrient gelatin deep and bacterial culture of

Staphylococcus aureus, Serratia marcescens, Streptococcus faecalis, or
Bacillis subtilis

c) Starch Hydrolysis: 1 starch agar plate and bacterial culture of Proteus

vulgaris, Escherichia coli, Bacillus subtilis

d) Casein Hydrolysis: 1 milk agar plate and bacterial culture of Bacillus

subtilis and Enterobacter aerogenes

59
 Environment e) Urea Hydrolysis: 1 urea slant and bacterial culture of Escherichia coli or
Science Lab
Course - 3 Proteus vulgaris

f) Citrate Utilization: 1 Simmon’s citrate slant and bacterial culture of

Escherichia coli or Serratia marcescens

g) Catalase Activity: 1 TSA slant and bacterial culture of Staphylococcus

aureus or Streptococcus faecalis

h) Tryptophan Hydrolysis: 1 tryptone broth (medium that contains

tryptophan) and bacterial culture of Escherichia coli or Enterobacter
aerogenes

i) Forceps
j) Incubators
k) Microscope and light source
l) Alcohol
m) Sterile water

Personal protective equipment

i) Gloves
ii) Apron
iii) Mask
iv) Shoe cover
v) Head cover

7.4 PROCEDURE
Always ensure that to follow aseptic protocols and also everything is properly
labelled. Use the inoculation method recommended for each media type;
these methods may vary. To ensure that only introducing of one bacterial
species into medium, disinfecting the loop or needle completely between
inoculations should be done. A positive result cannot be attributed to a single
bacterial species when a medium is infected with many types of bacteria. Use
the appropriate bacterial species for each test is important.

A. Carbohydrate Fermentation

• Take 1 tube of each of the broths:

• Lactose + phenol red, Sucrose + phenol red, and Glucose + phenol
red
• Proteus vulgaris, Escherichia coli, Bacillus subtilis, or
Streptococcus faecalis ( 1 of any bacteria).
• Inoculate the three types of fermentation broth with selected
bacteria. Make a note of any little bubbles in the Durham tubes
60
 Determination of
before inoculating the broths so that they are not misinterpreted as Metabolic
signs of gas generation during fermentation. Activities of
Bacteria Through
B. Gelatin Hydrolysis Biochemical Tests

• take 1 nutrient gelatin deep

• Use 1 of the following bacteria: Staphylococcus aureus, Serratia
marcescens, Streptococcus faecalis, or Bacillis subtilis
• To obtain some bacteria from stock culture, use an inoculation
needle (not a loop).
• Inoculate the gelatin deep by using the inoculating needle to stab
~3/4 of the way down into the nutrient gelatin and then drawing the
needle straight back up.

C. Starch Hydrolysis

• Take 1 starch agar plate

• Use all of the following bacteria: Proteus vulgaris, Escherichia coli,
Bacillus subtilis
• Divide starch agar plate into 4 areas using a wax pencil or a sharpie
marker.
• Make sure that the draw will be on the bottom of the petri dish (the
part that contains the agar) rather than on the lid.
• to indicate which bacterium will be inoculated into each area should
be properly labeled
• One area is left as a negative control. Using a loop, do spot
inoculations of each bacterial species in the areas. A spot inoculation
is used to ensure that a large number of bacteria will grow at a single
location and produce a concentrated amount of amylase.

D. Casein Hydrolysis

• take 1 milk agar plate

• Use both bacteria, Bacillus subtilis and Enterobacter aerogenes
• Divide milk agar plate into 3 areas using a wax pencil or a sharpie
marker.
• Label the plate to indicate which bacterium will be inoculated into
each area.
• One area is left as a negative control.
• Using a loop, do spot inoculations of each bacterial species in the
areas, just as you did on the starch agar plate.

E. Urea Hydrolysis

• Take 1 urea slant

• Use one of the bacteria, Escherichia coli or Proteus vulgaris 61
 Environment • To inoculate the urea slant, use a loop to obtain some bacteria from
Science Lab
Course - 3 the culture, and then carefully streak the surface of the slant.
• Do not stab down into the slant’s interior. Replace the cap on the
slant, but leave the cap somewhat loose.
• Wrap in the tube in aluminum foil.

F. Citrate Utilization
• Take 1 Simmon’s citrate slant
• Use one of the bacteria, Escherichia coli or Serratia marcescens
• Using an inoculation needle (not a loop), inoculate the Simmon’s
citrate by first stabbing the needle into the butt of the agar slant and
then streaking the surface of the slant before you pull the needle out
of the tube.

G. Catalase Activity
• Take 1 TSA slant

• Use one of the bacteria, Staphylococcus aureus or Streptococcus

faecalis
• Using a loop, inoculate the surface of the TSA slant. Make sure to
use a heavy inoculum.

H. Tryptophan Hydrolysis
• Take 1 tryptone broth (Note: this medium contains tryptophan)
• Use one of the bacteria, Escherichia coli or Enterobacter aerogenes
• Inoculate the medium with any chosen bacteria.

7.5 OBSERVATIONS
Results may record for the metabolic tests:

A. Carbohydrate Fermentation: look at the results of the carbohydrate

fermentation test. Compare inoculation tubes to the negative controls.
Record the findings as per table. When recording the outcomes of
fermentation studies, adopt the following convention. Acid = A G is the
letter G. The abbreviation AG denotes the presence of acid and gas.
"negative" can be used when there is neither acid nor gas is present.

Bacteria Glucose Lactose Sucrose

Proteus vulgaris
Escherichia coli
Bacillus subtilis
Streptococcus
faecalis

62
 Determination of
B. Gelatin Hydrolysis: Observe gelatin deeps and record results in the Metabolic
table Activities of
Bacteria Through
Biochemical Tests
Bacteria Liquid or solid Result (+/-)
Staphylococcus aureus
Serratia marcescens
Streptococcus faecalis
Bacillus subtilis

C. Starch Hydrolysis: Fill the plate with enough Gram's iodine to cover the
whole growing region as well as the agar surrounding it (one should also
add iodine to the negative control area). After added iodine, make sure
that don't tip the plate too much. Observe the appearance of plate and
record findings in the table.

Bacteria Color change? Starch present or Presence (+) or

(Y/N) absent? absence (-) of
amylase
Proteus
vulgaris
Escherichia
coli
Bacillus
subtilis
Negative
control

D. Casein Hydrolysis: Observe casein plate. It is helpful to hold it up in the

light to detect the clear zones. Indicate the location of the bacterial
growth and draw any clear zones that are present. Record your results in
the table.

Bacteria Clear zone? Result (+/-)

A. Enterobacter aerogenes
B. Bacillus subtilis
C. Negative control

E. Urea Hydrolysis: Observe urea slants. Record the results in the table.

Bacteria Clear zone? Result (+/-)

Escherichia coli
Proteus vulgaris

63
 Environment F. Citrate Utilization: Observe your Simmon’s citrate slants. Record the
Science Lab
Course - 3 results in the table.

Bacteria Color Growth? Result (+/-)

Escherichia coli
Serratia marcescens

G. Catalase Activity: Add a dropper full of H2O2 to the surface of the slant.
Record the results.

Bacteria Presence of Result (+/-)

bubbles
Staphylococcus aureus
Streptococcus faecalis

H. Tryptophan Hydrolysis: Perusal inoculated culture to stir up the

bacteria. Use a sterile swab to obtain some bacteria. Make sure to swirl
the swab around in the broth to pick up a large quantity of bacteria. Rub
the swab on one area of the BBL Dry Slide. Record any color changes
that occur between 30 – 60 seconds. Do not wait too long before
observation of result, as false positives can occur if results are not read
right away. Record results in the table.

Bacteria Color Result (+/-)

Escherichia coli
Enterobacter aerogenes

7.6 RESULTS
Results for Metabolic Tests performed in the laboratory:
Bacteria Lactose Gelatin Amyl Urea Citrate Catal Tryptopha
Fermenta ase ase se permea ase nase
tion se (Indole
(Simmo test)
n’s
citrate)
Bacillus
subtilis

Corynebacte
rim xerosis

Enterobacte
r aerogenes

Escherichia
coli

64
 Determination of
Micrococcus Metabolic
luteus Activities of
Bacteria Through
Biochemical Tests
Pseudomona
s aeruginosa

Proteus
vulgaris

Staphyloco
ccus
aureus
Streptococc
us faecalis
Serratia
marcescens

7.7 PRECAUTIONS
1) Always use the sterile glassware.

2) Before processing the samples in laminar flow bench, sterile the surface
to remove the contamination.

3) Always on the UV light before and after processing the samples in

laminar flow bench.

4) Always wear the lab coat, mask and gloves before start the analysis.

5) Always use the 70% alcohol for disinfect the working surface and after
complete dry the surface turn on the burner.

65
 Environment
Science Lab EXPT 8 DETERMINATION OF
Course - 3
BIOLOGICAL ACTIVITY OF SOIL
BY DEHYDROGENASE ASSAY

Structure

8.0 Introduction
8.1 Expected Learning Outcomes
8.2 Principle
8.3 Requirements
8.4 Procedure
8.5 Observations and Calculations
8.6 Result
8.7 Precautions

8.0 INTRODUCTION
Soil microorganisms produce wide range of enzymes and they constitute
natural catalysts for a range of important soil processes, including
decomposition of organic matter, formation and decomposition of humus, the
release of mineral substances and making them available to plants, molecular
nitrogen fixation, as well as the xenobiotics detoxification. Therefore,
enzymes may be useful in controlling the effects of pollution on the soil
environment. Soil quality and its degradation depend on a large number of
physical, chemical, biological, microbiological and biochemical properties,
the last two being the most sensitive since, they respond rapidly to changes.
The microbiological activity of a soil directly influences ecosystem stability
and fertility and widely accepted that a good level of microbiological activity
is essential for maintaining soil quality. The soil microbiological activity viz.,
the enzymatic activities play a key role in soil nutrient cycling, its activity is
essential in both the mineralization and transformation of organic matters and
plant nutrients in soil ecosystem.

Natural and anthropogenic disturbance to soil enzyme activity is exceedingly

sensitive, and they respond quickly. As a result, changes in soil quality
induced by environmental stress or management practices may be detected
using enzyme activity. These soil enzymes play an essential role in the
establishment process and development of plant cover in biogeochemical
cycles. Because it gives insights into the relative changes that occur in below-
ground system functioning as a plant community expands, therefore it is an
important aspect. In soil, enzyme activity is produced by the activity of
accumulated enzymes and from enzymatic activity of proliferating
microorganisms.

66
 Determination of
8.1 EXPECTED LEARNING OUTCOMES Biological Activity
of Soil By
Dehydrogenase
After studying and carrying out this experiment, you should be able to : Assay

1) Explain dehydrogenase enzyme;

2) Determine the biological activity of soil by dehydrogenase assay.

8.2 PRINCIPLE
There are lots of enzymes in the soil environment, such as Oxidoreductases,
Hydrolases, Isomerases, Lyases and Ligases. Each enzymes plays key role in
biochemical functions during the overall process of material and energy
conversion. Oil dehydrogenases are the major representatives of the class
Oxidoreductase enzymes. Among all enzymes in the soil environment,
dehydrogenases are one of the most important, and are used as an indicator of
overall soil microbial activity because they occur intracellularly in all living
microbial cells. Moreover, they are tightly linked with microbial
oxidoreduction processes. Determination of DHA in the soil samples gives us
large amount of information about the biological characteristic of the soil.
Although dehydrogenases are capable of using oxygen and other electron
receptors, most anaerobic microorganisms are produced. In other words, soil
DHA strongly increases under anaerobic conditions. Dehydrogenase Activity
(DHA) varies in different types of land use.

8.3 REQUIREMENTS
• Soil sample (fresh)-10 gm
• Sieve
• Test tubes
• Phosphate buffer
• Calcium carbonate
• Triphenyltetrazolium chloride (TTC)
• tetrazolium salts
• Methanol
• Mechanical Shekar
• Spectrophotometer
• NADH,
• INT,
• Tris,
• calcium chloride and
• tetrahydrofuran
• incubated soils from Period 1
67
 Environment • methanol
Science Lab
Course - 3 • fume hood
• 10 ml graduated cylinder
• 1 filtration funnel for each plastic vial
• Whatman® #42 filter paper
• stand for holding the funnels
• 50 ml volumetric flasks
• 5ml pipette
• pipette bulb
• cuvettes
• spectrophotometer
• tissues for wiping cuvettes
• container for collection methanol and TTC wastes

Personal protective equipment

• Gloves
• Apron
• Mask
• Shoe cover
• Head cover

8.4 PROCEDURE
Dehydrogenase enzymes are found in all living organisms. These enzymes
play active part in a number of reactions involving the transfer of electron
pairs. In catabolic processes, such as the transformation of complex or high-
energy molecules to simpler or low-energy molecules, dehydrogenases
catalyze the transfer of electron pairs from a substrate to NAD+, yielding
NADH. NADH, which works as an electron transfer intermediary, then
transfers the electrons to another molecule. NADP+ participates in anabolic
responses (the reverse of catabolic reactions). When two hydrogen atoms
"tag" along to keep the charge balanced, electrons are transferred.

Dehydrogenases are important in aerobic species in the electron transfer

pathway; hence their activity is a proxy for both respiration and total
metabolic activity. The assay involves incubating soil mixed with a solution
of the competitive NAD+ inhibitor 2,3,5-triphenyltetrazolium chloride (TTC)
and incubated with soil in air-tight containers to keep oxygen out. The TTC
serves as the last electron acceptor during respiration. The soil should be
freshly collected as dehydrogenase test results are adversely affected by
storage, even at 4°C. When oxygen is removed from TTC, electron transfer
occurs, resulting in the formation of the insoluble red dye triphenyl formazan
68
 Determination of
(TPF) from the pale yellow, water-soluble compound (Figure 8.1). The Biological Activity
results are compared against formazan standard curve generated in methanol of Soil By
Dehydrogenase
and reported in milligrams of formazan per gramme of soil. Assay

Figure 8.1 dehydrogenase facilitating an electron transfer

Normal Reaction In the presence of dehydrogenase, NAD+ is reduced to

NADH by transferring two electrons and two hydrogen ions from a variety of
molecules (only one of the two hydrogen ions actually participate in the
reaction and, therefore, only one is depicted here).

Natural Coenzyme The molecular changes of NAD+ after its reduction to

NADH are represented in this diagram. In the presence of the dehydrogenase
enzyme, the incoming H+ (in extra bold face font) is decreased. Other
chemicals are reduced using NADH.

Synthetic Coenzyme In a similar way as NAD+, analogue TTC 2,3,5-

Triphenyltetrazolium chloride (TTC) receives one H+ and two electrons. In
the presence of dehydrogenase, TTC is converted to triphenyl formazan,
which functions as a competitive NAD+ inhibitor. The triphenyl formazan,

69
 Environment unlike NADH, is a metabolic dead end. This poisons the system, but because
Science Lab
Course - 3 of the red hue, it is feasible to detect metabolic activity.

• For each soil, weigh 6 g (dry weight) in each of 4 plastic vials.

• Add 0.5 percent glucose to two duplicate vials (dry weight basis). There
will be no changes to two samples.
• By adding no soil to make a blank. This will provide four vials of soil
(one for each soil type) and one blank vial.
• Fill each vial with 1 mL of 3 percent TTC and 2.5 mL deionized water,
except the blank.
• Using a glass stirring rod, combine the soil and liquid, then cap the vials
(the vials need to be sealed against oxygen from the air). At the soil
surface, a tiny quantity of free liquid should occur.
• Leave the vials to incubate for one week.
• Add 10 mL methanol to each vial, mix, and transfer the suspension to a
funnel lined with Whatman #42 filter paper. In a 50 mL Erlenmeyer
flask, collect the filtrate.
• With two more 10 ml methanol rinses, wash the vial and the sediment-
containing funnel until the filtrate is clean of red colour. To make the
whole volume 50 ml, add extra methanol.
• The data for the standard curve may be found in Table. Because these
data are repeatable, standardization is not required each time the test is
run.
• If the absorbance values do not fall within the calibration range in Table,
dilute a sub-aliquot of the extract in a volumetric flask with methanol.
• Fill a cuvette with 5 mL of each sample. Using the blank as the zero,
read the absorbance of each sample using a spectrophotometer at 485
nm.
• Between samples, rinse the cuvettes with 1–2 ml methanol rinses.
• The results should be expressed in g TPF g-1 dry soil.
• Calculate the average of the two duplicates and report it.
• Display the data in a table showing the values for each soil by
amendment.

A standard curve can be developed from absorbance readings of standard

solutions.

Standard curve data and equation for (TPF) using visible absorption
spectrophotometry at l= 485nm. A is the absorbance value. X = the
concentration of TPF analyzed in the solution analyzed in the
spectrophotometer in mgmL-1

70
 Determination of
Table 2: Data for preparation of a standard curve. Biological Activity
of Soil By
X ([TPF] in mgml-1) Absorbance (485nm) Dehydrogenase
Assay
0.00 0.000
5.00 0.214
10.0 0.423
15.0 0.642
20.0 0.833
25.0 1.051
30.0 1.244

8.5 OBSERVATIONS AND CALCULATIONS

Observations:

S. No. Soil Sample Absorbance at 485nm (A)

1. Sample 1
2. Sample 2
3. Sample 3

The values obtained are compared against a formazan (Calbiochem) standard

curve prepared with methanol and they are reported as milligrams of
formazan per gram of soil.

Calculation:

Using the equation in Table 1,

X= A - 0 00629
0 0415
Where,
A= Absorbance of the soil sample at 485nm.
X = the concentration of TPF analyzed in mgTPFg-1 soil.

8.6 RESULTS
Dehydrogenase activity per gram dry soil is expressed in terms of milligram
formazan per gram dry soil.

The DHA activity in a given soil sample =………………….. mgTPFg-1 soil

8.7 PRECAUTIONS
1) Ensure all caps on the vials are tightly sealed to preclude oxygen entry.

71
 Environment 2) Do not contaminate the methanol extracts with soil during filtration. This
Science Lab
Course - 3 will affect spectroscopic readings
3) Avoid absorption of methanol through skin
4) Avoid ingestion of methanol through the mouth
5) Avoid skin contact with TTC.

