Micro

Contents i
MICROBIOLOGICAL TECHNIQUES
MICROBIOLOGICAL TECHNIQUES
N. Murugalatha
Assistant Professor and Head
Lali Growther J. Vimalin Hena
Assistant Professor Assistant Professor
N. Hema Shenpagam R. Anitha
D. Kanchana Devi G. Rajalakshmi
Department of Microbiology and Biotechnology

Hindusthan College of Arts and Science
Coimbatore, Tamil Nadu
MJP PUBLISHERS
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Cataloguing-in-Publication Data
Microbiological techniques / by N. Muruga-
Latha ... [et al.].- chennai: MJP
Publishers, 2012.
xiv, 442 p.; 24 cm.
Includes Appendix, References.
ISBN 978-81-8094-107-8 (hb.)
1. Microbiology 2. Techniques-Microbiology.
I. Murugalatha, N ... [et al.].
579 dx22 MIC MJP 142
ISBN 978-81-8094-107-8 MJP PUBLISHERS

© Publishers, 2012 New No. 5, Muthu Kalathy Street
All rights reserved Triplicane
Printed and bound in India Chennai 600 005
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This book has been published in good faith that the work of the author is original. All efforts have been taken to make the material error-free. However,
the author and publisher disclaim responsibility for any inadvertent errors.
PREFACE
Microbiological Techniques is designed for the students, to explore the world of microorganisms and
how the process of scientific discovery is carried out, with an ease. The study of microbiology is dynamic
because of the ubiquitous nature of the microbes and the variability inherent in every living organism. The
broad nature of the subject and diversity of topics from the fundamentals to its unique fields can make
the way of presentation a little difficult; but it is also a part of what makes microbiology an interesting
and challenging subject.
The book primarily focuses on the basic microbiological techniques with applications for undergraduate
and postgraduate students in diverse area of biological techniques. This book is the outcome of nearly a
decade of teaching and research experience. The manual comprises twelve parts in which exercises in first
three parts provide sequential developments of fundamental techniques. The remaining exercises are as
independent as possible to allow the instructor to select the desirable sequence. Exercises are pursued in a
normal scale providing maximum details so that one can perform the experiment independently and safely.
The style and simplicity of expression have been our twin objectives. All exercises have been thoroughly
tested in our laboratory by our students with wide variety of real talents and enthusiasm.
We wish to express our immense gratitude to the Management of Hindusthan College of Arts and
Science for allowing us to pursue this work, during our service in their Institution.
We have immense pleasure in expressing our deepest gratitude to our principal Dr. N. Baluswamy,
Hindusthan College of Arts & Science, for his valuable support which has been a great help in this endeavour.
We extend our gratefulness to all our colleagues in the Department of Biotechnology and Microbiology,
for encouraging us right through our work. We are most grateful to every soul involved in making up of
this manual spending their time, talent and interest in the work.
We look forward for the support, constructive ideas and valuable suggestions from the users of the book.
N. Murugalatha
Lali Growther
J. Vimalin Hena
N. Hema Shenpagam
R. Anitha
D. Kanchana Devi
G. Rajalakshmi
CONTENTS
1. Introduction to Microbiology 1
Introduction 1
Bacteria 1
Stages of bacterial growth 2
Factors affecting bacterial growth 3
Cyanobacteria 7
2. Tools of Microbiology 9
Microscopy 9
General principles of microscopy 9
Light microscopy 11
Electron Microscopy 18
Scanned-Probe Microscopy 20
Use and Care of Microscopes 22
Autoclave 26
Hot Air Oven 28
Incubator 29
Water Bath 31
BOD Incubator 32
Colony Counter 34
Haemocytometer 35
Oxygen Electrode 38
Lyophilizer 40
Laminar Air Flow (Vertical) 43
3. Fundamentals of Microbiology 45
Laboratory Precautions 45
Principles of Aseptic Techniques 46
Cleaning of Glassware 48
viii Contents
Safety 51
Control of Microorganisms 52
Sterilization 52
Disinfection 61
Culture Media Preparation 64
Different types of Media 65
Sterilization 68
Media Inoculation and Incubation 69
Pure Culture Techniques 71
Aerobic Culture Techniques 71
Anaerobic Culture Techniques 78
Robertson’s Cooked Meat (Rcm) Medium (Clostridium Sp.) 78
Anaerobic Jar (Total Anaerobes) 79
Wright’s Tube Method 82
Staining Techniques 84
Smear preparation 84
Types of staining techniques 85
Simple Staining 85
Gram Staining 86
Capsule Staining 89
Spore Staining (Schaeffer and Fulton Method) 90
Acid-Fast Staining (Ziehl–Neelsen’s Method) 92
Flagellar Staining 94
Negative Staining 96
Granule Staining 97
Giemsa stain for thin films (Blood) 97
Fontana’s stain for Leptospires 98
Periodic Acid—Schiff (PAS) Stain 98
Fungal wet mount—Lactophenol Cotton Blue Staining 99
Slide Culture Method 100
Micrometry 102
Micrometry and Measurement of Microorganisms 102
Motility Determination 105
Enumeration of bacteria, fungi and actinomycetes from soil 110
Phenol Coefficient Test 113
Maintenance and Preservation of Cultures 114
Contents ix
4. Microbial Physiology 117

Growth Curve 117
Direct Count 117
Viable Count 122
Turbidity Method 125
Biochemical Tests 128
IMViC Reactions 128
Indole Production Test 128
Methyl Red–Voges Proskauer Test 130
Citrate Utilization Test 131
Oxidase Test 133
Catalase Test 134
Urease Test 135
Hydrogen Sulphide Test 136
Triple Sugar Iron Agar Test 137
Nitrate Reduction Test 138
Polymer (Starch, Casein, Gelatin, Lipid) Degradation 140
Carbohydrate Fermentation Test 142
Amino Acid Decarboxylase Test 143
Phenylalanine Deaminase Test 145
Coagulase Test 146
Esculin Hydrolysis 147
ONPG ( O-Nitrophenyl β-d-Galactopyranoside) Test 149
OF Test for Carbohydrate Utilization 150
Malonate Utilization Test 152
Lecithinase Test 153
Pigment Extraction From Algae 154
Effect of Temperature on the Growth of Bacteria and Fungi 158
Effect of Osmotic Pressure on the Growth of Bacteria and Yeast 162
Effect of pH on the Growth of Bacteria and Fungi 164
5. Industrial Microbiology 167
Wine Production 167
Citric Acid Production 169
Production of Glutamic Acid 172
Protease Estimation 175
Production of Extracellular Cellulase by Solid-state Fermentation 180
x Contents
Mushroom Cultivation 183

Extracellular Enzyme Production—Amylase 185
Immobilization of Cells 186
6. Environmental Microbiology 189
Isolation of Nitrogen Fixers 189
Isolation of Symbiotic Nitrogen Fixers (Rhizobium Sp.) 189
Isolation of Free-living Nitrogen Fixers (Azotobacter Sp.) 190
Isolation of Phosphate Solubilizers 191
Standard Qualitative Analysis of Water by MPN Method 192
Quantitative Analysis of Water by Membrane Filter Technique 196
Dissolved Oxygen 200
Biological Oxygen Demand (BOD) 202
Chemical Oxygen Demand (COD) 204
Total Suspended Solids 206
Decolorization of Dye and Dye-containing Effluents 209
Nitrogen Cycle 210
Ammonification 211
Nitrification 212
Denitrification 213
Enumeration of Microorganisms from Wood and Paint 214
Assay of Microorganisms from Biomedical Waste 215
Isolation and Culture of Algae 216
Spraying 216
Single Cell/Colony/Filament Isolations 217
7. Food Microbiology 219
Methylene Blue Reduction Test 219
Phosphatase Test for Pasteurisation in Liquid Milk 221
Turbidity, Colony and Coliform Tests for Pasteurized Milk 223
Isolation of Food Spoilers 224
Analysis of Food Sample for Mycotoxin (Aflatoxin) 226
8. Genetics 229
Isolation of Chromosomal DNA from Bacteria 229
Isolation of Genomic DNA from Cauliflower by Supaquick Method 232
Isolation of Plasmid DNA 234
Agarose Gel Electrophoresis 235
Contents xi
Isolation of RNA 239

Isolation of Mutants 241
Induced Mutation—Isolation of Antibiotic-resistant Mutants 241
Isolation of Auxotrophic Mutants (Replica-plating Technique) 242
Restriction Digestion of Lambda DNA 244
Bacterial Conjugation 245
Bacterial Transformation 247
Southern Blotting 249
Western Blotting 253
Random Amplified Polymorphic DNA (RAPD) 255
Restriction Fragment Length Polymorphism (RFLP) 259
9. Immunology 265
Blood Grouping 265
Antistreptolysin O 267
Widal Test 269
Slide Agglutination Method 270
Tube Agglutination Method 271
Serological Tests for Diagnosis of Syphilis 272
VDRL (Venereal Disease Research Laboratory) test 273
RPR (Rapid Plasma Reagin) test 274
Enzyme-linked Immunosorbent assay (Elisa) 276
C-reactive Protein (CRP) 277
Rheumatoid Arthritis (RA) Factor 279
Countercurrent Immunoelectrophoresis 281
Immunodiffusion 282
Ouchterlony’s Double Diffusion (ODD) Technique 282
Radial Immunodiffusion (RID) Technique 285
Isolation and Characterization of Antigens 287
Purification of Immunoglobulins by Precipitation and Dialysis 288
10. Medical Microbiology 291
Isolation and Characterization of Pathogens from Clinical Samples 291
Sample Collection 291
Identification of Microorganisms 295
Sample Processing 295
Identification and Enumeration of Lymphocytes 310
xii Contents
Identification of Fungal Pathogens 313

Yeasts and Yeast-like Fungi 318
Morphology of Cryptococci 320
Germ Tube test 320
Growth on Cornmeal agar for Chlamydospore or True Hypae Production 321
Carbohydrate Assimilation Test 323
Carbohydrate Fermentation Test 323
Urease Test 324
Examination of Blood Smear for Malarial Parasite 325
Examination of Parasites From Faeces 326
Anti Microbial Susceptibility Testing (Kirby–Bauer Disc Diffusion Method) 329
Determination of Minimum Inhibitory Concentration 330
Antibiotic Susceptibility Testing 330
E-test 331
Broth Dilution Method 333
11. Biochemical Methodology 335
Centrifugation 335
Components 335
Types 336
Applications 338
Separation of Amino Acids 339
Paper Chromatography 339
Thin-layer Chromatography 343
Column Chromatography 345
Colorimeter and Spectrophotometer 347
Laws of Absorption 348
Visible Spectrophotometer 348
UV Spectrophotometer 351
Fluorscence Spectroscopy 352
Atomic Absorption Spectrophotometer (AAS) 356
Turbidometry 359
Bomb Calorimeter 362
Total Protein Estimation 364
Lowry’s Method 364
Bradford’s Method 367
Creatinine Estimation 368
Contents xiii
Fractionation and Size (Determination of Proteins Using SDS–PAGE 369