72
 Isolation of
EXPT 9 ISOLATION OF BIO- Bio-Surfactant
Producing Bacteria
SURFACTANT PRODUCING From
Environmental
BACTERIA FROM Samples

ENVIRONMENTAL SAMPLES

Structure

9.0 Introduction
9.1 Expected Learning Outcomes
9.2 Principle
9.3 Requirements
9.4 Procedure
9.5 Observations and Calculations
9.6 Result
9.7 Precautions

9.0 INTRODUCTION
Biosurfactants are amphiphilic compounds produced extracellularly by
microorganisms on cell surfaces, or excreted extracellularly. They have
hydrophilic and hydrophobic drips, which lower surface and interfacial
tension at the surface and interface, respectively.

Biosurfactants are surface active molecules that have several applications in

petrochemical, food and cosmetics industries, besides an important role in
environmental protection, oil spills control, biodegradation, and
detoxification of oil contaminated industrial effluents and soil.

Biosurfactants (BSs) as surface-active compounds have several unique

properties- when compared to other synthetic surfactants, it has a number of
advantages, including ease of manufacture, multifunctionality, better
biodegradability, and reduced toxicity. These compounds are primarily
biosynthesized as secondary metabolites and play critical roles in
microorganism growth and location. Based on the chemical structure of
hydrostatic component, BSs are classified into four categories: (1) glycolipid,
(2) fatty acid, (3) lipopeptide, and (4) polymer based. Glycolipid BSs are
potentially used commercially due to their high yield from renewable
resources and wide range of applications. The yields of glycolipid BSs
(sophorose lipids (sophorolipids, SLs) and mannosylerythritol lipids (MELs),
can exceed 100 g L-1 by the yeasts Starmerella bombicola and Pseudozyma
antarctica. In addition, glycolipid BSs show excellent surface-active
properties, including emulsifying, dispersing, solubilizing, foaming,
penetrating, and wetting actions, together with self-assembly and unique
biochemical properties.
73
 Environment Increasing environmental concerns, the advance in biotechnology and the
Science Lab
Course - 3 emergence of more stringent laws have led to biosurfactants being a potential
alternative to the chemical surfactants available on the market. Although
biosurfactants have promising use in bioremediation processes, their
industrial scale production is currently difficult due to high raw-material
costs, high processing costs and low manufacturing output. As a result, the
current research challenges are to increase the yield and to reduce the cost of
raw materials. Biosurfactants can potentially replace virtually any synthetic
surfactant and, moreover, introduce some unique physico-chemical
properties. Currently, the main application is for enhancement of oil recovery
and hydrocarbon bioremediation due to their biodegradability. The use of
biosurfactants has also been proposed for various industrial applications, such
as in food additives, cosmetics, detergent formulations and in combinations
with enzymes for wastewater treatment.

9.1 EXPECTED LEARNING OUTCOMES

After studying and performing this experiment you should be able to:

1) Explain isolation of Bio-Surfactant Producing bacteria.

2) Describe the Oil spreading test.
3) Perform the Drop collapse test.
4) Explain Bacterial adhesion to hydrocarbon (BATH) assay.
5) Explain the calculation of Emulsification index (E24) of isolated bacteria.

9.2 PRINCIPAL
Biosurfactants are surface-active biomolecules generated by microorganisms,
and they have a wide range of uses. Surface-active biomolecules have
received attention in recent years due to their unique features such as
selectivity, low toxicity, and relative simplicity of synthesis. For their
development, microorganisms need a variety of organic substances as a
source of carbon and energy. When the carbon source is in an insoluble form,
such as a hydrocarbon, microorganism produces a several substances,
biosurfactants and can spread into the cell. Ionic surfactants are excreted by
certain bacteria and yeasts, which emulsify the CxHy material in the growth
medium. Rhamnolipids generated by several Pseudomonas spp. and
sophorolipids produced by Torulopsis spp. are two examples of this group of
biosurfactant.

Other microbes have the ability to alter the structure of their cell walls by
creating nonionic or lipopolysaccharides surfactants in their cell walls.
Rhodococcus erythropolis, Mycobacterium spp., and Arthrobacter spp., all of
which create nonionic trehalose corynomycolates, are examples of this
category. Lipopolysaccharides such as emulsan generated by Acinetobacter

74
 Isolation of
spp., and lipoproteins such as surfactin and subtilisi, produced by Bacillus Bio-Surfactant
subtilis. Producing Bacteria
From
Environmental
Table 1. Major types of biosurfactants Samples

Bio surfactant class Microorganisms Applications

Glycolipids Rhamnolipids P. aeruginosa and Bioremediation
P. putida
P. chlororaphis Biocontrol agent
Bacillus subtilis Antifungal agent
Renibacterium Bioremediation
salmoninarum
Sophorolipids Candida Emulsifier,
bombicola and C. MEOR, alkane
apicola dissimilation
Trehalose lipids Rhodococcus spp. Bioremediation
Tsukamurella sp. Antimicrobial
and Arthrobacter agent
sp.
Mannosylerythritol Candida antartica Neuroreceptor
lipids antagonist,
antimicrobial
agent
Kurtzmanomyces Biomedical
sp application
Lipopeptides Surfactin Bacillus subtilis Antimicrobial
agent, biomedical
application
Lichenysin B. licheniformis Hemolytic and
chelating agent

Biosurfactants are involved in bioremediation in two ways : increasing the

surface area of hydrophobic water-insoluble substrates and increasing
the bioavailability of hydrophobic compounds.

9.3 REQUIREMENTS
• Contaminated Soil sample (fresh)-10 gm
• Non contaminated soil samples
• Water samples
• Nutrient broth
• Nutrient agar
75
 Environment • Autoclave
Science Lab
Course - 3 • Laminar Flow Bench
• pH meter
• Balance
• Bacterial Incubator
• Test tubes
• Petri plates
• Erlenmeyer flask (250ml)
• shaker incubator
• minimal salt medium
• Filter paper (0.45µm pore size)
• Triton X-100 as a positive control for Oil spreading test
• Crude oil 1 ml
• ZMA plates
• Kerosene, Hexadecane, Benzene and Toluene.
• 2-(4-iodophenyl)-3-(4-nitrophenyl)-5- phenyltetrazolium chloride (INT)
• Spectrophotometer

Personal protective equipment

• Gloves
• Apron
• Mask
• Shoe cover
• Head cover

9.4 PROCEDURE
A. Sample Collection:

Collect two water samples and two soil samples contaminated with any
hydrocarbon sources or samples nearby of petrol pumps.

A. Media composition and preparation

In order to isolate the biosurfactant producing bacteria, prepare nutrient broth

(dehydrated), nutrient agar medium (dehydrated as per strength required and
autoclave the media at 121 degree C for 15 Minutes.

Prepare minimal salt medium (in g/L) by adding 15g NaNO3, 1.1g KCl, 1.1g
NaCl, 0.00028g FeSO4.7H2O, 3.4g KH2PO4, 4.4g K2HPO4, 0.5g
MgSO4.7H2O, 0.5g yeast extract in 1 litre distilled water and sterile the

76
 Isolation of
media. For MSM agar media use 1.5 % agar powder in MSM liquid media Bio-Surfactant
and autoclave the media at 121 °C for 15 Minutes. Producing Bacteria
From
Environmental
B. Isolation of bacteria Samples

• Use water and soil samples and serially dilute and inoculate by spread
plate method on nutrient agar medium.
• Incubated under aerobic conditions at 37°C for 24 hours.
• Enrich at 37°C in shaker incubator (100 rpm) for 24 Hrs.
• Observe the colony morphology on nutrient agar.

C. Screening of bacteria

• Grow the bacterial isolates aerobically in 500 ml Erlenmeyer flask with

100 ml of mineral salt medium containing (gl-1) 1.0 K2HPO4, 0.2
MgSO4.7H2O, 0.05 FeSO4.7H2O, 0.1 CaCl2.2H2O, 0.001
Na2MoO4.2H2O, 30 NaCl and crude oil (1.0%, w/v).

• Inoculate a loopful of bacterial culture grown in crude oil containing

nutrient agar plates in sterilized mineral salt medium and keep in a
shaker for 7 days at 200 rpm and 30 °C.

• incubate for 7 days and centrifuge culture broth from each flask at 6000
rpm for 15 minutes at 4oC and filter the supernatant through 0.45µm pore
size filter paper.

• Use cell culture supernatant for drop collapse assay, oil spreading assay,
emulsification assay and surface tension measurement and the bacterial
cells will be used for BATH assay.

D. Oil spreading test: (Oil spreading experiment)

• Add 20 ml of distilled water in a Petri dish followed by the addition of

20 µl of crude oil to the surface of the water.

• Add 10 µl of cell free culture broth to the oil surface.

• Observe the presence of biosurfactant, if present in the cell free culture

broth, the oil will be displaced with oil free clearing zone and diameter
of this clearing zone indicates the surfactant activity, also called oil
displacement activity.

• Use distilled water (without surfactant), as a negative control, in which

no oil displacement or clear zone was observed and Triton X-100 as the
positive control.

77
 Environment
Science Lab
Course - 3

Oil Spreading Assay Drop collapse assay

Hydrocarbon overlay agar test

E. Drop collapse test: (Drop-collapse test)

• Add 2 microlitres of crude oil to the regions delimited on the covers of

96- well micro plates and leave to equilibrate for 24 hours.

• Transfer Five drops of culture after of the 48 hours to the oil-coated well
regions

• Observe drop size after 1 min with magnifying glass.

• Drop with flat surface consider as a positive for biosurfactant production

and cell culture with rounded drops as negative, indicative of the lack of
biosurfactant production.

F. Hydrocarbon overlay agar:

• Use Zobell Marine Agar (ZMA) plates coated individually with 40

microlitre of kerosene, hexadecane, benzene and toluene.

• Pure bacterial isolates spotted on these coated plates.

• Incubate the plates for 7-10 days at 28°C.

• Observe the colony surrounded by an emulsified halo consider positive

for biosurfactant production.

G. Bacterial adhesion to hydrocarbon (BATH) assay: Measure the cell

hydrophobicity by bacterial adherence to hydrocarbons.

• Wash the cell pellets twice and suspend in a buffer salt solution (g/L,
16.9 K2HPO4 and 7.3 KH2PO4) and diluted using the same buffer
solution to an optical density (OD) of ~ 0.5 at 610 nm.

78
 Isolation of
• Add 100 µl of crude oil in the cell suspension (2 ml) in test tubes (10 ml Bio-Surfactant
volume with 10 x 100 mm dimension) and vortex-shaken for 3 min. Producing Bacteria
From
• After shaking, allow to separate crude oil and aqueous phases for 1 hour. Environmental
Samples
• Measure OD of the aqueous phase at 610 nm in a spectrophotometer.
• From the OD values, calculate the percentage of cells attached to crude
oil by using the following formula:

% of bacterial cell adherence = (1-(OD shaken with oil/OD original)) x

100

Where:

OD shaken with oil - OD of the mixture containing cells and crude oil,

OD original - OD of the cell suspension in the buffer solution (before

mixing with crude oil)

• Add few drops of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-

phenyltetrazolium chloride (INT) solution to the above BATH assay
solution and

• Observe under a light microscope. The INT turned red if it reduces inside
the cells, indicating the viability of the cells adhered to the crude oil
droplets.

H. Calculation of Emulsification index (E24):

• Several colonies of pure culture are suspended in test tubes containing 2

ml of mineral salt medium, after 48 h of incubation;
• add 2 ml petroleum to each tube.
• Allow the mixture to vortex at high speed for 1 min and leave to stand
for 24 hours.
• Compare the results with SDS as positive control.

9.5 OBSERVATIONS AND CALCULATIONS

Observe the test perform by bio-surfactant producing bacteria isolated from
the sample collected from various studied location. Record the results of the
tests performed in the laboratory as below:

1) Oil spreading test:

2) Drop collapse test:
3) Hydrocarbon overlay agar
4) Bacterial adhesion to hydrocarbon (BATH) assay:
% of bacterial cell adherence = (1-(OD shaken with oil/OD original)) x
100

79
 Environment Where:
Science Lab
Course - 3 OD shaken with oil - OD of the mixture containing cells and crude oil,
OD original - OD of the cell suspension in the buffer solution (before
mixing with crude oil)
5) The emulsion index (E24)
Emulsification index= (Height of the emulsion layer/Total height) ∗100

9.6 RESULTS
1) Oil Spreading Assay:
2) Hydrocarbon overlay agar:
3) Bacterial adhesion to hydrocarbon (BATH) assay:
4) Emulsification index (E24):

Emulsification indexes (E24) of water isolate (W1) and soil isolate (S1)

Description E24 (%)

W1
W2
S1
S2
SDS (Positive
control)

9.7 PRECAUTIONS
1) Always use the sterile glassware.

2) Before processing the samples in laminar flow bench, sterile the surface
to remove the contamination.

3) Always on the UV light before and after processing the samples in

laminar flow bench.

4) Always wear the lab coat, mask and gloves before start the analysis.

5) Always use the 70% alcohol for disinfecting the working surface and
after completely drying the surface, turn on the burner.

80
 Isolation and
EXPT 10 ISOLATION AND ENUMERATION Enumeration of Air
Borne
OF AIR BORNE Microorganisms by
All Glass Impinger
MICROORGANISMS BY ALL Techniques

GLASS IMPINGER TECHNIQUES

Structure

10.0 Introduction
10.1 Expected Learning Outcomes
10.3 Principle
10.3 Requirements
10.4 Procedure
10.5 Observations
10.6 Result
10.7 Precautions

10.0 INTRODUCTION
Bioaerosols are microbes that are suspended in the air. For transit and
survival, pathogenic bioaerosols rely on atmospheric physicochemical
parameters such as temperature, humidity, sun radiation, wind, precipitation,
and air pressure. Today, airborne microbe concentration and composition are
of interest in a variety of situations, including agricultural and industrial
settings, hospitals, home and office environments, and so on. Microbes are
generally found in the atmosphere between 300 and 10,000 feet above the
ground. Tuberculosis is one of the most economically damaging infectious
illnesses in the world, owing to particularly high mortality rates in
underdeveloped nations. The causal agent, Mycobacterium tuberculosis, has
been proven to survive in air for up to 6 hours. When expelled from the lung,
the bacterium's tiny size (1-5m range) permits it to remain suspended in air
inside bioaerosols, where it is subject to prevailing aerodynamics and may be
transferred to a susceptible host by inhalation especially in a poorly ventilated
interior space.

Alternaria, Cladosporium, Penecillium, and Aspergillus are fungal spores

found in the air over 4000 feet from the surface, in both polar and non-polar
air masses. Respiratory allergies and pathogenic diseases of the respiratory
tract are induced by particulate matter in our environment. Due to the
presence of fungus, aeroallergens can trigger allergic responses in humans.
Allergies, asthma, and pathogenic infections of the respiratory tract are all
caused by airborne particles, which are a primary cause of respiratory
diseases in people. Fungal spores in the air are also key disease agents for
plants and the way by which many common saprotrophic (saprophytic)
fungus spread. Millions of small droplets of water and mucus are ejected at 81
 Environment around 200 miles per hour (100 meters per second) during a sneeze. The
Science Lab
Course - 3 droplets are roughly 10-100 micrometers in diameter at first, but they quickly
dry down to droplet nuclei of 1-4 micrometers, holding virus particles or
bacteria. This is a primary mode of human disease transmission.

Table 10.1 Some important diseases of humans transmitted from person to

person by inhaled airborne particles

Virus diseases (virus type) Bacterial diseases (bacterial name)

Chickenpox (Varicella) Whooping cough (Bordetella pertussis)

Flu (Influenza) Meningitis (Neisseria species)

Diphtheria (Corynebacterium
Measles (Rubeola)
diphtheriae)

Pneumonia (Mycoplasma pneumoniae,

German measles (Rubella)
Streptococcus species)

Tuberculosis (Mycobacterium
Mumps (Mumps)
tuberculosis)

Fungi are typically present as spores in the air. Spores can withstand harsh
environmental conditions because they have a thicker cell wall, protective
small molecules (sugars, amino acids, sugar alcohols, and betaine), and the
expression of heat shock proteins. These characteristics allow fungal spores
to survive in the air for longer than their vegetative counterparts.

Table 10.2: Some examples of air borne pathogenic microorganisms:

Type Disease Etiology

Pulmonary anthrax Bacillus anthracis
Bacteria Tuberculosis Mycobacterium tuberculosis
Legionella Legionella pneumophila
Coccidiodomycosis Coccidioides immitis
Fungi Cryptococcosis Cryptococcus neoformans
Blastomycosis Blastomyces dermatitis

10.1 Expected Learning Outcomes

After studying and performing this experiment you should be able to:

1) Identify the main types of organisms found in the air;

2) Describe the differences between aerosols, fomites, and droplets;
3) Explain testing of samples for airborne bacteria and fungus, using the
spread plate and membrane filtering procedures;

82
 Isolation and
4) Describe the process of calculating the number of bacteria and fungus in Enumeration of Air
each cubic meter of air. Borne
Microorganisms by
All Glass Impinger
10.2 PRINCIPLE Techniques

Microbes are omnivorous and they are generally found in soil, water, and the
living organism activities. Microflora of air is highly dynamic and is affected
by temperature, wind speed, moisture/humidity, pollution, and other human
and animal activity. They are vital because their presence in the air can harm
people's health, may cause spoilage, encourage composting, and
biodegradation, etc. Using sampling and isolation techniques the qualitative
and quantitative estimation of microorganisms present in the air is possible to
check. In the exposure plate technique, media plates are exposed to air for a
defined amount of time, after which the microbial flora settles on the plate.
When plates are incubated, microbe colonies form on the surface, which may
then be purified and identified. 'AIR' is one of the most important microbial
reservoirs. Microbes float freely in the air or are adhered to the surfaces of
small particles. Intact cells/colonies, spore bodies, and/or mycelium or
filament pieces are among them. Air samples or the exposure plate technique
can be used to isolate these components.