Buffer Solutions 374
Concentration Units (Molarity, Normality, Molality) 380
12. Virology 385
Isolation of Coliphages 385
Phage Titration 386
Egg Inoculation 389
Cultivation of Animal and Plant Viruses 395
Cultivation of Animal Viruses 395
Cultivation of Plant Viruses 401
Appendix 407
References 439
1
INTRODUCTION TO MICROBIOLOGY
INTRODUCTION
Microbiology is the study of microorganisms, that is, the organisms which are of microscopic
dimensions. These organisms are too small to be clearly perceived by the unaided human eye.
If an object has a diameter of less than 0.1 mm, the eye cannot perceive (or more correctly resolve)
it, and very little detail can be perceived in an object with a diameter of 1 mm. Roughly speaking,
organisms with a diameter of 1 mm or less are microorganisms and fall into the broad domain
of microbiology. Since most microorganisms are only a few thousandths of a millimetre in size,
they can be seen only with the aid of microscope.
These microorganisms are classified into protozoa, algae, fungi, bacteria and viruses. Viruses
are ultramicroscopic and have an obligate parasitic relationship, but for practical purposes these
still come under the domain of microbiology.
At present, there is a general agreement to include five major groups as microorganisms:
viruses, bacteria, fungi, algae and protozoa (the studies of which are called as virology, bacteriology,
mycology, phycology and protozoology respectively).
BACTERIA
Bacteria are unicellular organisms that lack chlorophyll and are among the smallest living things
on earth. Multiplying rapidly under favourable conditions, bacteria can aggregate into colonies
of millions or even billions of organisms within a space as small as a drop of water. The Dutch
merchant and amateur scientist, Antony Van Leeuwenhoek, was the first to observe bacteria and
other microorganisms. Using single-lens microscopes of his own design, he described bacteria and
other microorganisms (called animalcules) in a series of letters to the Royal Society of London
between 1674 and 1723.
Bacteria are classified under Prokaryotes. Broadly, this taxonomic ranking reflects the fact
that the genetic material of bacteria is contained in a single, circular chain of deoxyribonucleic
acid (DNA) that is not enclosed within a nuclear membrane. The word “prokaryote” is derived
2 Microbiological Techniques
from a Greek word meaning “prenucleus”. Moreover, the DNA of prokaryotes is not associated
with the special chromosome proteins called histones, which are found in higher organisms.
In addition, prokaryotic cells lack other membrane-bound organelles, such as mitochondria.
Prokaryotes belong to the kingdom, Monera. Some scientists have proposed splitting this
designation into the kingdoms, Eubacteria and Archaebacteria. Eubacteria, or true bacteria,
consist of more common species, while Archaebacteria (with the prefix “archae”, meaning
“ancient”) represent strange bacteria that inhabit very hostile environments. Scientists believe
that these bacteria are most closely related to the bacteria, which lived when the earth was formed.
Examples of archaebacteria are those bacteria which currently live in extremely salty environments
or extremely hot environments, like geothermal vents of the ocean floor. Some examples of
archaebacteria are Methanococcus jannaschiii, Haloferax, Thermus aquaticus and Thermococcus litoralis.
Outer membrane Storage granule

Cytoplasm Capsule
Periplasmic space (sometimes)
Cytoplasmic (inner)
membrane
Peptidoglycan
Flagellum
Basal body
Ribosome
Mesosome
Chromosome
Pilus
Figure 1.1 Structure of a bacterial cell
STAGES OF BACTERIAL GROWTH

Under ideal conditions, the growth of a population of bacteria occurs in several stages, termed
as lag, log, stationary and death phase. During the lag phase, active metabolic activity occurs
involving synthesis of DNA and enzymes, but no growth. Geometric population growth occurs
during the log or exponential phase, when metabolic activity is most intense and cell reproduction
exceeds cell death. Following the log phase, the growth rate slows and the production of new
cells equals the rate of cell death. This period, known as the stationary phase, involves the
establishment of an equilibrium in population numbers and a slowing of the metabolic activities
of individual cells. The stationary phase reflects a change in growing condition, for example, a
Introduction to Microbiology 3
lack of nutrients and/or the accumulation of waste products. When the rate of cell death exceeds
the number of new cells formed, the population equilibrium shifts to a net reduction in numbers
and the population enters the death phase, or logarithmic decline phase. The population may
diminish until only a few cells remain, or the population may die out entirely.
Stationary phase
Death
Log phase
Log phase
phase
Figure 1.2 Growth Curve
FACTORS AFFECTING BACTERIAL GROWTH

1. Temperature
The lowest temperature at which a particular species will grow is the minimum growth temperature,
while the maximum growth temperature is the highest temperature at which they will grow. The
temperature at which their growth is optimal is called the optimum growth temperature. In
general, the maximum and minimum growth temperatures of any particular type of bacteria are
about 30°F (–1°C) apart. Most bacteria thrive at temperatures at or around that of the human body,
i.e., 98.6°F (37°C), and some, such as Escherichia coli, are normal parts of the human intestinal
flora. These organisms are mesophiles (moderate temperature-loving), with an optimum growth
temperature between 77°F (25°C) and 104°F (40°C). Mesophiles adapt themselves to thrive in
temperatures close to that of their host. Psychrophiles, which prefer cold temperatures, are
divided into two groups. One group has an optimal growth temperature of about 59°F (15°C),
but can grow at temperatures as low as 32°F (0°C). These organisms live in ocean depths or Arctic
regions. Other psychrophiles that can also grow at 32°F (0°C) have an optimal growth temperature
between 68°F (20°C) and 86°F (30°C). These organisms, sometimes called psychrotrophs, are
often those associated with spoilage of food under refrigeration. Examples of pschychrotrophs are
Arthrobacter sp., Psychrobacter sp. and members of the genera Halomonas, Pseudomonas, Hyphomonas
and Sphingomonas. Thermophiles thrive in very hot environments, many having an optimum
growth temperature between 122°F (50°C) and 140°F (60°C), similar to that of hot springs in
Yellowstone National Park. Such organisms thrive in compost piles, where temperatures can rise
as high as 140°F (60°C). Extreme thermophiles grow at temperatures above 195°F (91°C), along
the sides of hydrothermal vents on the ocean bottom 217 mi (350 km) north of the Galapagos
Islands. Some bacteria grow in temperatures that can reach as high as 662°F (350°C). Some
examples of thermophiles are Thermus aquaticus and Thermococcus litoralis.
2. pH
Like temperature, pH also plays a role in determining the ability of bacteria to grow or thrive in
particular environments. Most commonly, bacteria grow optimally within a narrow range of pH
between 6.7 and 7.5. Acidophiles, however, prefer acidic conditions. For example, Thiobacillus
ferrooxidans, which occurs in drainage water from coal mines, can survive at pH 1. Other bacteria,
such as Vibrio cholerae, which causes cholera, can thrive at a pH as high as 9.0.
3. Osmotic Pressure

Osmotic pressure is another limiting factor for the growth of bacteria. Bacteria contain about
80–90% water; they require moisture to grow because they obtain most of their nutrients from
aqueous environment. Cell walls protect prokaryotes against changes in osmotic pressure over a
wide range. However, sufficiently hypertonic media at concentrations greater than those inside
the cell (such as 20% sucrose) cause water loss from the cell by osmosis. Fluid leaves the bacteria
causing the cell to contract, which, in turn, causes the cell membrane to separate from the
overlying cell wall. This process of cell shrinkage is called plasmolysis. Since plasmolysis inhibits
bacterial cell growth, the addition of salts or other solutes to a solution inhibits food spoilage by
bacteria, for example, in the salting of meat and fish. Some types of bacteria, called extreme or
obligate halophiles, are adapted to—and require—high salt concentrations, such as those found
in the Dead Sea, where salt concentrations can reach 30%. Facultative halophiles do not require
high salt environments to survive, but are capable of tolerating these conditions. Halophiles can
grow in salt concentrations up to 2%, a level that would inhibit the growth of other bacteria.
However, some facultative halophiles, such as Halobacterium halobium grow in salt lakes, salt flats,
and other environments where the concentration of salts is up to seven times greater than that
of the oceans. When bacteria are placed in hypotonic media with concentrations weaker than
the inside of the cell, water tends to enter by osmosis. The accumulation of this water causes the
cell to swell and then to burst, a process called osmotic lysis.
4. Carbon, Nitrogen and other Growth Factors

In addition to water and correct salt balance, bacteria also require a wide variety of elements,
especially carbon, hydrogen, nitrogen, sulphur, phosphorus, potassium, iron, magnesium and
calcium. Growth factors, such as vitamins, pyrimidines and purines (the building blocks of DNA),
are also necessary. Carbon is the fundamental building block of all the organic compounds needed
by living things, including nucleic acids, carbohydrates, proteins and fats. Chemoheterotrophs
are bacteria that use organic compounds such as proteins, carbohydrates and lipids as their carbon
source, and electrons from organic compounds as their energy source. Most bacteria (as well as
all fungi, protozoans and animals) are chemoheterotrophs. Chemoautotrophs (for example,
hydrogen, sulphur, iron and nitrifying bacteria) use carbon dioxide as their carbon source and
electrons from inorganic compounds as their energy source. Saprophytes are heterotrophs that
obtain their carbon from dead and decayed organic matter. Many different soil bacteria release
plant nitrogen as ammonia (ammonification). Bacteria, such as the Nitrosomonas, convert ammonia
to nitrite, while Nitrobacter convert nitrite to nitrate. Other bacteria, especially Pseudomonas, convert
nitrate to nitrogen gas. These bacteria complement the activity of nitrogen-fixing bacteria (for
example, Rhizobium, which fix nitrogen from the atmosphere and make it available to leguminous
plants, and Azotobacter, which are also found in fresh and marine waters). Together, the activities
of these bacteria are responsible for the nitrogen cycle by which the gas is taken up by living
organisms, used to make proteins and other organic compounds, returned to the soil during
decay, then released into the atmosphere to be reused by living things. Phototrophs use light as
their primary source of energy, but may differ in their carbon sources. Photoheterotrophs (purple
non-sulphur and green non-sulphur bacteria) use organic compounds as their carbon source, while
photoautotrophs (for example, photosynthetic green sulphur and purple sulphur bacteria) use
carbon dioxide as a source of carbon. Oxygen may or may not be a requirement for a particular
species of bacteria, depending on the type of metabolism used to extract energy from food (aerobic
or anaerobic). In all cases, the initial breakdown of glucose to pyruvic acid occurs during glycolysis,
which produces a net gain of two molecules of the energy-rich adenosine triphosphate (ATP).
5. Gaseous Requirements