Coughing, sneezing, wave action, splashing, wind, cooling towers,

ventilation systems, and other natural and human-made activities all
contribute to the formation of bioaerosols. Human exposure to airborne
microbes can occur by inhalation, ingestion, or skin contact, however
inhalation is the most common route that causes harm to humans. Aerosols
are known to transmit Legionella pneumophila, Mycobacterium TB, and
certain dangerous viruses. Exposure to bioaerosols has also been linked to
asthma, hypersensitivity pneumonitis, and other respiratory disorders.

The two most critical elements impacting microbe survival in the airborne
state are temperature and relative humidity. As a result, they are frequently
recorded during bioaerosol collection. As the rate of evaporation increases,
which occurs as relative humidity decreases and temperature rises, bacteria
and fungi become more stressed. Thus, higher relative humidity and lower
temperatures support improved survival. The effect of relative humidity
varies and greatly depend on the virus type, with certain viruses lasting better
at high relative humidity and others at low relative humidity.

There are three main ways for determining the number of microorganisms in
the air.

• Impingement- The entrapment of airborne practices in a liquid matrix

• Impaction- The forceful deposition of airborne particles on a solid
surface
• Filtration - the process of capturing airborne particles by size exclusion.

83
 Environment Gravity is a non-quantitative approach that involves exposing agar media to
Science Lab
Course - 3 the atmosphere and collecting airborne bacteria mostly by settling. This
approach is popular since it is affordable and simple to use.

10.3 REQUIREMENTS
• All glass impinger AGI-30
• 1 37-mm air monitoring cassette
• 20 ml of 0.1% peptone solution
• 1 500- or 1000-ml Erlenmeyer flask
• rubber or plastic tubing for connecting the impinger and cassette to the
vacuum source
• 1 vacuum pump or vacuum source
• 1 100 ml sterile graduated cylinder
• 1 dilution blank with 0.1% peptone or phosphate buffered saline
• 2 1-ml pipettes
• 1 10-ml pipette
• sterile 0.45m pore, 47-mm diameter membrane filters
• 1 filter unit (same as in Experiment 10)
• 2 sterile 37 mm 0.45mm pore filters
• vacuum pump or other source
• forceps
• gas burner
• pipette bulb
• vortex mixer
• 4 nutrient agar (NA)1 or trypticase soy agar (TSA)1 plates
• 4 Sabouraud dextrose agar (SDA)1 plates
• Culture media,
• Petri dishes,
• Stop watch,
• Glass marker,
• Water-bath, incubator.
• Colony counters, etc.

Personal protective equipment

• Gloves
• Apron
• Mask
84
 Isolation and
• Shoe cover Enumeration of Air
Borne
• Head cover Microorganisms by
All Glass Impinger
10.4 PROCEDURE Techniques

The AGI-30 is a popular liquid impinger (ACE Glass, Vineland, NJ). The
AGI-30 works by sucking air into a liquid through the intake. Any airborne
particles settle in the liquid, which may subsequently be tested for the
presence of microorganisms. Flow rates of 12.5 litres per minute are typical
for the AGI-30. In compared to many other sample devices, the AGI-30 is
simple to use, affordable, portable, dependable, and quick to sterilise. It also
has a high biological sampling efficiency. The normal collection amount is
20 mL, and the sample time is around 20 minutes. Longer sample durations
cause excessive evaporation of the liquid in the impinger, resulting in
inactivation or death of microorganisms in the liquid.

A sterilised petri dish is plated with 15-20 mL of appropriate melting culture

medium (Potato-dextrose-agar medium for fungi isolation, Nutrient agar
medium for bacteria isolation, Thornton media for actinomycetes isolation)
and left to settle for 30 minutes on the laboratory desk. Following that, each
petri dish is labelled with the kind of isolation used and the length of time it
was exposed to the air.

The petri dishes are exposed to air for 1, 2, 3, 4, and 5 minutes, respectively.
Respectfully (as per their marking). During each plate's exposure, the upper
cover is completely removed for the duration of the exposure. After that, each
petri dish is incubated for 2–5 days or more to see if colonies develop. The
colony counter then observes and counts the colonies (Fig. 10.1). There
should be at least five replicas for each re-enactment.

Various shapes of bacterial colony

A wide variety of bioaerosol sampling and analysis methods are available and
new methods are being developed.

A) Air Sampling

• Set up the AGI-30 all glass impinger

85
 Environment • Add 20 ml of 0.1% peptone to the reservoir followed by 0.1 ml of anti-
Science Lab
Course - 3 foam.
• Add 0.1 ml of anti-foam agent.
• Turn the vacuum source on for 10 minutes.

• With a 1-ml pipette remove 0.5 ml of fluid from the reservoir and place
0.1 ml each on one agar plate of either Nutrient Agar or Trypticase Soy
Agar and spread plate the samples as described in Experiment 5. Place
another 0.1 ml on a plate of Sabouraud dextrose agar for detection of
fungi.

• With a 10 ml pipette remove 6 ml of liquid from the reservoir and pass 5

ml through a 0.45mm membrane filter as described in Experiment 10.
Place the filter on an NA or TSA plate. Repeat the procedure, but place
the membrane filter on Sabouraud dextrose agar.

• Incubate the NA or TSA plates at 35°C for 24–48 hours.

• Incubate the SDA for 2 to 7 days.

10.5 OBSERVATIONS AND CALCULATIONS

Number of bacteria (NA or TSA) and fungi (SDA) colonies are examined on
the agar plates

The number of bacteria and fungi is calculated as per cubic meter. The AGI-
30 limiting orifice at the end of the glass tube, which is submerged into the
collection liquid, limits the amount of air passing through the liquid to 12.5
liters per minute. The concentration of microorganisms is usually reported as
numbers per cubic meter of air, which is calculated as follows.

The number of colonies appearing on dilution plates are counted, averaged

and multiplied by the dilution factor to find the number of cells/spores per
gram (milliliter) of the sample:

A) Volume of air (L) = Sample time (min)X 12.5 L/min

B) Number of organisms = Number of organisms in volume assayed

impinger after (CFU/ml) X ml remaining in impinger in operation

C) Number of organisms per L of air (CFU)= Number of organisms

collected by impinger Volume of air

10.6 RESULTS
Number of organisms per L of air is………………………………….. cfu/L

86
 Isolation and
10.7 PRECAUTIONS Enumeration of Air
Borne
Microorganisms by
1) Isolation of microorganisms should be done from a composite sample All Glass Impinger
collected randomly from a field. Techniques

2) Soil should be in a powdered form.

3) Each dilution must be thoroughly shaken before removing an aliquot for
subsequent dilution.
4) Use separate sterile pipettes for each dilution.
5) Immediately after the addition of the medium to a plate, inoculum is to
be mixed with circular and side to side movements of the petri plates.
6) Always use the sterile glassware.
7) Apply dilution factor before report the results
8) Before processing the samples in laminar flow bench, sterile the surface
to remove the contamination.
9) Always on the UV light before and after processing the samples in
laminar flow bench.
10) Always use the 70% alcohol for disinfect the working surface and after
complete dry the surface turn on the burner.
11) Prepare plates from three successive dilutions to ensure getting a
countable number of colonies
12) Change pipettes appropriately when plating different dilutions
13) Gently swirl the media during plating to distribute the soil inoculum
without getting media on the Petri dish lid
14) Check to make sure the agar has hardened before inverting the plates
15) Count only one dilution of the soil
16) Always add the streptomycin to the media
17) Don’t allow the media to cool too much since it will solidify, i.e., media
temperature should be comfortable to the touch
18) Don’t allow excess media to solidify in flasks as this makes clean up
difficult
19) Don’t press too hard on the tape mounts as this will destroy fungal
fruiting bodies
20) Always were the lab coat, mask and gloves before start the analysis.

87
 Environment
Science Lab EXPT 11 DETECTION OF COLIFORM IN
Course - 3
THE WATER SAMPLES BY
MEMBRANE FILTER TECHNIQUE

Structure

11.0 Introduction
11.1 Expected Learning Outcomes
11.2 Principle
11.4 Requirements
11.4 Media preparation
11.5 Procedure
11.6 Observations and Calculations
11.7 Result
11.8 Precautions

11.0 INTRODUCTION
Coliforms include bacteria that are found in the soil and water and has been
influenced by human or animal waste. Total coliforms are a group of related
bacteria that are (with few exceptions) not harmful to humans.

Pathogens, like bacteria, parasites, and viruses are known as potential cause
of health problems. As per Environment Protection Agency (EPA), total
coliforms are useful indicator of other pathogens for drinking water. Primary
Water Quality Criteria developed under the provision of Water Act, 1974,
total coliform in water and waste water is an important parameter to assess
the designated best uses of drinking water and outdoor bathing water quality.
Coliform bacteria are present in the environment and feces of all warm-
blooded animals and humans. The presence of Coliform bacteria in drinking
water indicates that disease-causing organisms (pathogens) could be in the
water system. Most pathogens which contaminate water come from the
feces of humans or animals.

Testing of drinking water for all possible pathogens is complex, time-

consuming, and expensive. Coliform bacteria test is easy and inexpensive. If
coliform bacteria present in a water sample, the source of water
contamination systems may be searched and restore safe drinking water.
Total coliform is a collection of different kinds of bacteria. Fecal coliform are
types of total coliform that exist in feces. E. coli is a subgroup of fecal
coliform. Drinking water samples is tested for total coliform test in
laboratories. If total coliform is present in water sample, test for presence of
E. coli is also checked.

88
 Detection of
11.1 EXPECTED LEARNING OUTCOMES Coliform in the
Water Samples by
Membrane Filter
After studying and performing this experiment you should be able to: Technique

1) Explain coliforms as an indicator microorganism;

2) Describe the media used for estimation of coliform from water samples
by membrane filtration technique;
3) Explain the importance of estimating total coliform;
4) Perform estimation of total coliform and fecal coliform in water samples;
5) Explain the precautions for microbiological analysis;
6) Describe the advantages and disadvantages of the Membrane Filter (MF)
technique.

11.2 PRINCIPLE
Coliform bacteria have long been used as water-quality indicators. Based on
the premise that, these organisms are present in the intestines of warm-
blooded animals, their presence in water could indicate that recent fecal
contamination has occurred.

This group of organisms has been defined by their ability to ferment lactose.
This group consists of bacteria from several genera belonging to the family
Enterobacteriaceae.

Several methods have been approved by Environmental Protection Agency

(EPA) for coliform detection: the multiple-tube fermentation technique; the
membrane filter technique and the presence/absence test (including the
ONPG-MUG test). The Membrane Filter (MF) technique is reproducible and
is used to test relatively large sample volumes. It usually yields numerical
results more rapidly than the multiple-tube fermentation procedure. The MF
technique is useful in monitoring drinking water and various natural waters
but has limitations, particularly when testing waters with high turbidity or
large numbers of non-coliform bacteria. The coliform group is defined as
facultative anaerobic, gram-negative, non-spore-forming, rod-shaped bacteria
that develop colonies with distinctive characteristics on specific media in MF
technique. When purified cultures of coliform bacteria are tested, they
produce negative cytochrome oxidase and positive galactosidase test
reactions.

11.3 REQUIREMENTS
a) Water sample
b) 20 ml tube of m-Endo broth
c) Pipets and graduated cylinders
d) Containers for culture medium
89
 Environment e) Culture dishes
Science Lab
Course - 3 f) Filtration units (base, funnel and clamps)
g) Membrane filters
h) m Endo agar
i) m FC agar
j) Absorbent pads
k) Forceps
l) Incubators
m) Microscope and light source
n) Alcohol
o) Sterile water

Personal protective equipment

i) Gloves

ii) Apron

iii) Mask

iv) Shoe cover

v) Head cover

11.4 MATERIALS AND CULTURE MEDIA

The need for uniformity dictates the use of commercial dehydrated media.
Never prepare media from basic ingredients when suitable dehydrated media
are available. Follow manufacturer’s directions for rehydration. Store opened
supplies of dehydrated media in a desiccator. Commercially prepared media
in liquid form (sterile ampule or other) may be used if known to give
equivalent results.

Agar preparation-Rehydrate product in 1 L water containing 20 mL 95%

ethanol. Do not use denatured ethanol, which reduces background growth and
coliform colony size. Avoid sterilizing by autoclave. Heat to near boiling to
dissolve agar, promptly remove from heat, and cool to between 45 and 50°C.
Dispense 5- to 7-mL quantities into 60-mm sterile glass or 4- to 6-mL
quantities into 50-mm plastic petri dishes. Final pH should be 7.2 ±0.2. A
precipitate is normal in Endo-type media. Refrigerate finished medium in the
dark, and discard unused agar after 2 weeks or sooner if there is evidence of
moisture loss, medium contamination, medium deterioration (darkening of
medium), or surface sheen formation].

Broth preparation- Prepare as above, omitting agar. Dispense liquid

medium (at least 2.0 mL per plate) onto sterile absorbent pads and carefully
remove excess medium by decanting plate. The broth may have a precipitate
90
 Detection of
but this does not interfere with medium performance if pads are certified free Coliform in the
of sulfite or other toxic agents at concentrations that could inhibit bacterial Water Samples by
Membrane Filter
growth. Refrigerated broth in screw capped bottles or flasks may be stored Technique
for up to 96 h.

11.5 PROCEDURE
a. Selection of sample size: Sample size will be governed by expected
bacterial density, degree of turbidity and, if applicable, regulatory
requirements.

Table 11.5: Suggested sample volumes for membrane filter Total

coliform test

Water source Volume (X) to be filtered (ml)

100 50 10 1 0.1 0.01 0.001 0.0001
Drinking water X
Swimming pools X
Wells, springs X X X
Lake, reservoirs X X X
Water supply X X X
intake
Bathing beached X X X
River water X X X X
Chlorinated X X X
sewage
Raw sewage X X X X

An ideal sample volume will yield 20 to 80 total coliform colonies and ≤200
colonies of all type on a membrane-filter surface. Analyze drinking waters by
filtering 100 mL or replicates of smaller sample volumes (e.g., duplicate 50-
mL portions or four replicates of 25-mL portions). Analyze other waters by
filtering three different volumes (diluted or undiluted), depending on the
expected bacterial density. When filtering <10 mL of sample (diluted or
undiluted), add approximately 10 mL sterile buffered dilution water to the
funnel and then add sample followed by another 25 to 50 mL dilution water
before filtration or pipet the sample volume into sterile dilution water and
then filter the entire contents of dilution bottle. This increase in water volume
helps disperse the bacterial suspension uniformly over the entire effective
filtering surface.

b. Filtration of sample:

• Use sterile filtration units at the beginning of each filtration series as a

minimum precaution to avoid accidental contamination.
91
 Environment • Using sterile forceps, place a sterile membrane filter (grid side up) over
Science Lab
Course - 3 porous plate of the base (Figure 11.1). Carefully place matched funnel
unit over base (lock it in place, if applicable).

• Thoroughly mix sample or dilution(s) of sample by vigorously shaking

(e.g., 25 times up and down in a 1 ft arc in 7 s) to break up clumps of
bacteria.

• Filter sample under partial vacuum (commonly used pressure: 81 kPa, 24

in. Hg, or 79% vacuum). With filter still in place, rinse interior surface of
funnel by filtering three 20- to 30-mL portions of sterile buffered dilution
water from a squeeze bottle (or another appropriate device). This is
satisfactory only if squeeze bottle and its contents do not become
contaminated during use. Do not reuse partially filled dilution water
bottles. Rinsing between samples prevents carryover contamination.

• When final rinse and filtration are complete, use aseptic technique to
disengage vacuum, unlock and remove funnel, immediately remove
membrane filter with sterile forceps, and roll filter onto selected medium
to avoid entrapping air.

• Incorrect filter placement is instantly obvious because patches of

unstained membrane indicate entrapped air. If such patches occur,
carefully reseat filter on agar surface.

• Place only one membrane filter per dish. Invert dish, and incubate at 35
±0.5°C for 22 to 24 h for m-Endo LES or m-Endo MF.

• Optionally, to sanitize funnels between samples after filter removal,

expose all surfaces of previously cleaned and sterilized assembly to UV
radiation for 2 min before reusing units for successive filtrations.

• If using broth, aseptically place a sterile pad in the culture dish, saturate
it with at least 2.0 to 3.0 mL of medium (depending on pad
manufacturer), and carefully remove excess medium by gently decanting
it from dish into a disposable terry towel or empty dish.

• Place sample filter directly on pad, invert dish, and incubate as specified
above. If loose-lidded dishes are used, place them in a humid chamber
(or humidified incubator). Differentiation of some colonies may be lost
if cultures are incubated >24 h.

• For non-potable water samples, funnels should be rinsed or sanitized

with UV after filter removal or after each sample because of the high
number of coliform bacteria and background flora present in these
samples.

92
 Detection of
Coliform in the
Water Samples by
Membrane Filter
Technique

Figure 11.1: Filtration apparatus needed in this experiment

• Check for sterility and coliform contamination at the beginning and end
of each filtration series, respectively, by filtering 20 to 30 mL of dilution
or rinse water through the filter (one funnel per sterilization batch). If
controls indicate contamination, reject all data from affected samples and
use new samples.

c. Counting: To count Colony-Forming Units (CFU) on Endo type

membrane filters, use a low-power (10 to 15X magnification) binocular
wide-field dissecting microscope or other optical device, with a cool
white fluorescent light source directed to provide optimal viewing of
shine. The typical coliform colony on Endo-type media has a pink to
dark-red color with a metallic surface shine. Count both typical and
atypical coliform colonies promptly after incubation. The shine area may
vary in size from a small pinhead to complete coverage of the colony
surface. A typical coliform colony may be dark red, mucoid, or nucleated
without shine. Generally pink, blue, white, or colorless colonies lacking
sheen are considered non-coliforms. A high count of non-coliform
colonies (>200 CFU) may interfere with the maximum development of
coliforms. Samples of disinfected water or wastewater effluent may
include stressed organisms that grow relatively slowly and produce
maximum sheen in 22 to 24 h.

d. Coliform verification: On Endo-type media, non-coliform organisms

may produce typical sheen colonies, and a typical colony (pink, dark red
93
 Environment or nucleated colonies without sheen) may be coliforms; thus verification
Science Lab
Course - 3 of all typical and atypical colony types is recommended (Figure 2).
Verify all colonies on Endo media by swabbing the entire membrane or
picking at least five typical colonies and five atypical colonies from a
given membrane filter culture.