Most bacteria may be placed into one of three groups based on their response to gaseous oxygen.
Aerobic bacteria thrive in the presence of oxygen and require it for their continued growth and
existence (Staphylococcus sp. Streptcoccus sp. Enterobacteriacae sp. Myobacterium tuberculosis). Other
bacteria are anaerobic (C. perfringens, C. botulinum), and cannot tolerate gaseous oxygen, such
as those bacteria which live in deep underwater sediments, or those which cause bacterial food
poisoning. The third group are the facultative anaerobes (Escherichia coli), which prefer growing
in the presence of oxygen, but can continue to grow without it. Let us discuss about each of
them in detail.
Aerobic bacteria Aerobic bacteria use oxygen to break down pyruvic acid, releasing much
more ATP than is produced during glycolysis, by the process known as aerobic respiration.
In addition, aerobic bacteria have enzymes such as superoxide dismutase, capable of breaking
down toxic forms of oxygen, such as superoxide free radicals, which are also formed by aerobic
respiration. During aerobic respiration, enzymes remove electrons from the organic substrate
and transfer them to the electron transport chain, which is located in the membrane of the
mitochondrion. The electrons are transferred along a chain of electron carrier molecules. At the
final transfer position, the electrons combine with atoms of oxygen—the final electron accept
or—which in turn combines with protons (H+) to produce water molecules. Energy, in the form
of ATP, is also produced. Along the chain of electron carriers, protons that are pumped across
the mitochondrial membrane re-enter the mitochondrion. This flow of electrons across the
membrane fuels oxidative phosphorylation, the chemical reaction that adds a phosphate group
to adenosine diphosphate (ADP) to produce ATP. Obligate aerobes must have oxygen in order
to live. Facultative aerobes can exist in the absence of oxygen also by using fermentative or
anaerobic respiration. Anaerobic respiration and fermentation occur in the absence of oxygen,
and produce substantially less ATP than aerobic respiration.
Anaerobic bacteria Anaerobic bacteria use inorganic substances other than oxygen as the
final electron acceptor. For example, Pseudomonas and Bacillus reduce nitrate ion (NO3-) to nitrite
ion (NO2-), nitrous oxide (N2O) or nitrogen gas (N2). Clostridium sp. which include those that
cause tetanus and botulism, are obligate anaerobes, that is, they are not only unable to use
molecular oxygen to produce ATP, but are also harmed by toxic forms of oxygen formed during
aerobic respiration. Unlike aerobic bacteria, obligate anaerobes lack the ability to synthesize
enzymes that neutralize these toxic forms of oxygen.
Microaerophilic bacteria Microaerophilic bacteria are a specific type of microorganism
(especially bacteria) that require oxygen to survive, but require environments containing
lower levels of oxygen than that are present in the atmosphere (~20% concentration). Many
microaerophiles are also capnophiles, as they require an elevated concentration of carbon dioxide.
In the laboratory they can be easily cultivated in a candle jar, a container into which a lit candle
is introduced before sealing the airtight lid. The flame burns until extinguished by oxygen
deprivation, creating a carbon dioxide-rich, oxygen-poor atmosphere.
Some of the examples are as follows.
 Borrelia burgdorferi, a species of spirochaete bacteria that causes Lyme disease in humans.
 Helicobacter pylori, a species of proteobacteria that has been linked to peptic ulcers and
some types of gastritis. Some do not consider it a true obligate microaerophile.
 Campylobacter has been described as microaerophilic.
 Streptococcus intermedius has also been described as microaerophilic.
CYANOBACTERIA
Cyanobacteria are photosynthetic, that is, they can manufacture their own food. Because they
are bacteria, they are quite small and usually unicellular, though they often grow in colonies
large enough to be seen. They have the distinction of being the oldest known fossils, more than
3.5 billion years old. They are one of the largest and most important groups of bacteria on earth.
The other great contribution of the cyanobacteria is the origin of plants. The chloroplast with
which plants make food for themselves is actually a cyanobacterium living within the plant’s cells.
Sometime in the late Proterozoic, or in the early Cambrian, cyanobacteria began to take up residence
within certain eukaryotic cells, making food for the eukaryote host in return for a home. This event
is known as endosymbiosis, and is also the origin of the eukaryotic mitochondrion.
Figure 1.3 Nostoc
Trichomes
Single trichome
dead cell
Figure 1.4 Oscillatoria
Cyanobacteria are often called blue-green algae because of their photosynthetic nature.
This name is convenient for talking about organisms that make their own food, but does not
reflect any relationship between the cyanobacteria and other organisms called algae. Cyanobacteria
are relatives of the bacteria, not eukaryotes, and it is only the chloroplast in eukaryotic algae to
which the cyanobacteria are related. Cyanobacteria are found throughout the world in terrestrial,
freshwater and marine habitats, but blooms typically occur in fresh water.
CHARACTERISTICS OF CYANOBACTERIA
1. Cyanobacteria or blue-green algae are gram-negative bacteria with a number of unusual
traits.
2. They are the largest and most diverse group of photosynthetic bacteria, which was previously
known as blue-green algae.
3. Cyanobacteria are true prokaryotes.
4. They vary greatly in shape and appearance.
5. They range in diameter from about 1 to 10 microns.
6. They may be unicellular and exist as colonies of many shapes, or form filaments called
trichomes.
7. They have normal gram-negative type cell wall.
8. They often use gas vesicles to move in the water, and many filamentous species have gliding
motility.
9. Their photosynthetic system closely resemble that of eukaryotes because they have
chlorophyll a and photosystem II. They carry out oxygenic photosynthesis, i.e., they use
water as an electron donor and generate oxygen during photosynthesis.
10. Cyanobacteria use pigments and electron transport chain components that are located
in thylakoid membranes linked with particles called phycobilisomes. They contain
phycocyanin pigment, and the CO2 in them is assimilated through the Calvin cycle.
11. Reserve food material in cyanobacteria is glycogen.
12. In cyanobacteria, hormogonia, akinetes and heterocysts are also produced.
2
TOOLS OF MICROBIOLOGY
MICROSCOPY
INTRODUCTION
Microscopy refers to the use of light or electrons to magnify objects. Microorganisms are
too small to be seen with the unaided eye, and hence they are observed with a microscope.
The word “Microscope” is derived from the latin word Micro which means “small”, and the
Greek word skopos, which means “to look at”. Modern microbiologists use microscopes that
produce with great clarity, magnification that ranges from 10–1000 times greater than those of
Leeuwenhoek’s single lens. This chapter describes how different types of microscopes function
and why one type is used in preference to the other.
Microorganisms and their structural components are measured in even smaller units, such as
micrometre and nanometre. A micrometre is equal to 0.000001 m (10–6 m).
GENERAL PRINCIPLES OF MICROSCOPY

Wavelength of Radiation
Visible light is one part of the spectrum of electromagnetic radiation that includes X-rays,
microwaves and radio waves. The beams of radiation may be referred to as either rays or waves.
These various forms of radiation differ in wavelength—the distance between two corresponding
parts of a wave. The human eye discriminates among different wavelengths of visible light and
sends patterns of nerve impulses to the brain, which interprets the impulses as different colours.
For example, we see wavelengths of 400 nm as violet and wavelengths of 650 nm as red.
White light, composed of many colours (wavelengths), has an average wavelength of 550 nm.
Using radiations of smaller wavelengths results in enhanced microscopy.
Magnification
A light microscope usually has at least three objective lenses: the low power, high power and
oil-immersion lenses. In general, these lenses magnify an object 10, 40 and 100 times, respectively
(Magnification is represented by the multiplication (×) sign).This intermediate image is remagnified
by the ocular lens with a standard 10X ocular lens, the total magnification achieved is
100×, 400× and 1000×, respectively. The total magnification is determined by the following formula:
Total magnification = Magnification of objective lens × Magnification of ocular lens
Resolution
Resolution, which is also called resolving power, is the ability to distinguish between objects that
are close together as distinct and separate. Leeuwenhoek’s microscopes had a resolving power of
about 1mm i.e., it could distinguish between objects if they were more than about 1mm apart,
and objects close together (1mm) appear as a single object.
Limit of resolution is the smallest distance by which two objects can be separated and still
be distinguishable as two separate objects. The better the resolution, the better the ability to
distinguish two objects that are close to one another. Modern microscopes can distinguish between
objects as close together as 0.2mm
The resolving power (RP) of a lens system is important in microscopy because it denotes the
size of the smallest object that can be seen clearly. The resolving power varies for each objective
lens and is calculated using the following formula:
λ
RP =
2 × NA
In this formula, the Greek letter l (lambda) represents the wavelength of light and is usually
set at 500 nm, the halfway point between the limits of visible light. The symbol NA stands for the
numerical aperture of the lens and refers to the size of the cone of light that enters the objective
lens after passing through the specimen. The numerical aperture of an objective lens is defined
by NA=n sin q where n is the index of refraction of the medium in which the lens is working
(1.0 for air, 1.33 for pure water, and up to 1.56 for oils), and q is the half-angle of the maximum cone
of light that can enter or exit the lens. This number generally is printed on the side of the objective
lens. For a low-power objective with a NA of 0.25, the resolving power is calculated as follows:
500 nm 550
RP = = = 1,000 nm or 1.0 µm
2 × 0.25 0.50
Since the resolving power for this lens system is 1.0mm, any object smaller than 1.0mm could
not be seen as a clear distinct object. An object larger than 1.0mm would be resolved. Resolving
Tools of Microbiology 11
power is calculated with the lens suspended in oil rather than air, a factor that increases the
numerical aperture to 1.25. Both low-power and high-power objectives are wide enough to capture
sufficient light for viewing. The oil-immersion objective, on the other hand, is so narrow that
most of the light would bend away and would miss the objective lens if oil were not used. The
index of refraction (or refractive index) is a measure of the light-bending ability of a medium.
Immersion oil has a refractive index of 1.5, which is almost identical to the refractive index of
glass. Because the refractive index is same for oil and glass, the light does not bend as it passes
from the glass slide and the specimen into the oil. By comparison, air has a refractive index of
1.0, which accounts for the abrupt bending of light as it enters. The oil, thus, provides a
homogeneous pathway for light from the slide to the objective, and the resolution of the object
increases. With the oil-immersion lens having a numerical aperture of 1.25, the highest resolution
possible with light microscope is 0.2mm (200 nm).
Contrast
Contrast refers to differences in intensity between two objects or between an object and its
background. Contrast is important in determining the resolution. For example, although you
can easily distinguish between two golf balls lying side by side on a green surface 15 m away, at
that distance it is much more difficult to distinguish between them if they lie on a white towel.
Most microorganisms are colourless and have very little contrast whether one uses light or
electrons. One way to increase the contrast between organisms and their background is to stain them.
LIGHT MICROSCOPY
1. Bright-field Microscopes
i. Simple microscope Leeuwenhoek first reported his observation of microorganisms
using a simple microscope in 1673. A simple microscope contains a single magnifying lens
(Figure 2.1).
ii. Compound microscope Simple microscopes have been replaced in modern laboratories
by compound microscopes. A compound microscope uses a series of lenses for magnification.
The microscope consists of a sturdy metal body or stand composed of a base and an arm to which
the remaining parts are attached. A light source, either a mirror or an electric illuminator, is
located at the base. Two focusing knobs, the fine and coarse adjustment knobs are located on
the arm, which can move the stage or the nosepiece to focus the image.
The stage is positioned halfway up the arm and holds the microscope slides by either simple
slide clips or a mechanical stage clip. A mechanical stage allows the operator to move a slide
around smoothly during viewing by use of stage control knobs. The substage condenser is mounted
within or beneath the stage and focuses a cone of light on the slide. Its position often is fixed in
simpler microscopes but can be adjusted vertically in more advanced models.
Lens
Folded Arm
Vertical limb
Stage
Stand
mirror
Foot
Figure 2.1 Simple microscope
The curved upper part of the arm holds the body assembly to which a nosepiece and one or
more eyepieces or oculars are attached. More advanced microscopes have eyepieces for both eyes
and are called binocular microscopes. The body assembly itself contains a series of mirrors and
prisms so that the barrel holding the eyepiece may be tilted for ease in viewing. The nosepiece
holds three to five objectives with lenses of differing magnifying power and can be rotated to
position any objective beneath the body assembly. (Figure 2.2)
The objective lenses on a typical microscope are: scanning objective lens (4×), low-power
objective lens (10×), high-power lens or high dry objective lens (40×) and oil-immersion
objective lens (100×). Ideally, a microscope should be parfocal, i.e., the image should remain
in focus when objectives are changed. Under usual operating conditions, the field of vision in a
compound light microscope is brightly illuminated. By focusing the light, the condenser produces a
bright-field illumination.
Eyepiece lenses
Revolving nosepiece
Objective lenses Arm
Stage with clips

Condenser Coarse and fine focus
Illuminator Stage Controls
Base
Figure 2.2 Compound microscope
2. Dark-field Microscope

Pale objects are best observed with dark-field microscopes. These microscopes utilize a dark-
field stop in the condenser that prevents light from directly entering the objective. Instead,
light rays are reflected inside the condenser so that they pass into the slide at such an oblique
angle that they miss the objective lens. Only those light rays that are scattered by the specimen
enter the objective lens and are seen, so the specimen appears light against a dark background.
This increases contrast and enables observation of more details than that are visible in
bright-field microscopy.
Dark-field microscopy helps in the diagnosis of diseases caused by spiral bacteria because
these organisms are near the limit of resolution of the light microscope and do not stain well.
For example, syphilis caused by the spiral bacterium Treponema pallidum has a diameter of about 0.15mm
This bacterium may be observed in scrapings taken from a lesion of a person who has the disease.
Dark-field microscopy also is the preferred way to study motility of live prokaryotic and eukaryotic
cells.
Objective lens
Light that strikes
specimen
Microbes on
slide
Condenser lens
Dark field ring
Light rays
Figure 2.3 Dark-field microscope
3. Phase Microscopes