Figure 11.2: Colonies visible after incubation period

11.6 OBSERVATIONS AND CALCULATIONS

Examine filter paper disc under a dissection microscope or by the colony
counter for the presence of coliform and count their number.

Calculation of Coliform Density

Select the membrane(s) with acceptable number of colonies and≤200 colony-

forming units (CFU) of all types per membrane, by the following equation:
Coliform colonies counted
(Total) coliforms, No./ 100 mL = ————————————— × 100
ml sample Filtered

= No. CFU/100 ml

For drinking water samples, if no total coliform colonies are observed, then
report the total coliform colonies counted as “<1 CFU/100 mL” or report
“total coliform bacteria absent per 100 mL sample.”

For non-potable water samples, if 10.0-, 0.1-, and 0.01-mL portions are
examined and all counts are 0, then calculate the number of coliforms per 100
mL that would have been reported if there had been 1 CFU on the filter
representing the largest filtration volume. For example, report <10 CFU/100
mL for a 10 mL sample volume with no coliform colonies, i.e, 1/10 X 100 =
<10 CFU/100 mL

94
 Detection of
Observations Coliform in the
Water Samples by
Examine all the inoculated plates/slants or in the broth for the amount of Membrane Filter
growth of the test organisms in the sterilized conditions by using the Technique

sterilized media.

Sample Sample volume Colonies No. of

source taken observed after CFU/100ml
incubation
1
2
3
4

11.7 RESULTS
The number of coliform estimated in a given environmental sample is
………….. cfu/100ml

11.8 PRECAUTIONS
1) Always use the sterile glassware.

2) Carefully prepare the dilution series when using the wastewater samples
or polluted samples

3) Apply dilution factor before report the results

4) Before processing the samples in laminar flow bench, sterile the surface
to remove the contamination.

5) Always on the UV light before and after processing the samples in

laminar flow bench.

6) Always were the lab coat, mask and gloves before start the analysis.

7) Always use the 70% alcohol for disinfect the working surface and after
complete dry the surface turn on the burner.

8) Use sterile filtration units at the beginning of each filtration series as

precaution to avoid accidental contamination.

95
 Environment
Science Lab EXPT 12 DETERMINATION OF THE
Course - 3
EFFECT OF INDUSTRIAL
EFFLUENTS ON SEED
GERMINATION AND SEEDLING
GROWTH OF RICE AND WHEAT

Structure

12.0 Introduction
12.1 Expected Learning Outcomes
12.3 Principle
12.4 Requirements
12.5 Procedure
12.6 Observations
12.7 Result
12.8 Precautions

12.0 INTRODUCTION
The chemical compounds found in industrial wastewater are usually well-
defined and easily identifiable. The effluent generated by various processes in
industries is discharged directly into nearby water bodies and agricultural
fields without any treatment. In addition to the aforementioned pollutants,
industrial effluents contain nitrates, nitrites, fluorides, arsenic, copper, zinc,
nickel, detergents, hydrocarbons, and radio nucleosides. Those factors can
lead to a decrease of pH, an increase in temperature, a rise in biological
oxygen demand (BOD), a fall in chemical oxygen demand (COD), turbidity,
heavy metals, and toxic chemicals. Untreated effluent released by any
industry into the surface or water bodies can lead to irreversible
environmental changes. The objective of the experiment is to determine the
effect of treated and untreated effluents on seed germination.

12.1 EXPECTED LEARNING OUTCOMES

After studying and performing this experiment you should be able to:

1) Describe the process of the seed germination experiment in the

laboratory;

2) Determine the effect of treated and untreated effluent on seed

germination.

96
 Determination of
12.2 PRINCIPLE The Effect of
Industrial Effluents
on Seed
Industrial waste water is generally used for irrigation purposes which is Germination and
highly warranted utility of water pollutants proportion. Utilizing waste water Seedling Growth of
Rice and Wheat
for irrigating crop plants has two goals. Safe disposal of industrial effluents is
the first and foremost important aspect. Leaking effluents may have a
detrimental effect on the environment and human health. The second aspect
is to recycle industrial effluents for irrigation purposes and as compost for
fertilizer. There is an adverse effects associated with the toxic effluent that
adversely affects germination, root elongation, root growth rate, shoot
elongation, stem growth, panicle emergence, as well as filling of grain (rice,
maize, wheat, etc).

12.3 REQUIREMENTS
Effluent samples (two different types) A, B.
i) Seeds ( Rice and wheat)
ii) Petri dishes(20 Nos.) or pots (20 Nos)
iii) Distilled wates (as a blank)
iv) Measuring cylinder

Personal protective equipment

i) Gloves
ii) Apron
iii) Mask
iv) Shoe cover
v) Head cover

12.4 PROCEDURE
Sample collection and preparation of dilutions:

• Collect two different Effluent/wastewater of untreated and one treated

effluent / wastewater from each type.
• Prepare two concentrations (100 and 50%) of untreated and treated
effluent along with control (distilled water) and run this experiment for
one week.
• Grow wheat and rice seeds in these effluent samples.
• Set four experiments as below:

First set (for germination of rice seeds)

A. Control (distilled water)

B. Untreated effluent (A) (50 and 100%)
97
 Environment C. Treated effluent (A) (50 and 100%)
Science Lab
Course - 3
Second set (for germination of wheat seeds)

A. Control (distilled water)

B. Untreated effluent (A) (50 and 100%)
C. Treated effluent (A) (50 and 100%)

Third set (for germination of rice seeds)

A. Control (distilled water)

B. Untreated effluent (B) (50 and 100%)
C. Treated effluent (B) (50 and 100%)

Fourth set (for germination of wheat seeds)

A. Control (distilled water)

B. Untreated effluent (B) (50 and 100%)
C. Treated effluent (B) (50 and 100%)

Pretreatment of seeds and setup the experiment:

• Wash rice and wheat seeds with hypochlorite solution then soaked
overnight in the test solutions (effluent).

• Conduct the experiment for concentrations of untreated and treated

effluent viz., 50 and 100% v/v for each treatment.

• Keep overnight soaked seeds on the petri plates (15 cm diameter)

containing butter paper soaked with respective test solutions for each set
of treatment.

• Add 2 ml of solution, each untreated and treated effluent of two different

source in in each Petri plates and 100% distilled water as a blank,
respectively.

• Repeat twice and germination in each experimental set.

• Plates were incubated at 28 ± 10C. After 48 hrs, calculate germination

percentage by counting the number of germinated seeds, which refers to
the normal seedlings along the plumule, radicle, and length
measurements. Record the results.

12.5 OBSERVATIONS
Observe the number of germinated seeds in each set of experiment and record
normal seedlings along the plumule, radicle length in the table below from 1
to 4.

98
 Determination of
Table 1: Effect of untreated and treated effluent (A) on seed germination The Effect of
of Rice seeds Industrial Effluents
on Seed
Germination and
Treatment 50% 100% Seedling Growth of
Rice and Wheat
Germina Plumule Radical Germina Plumule Radical
tion (cm) (cm) tion (cm) (cm)
(%) (%)

Untreated
effluent (A)
treated
effluent
(7d)
Control

Table 2: Effect of untreated and treated effluent (A) on seed germination

of wheat seeds

Treatme 50% 100%

nt Germination Plu Radical Germina Plumule Radi
(%) mule (cm) tion (cm) cal
(cm) (%) (cm)

Untreated
effluent
(A)
treated
effluent
(7d)
Control

Table 3: Effect of untreated and treated effluent (B) on seed germination

of Rice seeds
Treatment 50% 100%
Germination Plumule Radic Germination Plumule Radic
(%) (cm) al (%) (cm) al
(cm) (cm)

Untreated
effluent
(B)
treated
effluent
(7d)
Control
99
 Environment Table 4: Effect of untreated and treated effluent (B) on seed germination
Science Lab
Course - 3 of wheat seeds
Treatment 50% 100%
Germination Plumule Radic Germination Plumule Radic
(%) (cm) al (%) (cm) al
(cm) (cm)

Untreated
effluent
(B)
treated
effluent
(7d)
Control

Above figure showing the Germination in different wastewater treatments for

reference to perform the experiments.

12.6 RESULTS
1) The percent of seed germination was found maximum in ………dilution
2) The percent of seed germination was found minimum in ………dilution
3) Maximum growth in terms of plumule and radical length was found
maximum in …………………………………dilution
4) Minimum growth in terms of plumule and radical length was found
maximum in …………………………………dilution

12.7 PRECAUTIONS
1) Prepare the dilution of effluent with distilled water.
2) Before start the experiments, soaked the seeds overnight in their
respective dilutions.
3) Carefully measure the length of plumule and radical length due to very
sensitive.

100
 Composting of
EXPT 13 COMPOSTING OF Biodegradable
Waste and
BIODEGRADABLE WASTE AND Determination of
Compost Quality
DETERMINATION OF COMPOST
QUALITY

Structure

13.0 Introduction
13.1 Expected Learning Outcomes
13.2 Principle
13.3 Requirements
13.4 Procedure
13.5 Observations and Calculations
13.6 Result
13.7 Precautions

13.1 INTRODUCTION
Waste is now commonly considered as one of the world's most serious
environmental issues. Waste generated by collective activities of human
beings, waste generation and proper management is a need to focus on.
Appropriate waste management is one of the specific aim of environmental
protection. Waste is interdisciplinary problem and interlinked with many
other environmental and health issues.

Composting is a recent method of municipal waste treatment and

management. It is an exothermic biological oxidation process in which the
organic substrate undergoes aerobic biodegradation under the influence of
microorganisms in increasing temperature and humidity condition. The
organic substrate undergoes physical, chemical, and biological modifications
during biodegradation, resulting humus as stable end product. This product is
useful for agriculture purposes: it may be used as an organic fertilizer and as
a resource to improve the soil structure.

Composting is a natural phenomenon, where aerobic biological degradation

of organic materials involved under managed conditions. Composting is the
biological breakdown of organic components such as vegetative fragments
and livestock manures and /or agricultural waste etc. and produce a nutrient-
rich soil-like substance. Composting can increase soil productiveness,
magnitude of fertilizer, conserve water, minimize plant diseases, and promote
soil fertility. Composting is a rapidly growing industry as the recycling of
organic waste like vegetation and food waste minimizes the amount of
garbage and ultimately goes to landfill. The residual compost has been
described as a stable, sanitized, humus-like substance rich in organic matter
101
 Environment and devoid of objectionable odors that results after the composting process of
Science Lab
Course - 3 separating collected bio-waste.

Composting is the natural process of decomposition and recycling of organic

materials into humus rich soil which is known as compost.

13.1 EXPECTED LEARNING OUTCOMES

After studying and performing this experiment you should be able to:

1) Define compost;
2) Describe the type of waste is used for composting;
3) Explain the chemical reaction takes place in the process of composting;
4) Explain the management factors that control the effectiveness of
composting biodegradable household waste in small compost bins.
5) Determine the compost quality

13.2 PRINCIPLE
Composting is atypically a controlled natural process that makes a decent
conversion of degradable materials into stable and decomposable materials in
the presence of microorganism. Composting is simple but time taking
process. Composting has been around for quite long time, but it has some
flaws that have limited its pervasive usage efficiency. The transformation of
organic (degradable) wastes is either aerobic or anaerobic. Compost is
created when organic matter is converted under aerobic conditions. Biogas
and effluents, which can be utilized as bio-fertilizers, are produced when
anaerobically processed. Composting is a safe way to dispose of trash or
waste management. In aerobic process, microorganisms degrade and
transform complex degradable materials into organic and inorganic
byproducts. Composting is a way of changing diverse biodegradable wastes
into compounds that may be utilized safely and beneficially as bio-fertilizers.
When compared to the landfilling technique of waste disposal, which might
damage subterranean water, the composting process helps to safeguard
underground water from pollution. Organic matter from biodegradable wastes
is microbiologically decomposed during composting, resulting in a final
product with stable carbon, nitrogen, and other nutrients in the organic
fraction, with the stability varying depending on compost maturity.

The most acceptable composting approach rely on land availability, nature

and quantity of biodegradable waste to be treated, economic factors and
available workforce, the desired quality of the end product, and the amount of
the time available for processing. Composting is the microbial decomposition
of organic solid waste that comprises aerobic respiration and, in most cases, a
thermophilic stage. Home composting (HC) has long been thought and
considered traditionally as a horticultural recreational activity. Now a day it
has recently been identified as a major opportunity for managing a substantial
102
 Composting of
part of the domestic, biodegradable waste stream, reducing the amount of Biodegradable
waste collected for landfill disposal, and thus contributing to achieving the Waste and
Determination of
landfill directive's biodegradable waste disposal reduction goals. The Compost Quality
biodegradable waste is aerobically digested, resulting in a stabilized organic
proportion that may be recycled for agricultural use.

13.3 REQUIREMENTS
The quality of the compost and the time required for composting depends on
the technology to produce compost and also on the composition of the initial
compost mass.

a) Raw material: Composting materials and raw materials are divided into
six basic categories:

 food processing residues: compost substance produced from fruits,

vegetables, cereals, and meat after processing;

 agricultural and manure by-products formed in farrow, feedlots,

incubators, farms, greenhouses and the like;

 Forestry and wood industry residues- the byproduct obtained after

production of paper including bark, sawdust and fiber residues.

 organic waste or waste sludge - generated by treating the waste

sludge in plants for purification and recycling of waste water;

 yard and garden waste - such as leaves, shrubs, twigs and other
plant residues;

 municipal waste: Separated organic waste

Personal protective equipment

• Gloves
• Apron
• Mask
• Shoe cover
• Head cover

13.4 PROCEDURE
1) An open pile or a compost bin may be used. The size and kind of bin
required depends on the compostable material.

2) Select the area which is flat, well-drained, and sunny.

3) To allow for drainage and aeration, commence with a layer of coarse

materials (such as twigs). Leaves should be placed on top of this layer.
Then just alternate between layers of greens (nitrogen-rich materials) and
103
 Environment browns (non-nitrogen-rich materials) (carbon-rich material). Different
Science Lab
Course - 3 types of biodegradable waste brown and green to be used in the
composting process is mentioned in Table13.1

Table 13.1: Different types of biodegradable waste to be used in the

composting process

Browns Greens Don't Compost

Invasive weeds gone to
Evergreen needles Green leaves
see
Dried leaves Garden waste Meat/fish/bones
Paper egg cartons Flowers Fat/oil/grease
Paper towels/napkins Vegetables Dairy products
Cooked foods (attracts
Dried grass clippings Fruit peels
animals)
Shredded newsprint Scraps Pet waste
Bark Coffee grounds Plastics
Coffee filters Tea leaves/bags Metals
Straw Egg shells Glass
Sawdust (limited amt.) Flowers Toxic material
Dryer/vacuum lint Charcoal
Cardboard (cut into small
Chemical logs
pieces)
Dead house plants
Shredded brown paper bags

4) Also include biodegradable waste from the kitchen.

5) Continue adding until the Compost Bin is completely full. (As the
contents/pile of the bin decomposes, it will diminish.)

 The initial phase (Mesophilic)

 The temperature rise phase (Thermophilic)
 The phase of maximum (Thermophilic)
 Cooling

104
 Composting of
Biodegradable
Waste and
Determination of
Compost Quality

Figure 1: Composting process

Raw material for compost Finished compost

Figure 2

6) Examine your compost bin and make sure the following parameters are
satisfied to produce completed compost quickly:

• When adding new material, make sure to include it into the lower
levels.

• Materials should be as damp as a sponge that has been rung out. To

achieve this moisture level, add dry materials or water, as needed.

• Once a week, mix or turn the compost to aid in the decomposition

process and eliminate odours.

7) It takes four to six months to create completed compost. The finished

compost will be black, crumbly, and earthy smelling. The finished
compost will be placed on top of the compost bin or pile.

8) Remove all of the completed compost from the bin, leaving the
incomplete elements to decompose further. Before you use your
105
 Environment compost, be sure the decomposition process is complete; otherwise,
Science Lab
Course - 3 bacteria in the compost might extract nitrogen from the soil and hinder
plant development.

13.5 OBSERVATIONS AND CALCULATIONS

The ratio of the mixed material is adjusted until it reaches the desired range
of moisture content between 40% to 60%. The C: N ratio of the mixture can
be estimated using the selected materials based on moisture content

% Moisture Content =Wet weight-dry weight X100

Wet weight

Moisture Content (wt.) = % Moisture

100

Dry Weight = Total weight - Weight of Water

Proportions of dry materials can be calculated on the basis of the carbon and
nitrogen contents since it’s relatively easy to add moisture to the composting
mix. Because the C: N ratio is calculated on a dry weight basis, it's critical to
understand if the laboratory results are calculated on a wet or dry basis.

Nitrogen Content = Dry Weight X (%N/100)

Carbon Content = Dry Weight X (%C/100)

Carbon Nitrogen Ratio (C/N) = Carbon Content .

Nitrogen Content

Mix Moisture = Wt. of Water in material A + water in B + water in C+

Total Weight of all Material

Weight of water to be added, Mx = W(Md – Mi .

(1 - Md)

Symbols:

Mx = Amount of water to be added, tons

Md = Desired moisture content, % total weight/100
Mi = Initial material moisture content/100
W = Initial feedstock weight, tons including water
Record the temperature of the composting days

106
 Composting of
13.6 RESULTS Biodegradable
Waste and
Determination of
The Nitrogen Content in the finished compost is Compost Quality
………………………………………………
The Carbon Content in the finished compost is
………………………………………………..
Carbon Nitrogen Ratio (C/N) in the finished compost is
…………………………………….
% Moisture Content of final
compost…………………………………………………………
Moisture Content
(wt.)…………………………………………………………………………
Temperature of the composting days

Composting days Temperature

degree C
1
2

13.7 PRECAUTIONS
1) Be sure the decomposition process is complete before you use your
compost; otherwise, microbes in the compost could take nitrogen from
the soil and harm plant growth.