There are two types of phase microscopes: phase-contrast microscopes and differential
interference contrast microscopes.
i. Phase-contrast microscope Another way to observe microorganisms is by using a
phase-contrast microscope. Phase-contrast microscope is the simplest phase microscope that
produces sharply defined images in which fine structures can be seen in living cells. These
microscopes are particularly useful for observing cilia and flagella. Phase-contrast microscopy
is especially useful because it permits detailed examination of internal structures in living
microorganisms. In addition, it is not necessary to fix (attach the microbes to the microscope
slide) or stain the specimen, a procedure that could distort or kill the microorganisms.
The principle of phase-contrast microscopy is based on the wave nature of light rays, and
the fact that light rays can be in phase (their peaks and valleys match) or out of phase. If the
wave peak of light rays from one source coincides with the wave peak of light rays from another
source, the rays interact to produce reinforcement (relatively brighter image). However, if the
wave peak from one light source coincides with the wave trough from another light source, the
rays interact to produce interference (relatively darker image).
Light rays passing through a specimen naturally slow down and are shifted about
1/4 wavelength out of phase. A special filter called a phase plate, which is mounted in a phase
objective lens, retards these rays another 1/4 wavelength, so that they are 1/2 wavelength out of
phase with their neighbours. When the phase microscope lens brings the two sets of rays together,
troughs of one wave interfere with the crests of the other because they are out of phase, and thus,
contrast is created (Figure 2.4).
Elevated (or excavated) ring on phase plate is coated to

reduce the intensity of direct light. Differences in length of light
path for direct and indirect light creates a 41 λ phase shift.
Diffracted light
1
λ
4
Undeviated
light
Objective Phase plate

lens
Specimen
Condenser
Phase annulus
Phase annulus creates a ring of oblique

illumination
Dim, indirect light deviated by passage through specimen

1
has been delayed 4 λ due to diffraction.
Direct light
(undeviated)
Now the direct light has been dimmed

1
and delayed 4 λ by passage through
phase plate elevations. Interference
with deviated light is now accentuated.
Figure 2.4 Phase-contrast microscope
ii. Differential interference contrast microscope In the mid-1950s, a French optics

theoretician named Georges Nomarski modified the Wollaston prism used for detecting optical
gradients in specimens and converting them into intensity differences. Today there are several
implementations of this design, which are collectively called differential interference contrast
(DIC). Living or stained specimens, which often yield poor images when viewed in bright-field
illumination, are made clearly visible by optical rather than chemical means.
Light detector
Polarizing filter
Wollaston prism
Objective lens
Sample
Condenser lens
Wollaston filter
Polarizing filter
Light source
Figure 2.5 Differential interference contrast microscope
In transmitted light DIC, light from the lamp is passed through a polarizer located beneath
the substage condenser, in a manner similar to polarized light microscopy. Next in the light path
(but still beneath the condenser) is a modified Wollaston prism that splits the entering beam of
polarized light into two beams travelling in slightly different directions. The Wollaston prism is
composed of two quartz wedges cemented together, from which emerging light rays vibrate at
90 degrees relative to each other with a slight path difference. A different Wollaston prism is
needed for each objective of different magnification. Wollaston prisms are usually loaded into a
revolving turret on the condenser, which allows the microscopist to rotate the appropriate prism
into the light path when changing magnifications.
The plane-polarized light vibrating only in one direction (East-West) perpendicular to the
propagation direction of the light beam, enters the beam-splitting modified Wollaston prism and
is split into two rays vibrating perpendicular to each other. These two rays travel close together
but in slightly different directions. The rays intersect at the front focal plane of the condenser,
where they pass travelling parallel and extremely close together with a slight path difference,
but they are vibrating perpendicular to each other and are therefore unable to cause interference.
The distance between the rays, called the shear, is so minute that it is less than the resolving
ability of the objective.
The split beams enter and pass through the specimen where their wave paths are altered in
accordance with the specimen’s varying thickness, slopes, and refractive indices. These variations
cause alterations in the wave path of both beams passing through areas of any specimen details
lying close together. When the parallel beams enter the objective, they are focused above the rear
focal plane where they enter a second modified Wollaston prism that combines the two beams at a
defined distance outside of the prism itself. This removes the shear and the original path difference
between the beam pairs. As a result of having traversed the specimen, the paths of the parallel beams
are not of the same length (optical path difference) for differing areas of the specimen.
In order for the beams to interfere, the vibrations of the beams of different path length must be
brought into the same plane and axis. This is accomplished by placing a second polarizer (analyser)
above the upper Wollaston beam-combining prism. The light then proceeds toward the eyepiece
where it can be observed as differences in intensity and colour. The design results in one side of
the image appearing bright (or possibly in colour) while the other side appears darker (or another
colour). This shadow effect bestows a pseudo three-dimensional appearance to the specimen.
There are numerous advantages in DIC microscopy as compared to phase-contrast microscopy.
With DIC, it is possible to make fuller use of the numerical aperture of the system and to provide
optical staining (colour). DIC also allows the microscope to achieve excellent resolution. Use of
full objective aperture enables the microscopist to focus on a thin plane section of a thick specimen
without confusing images from above or below the plane.
4. Fluorescence Microscope

Fluorescence microscopy takes advantage of fluorescence, the ability of substances to absorb
short wavelengths of light (ultraviolet) and give off light at a longer wavelength (visible).
Some organisms fluoresce naturally under ultraviolet light. If the specimen to be viewed does
not naturally fluoresce, it is stained with one group of fluorescent dyes called fluorochromes.
The fluorescence microscope exposes a specimen to ultraviolet, violet or blue light and
forms an image of the object with the resulting fluorescent light. A mercury vapour arc lamp or
other source produces an intense beam, and heat transfer is limited by a special infrared filter.
The light passes through an exciter filter that transmits only the desired wavelength. A dark-
field condenser provides a black background against which the fluorescent objects glow.
The microscope forms the image of the fluorochrome-labelled microorganisms from the light
emitted when they fluoresce. A barrier filter positioned after the objective lenses removes any
remaining ultraviolet light, which would damage the viewer’s eyes, or blue and violet light, which
would reduce the contrast of the image (Figure 2.6).
An important application of fluorescence microscopy is the fluorescent antibody technique
used to identify an unknown organism. In one variation of this procedure, fluoresein is
chemically attached to antibodies, the protein molecules produced by the body’s immune system.
These “tagged” antibodies are mixed with a sample of unknown organism. If the antibodies are
specific for that organism, they will bind to it and coat the cells with the dye. When subjected to
UV light, the organism will fluoresce. The organism fails to fluoresce, if the antibodies are not
specific to that organism and a different tagged antibody is tried.
Eyepiece
Barrier filter
HBO
lamp
house Dichroic beam splitter
Excitation
filter
Exciting Objective
light
Emitted
fluorescence
light Specimen
Figure 2.6 Fluorescent microscope

Fluorescent microscope is also used to stain certain pathogens. For example, the dye fluorescein
isothiocyanate attaches to cells of Bacillus anthracis, the causative agent of anthrax, and appears
apple green. Another dye, auramine O, which fluoresces yellow, stains Mycobacterium tuberculosis.
ELECTRON MICROSCOPY
Objects smaller than about 0.2mm such as viruses or the internal structures of cells, must be
examined with an electron microscope. In electron microscopy, a beam of electrons is used,
instead of light. Free electrons travel in waves. The resolving power of the electron microscope
is far greater than that of the other light microscopes.
The better resolution of electron microscope is due to the shorter wavelengths of electrons;
the wavelength of the electrons are about 100,000 times smaller than the wavelength of visible
light. Thus, electron microscopes are used to examine structures too small to be resolved with
light microscopes. Images produced by electron microscope are always black and white, but they
may be coloured artificially to accentuate certain details.
Instead of using glass lenses, an electron microscope uses electromagnetic lenses to focus a
beam of electrons onto the specimen. There are two types of electron microscopes: the transmission
electron microscope and the scanning electron microscope.
1. Transmission Electron Microscope

In the transmission electron microscope (TEM), (Figure 2.7(a)) a finely focused beam of electrons
from an electron gun passes through a specially prepared ultrathin section of the specimen.
The beam is focused on a small area of the specimen by an electromagnetic condenser lens that
performs roughly the function of the condenser of the light microscope, directing the beam of
electrons in a straight line to illuminate the specimen.
Electron microscope uses electromagnetic lenses to control illumination focus and
magnification. Instead of being placed on a glass slide, as in light microscopes, the specimen
is usually placed on a copper mesh grid. The beam of electron passes through the specimen
and then through an electromagnetic objective lens, which magnifies the image. Finally, the
electrons are focused by an electromagnetic projector lens, rather than by an ocular lens as in
a light microscope, onto a fluorescent screen or photographic plate. The final image, called a
transmission electron micrograph, appears as many light and dark areas, depending on the
number of electrons absorbed by different areas of the specimen.
In practice, the transmission electron microscope can resolve objects as close together as
2.5 nm and objects are generally magnified 10,000–100,000×. TEM is used to view and record
detailed structures, such as organelles within cells. Ultrathin sections of the object must be prepared
because the electron beam can penetrate matter only for a very short distance. After embedding
the specimen in a suitable plastic mounting medium or freezing it, scientists cut the specimen into
sections with a diamond knife. In this manner, a single bacterium can be sliced, like a loaf of bread,
into hundreds of thin sections. Several of the sections are placed on a small grid and stained with
heavy metals such as lead and osmium, to provide contrast (positive staining) or used to increase
the electron opacity of the surrounding field (negative staining). Negative staining is useful for
the study of the smallest specimens, such as virus particles, bacterial flagella and protein molecules.
In addition to positive and negative staining, a microbe can be viewed by a technique called
shadow casting. In this procedure, a heavy metal, such as platinum or gold, is sprayed at an
angle of about 45 degrees so that it strikes the microbe from only one side. The metal piles up on
one side of the specimen and the uncoated area on the opposite side of the specimen leaves a clear
area behind it as a shadow. This gives a three-dimensional effect to the specimen and provides
a general idea of the size and shape of the specimen. Transmission electron microscopy has high
resolution and is extremely valuable for examining different layers of specimens. However, it
does have certain disadvantages. Because electrons have limited penetrating power, only a very
thin section of specimen (about 100 nm) can be studied effectively. Thus, the specimen has no
three-dimensional aspect. In addition, specimens must be fixed, dehydrated and viewed under
a high vacuum to prevent electron scattering. These treatments not only kill the specimen but
also cause some shrinkage and distortion, sometimes to the extent that there may appear to be
additional structures in a prepared cell. Such additional structures that appear as a result of the
method of preparation are called artifacts.
2. Scanning Electron Microscope

The scanning electron microscope (SEM) was developed in the late 1960s to enable researchers
to see the surfaces of objects in the natural state and without sectioning. In scanning electron
microscopy, an electron gun produces a finely focused beam of electrons called the primary electron
beam (Figure 2.7 (b)). These electrons pass through electromagnetic lenses and are directed over
the surface of the specimen. The primary electron beam knocks electrons out of the surface of the
specimens and the secondary electrons thus produced are transmitted to an electron collector,
amplified and used to produce an image on a viewing screen or photographic plate. The image
is called a scanning electron micrograph.
This microscope is especially useful in studying the surface structures of intact cells and
viruses. In practice, it can resolve objects as close together as 20 nm, and magnication produced
is generally of 1,000–10,000×.
Electron gun
Primary electron beam
Electron beam
Electromagnetic
condenser lens Electromagnetic lenses
Viewing screen
Electromagnetic Viewing
objective lens eyepiece
Electron
collector
Electromagnetic Secondary
projector lens electrons
Fluorescent Specimen
screen or photo-
graphic plate Amplifier
(a) Transmission electron microscope (b) Scanning electron microscope
Figure 2.7 Electron microscope
SCANNED-PROBE MICROSCOPY
Since the early 1980s, several new types of microscopes, called scanned-probe microscopes,
have been developed. They use various kinds of probes to examine the surface of a specimen at
a very close range, and they do so without modifying the specimen or exposing it to damaging
high-energy radiation. Such microscopes can be used to map atomic and molecular shapes, to
characterize magnetic and chemical properties and to determine temperature variations inside
cells. Among the new scanned-probe microscopes are the scanning tunnelling microscope and
the atomic force microscope.
1. Scanning Tunnelling Microscope

Scanning tunnelling microscope (STM) uses a thin metal (tungsten) probe that scans a specimen
and produces an image revealing the bumps and depressions of the atoms on the surface of the
specimen (Figure 2.8). The resolving power of a STM is much greater than that of an electron
microscope, it can resolve features that are only about 1/100th the size of an atom. Moreover,
special preparation of the specimen for observation is not needed. STMs are used to provide
detailed views of molecules, such as DNA.
Control voltages for piezotube