2) Remove all the finished compost from the bin, leaving unfinished
materials in the bin to continue decomposing.

107
 Environment
Science Lab EXPT 14 DETERMINATION OF
Course - 3
PESTICIDES IN
ENVIRONMENTAL SAMPLES
USING GAS CHROMATOGRAPHY

Structure

14.0 Introduction
14.1 Expected Learning Outcomes
14.2 Principle
14.3 Requirements
14.5 Sample collection, Preservation and Pretreatment
14.6 Cleaning of laboratory glassware
14.7 Procedure
14.8 Observations and Calculations
14.9 Result
14.10 Precautions

14.1 INTRODUCTION
Pesticides are materials that are destined to regulator pests. A pesticide is any
ingredient or mixture of substances intended for preventing, destroying,
repelling or mitigating any pest. A pesticide can be a chemical or a biological
agent such as a virus, bacterium, antimicrobial, or disinfectant that deters,
incapacitates or kills the pests. The wide-ranging use of pesticides troubles
the soil, air, food, surface and ground waters, and quality causing serious
impacts on the ecosystem functions, environment and on human health. In
natural waters pesticide residues are present at very low levels and can be
degraded when submitted to lower pH levels or exposed to solar radiation. It
is commonly used to eliminate or control a variety of agricultural pests that
can damage crops and livestock and reduce farm productivity. The most
commonly applied pesticides are insecticides to kill insects, herbicides to kill
weeds, rodenticides to kill rodents, and fungicides to control fungi, mould,
and mildew. The toxic chemicals in these are designed to deliberately
released into the environment. Though each pesticide is meant to kill a
certain pest, a very large percentage of pesticides reach a destination other
than their target. Instead, they enter the air, water, sediments, and even end
up in our food.

Pesticides have been linked with human health hazards, from short-term
impacts such as headaches and nausea to chronic impacts like cancer,
reproductive harm. The use of these also decreases the general biodiversity in

108
 Isolation and
the soil. If there are no chemicals in the soil there is higher soil quality, and Characterization of
this allows for higher water retention, which is necessary for plants to grow. Bacteria From Soil
Samples
Surface and ground water contamination due to extensive use of pesticide for
agricultural purposes is a serious threat to the environment. Most important
among them is the pollution due to OCPs, because of their high toxic effect
and persistence for a long time in the environment. Pollution by some
common pesticides, such as Organophosphorous pesticides (OPPs) due to
mistreatment, direct run off, empty containers careless discarding, leaching,
and utensils and apparatus washings, etc. is also important. Chlorinated
pesticides have fatal toxic effects for aquatic life forms such as fish. These
pesticides are counted hazardous for the environment and human beings as
well, due to bioaccumulation and biomagnifying effects in the food chain and
because they may produce reproductive and carcinogenic effects in animals
and human beings.

14.1 EXPECTED LEARNING OUTCOMES

After studying and performing this experiment you should be able to:

1) Explain the principle of gas chromatography;

2) Determine the concentration of pesticides in an environmental sample;
3) Explain suitable solvent for extraction of samples for pesticides analysis;
4) Explain stationary and mobile phase in Gas chromatography;
5) Describe chromatogram.

14.2 PRINCIPLE
The basic principle of gas chromatography is that the greater the affinity of
the compound for the stationary phase, more the compound will be retained
by the column and the longer it will be before it is eluted and detected. The
heart of the gas chromatograph is the column in which separation of the
component takes place. The source and control of the carrier gas flow
through the column, a mean of sample introduction and a means of detection
of the components as they elute from the end of the column must be added.

Since temperature will influence the volatility of the analytes, the column is
placed in a thermostatically controlled oven. Pesticides are chemical or
biological products which are used in direct form, in aqueous solutions or as
mixtures for preventing, fighting and killing a variety of pests, such as weeds,
insects, rodents, and fungi. The widespread occurrence of pesticides in
natural waters is due to their widespread application in agriculture throughout
the world.

Human being is constantly exposed to a large number of pesticides present in

the environment. The pesticides present in water, wastewater and soil
samples are extracted with methylene chloride (HPLC grade). The extract is
109
 Environment concentrated by evaporation, solvent exchanged to hexane and if necessary,
Science Lab
Course - 3 cleaned by column adsorption chromatography. The individual pesticides are
determined by gas chromatography using a suitable analytical column.
Pesticide compounds are separated in the column and reach the detector. A
quantitatively proportionate change occurs in electrical signal which is
measured on an integrator or personal computer and each component is
observed as a peak in the chromatogram recorded on the recorder. The
retention time of particular peak is indicative of the particular pesticide and
peak height/peak area of that particular peak is proportional to its quantitative
presence. Detector system in the Gas chromatograph is selected on the basis
of the specificity and sensitivity needed in determination of specific
pesticides.

This gas chromatographic method is applicable for determination of

organochlorine pesticides (Aldrin, α-BHC, β-BHC, γ-BHC, Dieldrin, o,p’-
DDT, p,p’-DDT, p,p’-DDE, α-Endosulfan & β-Endosulfan) using
electron capture detector (ECD) and organo-phosphorus pesticides
(Chloropyriphos, Dimethoate, Ethion, Malathion, Parathion Methyl) residues
in water and wastewater samples using capillary columns with a flame
photometric detector (FPD) or a nitrogen phosphorus detector (NPD).

Gas chromatography
110
 Isolation and
INTERFERENCE Characterization of
Bacteria From Soil
Some organic compounds other than chlorinated compounds respond to ECD Samples
and thus create interference during determination of pesticide. Among these
are oxygenated and unsaturated organic compounds. Polychlorinated
biphenyl (PCB) and phthalate esters cause interference in normal
measurement of pesticides. These organic compounds are widely used as
plasticizer and respond to ECD. These interferences can be removed by
column chromatography of extracted sample.

14.3 REQUIREMENTS
Apparatus

1) Separatory funnel shaker.

2) Rotary vacuum evaporator.
3) Evaporatory round bottom flasks (500, 250 and 125 ml).
4) Gas chromatography equipment with suitable detector.
5) Chromatography column: Suitable for pesticides analysis
6) Sampling bottle (1L Capacity), amber glass with TFE- lined screw cap.
7) Separatory funnel (2L capacity).
8) Chromatography glass column for clean-up (300 mm x 20 mm ID).
9) Graduated cylinder (1L capacity).
10) Silanized glass wool.
11) Filter paper: Whatman 41.
12) Glass funnels.
13) Microlitre syringe 10 μl capacity (Autosampler) for sample injection.
14) Graduated pipettes for dilutions.
15) Hot air oven.

Reagents

1) Hexane (HPLC grade)

2) Acetone (HPLC grade)
3) Petroleum ether, boiling range 40-60 °C.
4) Diethyl ether.
5) Methylene chloride (HPLC grade).
6) Florisil, PR grade, 60-100 mesh.
7) Sodium sulphate, anhydrous (AR grade).
8) Nitrogen as carrier gas (99.99% pure), free from moisture and oxygen.
9) Certified pesticides standard in solutions or neat.
111
 Environment Personal protective equipment
Science Lab
Course - 3
1) Gloves
2) Apron
3) Mask
4) Shoe cover
5) Head cover

14.4 SAMPLE COLLECTION, PRESERVATION

AND PRETREATMENT
The most important step in water quality monitoring is the collection of a
representative sample, which includes the selection of sampling sites, types of
samples (grab, mixed or composite), sample container; volume, collection
method, sample handling (transportation), preservation and storage.

Sample Container

For pesticide residue analysis, sample container (1L litre) of high quality
amber glass with Teflon lined stopper is suitable. Plastic or polyethylene
containers must not be used, as pesticides present in water samples may get
adsorbed on inner walls of container. The glass containers before use need to
be cleaned properly. Washing with acid, detergent, tap water, distilled water,
acetone and finally with the working organic solvent is appropriate.

Sample volume

One litre of water sample is required to be collected.

Storage of Samples

The samples should be stored in amber glass bottles at 4 °C. Ensure that the
samples do not absorb or lose moisture during storage. Avoid long storage
period. Samples should be analyzed as early as possible. Do not hold samples
for more than 7 days.

The sample after extraction may be stored at 4 °C until analyzed on gas

chromatograph. The analysis should be completed within a maximum period
of 40 days.

14.5 CLEANING OF LABORATORY

GLASSWARE
Since pesticides present in environmental samples are in parts per billion
(PPB), therefore, thorough cleaning and washing of glassware used in
extraction and analysis is mandatory. Failures to do so can lead to the
problems in the interpretation of final chromatograms, due to the presence of
unidentified additional peaks resulting from contamination of glassware.
112 Particular care must be taken with glassware such as Kuderna-Danish flasks
 Isolation and
or Flash Evaporator, evaporative concentration tubes, or any other glassware Characterization of
coming in contact with an organic extract evaporated and concentrated to Bacteria From Soil
Samples
lesser volume. The process of concentrating the pesticides in this operation
may similarly concentrate the contaminating substance, resulting in extra new
chromatographic peaks, which in extreme cases, may completely overlap and
mask the pattern of pesticide peaks.

Basic cleaning steps of laboratory glassware used for pesticide analysis are:

• Removal of surface residuals immediately after work by using acetone.

• Soak with deep penetrate or oxidizing agent to destroy traces of organics.
• Hot water rinse to flush away material loosened by deep penetrant soak.
• Distilled water rinse to remove metallic deposit from the tap water.
• Dry in hot air oven.
• A preliminary flush of the glassware just before using with the same
solvent to be used in the analysis.

14.6 PROCEDURE FOR ANALYSIS

A. Extraction procedure for Organo-chlorine (OCPs) and Organo-
phosphorous pesticides (OPPs)

1) Take out the samples from storage and wait for samples to achieve
ambient temperature.

2) Shake sample well in the sample bottle and pour the whole sample into a
2-L separatory funnel.

3) In case the sample contains high concentration of suspended solids, add

5 ml conc. HCl or one spatula of NaCl, to avoid colloid formation. If
sample is alkaline, add concentrated Hydrochloric acid (HCI) to
neutralize the pH.

4) Rinse the sample bottle and cylinder with 50 ml Methylene chloride,

pour this solvent into separatory funnel and shake for 2 min.

5) Let stand for at least 15 min to separate organic and aqueous phases.

6) Collect organic phase through a funnel containing a layer of anhydrous

sodium sulphate on filter paper/glass wool into a 250 ml round bottom
flask.

7) Repeat step 5 and two times and collect organic phase into flask
containing organic phase of step 6.

8) Reduce the pooled volume of organic phase extract in Rotary vacuum

evaporator to about 5-10 ml.

9) Add 25 ml hexane to concentrated sample extract to remove traces of

dichloromethane and concentrate to less than 2 ml.
113
 Environment 10) If cleanup is not required, then make final volume to 2.0 ml after
Science Lab
Course - 3 washing of flask with hexane.

11) If samples were from clean matrix, then generally clean-up of extract is
not required. Otherwise follow cleanup procedure using glass column
chromatography.

B. Absorption chromatography for sample extract cleanup

Plug the column with glass wool.

1) Add 20 g activated Florisil to a glass chromatographic column. Settle the

Florisil by tapping the column. Add anhydrous sodium sulphate to the
top of the Florisil to from a layer 1 to 2 cm deep.

2) Pre-elute the column with 60 ml of hexane and discard the eluate. Just
prior to exposure of the sodium sulphate to air,

3) Quantitatively transfer the concentrated sample extract onto the column,

with two 2 ml rinses of flask with hexane.

4) Place a round bottom flask (500 ml) under the chromatographic column.
Drain the column into the flask until the sodium sulphate layer is nearly
exposed.

5) Elute the column with 200 ml of 6 % diethyl ether in hexane using a drop
rate of about 5 ml/min.

6) Elute the column again; using 200 ml of 15 % diethyl ether in hexane

into a same flask.

7) Perform a third elution using 200 ml of 50 % diethyl ether in hexane into

in same flask.

8) Perform a final elution with 200 ml of 100 % diethyl ether into same
flask.

9) Concentrate the combined all four elutes to the known volume of 2-5 ml
using Rotary vacuum evaporator and then analyze by gas
chromatography.

C. Preparation of Calibration Standards

1) Stock pesticides standards: Certified standard solutions or neat,

traceable to their purity.

2) Intermediate standard (A): After dilution of each stock pesticide

standards, make mixture of intermediate standard (A) in hexane to get
concentration of each compound approximately 50 ppm. This standard
solution is stable for at least six months.

3) Intermediate standard (B): Make dilution of intermediate standard (A)

in hexane to get mixture of intermediate standard (B) of approximately 1
114
 Isolation and
ppm (mg/L) or 1000 ppb (µg/L). Prepare this solution at the time of Characterization of
instrument calibration. Bacteria From Soil
Samples
4) Working standards: Make serial dilution of intermediate standard (B)
to get concentrations of working standard solutions in the ranges as
detailed below. Prepare fresh when required for instrument calibration.

Chromatogram

Approximate concentration ranges of each compound in the working standard

for five level calibrations:

a) Organophosphorous Pesticides:
Chloropyriphos : 0.2 – 5.0 ng/µl
Dimethoate : 0.2 – 5.0 ng/µl
Ethion : 0.2 – 5.0 ng/µl
Malathion : 0.2 – 5.0 ng/µl
Parathion Methyl : 0.2 – 5.0 ng/µl
b) Organochlorine Pesticides:
Aldrin : 0.02 - 0.1 ng/µl
α-BHC : 0.02 - 0.1 ng/µl
β-BHC : 0.02 - 0.1 ng/µl
γ-BHC : 0.02 - 0.1 ng/µl
Dieldrin : 0.02 - 0.1 ng/µl
115
 Environment o,p’-DDT : 0.02 - 0.1 ng/µl
Science Lab
Course - 3 p,p’-DDE : 0.02 - 0.1 ng/µl
p,p'-DDT : 0.02 - 0.1 ng/µl
α-Endosulfan : 0.02 - 0.1 ng/µl
β-Endosulfan : 0.02 - 0.1 ng/µl

D. Instrumental Quantification of Pesticides

Gas Chromatography Conditions

A Gas Liquid Chromatograph (GLC) equipped with ECD is used for organo-
chlorine pesticides, while FPD is used for organo-phosphorous pesticides for
their analysis. The operating parameters suggested may be varied according
to the available facilities, provided standardisation is done.

Organochlorine Pesticides

For quantification of pesticides (Aldrin, α-BHC, β-BHC, γ-BHC, Dieldrin,

o,p’-DDT, p,p’-DDT, pp’-DDE, α-Endosulfan & β-Endosulfan), following
Gas chromatographic conditions may be used:

Detector: Electron Capture Detector (ECD)

Analytical column: Elite-1 (30 m x 0.25 mm x 0.33 µm film) or equivalent

may be used.

Temperature:

Injector - 250 °C
Detector - 300 °C
@7 °C/min @5 °C/min
Oven - 190 °C ----------- 222 °C -------------- 250 °C
(0 min) (0 min) (5 min)

Carrier gas: Purified laboratory grade Nitrogen @ flow of 1.0 ml/min.

Organophosphorous pesticides

For quantification of and organo-phosphorus pesticides (Chloropyriphos,

Dimethoate, Ethion, Malathion & Parathion Methyl), following Gas
chromatographic conditions may be used:

Detector: Flame Photometric Detector (FPD)

Analytical column: DB-5 (30 m x 0.25 mm ID x 0.25 µm film) or equivalent

Temperature:

Injector : 250 °C
Detector (FPD) : 290 °C
116
 Isolation and
Carrier Gas Flow : 9.0 ml/min Characterization of
Bacteria From Soil
Column Flow : 1.0 ml/min Samples
Hydrogen Flow : 80 ml/min
Air Flow : 120 ml/min

Column oven temperature programme:

Rate Temp Hold time

- 150 °C 1.0 min
3 °C/min 220 °C 1.0 min
15 °C/min 260 °C 5.0 min

Procedure (Orqano-chlorine & Organo-phosphorous Pesticides)

1) Inject 1 µI of working standard solution of pesticides to be analyzed into

Gas chromatograph kept ready at appropriate temperature programming.
The chromatogram and result report provided by Gas chromatograph
integrator/ microprocessor/computer is examined. If peaks of different
organic compounds are well resolved and precision in peak area is
satisfactory, calibrate the instrument using integration parameters of the
chromatographic software.

2) Inject 1 μl of sample extract. Chromatographic software will calculate

the concentration of identified compound on the basis of area of the
compound characteristic peak and calibration factors established from
the multi-level calibration.

14.7 OBSERVATIONS AND CALCULATIONS

CALCULATIONS

Concentration of pesticides is calculated as given below:

When the GC instrument gives concentration directly:
C x VE
Pesticide concentration µg/L = ------------------
(Microgram/L) VS
Where:
C = Quantity of pesticide in injected volume (ng)
VE = Extracted sample volume (ml)
VS = Volume of sample taken for extraction (Litre)
Note: If the sample extract was diluted, then apply Dilution Factor

14.8 RESULTS
The concentration of OCPs and OPPs in a given sample (water) is as below:
117
 Environment a) Organophosphorous Pesticides:
Science Lab
Course - 3 Chloropyriphos : ………………………………..ng/µl
Dimethoate : ………………………………. ng/µl
Ethion : ………………………………. ng/µl
Malathion : ………………………………. ng/µl
Parathion Methyl : ………………………………. ng/µl

b) Organochlorine Pesticides:

Aldrin : ………………………………. ng/µl

α-BHC : ………………………………. ng/µl
β-BHC : ………………………………. ng/µl
γ-BHC : ………………………………. ng/µl
Dieldrin : ………………………………. ng/µl
o,p’-DDT : ………………………………. ng/µl
p,p’-DDE : ………………………………. ng/µl
p,p'-DDT : ………………………………. ng/µl
α-Endosulfan : ………………………………. ng/µl
β-Endosulfan : ………………………………. ng/µl

4.9 PRECAUTIONS
• Always use the clean glassware.
• Adjust calculated concentrations of detected analytes to reflect initial
sample volume and any dilutions performed.
• Before reporting the data, review the chromatogram for any incorrect
peak identification or poor integration.
• If an analyte peak area exceeds the range of the initial calibration curve,
the extract may be diluted with buffered reagent water.

118
 Determination of
EXPT 15 DETERMINATION OF BIOGAS Biogas Production
From Organic
PRODUCTION FROM ORGANIC Waste By Liquid
Displacement
WASTE BY LIQUID Method

DISPLACEMENT METHOD

Structure

15.0 Introduction
15.1 Expected Learning Outcomes
15.2 Principle
15.4 Experimental Setup (Design, Fabrication of the Reactor)
15.5 Determination of Biogas Production by Liquid Displacement Method
15.6 Observations and Calculations
15.7 Result
15.8 Precautions

15.0 INTRODUCTION
Biogas is the mixture of gases produced by the breakdown of organic matter
in the absence of oxygen (anaerobically), primarily consisting of methane and
carbon dioxide. Biogas can be produced from raw materials such as
agricultural waste, manure, municipal waste, plant material, sewage, green
waste or food waste. Biogas is an energy-rich gas produced by anaerobic
decomposition or thermochemical conversion of biomass. Biogas is
composed mostly of methane (CH4), the same compound in natural gas, and
carbon dioxide (CO2). Biogas is a renewable energy source and produced
biologically through anaerobic digestion and is differs from natural gas or
fossil fuel that are produced by geological processes. Biogas is primarily
composed of methane gas, carbon dioxide, and trace amounts of nitrogen,
hydrogen, and carbon monoxide. When manure is anaerobically digested,
the biogas produced is primarily composed of methane and carbon dioxide,
with lesser amounts of hydrogen sulfide, ammonia, and other gases. Each of
these gases has safety issues. Overall, biogas risks include explosion,
asphyxiation, disease, and hydrogen sulfide poisoning.

15.1 EXPECTED LEARNING OUTCOMES

After studying and performing this experiment you should be able to:

1) Explain the set up an experiment for biogas production from organic

waste;
2) Describe the principle of biodegradation of organic waste;
3) Explain the type of chemical reactions takes place in the whole process;
119
 Environment 4) Determination of the composition of biogas produced.
Science Lab
Course - 3
15.2 PRINCIPLE
Biogas is a type of biofuel that is naturally produced from the decomposition
of organic waste. When organic matter, such as food scraps and animal
waste, break down in an anaerobic environment (an environment absent of
oxygen) they release a blend of gases, primarily methane and carbon
dioxide. Because this decomposition happens in an anaerobic environment,
this process of producing biogas is also known as anaerobic digestion.
Biogas is a renewable, as well as a clean source of energy. Gas generated
through biodigestion is non-polluting; it actually reduces greenhouse
emissions (i.e. reduces the greenhouse effect). In addition, the raw materials
used in the production of biogas are renewable, as trees and crops will
continue to grow.

In the biogas reactor, microbial action begins and the biomass enters a
gradual process of fermentation, which means that microbes feed on organic
matter, such as proteins, carbohydrates and lipids, and their digestion turns
these into methane and carbon dioxide.

The biogas production technology is relatively inexpensive, so it can be

easily employed in domestic settings. Small bio-digesters can be used in the
home. Kitchen waste and animal manure are used as sources of methane. The
produced gas can be used as a fuel for cooking and electricity production.
Green energy, such as biogas, is used as a source of electricity and heat for
local utilities also. Environmental advantages are reduction in emissions of
greenhouse gases such as methane, CO2, and nitrous oxide. This
environmentally friendly recirculation of organic waste from industry and
households also reduces greenhouse gases.

The fermentation of organic matter takes place with the assistance of

bacterial communities to produce biogas. During fermentation, the organic
material changes from its original composition into biogas. A brief overview
of the chemical reactions taking place throughout the entire process is given
below:

1) Hydrolysis: A digestion process begins with the hydrolysis stage.

Hydrolysis happens when insoluble organic polymers (such as
carbohydrates) are broken down into their component parts, making
them available to the next stage of bacterial activity called acidogenic
bacteria.

(C6H10O5) n+ nH2O→n C6H12O6+ nH2

2) Acidogenesis: Sugars and amino acids are transformed into carbon

dioxide, hydrogen, ammonia, and organic acids by acideogenic bacteria.

C6H12O6↔2CH3CH2OH+2CO2
120
 Determination of
C6H12O6+2H2↔2CH3CH2COOH+2H2O Biogas Production
From Organic
C6H12O6→3CH3COOH Waste By Liquid
Displacement
3) Acetogenesis: During the third stage, acetogenic bacteria convert Method

organic acids into acetic acid, hydrogen, ammonia, and carbon dioxide,
which sets the stage for the final stage - methanogens.

CH3CH2COO−+3H2O↔CH3COO−+H+HCO3−+3H2

C6H12O6+2H2O↔2CH3COOH+2CO2+4H2

CH3CH2OH+2H2O↔CH3COO−+3H2+H+

4) Methanogenesis: As a result of these aerobic reactions, methanogens

produce methane and carbon dioxide, which can then be used as a green,
flammable energy.

CH3COOH→CH4+CO2

CO2+4H2→CH4+2H2O

2CH3CH2OH+CO2→CH4+2CH3COOH

Organic waste

Organic Matter (complex)

Carbohydrates, fats, proteins

Hydrolysis
Soluble Organic Molecule
(Sugar, fatty acids, amino acids)

Fermentation

Soluble Organic Molecules

(Sugar, fatty acids, amino acids)

Fatty acids

Acetate Methanogens H2 +
CO2

CH4+CO2
Figure 15.1 : Process flow chart of biogas production from organic waste

121
 Environment Table 15.1: The percentage composition of the products of biogas
Science Lab
Course - 3
Products Composition (%)
Methane (CH4 ) 50–57
Carbon di oxide (CO2 ) 25–50
Nitrogen (N2 ) 0–10
Hydrogen (H2 ) 0–1
Hydrogen sulfide(H2S) 0–3
Oxygen (O2) 0–2

15.3 REQUIREMENTS
Apparatus

1) Plastic container with a capacity of 20 liter volume.

2) Organic waste (any organic waste such as kitchen waste, agriculture
waste etc.)
3) Measuring cylinder
4) Rubber tubing
5) Temperature device
6) Incubator to perform the experiments
7) Gas liquid chromatography
8) Duration of the experiment=40 to 50 days

Personal protective equipment

1) Gloves
2) Apron
3) Mask
4) Shoe cover
5) Head cover

15.4 EXPERIMENTAL SETUP (DESIGN,

FABRICATION OF THE REACTOR)
1) A plastic container with a capacity of 20 liters is used for designing a
bench size reactor.

2) The reactor has an input at the bottom for upflow reactors and an exit at a
height of 27.5 cm from the bottom for waste water discharge. An
opening on the top of the digester might be used to collect biogas from
the reactor.

122
 Determination of
3) To prepare biowaste for anaerobic digestion, it is crushed into smaller Biogas Production
bits and slurred. Slurring biowaste involves adding liquid to make it From Organic
Waste By Liquid
simpler to process. Displacement
Method
4) At the initial stage, cow dung is mixed with water in 1:2 ratios in the
digester/reactor and is closed at the top using floating drum and some
weights are kept on the drum to increase the pressure in the gas. Feed the
reactor with 15-lit (organic wastes plus water or slurry), and 15 % of
anaerobic bacterial inoculum which is inoculated with 15% (10 + 5) seed
culture and purge by liquid N2 gas to remove O2 from reactor.

5) Use M-seal to close the reactor and maintain anaerobic conditions.

6) A biogas outlet is located at the top of the reactor and it is linked to a

calibrated water displacement bottle through a rubber (plastic) hose for
measuring gas produced by the reactor.

7) The minimum temperature ranges require from 35°C- 55°C. As a result,

maintaining this temperature gradient is important for the system
efficiency of the process.

8) Leave the reactor either in incubator or ambient environment as per

required temperature for stabilization of anaerobic digestion.

9) The biogas is production takes place by anaerobic digestion in massive

tanks about a three- week period.

Fig.15.2 : Experimental setup for biogas production

15.5 DETERMINATION OF BIOGAS

PRODUCTION BY LIQUID DISPLACEMENT
METHOD
Gas liquid chromatography was used to determine the composition of biogas.
The biogas compositions of methane and carbon dioxide is determined using
the following parameters for measuring biogas compositions:

 Column–stainless steel, 180 long, 3mm diameter, 60/80 mesh, Porapak Q

 Detector -thermal conductivity detector
123
 Environment  Carrier gas- hydrogen detector current – 74-mA
Science Lab
Course - 3  Attenuation – 3x
 Carrier gas flow rate- 60 ml \min
 Oven temperature: 500C
 Injector temperature: 1100C
 Detector temperature: 1100C
 Sample size: 1ml

The standard calibration curve for methane is obtained by injecting standard

methane sample containing 4.95% methane in nitrogen.

15.6 OBSERVATIONS AND CALCULATIONS

Observation

Following parameters to be analyzed for the experiments after following the

S. No 1- 7:

1) To optimize the temperature till the end of the experiment up to 50 days.

Day 1 Day Day 3 Day 4 Day 4 Day 5

2
Temperature
(Degree C)
Biogas (l)

2) Determine the pH of the feeding substrates and effluent, the ammonia

nitrogen content in the digester, the conductivity of the salt
concentration, total solids (TS), volatile solids (VS), and the ratio of
volatile organic acids to total inorganic carbon in the effluent.

During the feeding phase, the pH, TS, and VS were monitored every day,
while the others were tested twice a week. The DIN EN 15934 and DIN
15935 standards were used to define TS and VS. An ammonia nitrogen
analyzer was used to determine the amount of nitrogen in the ammonia.

3) To calculate the Rate of organic load and the biogas yield: Biogas
production is proportional to the organic load. It represents the volume of
biogas produced from a mass of waste:

Biogas yield = Biogas volume / quantity of waste

Example to calculate the biogas yield as below:
For 2.129 L of biogas from 25g of waste.
Therefore, Biogas yield is 85.16 l/Kg of fresh waste (FW).
If, Dry matter DM = 20% FW and Organic matter OM = 17% FW,
Then,
124
85.16 l/kg FW Equivalent to 85.16 l / 0.20 Kg DM = 425.8 l/kg DM. Determination of
Biogas Production
Thus 85.16 L/ Kg FW Equivalent to 85.14 l / 0.17 Kg OM = 500.82 l / Kg From Organic
OM Waste By Liquid
Displacement
So, the biogases yield: Method

Biogas yield = 85.16 L/Kg (FW) = 425.7 l/Kg DM = 500.82 l/Kg OM

Observe the temperature and biogas production from day 1 to day 50 and
record the result at what temperature, maximum biogas is produced.

15.7 RESULTS
Biogas production days wise observed in the experiment performed in the
laboratory is as below Table :

Day Day 2 Day Day Day Day

1 3 4 4 5
Temperature
(Degree C)
Biogas (l)
• The composition of biogas produced in the laboratory is:
CH4…………………………………… and
CO2……………………………………….
• pH of Feeding substrate and effluent:……….
• Total solids (TS), the volatile solids (VS) of Feeding substrate and
effluent:……….
• ratio of volatile organic acids to total inorganic carbon of Feeding
substrate and effluent:………………….
• Ammonia nitrogen of Feeding substrate and effluent:………………….

15.8 PRECAUTIONS
1) The optimal conditions should be maintained for anaerobic digestion in
small bioreactors.

2) The reactor should be filled two-thirds of the maximum volume with a

suspension.

3) After stabilization of the process, observe and record the volume of

water displaced on daily basis.

4) The reactor should be airtight.

125
 Environment
Science Lab EXPT 16 DETERMINATION OF TRACE
Course - 3
METALS IN ENVIRONMENTAL
SAMPLES BY INDUCTIVELY
COUPLED PLASMA OPTICAL
EMISSION SPECTROSCOPY
(ICP-OES)

Structure

16.1 Introduction
16.1 Expected Learning Outcomes
16.2 Principle
16.3 Requirements
16.4 Sample collection
16.5 Sample Digestion
16.6 Procedure
16.7 Observations and Calculations
16.8 Result
16.9 Precautions

16.1 INTRODUCTION
The detection of trace metal pollution in wastewater is a crucial method of
protecting human and environmental health. Countries around the world
manage wastewater in different ways, but the objective is to reduce the
pollution from natural waterways. The examination of trace metals and heavy
metals in a sample detects and quantifies extremely minute amounts of metals
and heavy metals. Although certain trace metals are necessary for our health,
many metals are poisonous and have detrimental consequences for human,
animal, and plant health, as well as the environment.

ICP optical emission spectrophotometers (ICP-OES) have been widely

utilized for 25 years and are currently one of the most flexible techniques of
inorganic analysis. Its properties are likened to those of atomic absorption
spectrophotometers. When compared to Atomic Absorption
Spectrophotometer, where the excitation temperature of an air-acetylene
flame is between 2000 and 3000 K, the excitation temperature of an argon
ICP is between 5000 and 7000 K, which efficiently excites a wide range of
elements. In addition, employing an inert gas (argon) makes the production of
oxides and nitrides harder to be generated.

Extremely sensitive analytical equipment is required to determine relatively

small quantities of potentially harmful elemental contaminants such as lead
126
 Determination of
(Pb), mercury (Hg), arsenic (As), cadmium (Cd), copper (Cu), nickel (Ni), Trace Metals in
zinc (Zn) and others elements. Depending on the complexity of the sample Environmental
Samples by
(i.e. the sample matrix) and the analytical technique employed, the metals in Inductively
a sample are commonly quantified in parts per million (ppm), parts per Coupled Plasma
Optical Emission
billion (ppb), or even parts per trillion (ppt). Atomic Absorption Spectroscopy
Spectroscopy (AAS), Inductively Coupled Plasma Optical Emission (ICP-OES)
Spectroscopy (ICP-OES), and Inductively Coupled Plasma Mass
Spectrometry (ICP-MS) are common analytical procedures for trace metal
detection. A number of inorganic methods, such as Atomic Absorption
Spectroscopy (AAS), Inductively Coupled Plasma Optical Emission
Spectroscopy (ICP-OES), and Inductively Coupled Plasma Optical Emission
spectroscopy (ICP-OES), can be used to analyze trace elements in waste
water.

16.1 EXPECTED LEARNING OUTCOMES

After studying and performing this experiment you should be able to:

1) Explain the principle of Inductively Coupled Plasma Optical Emission

Spectroscopy (ICP-OES);
2) Determine the concentration of trace metals in an environmental sample;
3) Explain the processing of samples for trace metal analysis;
4) Explain the digestion process for sample analysis;
5) Determine the trace metal through Inductively Coupled Plasma Optical
Emission Spectroscopy (ICP-OES).

16.2 PRINCIPLE
Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) is an
analytical technique for determining the amount of specific elements in a
sample. The ICP-OES principle uses the fact that atoms and ions can absorb
energy and use it to transfer electrons from a ground to an excited state. The
source of energy in ICP-OES is heat from an argon plasma operating at
10,000 kelvin.
As excited atoms transition to a lower energy state, the ICP-OES principle
relies on their emitting light at specific wavelengths.

127
 Environment
Science Lab
Course - 3

Fig 16.2 : Atomic emission spectrometry (AES)

When an electron transitions from a higher to a lower energy level, generally

the ground state, it produces light with a certain specific wavelength. The
wavelength of emitted light is determined by the sort of atom or ion (i.e.,
whose element it is) and the energy levels the electron is travelling between.
The quantity of atoms or ions that make the transition determines how much
light is emitted at each wavelength. The Beer Lambert law describes the link
between the intensity of light and the element's concentration.

Another technique of Optical Emission Spectrometry is ICP, which stands for

Inductively Coupled Plasma. The component elements (atoms) are stimulated
when plasma energy is applied to an analytical sample from the outside.
Emission rays (spectrum rays) are emitted when excited atoms return to a low
energy state, and the emission rays that correspond to the photon wavelength
are detected. The strength of the photon beams determines the element type,
and the position of the photon rays determines the content of each element.
To generate plasma, argon gas is introduced into the torch coil, and a high-
frequency electric current is passed through the work coil at the torch tube's
tip. Argon gas is ionized and plasma is formed using the electromagnetic
field generated in the torch tube by the high frequency current. This plasma
has a high electron density and temperature (10000K), which is employed in
the sample's excitation and emission. Through the thin tube in the centre of
the torch tube, solution samples are atomized and injected into the plasma.

128
 Determination of
Trace Metals in
Environmental
Samples by
Inductively
Coupled Plasma
Optical Emission
Spectroscopy
(ICP-OES)

Fig 16.2 Schematic diagram of Inductively Coupled Plasma Optical Emission

spectroscopy (ICP-OES)

16.3 REQUIREMENTS
1) Plastic (polyethylene or equivalent) bottles for sample collection
2) Water sample (500 ml)
3) Solid sample (250gms)
4) Measuring cylinder (250ml)
5) Volumetric flask (250ml)
6) Glass funnel
7) Nitric acid
8) Electric Hot Plate
9) filter paper Whatman No.-42
10) deionized water
11) Inductively Coupled Plasma Optical Emission spectroscopy (ICP-OES)
12) Argon gas
13) Nitrogen gas
14) Air compressor
15) Certified reference materials (single element / multi element)
16) Plastic bottles (100 ml)
17) Micropipettes
18) Sample tube for analysis.