Piezoelectric tube
with electrodes
Tunneling Distance control

Current amplifier and scanning unit
D Tip
Sample
Tunnelling
Voltage
Data processing
and display
Figure 2.8 Schematic diagram of scanning tunneling microscope
2. Atomic Force Microscope

In atomic force microscopy (AFM), a metal and diamond probe is gently forced down onto a
specimen. As the probe moves along the surface of the specimen, its movements are recorded
and a three-dimensional image is produced. The deflection of a cantilever holding the tip is
monitored by a photodiode. As with STM, AFM does not require special specimen preparation.
AFM is used to image both biological substances (upto atomic level) and molecular processes
(such as the assembly of fibrin, a component of a blood clot).
Recent studies using atomic force microscopes have provided insight into the molecular folding
of lipopolysaccharides on the surfaces of gram-negative pathogenic bacteria.
Laser
Photo diode
Cantilever
Tip
t
un
Sample
mo
le
mp
Sa
Z
X
Piezoelectric
scanner
Piezo movement

Figure 2.9 Atomic force microscope
The various types of microscopes described above are summarized in Table 2.1.
USE AND CARE OF MICROSCOPES

 Always carry the microscope with two hands, one hand on the arm and one hand placed
under the base for support.
 Remove cover, plug into outlet, turn on light.
 Place slide in mechanical slide holder.
 Set the stage in the “all the way up” position.
 Using scanning objective, locate the object; using the coarse adjustment knob, focus to obtain
the clearest image possible.
 Using the fine adjustment knob, bring the object into the clearest image possible; centre the
object in the field of view.
 Bring the low-power objective into place.
 Focus the object using minor adjustments with the fine adjustment knob.
 Centre object in field of view; adjust light source, if needed.
 Bring the high-power objective into place.

 Using only the fine adjustment knob, bring the object into the clearest focus possible. Adjust
light, if needed and centre the object in the field of view.
 When the work is finished with your microscope:
 Remove the slide.

 Centre the mechanical stage holder.
 Place the scanning objective in place.
 Return the stage to the “all the way up” position.
 Turn off the light.
 Fold up and tie the cord.
 Replace the cover.
 Return the microscope to the cabinet.
Cleaning the Microscope
 Do not let the microscope get too dirty—always use the dust cover when not in use.
 To clean the eyepiece, use a high-quality lens paper. First, brush any visible dust from the
lens, and then wipe the lens.
 Do not use facial tissues; they are made from ground-up wood fibres and could damage the
lenses.
 To clean the objective lenses, use a fresh piece of the lens paper each time so that you do not
transfer dust from one lens to another.
 Use lens paper on all glass parts of the microscope. (A cotton swab (Q-tipTM) can be used in
place of lens paper).
Things to Remember
 Never use the coarse adjustment knob when on high power.
 Never swing the oil-immersion objective into place.
 If you lose your object (microscope gets bumped, slide is displaced) always start from the
beginning (scanning objective, stage “all the way up”) to relocate your object.
 Total magnification = Magnification of the eyepiece × Magnification of the objective
10× 4 (scanning) = 40×
10× 10 (low) = 100×
10× 40 (high) = 400×

Table 2.1 A summary of various types of microscopes
Microscope type Distinguishing features Principal use

I. Light microscopes
1. Bright-field microscope Uses visible light as a source of illumination. To observe various stained specimens and to
Cannot resolve structures smaller than about count microbes. Does not resolve very small
0.2 µm; specimen appears against a bright specimen, such as viruses.
background; inexpensive and very easy to use.

2. Dark-Field microscope Uses a special condenser with an opaque disc To examine living microorganisms that are
that blocks light from entering the objective invisible in bright-field microscopy; do not
lens directly; light reflected by specimen enters stain easily or are distorted by staining.
the objective lens and the specimen appear Frequently used to detect Treponema
light against dark background. pallidum in the diagnosis of syphilis.
3. Phase contrast Uses a special condenser containing an annular To facilitate detailed examination of the
microscope (ring-shaped) diaphragm. The diaphragm internal structures of the living specimens.
allows direct light to pass through the
condenser, focusing light on the specimen and
a diffraction plate in the objective lens. Direct
and reflected or diffracted light rays are
brought together to produce the image; no
staining is required.
4. Differential interference Like phase-contrast, this uses differences in To provide three-dimensional images.
contrast (DIC) refractive indices, to produce image; uses two
microscope beams of light separated by prisms. The
specimen appears coloured as a result of the
prism effect; no staining required.
5. Fluorescent microscope Uses an ultraviolet or near ultraviolet source of To rapidly detect and identify microbes in
illumination that causes fluorescent microbes tissues or clinical specimens, for
(green-coloured) in a specimen to emit light. fluorescent–antibody technique (immuno-
fluorescence).
(Contd.)
Table 2.1 (Continued)
Microscope type Distinguishing features Principal use

II. Electron microscopes
1. Transmission electron Uses a beam of electrons instead of light; To examine viruses or the internal ultra
microscope electron passes through the specimen structures structure in thin sections of cells (usually
smaller than 0.2µm can be resolved. The magnified 10,000–100,000×).
image produced is two-dimensional.
2. Scanning electron Uses a beam of electrons instead of light; To study the surface features of cells and
microscope electrons are reflected from the specimen viruses (usually magnified
structures smaller than 0.2µm can be resolved. 1,000–10,000×).
The image produced appears
three-dimensional.
III. Scanned-probe microscopes
1. Scanning tunneling Uses a thin metal probe that scans a specimen To provide detailed view of molecules
microscope and produces an image revealing the bumps inside cells.
and depressions of the atoms on the surface of
the specimen. Resolving power is much greater
than that of the electron microscope. No
special preparation required.
2. Atomic force Uses a metal and diamond probe gently forced To provide images of biological molecules
microscope down along the surface of the specimen. and molecular processes.
Produces a three-dimensional image. No special
preparation required.
AUTOCLAVE
Definition
Autoclave is a device used for sterilization. It uses moist heat for killing all the microbes, including
heat-resistant spores. It was discovered by Chamberland in 1884.
Principle
One of the important agents used in sterilization is heat. Autoclave uses moist heat
(121°C at 15 lb/in2 pressure for 15 minutes). The moist heat is highly penetrative. Heat coagulates
organisms. Autoclave has the mechanism for regulating steam pressure in ensuring complete
evacuation of air from the chamber. A suitable safety valve is included to avoid explosion.
The pressure of air allows increase in pressure within the chamber without increase in temperature.
Hence, all the air is expelled out of the chamber.
Structure
It has a double-walled, high gauze steel jacket in a hollow cylindrical manner (Figure 2.10).
Three sides are closed with double-walled steel. Upper side is closed with an air-tight thick door.
The door is fixed with bakelite handle. There is a safety lock within the door. Inside the cylinder,
the working space is found. There are many types of autoclaves—vertical and horizontal—of
varying size.
A water tank with an electric heater is found to be fixed at the bottom of the chamber. In it,
the water is heated and steam is produced, which is sent into the chamber. The steam of correct
pressure is sent into the chamber. The pressure regulator is fixed with the steam line pipe to
cut off the steam. The steam intake pipe from the tank is split into 2 or 3 small pipes so that
steam is supplied to all parts of the chamber in a uniform manner with the same pressure. Two
pipelines from the chamber are fitted with the chamber pressure gauze to measure the pressure
and another to the thermometer, to measure the temperature. The pressure is measured in lb/in2
and temperature in °C. On the bottom side of the chamber, a drain pipeline is seen that removes
excess water. Another vent is seen that removes excess steam faster after sterilization. The vent
lines are connected to a waste line, which is let out of the autoclaving room. The vent pipe lines
are fixed with the operating valves which should be in a closed condition to build the pressure
inside the chamber while autoclaving. At the top of the autoclave, safety air valves are fixed.
If the pressure goes above 15 lb/in2, it automatically bursts off to release the steam pressure
inside the autoclave. The materials should not touch the walls, as it might lead to breakage of
the glasswares.
In autoclaves, vertical and horizontal types with built-in water tanks and electric heater are
also available.
Steam exhaust pipe
Steam from jacket into chamber
Inner chamber Door
Steam in
Culture
medium
Jacket
To drain Steam supply
Figure 2.10 Autoclave
Method of Operation
The temperature is set by turning the temperature setting knob at the bottom of the water bath.
It raises the temperature of the thermostat. The pilot indicator lamp starts to glow and indicates
that the temperature has risen. The temperature in the thermometer is watched till it reaches
the desired temperature.
Checking the Efficiency of Autoclave

To check the efficiency, strips containing Bacillus stearothermophilus are placed along with the
material to be sterilized. After sterilization, the strips are placed in the nutrient agar medium to
check for the survivors. Chemical sterilization check tapes also can be used.
Uses
1. Used to sterilize media, nutrient solution.
2. Used to sterilize clothes, cotton and other easily charable substances.
HOT AIR OVEN

Definition
Hot air oven is a device that is used to sterilize objects with the help of dry heat as the sterilizing agent.
Principle
This is an air-dry type of sterilizer. It is used for the sterilization of the articles made of glass or
metals. It is used to sterilize pipettes, Petri dishes and flasks. Direct contact of glassware with
the walls of hot air oven should be prevented. Otherwise, it will lead to breakage of glasswares.
Hence, glasswares are kept in Petri cans and pipette cans. Dry heat is less penetrative than moist
heat. Their ability to attack the enzymes in microbes is very slow. Hence, a high temperature
treatment for a long time is required for killing all organisms. All the materials are treated at
160°C for one hour. Laboratory coats, rubber, culture media would burn or boil to dryness, and
hence, cannot be sterilized by hot air oven.
Figure 2.11 Hot air oven

Structure
It is a cube-like structure, approximately of equal height × length × breadth (Figure 2.11).
It is made of three walls with two air spaces. The outer wall is covered with asbestos pad to reduce
the radiation by heat. A burner manifold runs along both sides. The coil is connected to an electric
circuit and conventional current travels in a complete circuit through the wall spaces and interior
of the oven. Two openings are present on the two sides for the products of combustion to escape
through. At the point where the electric circuit enters into the coil, a thermostat is fixed. It regulates
the current flow. The thermostat is operated on both directions by adjusting a temperature setting
knob. A rise in temperature is indicated by the thermostat by the glow of pilot lamp. Adjustable
shelves made up of perforated steel plates are present for keeping the materials to be sterilized. The
whole instrument is kept in use or out of use by an “On/Off” switch.
Method of Operation
The temperature is set by turning the temperature setting knob at the bottom of the oven.
the desired temperature of 160ºC. Once the temperature is set, the setting knob should not be
disturbed so that the temperature is maintained.
Checking the Efficiency of Hot Air Oven

To check the efficiency, strips containing Bacillus stearothermophilus are placed along with the
material to be sterilized. After sterilization, the strips are placed in the nutrient agar medium to
check for survivors. Chemical sterilization check tapes also can be used.
Use
It is used to sterilize all glassware and equipment.
INCUBATOR
Definition
Incubator is an instrument that is used to maintain optimum temperature for organisms to grow
and perform maximum metabolic activity.
Principle
Temperature is considered to be an important factor for the growth of microorganisms. In the
energy-producing process in an organism, the metabolic pathway consists of many complex
enzymes by which the complex substrates are converted into simpler ones. The enzymes catalyse
chemical reactions in which the energy-rich compounds, called the ATP molecules, are produced.
These enzymes have their own speed of catalysing the reactions. Many external factors are found
to be playing a major role in the determination of speed of an enzyme. When the temperature
is found to be optimum, the enzyme activity is found to be at peak level, which results in faster
growth and multiplication of the organism. Each organism produces different extracellular enzymes
and each enzyme has its own temperature for maximum activity. This temperature is known as
optimum temperature of the organisms. By maintaining the optimum temperature, enzymes
are activated at high speed, and growth can be seen in large amounts.
Structure
It is an instrument similar to that of hot air oven in structure (Figure 2.12). The usual temperature
of a bacteriological incubator is 37°C since most of the bacteria are mesophilic in nature.
For fungal cultures, the optimum temperature is around 25ºC. It is constructed with two walls.
The outer wall is covered with asbestos pad to reduce the loss of heat. The incubator is fitted with
an insulator door. In the door, plexi glass seal is fixed, which helps in inspecting the specimen
without disturbing the temperature. The inner chamber is made of stainless steel with trays to
keep the materials to be incubated. The shelf positions of the trays are adjustable. All the controls
are mounted at the bottom of the incubator, which comprises of self-illuminated “On/Off ” switch,
heat energy regulator, solid-state fully automated temperature controller cum indicator, indicator
lamps and protective fuses.
Figure 2.12 Incubator
Method of Operation
The heat energy is radiated manifold through a coil burner running along the rear side.
The electric current is passed through a thermostat of low range. The thermostat is connected
with capacitors for the continuous usage of the instrument. A thermometer is seen at the top
to know the temperature inside the incubator. The power supply can be checked through pilot
lamps. Many incubators of different ranges are used for the growth of organisms.
Uses
Incubator is used to incubate inoculated Petri plates. Various types of microbes can be incubated
by setting various temperatures.
WATER BATH
Definition
Water bath is a covered glass or metal tank containing water at a thermostatically controlled
temperature, which is used in the incubation of microbes.
Principle
The contents placed in a water bath are raised to the required temperature much more rapidly
than in an incubator. In an incubator, the heat is radiated through air molecules, where a
loss of energy is seen. The penetration of heat energy is slow. Hence, heat waves are radiated
through water molecules in a water bath. These water baths are used for short-term incubation.
The material to be incubated is placed in a vessel suspended in the water. The level of water has
to be maintained at one-half to two-thirds of liquid in containers to be incubated. This results
in convention current, which heats the contents of the vessel, and hastens reactions, such as
agglutination. A motor-driven propeller ensures rapid mixing of the water and the maintenance
of the given temperature in all parts of the tank. Greater temperature stability can be achieved
in a water bath than in a hot air incubator. By maintaining the incubation temperature in water
bath, the enzyme action is found to be maximum than in an incubator.
Structure
Water bath is made up of thick steel with heat resistant material (Figure 2.13). Bakelite present
in between the two walls prevents heat loss through walls. The water bath is constructed with
electrical heating coils at the bottom which is connected with a thermostat. The heating coils are
covered with perforated steel stands to prevent the direct contact of vessels with the coils. The
thermostat is controlled with a temperature control knob. The temperature range is marked in
°C in the knob. By turning the knob left or right, the temperature can be lowered or increased.
The turning of the knob alters the current supply to the thermostat, which in turn heats the
electric coil to increase the temperature of water or cuts the electric supply to the electric heating
coil. This reduces the temperature of the water. A pilot lamp indicates the increase in temperature
and is seen near the temperature setting knob. The thermostat is connected with a capacitor
for continuous use of water bath. The water bath is fitted with lids to prevent the heat loss and
evaporation. Distilled deionized water should be used in water bath to avoid chalky deposits.
A thermometer is attached in such a way that its temperature-sensitive region is in touch with
the water. The current temperature inside the water bath can be determined.
Figure 2.13 Water bath
Refrigerated Water Bath

It is used for maintaining temperature below the ambient temperature. It includes a cooling coil,
which operates continuously and maintains a steady temperature by means of the thermostat
and heater of the water bath.
Method of Operation
The temperature is set by turning the temperature setting knob at the bottom of the water bath.
the desired temperature.
Uses
1. It is used for short-time incubation.
2. It is used to sterilize temperature-sensitive compounds at high temperature.
3. It is used to incubate organisms at correct temperature, so that its enzyme activity is at
maximum level and hence, can be used for enzyme extraction.
BOD INCUBATOR
Definition
BOD incubator is an instrument used for measuring the biological oxygen demand, i.e., the
amount of oxygen used in the respiratory process of microorganisms in oxidizing the organic
matter in sewage.
Principle
The amount of oxygen used is measured by calculating the dissolved oxygen. These environmental
test chambers are devised so that it can satisfy a variety of environmental conditions. They are
especially suitable for the stimulation of fixed temperature and relative humidity values above
freezing point.
Structure
They have double walls, the exterior wall is made of sheet steel and the interior is made of
stainless steel, to avoid corrosion (Figure 2.14). A flush fitting insulated door has leak gasket.
The hand-operated wiper allows a clear view of the interior. To the inside of the door, a glass door
with tight closure is seen. The rear chamber of the interior is fitted with a refrigeration evaporator
to decrease the temperature; heater is seen to increase the temperature of powerful air-circulating
fan to create a positive air flow in the chamber. A thermostatically sealed compressor below the
chamber provides temperature below ambient. The same compartment houses a boiler with an
immersion heater and water level controller for creating humidity. The control panel comprises
of an “On/Off ” switch, heat energy regulators, fully automatic solid-state digital electronic
controller cum indicators, indicator lamps and protective fuses.
Figure 2.14 BOD Incubator

Method of Operation
The temperature is set by turning the temperature setting knob at the bottom of the BOD
incubator. It raises the temperature of the thermostat. The thermostat is connected with capacitors
for the continuous usage of the instrument.
Use
It is used to estimate the amount of oxygen consumed. If all the oxygen is utilized in the given
sample, it indicates a high level of sewage pollution.
COLONY COUNTER
Definition
A colony counter is an instrument that is used to count colonies of bacteria or other microorganisms
growing on an agar plate. Early counters were merely lighted surfaces on which the plate was
placed, with the colonies marked off with a felt-tipped pen on the outer surface of the plate
while the operator kept the count manually. More recent counters attempt to count the colonies
electronically, by identifying individual areas of dark and light according to automatic or user-set
thresholds, and counting the resulting contrasting spots.
Principle and Method of Operation

An appropriate dilution or several dilutions within the estimated appropriate range, is spread
using sterile technique on the agar plate, which is then incubated under the appropriate conditions
for growth until individual colonies appear. Each colony marks the spot where a single organism
was originally placed, thus the number of colonies on the plate equals the number of organisms
within the volume of liquid spread on the plate. That concentration is then extrapolated by the
known dilution from the original culture, to estimate the concentration of organisms within that
original culture.
The maximum number of colonies which may be effectively counted on a single plate is
somewhere between 100 and 1,000, depending on the size of the colony and the type of organism.
Structure
The machine is operated by “On/Off ” switch. It also has a light switch to switch on the light
source. The colony counter has a centring device at the centre which has an illumination screen
holder to place the plate to be counted. There is a reset button to reset the colony counter. It has
digital counter which shows the number of colonies counted. A felt-tipped pen marks off the
colony. A magnifying glass is attached to the centring device by a rod (Figure 2.15).
Use
Colony counter is used to estimate the density of microorganisms within a culture.
2
3
4 Front part
5 1. Threaded hole for holder rod
2. Reset push-button
3. Digital counter
4. IIIuminated screen holder for reticule
5. Centring device
Side part
6 6. Magnifying glass holder
7. Feeder for top light
7 8. Bracket
Rear part
9. Socket for contact pointer
10. Socket for fiber marker
11. Light switch
. 8 12. On/off switch
13. Fuse
14. Mains cable
9 13
10 14
12
11
Figure 2.15 Colony counter
HAEMOCYTOMETER
Definition
The simplest, most convenient and cheapest means for accurately determining the numbers of cells
in a sample is to use a haemocytometer and a microscope. A haemocytometer is a specialized
slide that has a counting chamber with a known volume of liquid.
Principle and Method of Operation

 The haemocytometer consists of a heavy glass slide with two counting chambers, each of
which is divided into nine large 1 mm squares, on an etched and silvered surface separated
by a trough.
 A coverslip sits on top of the raised supports of the ‘H’-shaped troughs enclosing both chambers.
There is a ‘V’ or notch at either end where the cell suspension is loaded into the haemocytometer.
When loaded with the cell suspension, it contains a defined volume of liquid.
 The engraved grid on the surface of the counting chamber ensures that the number of particles
in a defined volume of liquid is counted.
 The haemocytometer is placed on the microscope stage and the cell suspension is counted.
Figure 2.16 shows the haemocytometer system.
Figure 2.16 Haemocytometer system
1 mm
corner square
Middle square
Figure 2.17 Standard haemocytometer chamber

One-half (i.e., one chamber) of a haemocytometer with Neubauer ruling is shown in

Figure 2.17.
 This is one of the two chambers in the haemocytometer.
 The entire chamber has nine 1.0 mm × 1.0 mm large squares separated from one another
by the triple lines. Area of each is 1 mm².
 Within each of the larger corner 1 mm² squares are 16 small squares that are there
to help orient you during counting, to help avoid counting a given cell more than once.
 The central 1 mm² area is divided into 25 small squares and each of these is marked into
a further 16 tiny squares.
 Each of the large square (divided into 25 small squares) is 1 mm on each side or
1mm×1mm =1mm2 in area. The depth of the chamber is 0.02 mm3. One cubic
millimetre is equivalent to 1/1000 cubic centimetre (cc or ml) or 10–3 ml. Therefore,
0.02 cu.mm = 1/1000× 0.02 cc or 1/5 × 10– 4ml.
Therefore, the number of cells counted in this one large square should be multiplied by
5× 104 to calculate the cell number per ml of sample used for counting, i.e., average number
of cells in one large square × dilution factor × 104 (104 = conversion factor to convert
10– 4 ml to 1 ml).
Disadvantages of direct counting