Personal protective equipment

1) Gloves
2) Apron 129
 Environment 3) Mask
Science Lab
Course - 3 4) Shoe cover
5) Head cover

16.4 SAMPLE COLLECTION

Water and wastewater samples are collected in plastic (polyethylene or
equivalent) bottles and acidified with approximate 2 ml Nitric acid and also
maintaining pH less than 2 and sample volume should be 500 ml. The solid
samples are collected minimum 250 gm in zip lock polyethylene bag and ice
preserved in filed, then stored it in refrigerated conditions at approximately
4oC to minimize losses of volatile metals & metalloids.

16.5 SAMPLE DIGESTION

100 ml sample of water is digested by adding 10 ml of Nitric acid (Reagent
Grade) and in case of solid sample, 1 gm of oven dried sample is used for
digestion by adding 10 ml of Nitric acid or a suitable acid mixture using
Electric hot plate (adjustable & capable to maintain temperature) at a
temperature of 900C - 950C until sample volume has been reduced till 15-20
ml then, remove the flask from hotplate & allow it for cool at room
temperature. Filter the sample using filter paper (Whatman No.-42). Final
volume has been adjusted to 100 ml using deionized water. Parallelly a blank
(i.e.- deionized water and Nitric Acid) sample should be kept for digestion
during each batch of sample digestion. (Caution: Do not boil the sample).
Sample digestion is processed as per APHA: 3030 E for water samples &
USA EPA: 3050b for soil, sludge & sediment Samples.

Complex Matrixes of samples like siliceous matrixes, organicand geological

samples are digested using Microwave assisted acid digestion method (Close
Digestion Method) as per US EPA: 3052. Aqueous sample and extracts are
digested using Microwave assisted acid digestion method US EPA: 3015a.
Oil, Soil & Sediment samples are digested using Microwave assisted acid
digestion method US EPA: 3051a.

16.6 PROCEDURE FOR ANALYSIS

Preparation of working standards for Calibration

It is essential to use highly pure metals or chemicals to

• Prepare the working standard (10ppm) from the high quality certified
reference material (stock solution) of trace metals (single element or
multi-element as per the requirement) in the suprapure or ultrapure Nitric
acid.

• Working standard solutions are made by diluting the stock solution with
an appropriate solvent until the required concentration is obtained, within
130
 Determination of
the detection limits of the analyzing equipment. Working standards are Trace Metals in
frequently developed just as-needed, depending on the stability of the Environmental
Samples by
stock solution. Inductively
Coupled Plasma
• Prepare the 5 working standard of 0.2, 0.5, 1.0, 2.0, 5.0 ppm for analysis Optical Emission
Spectroscopy
in ICP-OES. (ICP-OES)

• Solutions containing known concentrations of each element are

measured to calibrate an ICP-OES. A calibration curve is generated
using this data. The calibration curve establishes the relationship
between the intensity of light emitted at a particular wavelength and the
concentration of the element in the solution.

Fig.16.2 :Sample Analysis Using the Selected Analytical Method

Metal atoms in the sample solution are transformed to metal ions using the
Inductively Coupled Plasma (ICP) analytical technique. Optical emission
spectroscopy (ICP-OES) or plasma mass spectrometry are used to separate
and identify the various metal ions (ICP-MS).

The main steps in an ICP-OES analysis are:

1) The metal elements in the sample to be measured are selected, such as

As, Pb, Co, Cr etc.

2) Solutions of the samples are prepared, using the conventional techniques

of quantitative chemical analysis.

3) A set of calibrating solutions is prepared. Each solution contains

accurately known concentrations of the analyte elements. The range of
concentrations for each element in the set is chosen to include the
expected concentration of that element in the sample solutions (if this is
known).

4) The calibrated and sample solutions are then delivered into the plasma,
and the intensities of light at relevant analytical lines are measured. The
131
 Environment analytical line for S might be 181.972 nm, 220.253 nm for Pb, and
Science Lab
Course - 3 213.618 nm for Pb.

5) Calibration graphs are prepared for each element from the emission
intensities of the calibrating solutions.

6) The concentrations of the metal elements in every sample solution are

determined from the calibration graphs. The concentrations in the
original sample are then calculated from the measured concentrations of
the elements in the sample solution and the known dilution factor. ICP-
OES analysis delivers results that list the concentration of the selected
elements, typically in µg/L or mg/L.

16.7 OBSERVATIONS AND CALCULATIONS

In water or solid samples, the concentration of Trace metals is determined in
mg/kg.
Trace metals concentration mg/L (water) or mg/Kg (solid) = ((A*B)/C)
A= Concentration of metal in digested solution mg/L
B= Final volume of digested solution, ml
C= Sample size ml (water) or gm (Solid)

16.8 RESULTS
The concentration of trace metals in a given sample (water) is as below:
The conc. of As = ………………
Conc. of Co = ………………….
Conc. of Pb = ……………..
Conc. of Fe = ……………….
The concentration of trace metals in a given sample (Soil)is as below:
The conc. of As =………….
Conc. of Co =………..
Conc. of Pb=…………….
Conc. of Fe …………...=

16.9 PRECAUTIONS
1) Always use clean glassware for the preparation of working standard for
calibration.
2) Carefully handle the samples during digestion of samples.
3) Working standard should be prepared only in suprapure or ultrapure
Nitric acid.
4) Sample should be diluted with distilled water or ultra-pure water.
5) Apply dilution factor before report the results.

132
 Determination of
EXPT 17 DETERMINATION OF Alcoholic
Fermentation From
ALCOHOLIC FERMENTATION Grape Juice by
Saccharomyces
FROM GRAPE JUICE BY Cerevisiae

SACCHAROMYCES CEREVISIAE

Structure

17.0 Introduction
17.1 Expected Learning Outcomes
17.2 Principle
17.3 Requirements
17.4 Media prepared
17.5 Procedure
17.6 Observations and Calculations
17.7 Result
17.7 Precautions

17.0 INTRODUCTION
Alcoholic fermentation is a complex biochemical process during which
yeasts convert sugars to ethanol, carbon dioxide, and other metabolic
byproducts that contribute to the chemical composition and sensorial
properties of the fermented foodstuffs. Alcoholic fermentation is the basis for
the manufacturing of alcoholic beverages such as wine and beer. Alcoholic
fermentation is the best known of the fermentation processes, and is involved
in several important transformations, stabilization, and conservation
processes for sugar-rich substrates, such as fruit, and fruit and vegetable
juices. Alcoholic fermentation is carried out by yeasts and some other fungi
and bacteria. The first step of the alcoholic fermentation pathway
involves pyruvate, which is formed by yeast via the EMP pathway.

17.1 EXPECTED LEARNING OUTCOMES:

After studying and performing this experiment you should be able to:

1) Describe alcoholic fermentation;

2) Describe the process involved in alcoholic fermentation;
3) Explain the requirements for alcoholic fermentation experiment;
4) Determine the parameters to be analyzed in alcohol production.

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Science Lab 17.2 PRINCIPLE
Course - 3
Fermentation is a term used for any process in which chemical changes are
brought about by the action of enzymes produced by living microbes in an
organic substrate, whether carbohydrate, fat, protein or other type of organic
material. Fermentation is a metabolic process that some bacteria use to break
down glucose when O2 is not available. Fermentation includes the reactions
of glycolysis (where a single molecule of glucose is broken down into 2
molecules of pyruvate), as well as additional reactions that produce a variety
of end products (acids, alcohols, gases). The end products are characteristic
of individual bacterial species. Ethanol fermentation, also called alcoholic
fermentation, is a biological process which converts sugars such as glucose,
fructose, and sucrose into cellular energy, producing ethanol and carbon
dioxide as by-products. Because yeasts perform this conversion in the
absence of oxygen, alcoholic fermentation is considered an anaerobic
process. It also takes place in some species of fish (including goldfish
and carp) where (along with lactic acid fermentation) it provides energy when
oxygen is scarce.

The vital substrate molecule for fermentation is always glucose. Some

bacteria use additional chemical reactions to convert other monosaccharides
and disaccharides into glucose. Some bacteria produce gases when
fermenting a carbohydrate. If gases (typically CO2) are produced during the
fermentation process, a bubble will form at the top and the medium will also
be acidic. Carbohydrate fermentation media are frequently used to distinguish
members of the family Enterobacteriaceae (e.g., Escherichia coli,
Enterobacter aerogenes) from each other.

Figure 17.1: Alcoholic fermentation

134
 Determination of
17.3 REQUIREMENTS Alcoholic
Fermentation From
Grape Juice by
a) Grape juice broth culture of Saccharomyces cerevisiae var. ellipsoideus Saccharomyces
Cerevisiae
b) Pasteurized commercial Grape juice (500 ml)
c) 1 per cent phenolphthalein solution
d) N Sodium hydroxide
e) Sucrose (20g)
f) One litghre sterile Erlenmeyer flask
g) Rubber stopper (fitted with a 5 cm glass tube plugged with cotton)
h) Sterile 10 ml pipettes
i) Burette
j) Spatula
k) Glassine paper
l) Alcoholometer
m) Distillation apparatus
n) Wire gauze
o) Glass beads
p) Ring stand
q) Bunsen burner

Personal protective equipment

• Gloves
• Apron
• Mask
• Shoe cover
• Head cover

17.4 PROCEDURE
1) Take 500 ml of the grape juice into a sterile flask

2) Add 20ml of sucrose and 50 ml of culture of the yeast (10% starter

culture) to the flask and close the flask with the rubber stopper.

3) Incubate the flask at 25 0C for 21 days and add 20 g of sucrose to the

fermenting juice after 2 and 4 days of incubation

4) Determine total acidity, volatile acidity and alcohol at different intervals.

Titration method for determination of total acidity, volatile acidity:

a) Take 10 ml of fermenting juice to a flask

135
 Environment b) Add 10 ml of distilled water and 5 drops of phenolphthalein indicator
Science Lab
Course - 3 (11%).
c) Mix the content
d) Titrate it against 0.1N NaOH taken in a burette to the first persistent pink
colour.
e) Note the volume of NaOH used.

Repeat step ‘a’ through ‘e’ taking fermenting juice and also for control after
14 and 21 days.

1) Determine the % alcohol by dipping alcoholometer in the fermenting

juice at 7 day intervals.
2) Use distillation apparatus and pour 100 ml of fermented grape juice in
the distillation flask, add a few glass beads and put thermometer in the
hole.
3) Start circulation of water through the condenser.
4) Light the Bunsen burner and allow the liquid to drip in the receiver flask.
5) Adjust the heat in such a way that the drops come at the rate of 1 per
second.

Figure 17.2 : Distillation assembly for recovery of alcohol

17.5 OBSERVATIONS AND RESULTS

Note and record the appearance, odour, presence of turbidity or sediments of
the contents of the flask.

By applying the formula, total acidity and volatile acidity can be calculated:
Volume of alkali used X Normality of alkali
Total Acidity = ——————————————————— × 7.5
(expressed as % tartaric acid) Volume of sample in gm (1ml=1gm)
Volume of alkali used X Normality of alkali
Volatile Acidity = ——————————————————— × 6.0
(expressed as % acetic acid) Volume of sample in gm (1ml=1gm)

136
 Determination of
Total acidity, volatile acidity and percent alcohol of the fermenting juice at 7- Alcoholic
days intervals may be recorded. Also determine the total alcohol per ml of Fermentation From
Grape Juice by
the grape juice produced under laboratory conditions at 12 hr, 24 hr or 7 day Saccharomyces
intervals as per experimental requirement. Cerevisiae

17.6 RESULTS
Results for Metabolic Tests performed in the laboratory is as below in the
table:

Total Acidity (expressed as % tartaric acid) = ………………………………

Volatile Acidity (expressed as % acetic acid) = ……………………………
Total alcohol per ml of the grape juice at 7 days intervals = …………………

17.8 PRECAUTIONS
1) Always wear lab coat and gloves when you are in the lab.
2) Always use the sterile glassware.
3) The thermometer bulb should always be placed just below the side arm
of the distillation flask.
4) Joints must be airtight and should be checked before lighting the
Bunsen burner.
5) Add drop wise during the titration.
6) Stop when light pink colour appeared during titration.
7) Note proper reading with accurate time intervals.

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Science Lab EXPT 18 SEPARATION OF AMINO ACIDS
Course - 3
BY THIN LAYER
CHROMATOGRAPHY

Structure

18.0 Introduction
18.1 Expected Learning outcomes
18.2 Principle
18.3 Requirements
18.4 Procedure
18.5 Observations and Calculations
18.6 Result
18.7 Precautions

18.0 INTRODUCTION
Chromatography is the most useful general technique for the separation of
closely related compounds in a mixture. The separation is affected by
differences in the equilibrium distribution of the components between two
immiscible phases, viz., the stationary and the mobile. Differences in the
equilibrium distribution are a result of nature and degree of interaction of the
components with these two phases. The stationary phase is a porous medium
like silica or alumina, through which the sample mixture percolates under the
influence of a moving solvent (the mobile phase). There are a number of
interactions between the sample and the stationary phase and affect the
separation of compounds. Separation of amino acids are one of the important
applications of Thin Liquid Chromatography (TLC). Amino acids contain
both the amino groups as well the carboxylic groups. A- amino acids are the
most important as these are the units from which proteins are made.

18.1 EXPECTED LEARNING OUTCOMES

After studying and performing this experiment you should be able to:

1) Explain the basic principle of TLC;

2) Perform the Separation of amino acids;
3) Explain the major application of Thin Layer Chromatography (TLC);
4) Define TLC - Rf value

138
 Separation of
18.2 PRINCIPLE Amino Acids by
Thin Layer
Chromatography
Thin layer chromatography (TLC) technique is used for separation and
identification of compounds. TLC plate is made up of a thin layer of silica
adhered to glass or aluminum for support. Thin layer chromatography (TLC)
technique is used for separation and identification of compounds. TLC plate
is made up of a thin layer of silica and supported by glass or aluminum. The
silica gel is considered as the stationary phase and the solvent mixture acts as
the mobile phase. The compounds are soluble to different degrees is the
characteristic of the ideal solvent system. Separation results from the
partition equilibrium of the components in the mixture.

In this simplest form of the technique, a spot of the sample mixture is

applied near one end of the TLC plate and allowed to dry. The strip or plate is
then placed with dipping in to the solvent mixture, taking care that the sample
spot/zone is not immersed in the solvent. The solvent moves towards the
other end of the strip and the test mixture separates into various components.
This is called as the development of TLC plates.

The separation depends on several factors:

a) Solubility: the more soluble a compound is in a solvent, the faster this

will move up the plate.

b) Attractions between the compound and the silica: the more the
compound interacts with silica, the lesser it moves

c) Size of the compound: the larger the compound, it moves up the plate
slowly.

The plate is removed after an optimal development time. Plate is dried and
the spots/zones are detected using a suitable location reagent. The
components of a mixture are identified by the calculation and comparison of
Rf values.

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Science Lab
Course - 3

Fig 18.1: TLC technique

18.3 REQUIREMENTS
Reagents:

1) 2% solution of individual amino acids.

2) Solvent mixture of normal butanol, acetic acid and water in the ratio
12:3:5 by volume.
3) Ninhydrin reagent.

Requirements:

1) TLC plate
2) TLC chamber
3) Capillary tubes
4) Reagent spray bottle
5) Conical flasks
6) Beakers

18.4 PROCEDURE
Chromatographic Separation of Amino acids:

Thin Layer Chromatography is used to separate the amino acids in a given

mixture. All 20 of the common amino acids (standard amino acids) are a-
amino acids. Carboxyl group and an amino group bonded to the same carbon
atom (the α- carbon) and differ from each other in their side chains, or R
groups, which vary in structure, size, and electric charge. Interaction of the
amino acids with the stationary phase like silica varies depending on their 'R'
groups. The amino acid that interacts strongly with silica will be carried by
the solvent to a small distance, whereas the one with less interaction will be
moved further. By running controls alongside, identification of the
components of the mixture is possible.

140
 Separation of
Amino Acids by
Thin Layer
Chromatography

Fig 18.1: Amino acids

Since amino acids are colourless compounds, ninhydrin is used for detection.
Ninhydrin reagent is sprayed after development of the TLC plate and dried
in an oven, at 105°C for about 5 minutes.

Ninhydrin reacts with α- amino acids that results in purple-coloured spots [

due to the formation of the complex - Rheuman's purple]. Ninhydrine is 2-
hydrate of indane-l,2,3 trione (or triketohydrindene hydrate). It reacts with
amino acids to yield highly coloured products.

heat

Amino acid + Ninhydrin coloured product

The formation of visible colour with ninhydrin has limits of detection which
may vary from 0.01-0.5 pg depending on the particular amino acids.

Rf values be calculated and compared with the reference values to identify

the amino acids. [The Rf value for each known compound should remain the
same provided the development of plate is done with the same solvent, type
of TLC plates, method of spotting and in exactly the same conditions].

1) Pour the solvent mixture in to the TLC chamber and close the chamber.

2) The chamber should not be disturbed for about 30 minutes so that the
atmosphere in the jar becomes saturated with the solvent.

3) Cut the plate to the correct size and using a pencil, gently draw a straight
line across the plate approximately 2 cm from the bottom.

4) Using a capillary tube, a minute drop of amino acid spotted on the line.

5) Allow the spot to dry.

6) Spot the second amino acid on the plate [enough space should be
provided between the spots].

7) Repeat the above step for spotting the unknown acid.

8) Place the plate in the TLC chamber as evenly as possible and lean it
against the side (immerse the plate such that the line is above the
solvent). Allow capillary action to draw the solvent up the plate until it is
approximately 1 cm from the end.

141
 Environment 9) Remove the plate and immediately draw a pencil line across the solvent
Science Lab
Course - 3 top.

10) Under a hood dry the plate with the aid of a blow dryer.

11) Spray the dry plate with ninhydrin reagent.

12) Dry the plates in hot air oven at 105°C for 5 min. Ninhydrin will react
with the faded spots of amino acids and make them visible as purple-
coloured spots.

13) After some time, mark the centre of the spots, then measure the distance
of the centre of the spots from the origin and calculate the Rf values.

18.5 OBSERVATIONS AND CALCULATIONS

Formula for calculating Rf value:

Amino acid ds dm Rf
Alanine
Glutamic acid
Glycine
Glycine
Leucine
Valine
Lysine
Tyrosine
Unknown Rf resemble
sample 1 with
……………
Unknown Rf resemble
sample 2 with ................