 Since only a small volume is of fluid sampled, the microbial population must be fairly large
enough for accuracy.
 It is also difficult to distinguish between living and dead cells.
Errors
Haemocytometer counts are, however, subject to the following sources of error:
1. Non-uniform suspensions It is assumed that the volume of cell suspension placed into
the chamber represents a truly random sample. This will not be a valid assumption unless the
suspension is well dispersed and free of cell clumps. Distribution in the haemocytometer chamber
depends on the number of particles, rather than particle mass. Thus, cell clumps will distribute in
the same way as single cells, distorting the final result. Unless 90% or more of the cells are free
from contact with other cells, the count should be repeated with a fresh sample from the original
culture. The cell suspension must always be mixed thoroughly before sampling!
2. Improper filling of chambers In order to fill properly by capillary action, a chamber must
be scrupulously clean. This also applies to the Pasteur pipette used to fill the chamber. New
pipettes may be dry-heat sterilized. The chamber and coverslip are cleaned first with distilled
water, then by ethanol and then wiped dry with a Kim wipe. If the cell suspension does not flow
in immediately and smoothly, it means the chamber is dirty and should be recleaned.
3. Failure to adopt a convention for counting cells in contact with boundary lines or with each
other Some cells can settle on the border gridlines and so it becomes difficult to decide whether
or not to count such a cell. In order to determine whether to count or not to count, concerning a
cell on a border, a convention should be developed in which half of the cells that touch a border
is not counted. For example, one may decide not to count cells if they touch the bottom and right
border (or the top and left border). Each one can decide their own convention but whichever is
chosen, they MUST be consistent.
OXYGEN ELECTRODE
INTRODUCTION
In 1954, Dr. Leland Clark invented the first membrane-covered electrode, designed to measure
the concentration of oxygen in blood, solution and gases. This electrode was innovative because
it was the first electrode to have both the anode and the cathode under the same non-conductive
polyethylene membrane.
Principle
The oxygen electrode has two main components:
1. The electrode base which houses the central platinum working electrode and the surrounding
silver/silver chloride counter/reference electrode. Conduction between these electrodes is by 3
M potassium chloride with a semi-permeable membrane that is used to separate the sample.
2. The incubation or monitoring chamber mounted on the base with a built-in water jacket.
This ensures that measurements are conducted at a constant temperature, which is essential
for accurate readings. A plunger fits into the top of the chamber and has two functions.
The first is to seal the sample from the atmosphere apart from a very small hole that is used to
inject materials with a syringe. The second function is to provide a reduction of the specified
sample volume by upto 6:1.
The standard dissolved oxygen electrode can be supplied with either a Perspex or a glass
incubation chamber (Figure 2.18 (a), (b)) and has a sample volume of approximately 1–7 ml.
It should be noted that the bases for the two electrodes (Perspex electrode or glass electrode) are
not interchangeable. Alternative incubation chambers ranging from 1 ml to 50 ml are available,
however, glass units are limited to standard precision bore glass tube sizes. Another option
available is a flow-through plunger with two holes drilled, instead of one, enabling the dissolved
oxygen electrode to sample from a process or flowing system.
Method of Operation
Much of our knowledge of electron transport in mitochondria and chloroplasts comes from oxygen
electrode recordings. The oxygen concentration in a sealed incubation chamber is continuously
monitored, and the effects of making various additions to the chamber can be observed.
 The upper section containing a transparent, thermostatted sample chamber is secured to the
lower electrode assembly with a screwed ring.
 A thin teflon membrane is trapped between the two sections and separates the isotonic
incubation medium from the strong KCl electrolyte in the electrode compartment.
 The adjustable stopper is used to seal the incubation chamber and prevent the room air from
dissolving during the experiments.
 A small hole at the centre of the stopper permits the expulsion of air bubbles and allows small
volumes of reagents to be added with a microlitre syringe. The contents of the chamber are
stirred continuously with a magnetic stirrer.
 A small polarizing voltage ( 0.6 volt) is applied between the silver anode (+) and the platinum
cathode (–). Oxygen diffuses through the teflon membrane and is reduced to water at the
platinum cathode:
O2 + 4H + + 4e – → 2H2O
The circuit is completed at the silver anode, which is slowly corroded by the KCl electrolyte
Ag + Cl – → AgCl + e –
The resulting current is proportional to the oxygen concentration in the sample chamber.
This signal can be amplified and recorded.
Plunger
Locking ring
‘O’ ring
Incubation Chamber
‘O’ ring
Teflon mernbrane
Electrode base
Connecting lead
Working electrode
Clamping screw
(a)
Plunger
Locking ring
‘O’ ring
Water out-let
Incubation chamber with ‘O’ ring
Water outlet with ‘O’ ring
Locking ring
‘O’ ring
Teflon membrane
Electrode base
Working electrode Connecting lead
Clamping screw
(b)
Figure 2.18 Standard dissolved oxygen electrode with (a) Glass incubation
chamber (b) Perspex incubation chamber
LYOPHILIZER
Definition
Lyophilization is a technique that is used to preserve microbial cultures by freeze-drying.
Principle
Most microbial laboratories maintain a large collection of strains referred to as stock culture collection.
These organisms can be used as reference strains for taxonomic studies, research work, etc.
The cultures are maintained or preserved for a short period, say a few months to many
years, by:
1. Periodic transfer of mother culture to a fresh medium which favours slow growth
(e.g., nutrient agar) at a particular temperature. Frequent transfer should be avoided as
it leads to the development of mutants and variants.
2. Covering the growth on agar slant with sterile mineral oil, at least 12–15 mm above the
tip of the slant.
3. Revival of cultures by opening the vials, adding liquid medium and transforming the
dehydrated medium to a suitable growth medium.
4. Preservation by liquid nitrogen at a very low temperature.
Lyophilization or freeze-drying can be used to preserve many kinds of bacteria that would
be killed by ordinary drying. In this process, a dense cell suspension is placed in small vials and
frozen at –60°C to –78°C. The vials are then subjected to rapid dehydration and high vacuum.
This results in minimum damage to delicate cell structures. The vials are then stored under
high vacuum in refrigerator. By this method, one can preserve a culture for more than 30 years.
It requires minimum storage space. Hundreds of lyophilized cultures can be stored in a small
area. Culture vials can be conveniently transported.
Method of Operation
1. Freeze-dry the sample at –80°C or with liquid nitrogen.
2. The switches for both the vacuum and refrigeration should be set to “Auto” (switches are
both down).
3. The lights for vacuum and refrigeration should both be “On” (green lights).
4. The temperature should read – 40°C.
5. The pressure should read ~100× 10–3 M bar.
6. If temperature and vacuum are not “On”/set, turn “On” refrigerator and pump, and allow
the temperature and pressure to reach appropriate levels.
7. Place the frozen sample in a round bottom lyophilizer tube.
8. Place the tube into the rubber gasket, attached to the lyophilizer.
9. Turn the valve attached to the rubber gasket 180° clockwise to open the chamber to the
pump.
10. At this point, the vacuum reading should go down and then come back up as the
vacuum pumps down the tube.
11. To remove the tube, turn the valve counter-clockwise 180° and remove the lyophilizer tube.
Components
The lyophilizer consists of a cabinet that encloses a vacuum pump, and drying and refrigerating
units. (Figure 2.19) The chamber is closed with a chamber lid. The product tray is placed to collect
the lyophilized sample. It is operated by “On/Off ” switch. The liquid sample is freeze-dried by
the lyophilizer which can be stored for years.
(a) (b)
Product Tray
Vapor Screen
Stoppering system
(C)
Figure 2.19 Lyophilizer
LAMINAR AIR FLOW (VERTICAL)
Laminar air flow cabinets are designed to provide a work area confirming to class 100 of revised US
Federal Standard 209 B. The unrestricted open front work area allows the use of equipment under
biologically clean conditions. It includes horizontal laminar airflow and vertical laminar airflow.
Our vertical laminar airflow has vertical direction flow and particle retention above
0.5 micron. These are appreciated for their low noise and versatile usage. These have been designed
with latest technology and can also be designed as per the specifications, provided by our clients.
Application
The vertical laminar airflow cabinets is designed to provide a high degree of protection for
process products in laboratories and production facilities. Many critical applications in the
medical, pharmaceutical, nuclear-power, and micro-electronic fields demand an ultra-clean work
environment which is free from biological and particulate contamination.
Operation
Ambient air is drawn in through pre-filter and is introduced into the work zone through
the HEPA filter. The average velocity of air in the work zone is maintained between
0.4 and 0.5 m/s.
Construction
The cabinets are fabricated out of thick board duly sunmica-clad or mild steel cabinet duly
epoxy coated (Figure 2.20). The work table is made of thick board which is sunmica-clad at top
or a stainless steel table top will be provided as optional accessories. All joints are sealed with
silastic sealant. Side panels are provided of thick transparent acrylic sheets. The unit is fitted with
pre-filter and is pre-filtered air made to pass through highly effective HEPA (High efficiency
particulate air) filter having efficiency rating as high as 99.997%, thus retaining all air-borne
particles of size 0.3 micron and larger. Using a dynamic machine, the blower and motor assembly
is statically and dynamically balanced. Blowers are mounted on vibration isolation mounts to
minimize vibration. The working table is illuminated by fluorescent lightings fitted to the unit.
The height of the working table provides comfortable “sit down” working position for the operator
to work on 220/230 volts AC supply.
HEPA filter
Zone heaters
Rear wall
Air return
plenum
A/C
module
Figure 2.20 Laminar air flow chamber

3
FUNDAMENTALS OF MICROBIOLOGY
LABORATORY PRECAUTIONS
Strict adherence to prescribed rules is necessary for personal and environmental safety. Because
most microbiological laboratory procedures require the use of living organisms, an integral part
of all laboratory sessions is the use of aseptic techniques. All microorganisms should be treated
as potential pathogens (organisms capable of producing disease). Thus, microbiology students
must follow aseptic techniques in the preparation of pure cultures.
The following basic steps should be observed at all times to reduce contamination, and to
prevent accidental injury and infection:
1. Laboratory coats or aprons must be worn at all times in the laboratory. This is to ensure
that culture material is not accidentally deposited on our clothes or skin, and as a
safeguard to protect our clothes and ourself from chemical spills and stains.
2. Long hair must be tied back to minimize its exposure to open flames.
3. Closed shoes should be worn at all times in the laboratory setting.
4. Only those materials pertinent to the laboratory work, such as laboratory manuals,
laboratory notebooks and other laboratory materials, should be brought to the laboratory
work space. All other items, such as books and bags, should be kept away from the
work area.
5. At the beginning and termination of each laboratory session, bench tops should be
wiped with a disinfectant solution.
6. Contaminated instruments, such as inoculating loops, needles and pipettes should not
be placed on bench tops. Loops and needles should be sterilized by red heat and pipettes
should be disposed off in disinfectant solutions.
7. All culture material and chemicals should be properly labelled with the name, class,
date and experiment. Labelling is critical to avoid improper use or disposal of materials.
8. Care must be taken, while working with Bunsen burners. To avoid injuries, burner
should be turned off when not in use. When reaching for objects, care must be taken,
not to place hands into the flame.
9. All contaminated materials must be autoclaved before disposal or reuse.
10. Rapid and efficient manipulation of fungal cultures is required to prevent the
dissemination of their reproductive spores in the laboratory environment.
11. Cultures must be carried in a test tube rack when moving around the laboratory.
Likewise, cultures must be kept in a test tube rack on the bench tops when not in
use. This serves a dual purpose—prevents accidents and avoids contamination of the
environment.
12. Spilled cultures or broken culture tubes should be covered with paper towels, and
saturated with disinfectant solution. After 15 minutes of reaction time, the towels
should be removed and disposed off in a proper manner.
13. Mouth pipetting of broth cultures or chemical reagents should not be done. Pipetting
must be carried out with the aid of a mechanical pipetting device only.
14. Unnecessary movement around the laboratory must be avoided to prevent distractions
that may cause accidents.
15. Application of cosmetics or use of contact lenses in the laboratory is not advisable.
16. Smoking, eating and drinking in the laboratory are absolutely prohibited.
17. After the laboratory session, hands should be washed before leaving the laboratory.
Specific precautions must be observed when handling body fluids of unknown origin due to
the possible transmission of the HIV and hepatitis B viruses in these test specimens. They are:
i. Disposable gloves must be worn during the manipulation of these test materials.
ii. Immediate washing of hands is required, if contact with any of these fluids occurs and
also upon removal of the gloves.
iii. Masks, safety goggles and laboratory coats should be worn, if aerosol formation and
splattering of fluids are likely to occur.
iv. Spilled body fluids should be decontaminated with a 1:10 dilution of household bleach,
covered with paper towel and then allowed to react for 10 minutes before removal.
v. Test specimens and supplies in contact with these fluids must be placed into a container
of disinfectant prior to autoclaving.
PRINCIPLES OF ASEPTIC TECHNIQUES