18.6 RESULTS
Following amino acids are present in a given mixture sample:

1) ………………………………
2) ………………………………

142
 Separation of
18.7 PRECAUTIONS Amino Acids by
Thin Layer
Chromatography
1) Always wear lab coat and gloves when you are in the lab. When you
enter the lab, switch on the exhaust fan and make sure that all the
reagents required for the experiment are available. If it is not available,
prepare the reagents using the components shown in the reagent
preparation.

2) Care should be taken while handling reagents like Ninhydrin reagent.

This reagent is a strong oxidizing agent and should not be inhaled or
spilled on hands or other body parts.

3) Accidental spill of reagent may cause severe itching sensation. Wash the
spilled area with cold water and inform the lab assistant immediately.

4) Hold the TLC plates by their side. Ensure that you do not touch the
developing part of the TLC plate, because your finger prints will also get
developed and result will be unclear.

5) Make certain that the spots applied to the plate are above the surface of
the eluting solvent.

6) Before applying the second spot make sure that the previously applied
spot is dried.

7) Spot the components with proper space in between.

8) Ensure that the chamber is saturated with the solvent vapour before you
place the TLC plate in it.

9) provide enough time for the solvent to advance up the plate.

10) The top of the solvent must not advance up to or beyond the edge of the
plates.

11) Measure proper value.

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 Environment
Science Lab EXPT.19 DETERMINATION OF SIDIUM
Course - 3
AND POTASIUM IN WATER BY
USING FLAME PHOTOMETER

Structure

19.0 Introduction
19.1 Expected Learning Outcomes
19.2 Principle
19.3 Requirements
19.4 Procedure
19.5 Observations and Calculations
19.6 Result
19.7 Precautions

19.0 INTRODUCTION
High concentration of sodium and potassium in water samples are
responsible for kidney-related problems. Study of the amount of sodium and
potassium contents in the water of the water bodies is important. Water
quality index (WQI) of the collected water samples is calculated to
understand the influence of different water quality. Water and food are the
source of sodium. Sodium could be harmful to a human if it exceeds the
normal levels. Sodium intake is a concern for those who are suffering from
heart and kidney problem or insomnia. Low sodium content food is advisable
to avoid problems with sodium. Potassium is the sodium’s partner and stays
inside the cells with cell barrier at the other side. Potassium is considered as
an electrolyte which does not circulate freely inside the human body. The
concentration of potassium is responsible for withstanding pressure of
sodium and the pressure generated due to water movement. Inside the cell,
Sodium/potassium are inseparable, and help our body in controlling neural
transmission and contraction of muscles. Potassium level in the body is vital
as it maintains a balance water level inside the body. It controls blood
pressure and plays a pivotal role in neural transmission and contraction of
muscle. Therefore, Water Quality Index (WQI) calculation of water sample
is important to get an idea of quality of drinking water. Water quality can be
checked whether it is fit for human consumption in reference to the presence
of sodium and potassium.

19. 1 EXPECTED LEARNING OUTCOMES

After studying and performing this experiment you should be able to:

1) Determine the concentration of sodium and potassium in water sample;

144
 Determination of
2) Explain the principle of Flame photometric method; Sidium and
Potasium in Water
3) Perform a flame photometric determination; by Using Flame
Photometer
4) Determine the concentration of sodium and potassium ions in a given
sample.

19.2 PRINCIPLE
Methods for the determination of sodium and potassium include flame atomic
absorption, inductively coupled plasma, flame photometry, and selective ion
electrode. The inductively coupled plasma/mass spectrometric method
usually may be applied successfully (with lower detection levels)

In the field of analytical spectrophotometers, the concept of the introduction

of photomultiplier tube in the grating-based spectrophotometers to AAS was
significant. Flame photometer has a high resolution for the detection of alkali
metal ions such as sodium (Na+) and potassium (K+). Flame photometer
works by atomizing a solution sample into a flame and separates the
characteristic spectra of an element along with measurement of emission.
Low excitation is obtained from flame-based devices ranging from the
temperature of 1000–3000 °C. Due to low excitation temperature from flame
than arc flame, this method is well suited for alkali metal.

ICP-AES is superior in metal ions detection over flame photometry

technique, but the efficiency of sodium and potassium detection by this is
very low compared to flame photometry. On the introduction of group II
compounds consisting of alkaline earth and alkali metals into the flame, they
dissociate into separate atoms.

On dissociation, the atoms get excited to higher states. But at higher states,
they are not stable, so they return back to the ground state with the emission
of characteristics wavelength. As they return back to the ground state, they
emit colour in the range of visible spectrum. Each alkali metal and alkaline
earth metals have a characteristic wavelength. When a solution containing
Na+ and K+ is aspirated into the flame the light emitted is of different
wavelengths (colours), for the two ions. These can be measured by using
appropriate Coloured filters.

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Science Lab
Course - 3

Instrumentation of Flame Photometer Digital Flame Photometer

19.3 REQUIREMENTS
APPARATUS

a) Flame photometer (either direct-reading or internal standard type)

b) Glassware

REAGENTS

a) Reagent water: water for preparing all reagents and calibration

standards, and as dilution water.

b) Stock potassium solution: Dissolve 1.907 g KCl dried at 110°C and

dilute to 1000 mL with water; 1 mL = 1.00 mg K. and Stock sodium
solution: Dissolve 2.542 g NaCl dried at 140°C to constant weight and
dilute to 1000 mL with water; 1.00 mL= 1.00 mg Na.

c) Intermediate potassium solution: Dilute 10.0 mL stock potassium

solution with water to 100 mL; 1.00 mL =0.100 mg K. Use this solution
to prepare calibration curve in potassium range of 1 to 10 mg/L.

Intermediate sodium solution: Dilute 10.00 mL stock sodium solution

with water to 100.0 mL; 1.00 mL = 0.10 mg Na (1.00 mL = 100 g Na).
Use this intermediate solution to prepare calibration curve in sodium
range of 1 to 10 mg/L.

d) Standard potassium solution: Dilute 10.0 mL intermediate potassium

solution with water to 100 mL; 1.00 mL = 0.010 mg K. Use this solution
to prepare calibration curve in potassium range of 0.1 to 1.0 mg/L.

Standard sodium solution: Dilute 10.00 mL intermediate sodium

solution with water to 100 mL; 1.00 mL 10.0 g Na. Use this solution to
146 prepare calibration curve in sodium range of 0.1 to 1.0 mg/L.
 Determination of
19.4 PROCEDURE Sidium and
Potasium in Water
by Using Flame
Trace amounts of sodium or potassium can be determined by flame emission Photometer
photometry at 589 nm or 766.5 nm, respectively. Sample is nebulized into a
gas flame under carefully controlled, reproducible excitation conditions. The
sodium resonant spectral line at 589 nm is isolated by interference filters or
by light-dispersing devices such as prisms or gratings. Emission light
intensity is measured by a phototube, photomultiplier, or photodiode.
Alignment of the wavelength dispersing device and wavelength readout may
not be precise. The appropriate wavelength setting, which may be slightly
more or less than 589 nm or 766.5 nm, can be determined from the maximum
emission intensity when aspirating a sodium standard solution, and then used
for emission measurements. The calibration curve may be linear but has a
tendency to level off or even reverse at higher concentrations. Linear to near-
linear range is advisable.

Instrument operation: Due to differences between makes and models of

instruments, it is not possible to formulate detailed operating instructions for
instrument operation. Users may follow the procedure as per manufacturer’s
recommendations for selecting proper photocell and wavelength, adjusting
slit width and sensitivity, appropriate fuel and oxidant gas pressures, and the
steps for warm-up, correcting for interferences and flame background, rinsing
of burner, igniting flame, and measuring emission intensity.

Steps to follow:

• Ensure the photometer drain is leading into a sink and that the instrument
is connected to gas, air and electricity supplies.

• Ensure the mains supply gas tap is off.

• Insert the sodium optical filter.

• Switch on the instrument and unclamp the galvanometer by turning

counterclockwise.

• Open the mica window, turn on the mains gas supply, light the gas and
close the window

• Turn on the air supply control and adjust the air pressure to 10 lb/in2.
Leave for 1-2 minutes to stabilize.

• Place a beaker of distilled water into position at the left-hand side of the
instrument and insert the narrow draw tube into it to allow water to pass
through the photometer. (NOTE: once set up, the photometer must have
water running through it at all times when a salt solution is not being
measured. The rate of uptake is fast, so make sure there is always enough
water in the beaker).

147
 Environment • Adjust the gas control to give a flame with a large central blue cone then,
Science Lab
Course - 3 with water passing through the instrument, slowly close the gas control
until ten separate blue cones just form.

• Set the galvanometer to zero using the "Set zero" control.

a) Direct-intensity measurement:

• Prepare a blank and sodium & potassium calibration standard in stepped

amounts in any of the following applicable ranges: 0 to 1.0, 0 to 10, or 0
to 100 mg/L.

• Determine emission intensity at 589 for sodium nm and at 766.5 nm for

potassium.

• Aspirate calibration standards and samples enough times to secure a

reliable average reading for each.

• Construct a calibration curve from the sodium & potassium standards,

separately.

• Draw a graph between the concentration of the standard Na+ (or K+ )

ion (on the X-axis) versus the intensity ratio of the Na+ (or K+) ion (on
the Y-axis) in the graph.

• Determine the concentration of the given sample solution with the help
of the calibration curve.

• Calculate the concentration of the sodium/potassium ions present in the

unknown sample solution by accounting for the dilution factor and report
the value.

19.5 OBSERVATIONS AND CALCULATIONS

a) Observe the emission intensity data for the standard and sample
solutions.

b) Plotting the calibration curve: Plot the graph between the intensity ratio
of Na+ or K+ ion on Y-axis and the concentration of standard Na+ (or
K+) ion as per Figure below:

Fig 19.1 Calibration curve for Sodium and potassium, observe the reading with respect
of the concentration.
148
 Determination of
Concentration of Intensity ratio of Sidium and
Sodium / Potassium Na+ (or K+ ) Potasium in Water
by Using Flame
ion (PPM) Photometer

Sodium ion 2
(Na+)
4
6
8
10
Sodium ion (K+) 2
4
6
8
10

c) Determining the concentration of the Na+ or K+ ion in the sample from

calibration curve.

Plot the value of intensity of the sample solution on the calibration curve
obtained and determine the corresponding concentration value in ppm.

Let the concentration of Na+ or K+ ion in the sample solution obtained

from the graph = A ppm. The actual concentration of Na+ or K+ ion in
the sample solution would be: = A× D = ……ppm

Where, A=direct reading from the calibration curve,

D= dilution factor, if any.

Determining the concentration of the Na+ or K+ ion in the sample by the

following formula:

mg Na/L= (mg Na/L in portion) X D

mg K/L= (mg K/L in portion) X D

19.6 RESULTS
The concentrations of Na+ and K+ ions in a given solution:
[Na+ ] (PPm) =…………..
[K+ ] (PPM) =…………..

19.7 PRECAUTIONS
1) Deionized water should be used.

149
 Environment 2) Wavelength of standard line should be close to the wavelength of analyte
Science Lab
Course - 3 line.

3) Do not store samples in soft-glass bottles because of the possibility of

contamination from leaching of the glass.

4) Use acid-washed polyethylene or borosilicate glass bottles. Adjust

sample to pH < 2 with nitric acid. This will dissolve potassium salts and
reduce adsorption on vessel walls.

5) When the instrument is no longer required, switch off in the following

sequence:

i) Turn off the gas control and the mains gas supply
ii) Wait for the flame to die out.
iii) Turn off the air supply.
iv) Switch off the electricity
v) Clamp the galvanometer.

6) Do not lean over the instrument or you will set your hair alight.

7) Clean properly the instrument after use.

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 Determination of
EXPT 20 DETERMINATION OF Suspended
Particulate Matter
SUSPENDED PARTICULATE (Spm) in Ambient
Air Using High
MATTER (SPM) IN AMBIENT AIR Volume Sampler
(HVS)
USING HIGH VOLUME SAMPLER
(HVS)

Structure

20.0 Introduction
20.1 Expected Learning Outcomes
20.2 Principle
20.3 Requirements
20.4 Procedure
20.5 Observations and Calculations
20.6 Result
20.7 Precautions

20.1 INTRODUCTION
Air pollution became major pollution all over the world. Air pollution
became major pollution all over the world. The presence of particulate matter
(PM) in the ambient air is the leading cause of many diseases in different
metropolitan and industrial regions throughout the world. The air quality we
breathe depends on the quality of air which is primarily determined by the
amount of gaseous pollutants and particulate matter present in the air. This
particulate matter (PM) inhaled into the human respiratory system is related
to the most serious health effects including pulmonary and cardiovascular
illnesses. The construction of roads and road side area increases the
particulate matter in to the environment. Suspended Particulate Matter (SPM)
is usually defined as comprising particles less than 10 μm in diameter
suspended in the atmospheric environment. Air particulates which has less
than 2 μm in diameter mainly originate from incinerators, boilers, and
automobiles and may reach deeply into human lungs during respiration cause
respiratory diseases. SPM may also be produced by photochemical reactions
of gaseous substances in the atmosphere. Therefore, there is necessity to
know the concentration of particulate matter (PM) in to the atmosphere.

20.1 EXPECTED LEARNING OUTCOMES

After studying and performing this experiment you should be able to:

1) Determine the particulate matter (PM) in the ambient air;

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 Environment 2) Explain the interference during sampling and processing of samples for
Science Lab
Course - 3 analysis of particulate matter (PM) in the ambient air;

20.2 PRINCIPLE
Air is drawn through a size-selective inlet and through a 20.3 X 25.4 cm (8 X
10 in) filter at1132 L/min flow rate. Particles with aerodynamic diameter less
than the cut-point of the inlet are collected, by the filter. The mass of these
particles is determined by the difference in filter weight of prior and after
sampling. The concentration of PM10 in the designated size range is
calculated by dividing the weight gain of the filter by the volume of air
sampled.

Figure20.1: SPM samplers

Interferences

When filters are heavily loaded with aerosols, particle loss happens during
transport. Particle loss is minimized by folding the filter and putting in to a
proper container envelope prior to transport.

20.3 REQUIREMENTS
1) Analytical balance
2) Sampler: High Volume Sampler (HVS) capable of drawing ambient air
through a portion of a clean glass fibre filter of (8 X 10 in).
3) Orifice Transfer Standard (Top loading orifice kit) for calibration of flow
of HVS
4) Filter Media - A Glass fiber filter of 20.3 X 25.4 cm (8 X 10 in)
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 Determination of
20.4 PROCEDURE Suspended
Particulate Matter
(Spm) in Ambient
1) Filter preparation Air Using High
Volume Sampler
Filter inspection: Inspect the filter for pin holes. Remove loose particles with (HVS)
a soft brush. Apply the filter identification number or a code to the filter.
Condition the filter in conditioning room maintained within 20-30° C and 40-
50% relative humidity or in an airtight desiccator for 24 hours. Take initial
weight of the filter paper (Wi) before sampling.

2) Calibration

Periodical calibration of the sampler is being done by a calibrated Orifice

Transfer Standard - The PM10 sampler calibration orifice consists of a 3.175
cm (1.25 in) diameter hole in the end cap of 7.62 cm (3 in) diameter by 20.3
cm (8 in) long hollow metal cylinder. This orifice is mounted tightly to the
filter support in place of the inlet during calibration. A small tap on the side
of the cylinder is provided to measure the pressure drop across the orifice.
The relationship between pressure difference and flow rate is established via
a calibration curve derived from measurements against a primary standard at
standard temperature and pressure. Flow resistances that simulate filter
resistances are introduced at the end of the calibrator opposite the orifice by a
set of perforated circular disks.

Figure 20.2: Top loading Orifice kit

3) Field Sampling

Tilt back the inlet and secure it according to manufacturer's instructions.

Loosen the faceplate wing nuts and remove the faceplate. Remove the filter
from its jacket and centre it on the support screen with the rough side of the
filter facing upwards. Replace the faceplate and tighten the wing nuts to
secure the rubber gasket against the filter edge. Gently lower the inlet. For
automatically flow-controlled units, record the designated flow rate on the
data sheet. Record the reading of the elapsed time meter. The specified length
of sampling is commonly 8 hours or 24 hours. During this period, several
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 Environment readings (hourly) of flow rate should be taken. The sampling flow rate shall
Science Lab
Course - 3 not be less than 0.8 m3/min. After the required time of sampling, record the
flow meter reading, take out the filter media from the sampler, and put in a
container or envelope.

4) Analysis

Condition the filter after sampling in the conditioning room maintained

within 20-30° C and 40-50% relative humidity or in an airtight desiccator for
24 hours. Take final weight of the filter paper (Wf).

20.5 OBSERVATIONS AND CALCULATIONS

Observe and record the initial and final weight of filter paper and put these
values in the formula for determination of concentration of Suspended
Particulate Matter (SPM) as below:
Concentration of SPM (µg/m3 ) in ambient air sample = (Wf – Wi) x 106/V
Where,
C SPM = Concentration of SPM, µg/m3
Wf = Final weight of filter in g
Wi = Initial weight of filter in g
6
10 = Conversion of g to µg
V = Volume of air sampled, m3

20.6 RESULTS
The concentration of Suspended Particulate Matter (SPM) in a given location
= ………………….. µg/m3

20.7 PRECAUTIONS
1) Loose particles remove completely.
2) Proper calibrate the instrument.
3) Carefully transfer the filter paper for processing and analysis in the
laboratory.
4) Filter media should collect more than 99% of 0.3 urn particles, have an
alkalinity of less than 25 micro equivalents/gram, and should not gain or
lose weight equivalent to more than 5 microgram/m3, estimated from the
nominal volume sampled over a 24-hour period.
5) Prior to weighing, filters should be equilibrated at a constant temperature,
within ±3% between 15°C and 30°C, and at constant relative humidity,
within ±5% between 20% and 45%.

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