Microorganisms are ubiquitous. They are found in soil, air, water, food, sewage and on body
surfaces. In short, every area of our environment is replete with them. The microbiologists separate
these mixed populations into individual species for study. A culture containing a single species
Fundamentals of Microbiology 47
of cells is called a pure culture. If another species of microorganisms enters a pure culture, it is
said to be contaminated and is called a mixed culture.
To isolate and study microorganisms in pure culture, the microbiologist requires the application
of specific techniques. These methods of obtaining and maintaining pure cultures are collectively
called aseptic techniques. Proper aseptic transfer technique protects the microbiologist from
contamination with the culture, which should always be treated as a potential pathogen. Aseptic
technique involves avoiding any contact of the pure culture, sterile medium and sterile surfaces
of the growth vessel with contaminating microorganisms. To accomplish this task,
 The work area is cleansed with disinfectant to reduce the number of potential contaminants.
 The transfer instruments are sterilized.
 The work is accomplished quickly and efficiently to minimize the time of exposure during
which the contamination of the culture or laboratory worker can occur.
The typical steps for transferring a culture from one vessel to another are as follows:
1. The inoculation loop or needle is always sterilized by flaming before using it on any
culture material. An inoculation needle or loop must always be sterilized by holding it
in the hottest portion of the Bunsen burner flame, the inner blue cone, until the entire
wire becomes red hot. Then, the upper portion of the handle is rapidly passed through
the flame. Once flamed, the loop is never put down but is held in the hand and allowed
to cool for 10 to 20 seconds.
2. The tip of the culture tube is always flamed before inserting the sterile loop into the
culture. This destroys any contaminating cells that may have been inadvertently deposited
near the tip of the tube during previous transfer or by any other means.
3. Depending on the culture medium, a loop or needle is used for removal of the inoculum.
Loops are commonly used to obtain a sample from a broth culture. Either of the
instruments can be used to obtain the inoculum from a slant culture by carefully touching
the surface of the solid medium in an area exhibiting growth so as not to gauge into the
agar. A straight needle is always used when transferring microorganisms to an agar deep
tube from both solid and liquid cultures.
4. The cell-laden loop or needle is inserted into the subculture tube, in case of a broth, to
dislodge the organisms. With an agar slant medium, it is drawn lightly over the hardened
surface in a straight or zig-zag line. For inoculation of an agar deep tube, a straight needle
is inserted to the bottom of the tube in a straight line and rapidly withdrawn along the
line of insertion. This is called stab inoculation.
5. Following inoculation, the instrument is removed, the necks of the tubes are reflamed
and the caps are replaced on the same tube from which they were removed.
6. The needle or loop is again flamed to destroy remaining organisms.
7. All the culture materials are kept covered with their respective caps and lids when not
making transfers. The tube caps or Petri dish lids must not lie on the tabletop, thereby
exposing cultures to possible contamination. When transferring colonies from Petri plates,
the lid is used as a shield by slightly raising it enough so that the loop can be inserted
while the agar surface is still protected from contaminants falling on it.
8. The tube closures or the Petri dish lids must not be allowed to touch anything except their
respective culture containers. This will prevent contamination of closures and therefore
of cultures.
9. The used pipettes should be placed in disinfectant solution immediately.
All the above principles should be followed strictly to avoid contamination.
CLEANING OF GLASSWARE
In microbiology, clean glassware is crucial to ensure valid results. Previously used or new glassware
must be thoroughly cleaned. Laboratory ware and equipment that are not chemically clean are
responsible for considerable losses in personnel time and supplies in many laboratories. Chemical
contaminants that adversely affect experimental results are not always easily detected. This section
describes the procedures for producing chemically clean glassware.
Glassware for use in microbiological laboratory work should not be merely clean, but
chemically clean. Test tubes, Petri dishes, flasks, etc., are the receptacles used in the microbiological
laboratory for containing the different nutrient substances upon which microorganisms are to
subsist.
Very frequently free alkali may be present on new glassware in sufficient quantity to prevent
microbial growths in the nutrients contained therein. Glassware which looks clean may have been
used previously and should be given a thorough cleaning to get rid of possible traces of chemicals,
or other substances having germicidal properties. Directions are to be carefully followed and all
new and apparently clean glassware should be cleaned in the order given.
Cleaning New or Apparently Clean Glassware

All new glassware should first be treated with germicidal cleaning solutions (1% HCl or 3%
lysol) before proceeding with the directions for cleaning glassware.
Small amounts of organic matter adhering to glassware may be oxidized by these solutions,
but will not disappear until removed by a suitable brush and cleaning powder.
Cleaning Test Tubes

New test tubes should be filled with cleaning solution, placed in a wire basket and heated for at
least fifteen minutes in the autoclave with steam and pressure.
After removing test tubes from the cleaning solution:

1. Wash them in water with a test-tube brush, using cleaning powder if necessary.
2. Rinse with tap water till clean and free from cleaning powder.
3. Rinse with distilled water.
4. Drain.
5. Rinse test tubes and other glassware, flasks, pipettes, etc., with alcohol to facilitate drying,
then drain.
6. Autoclave if possible.
Cleaning Bacterial Culture Flasks
After treating flasks with cleaning solution:
1. Wash them as clean as possible with tap water and a flask brush; use cleaning powder if
necessary. (When using cleaning powder, empty all water out of the flask, wet the flask brush
with tap water, dip it in the cleaning powder and then rub the soiled portions vigorously.)
2. Rinse with tap water till clear and free from cleaning powder.
3. Rinse with distilled water.
4. Drain.
5. Autoclave.
Cleaning Petri Dishes
After removing Petri dishes from the cleaning solution:
1. Wash them in water, using cleaning powder if necessary.
2. Rinse with tap water. (It is not necessary to use alcohol or distilled water.)
3. Wipe immediately with a clean cloth.
Cleaning Pipettes
1. Place pipettes delivery end down, in a glass cylinder in cleaning solution and allow them to
stand overnight.
2. Pipettes which have been used should be washed immediately. Grease which cannot be
removed with water should be treated with 10% NaOH and then with cleaning solution.
3. Rinse with tap water, followed by distilled water.
4. Rinse with alcohol. (Alcohol may be used repeatedly.)
5. Drain.
Cleaning Fermentation Tubes

1. Rinse with tap water/distilled water.
2. Fill with cleaning solution and heat for fifteen minutes in steam or allow to stand overnight
if more convenient.
3. Wash thoroughly in tap water, using a test-tube brush if necessary.
4. Rinse in distilled water and drain.
Cleaning Cover Glasses and Slides
1. Immerse the cover glasses or slides, one by one in a 10% solution of sodium hydroxide (NaOH)
for thirty minutes only. This strength of NaOH will etch the glassware if left longer.
2. Wash separately in tap water, handling with ordinary forceps.
3. Put, one at a time, in cleaning solution, and leave overnight as convenient.
4. Wash separately in water.
5. Immerse in clean alcohol (95%).
6. Wipe with a clean cloth.
7. Always handle cover glasses and slides with forceps.
Cleaning Other Glassware
Some modification of these methods will be adaptable to nearly all glassware.
Note 1 Glassware containing liquiefiable solid media is best cleaned by heating and pouring
out the material while in liquid condition, then treating as above. (Solid media when liquefied
by heat should never be thrown in the sink, as it will solidfy when cold and clog up the taps
and drains.
Note 2 Flasks, test tubes, Petri dishes, etc., containing cultures, must be heated one hour
in flowing steam before cleaning.
Cultures containing spores should be autoclaved previous to cleaning.
Note 3 If cultures or media have become dry, add water before heating. Special care must be
used in cleaning glassware in which mercuric chloride or any other disinfectant has been used.
Stain Removal
Permanganate stains: Immerse the glassware in a solution of 3% sulphuric acid with 3%
hydrogen peroxide. Rinse with tap water, followed by distilled water. Rinse with alcohol. Drain.
Iron stains: Immerse the glassware in a solution containing one part hydrochloric acid with one
part water. Rinse with tap water, followed by distilled water. Rinse with alcohol. Drain.
Washing Out Common Lab Chemicals

 Water-soluble solutions (e.g., sodium chloride or sucrose solutions) Rinse 3–4 times with
deionized water then put the glassware away.
 Water-insoluble solutions (e.g., solutions in hexane or chloroform) Rinse 2–3 times with
ethanol or acetone, rinse 3–4 times with deionized water, then put the glassware away. In
some situations other solvents need to be used for the initial rinse.
 Strong acids (e.g., concentrated HCl or H2SO4) Under the fume hood, carefully rinse the
glassware with copious volumes of tap water. Rinse 3–4 times with deionized water, then
put the glassware away.
 Strong bases (e.g., 6 M NaOH or concentrated NH4OH) Under the fume hood, carefully
rinse the glassware with copious volumes of tap water. Rinse 3–4 times with deionized water,
then put the glassware away.
 Weak acids (e.g., acetic acid solutions or dilutions of strong acids such as 0.1 M or 1 M HCl
or H2SO4) Rinse 3–4 times with deionized water before putting the glassware away.
 Weak bases (e.g., 0.1 M and 1 M NaOH and NH4OH) Rinse thoroughly with tap water
to remove the base, then rinse 3–4 times with deionized water before putting the glassware
away.
Washing for Biochemical Work
For biochemical work, first wash the glassware in warm tap water and then immerse in dichromate-
sulphuric acid cleaning solution for 12–24 hours. Then remove, wash and rinse in hot tap water
for at least four times and in distilled water twice. In case of pipettes, place the used pipettes in
3% lysol. If necessary, keep them overnight in detergent or dichromate sulphuric acid cleaning
solution. Wash with tap water followed by deionized water.
Dichromate sulphuric acid cleaning solution Dissolve 400.0 g of potassium dichromate in
4000.0 ml of distilled water. Slowly add sulphuric acid (400.0 ml) until solution turns dark brown.
SAFETY
Sulphuric acid Toxic on inhalation of fumes. Affects the skin, respiratory and reproductive systems
and foetal tissue. Irritant to skin, eyes and respiratory tract.
Potassium dichromate Toxic by inhalation and ingestion. Affects the reproductive system and
foetal tissue. Corrosive to skin, eyes and mucous membranes.
Hydrochloric acid Strong irritant to skin, eyes and respiratory system. Corrosive.
Difference between sterilization and disinfection
Sterilization Disinfection
It is the complete destruction or Disinfection describes a process that eliminates
elimination of all viable organisms many or all pathogenic microorganisms, except
including spores. bacterial spores from inanimate objects.
Procedures involve the use of heat, Procedures involve the use of chemical
radiation or chemicals. disinfectants.
Sterilization is absolute. It means that Disinfectants in terms of their activity are
ALL of the microorganisms have either classified as:
been removed or killed. • High-level disinfectants that are
chemical sterilants, which when used for
a shorter exposure period than would be
required for sterilization, kill all
microorganisms with the exception of
high numbers of bacterial spores, e.g.,
ethylene oxide, glutaraldehyde,
formaldehyde
• Intermediate-level disinfectants that
may kill mycobacteria, vegetative
bacteria, most viruses, and most fungi
but do not necessarily kill bacterial
spores, e.g., phenolics, halogens.
• Low-level disinfectants that may kill
most vegetative bacteria, some fungi,
and some viruses, e.g., alcohols,
quaternary ammonium compounds.
CONTROL OF MICROORGANISMS
Control of microorganisms is essential in the home, industry and medical fields to prevent and
treat diseases, and to inhibit the spoilage of foods and other industrial products. Common methods
of control involve chemical and physical agents that adversely affect microbial structures and
functions, thereby producing a microbicidal or microbistatic effect. A microbicidal effect is one
that kills the microbes immediately. A microbistatic effect inhibits the reproductive capacities
of the cells and maintains the microbial population at a constant size. Chemical methods for control
of microbial growth include the use of antiseptics, disinfectants and chemotherapeutic agents.
STERILIZATION
Sterilization is defined as the process by which an article, surface or medium is freed of all
microorganisms, either in the vegetative or spore state.
Preparation of Glassware for Sterilization

Plug the pipettes at the mouth end with a little bit of cotton. Using a needle, place this plug
at about an inch from the end. Place them in a pipette can for sterilization. Similarly, place Petri
dishes in Petri cans.
Plug the test tubes with cotton and then place in wire baskets and cover with paper. Similarly,
plug the flasks and cover it with a paper before sterilization. A properly plugged tube can hold
its own weight when suspended from the plug. The plug should be inserted to a depth of 3–4
cm into the tubes and enough cotton should project out of the mouth to protect the rim from
dust and also to give a good hold for removing and inserting the plug.
Methods Used for the Control of Microbial Growth

I. Temperature Temperature has an effect on cellular enzyme systems and therefore, a marked
influence on the rate of chemical reactions and thus, on the life and death of microorganisms.
Low temperatures will inactivate enzymes and produce a static effect. High temperatures destroy
cellular enzymes, which become irreversibly denatured. The application of heat is a common means
and is effective. However, moist heat (because of the hydrolysing effect of water and its greater
penetrability) causes coagulation of proteins and kills cells more rapidly at lower temperatures
than does dry heat.
1. Dry heat Dry heat is suitable for glassware, instruments and articles not affected by very
high temperature. Water, impermeable oils and waxes can be sterilized by dry heat. Dry heat
sterilization includes the following:
i. Red heat Red heat is used to sterilize inoculating wires and loops by holding them
almost vertically in Bunsen flame until red hot along their whole length, almost to the
tip of the metal holder. Points of forceps and the surface of shearing spatula may also
be heated to redness.
ii. Flaming Flaming is used to destroy vegetative organisms (by slow passage through
Bunsen flame) on the surface of scalpel, blades, glass slides, coverslips and the mouths
of culture tubes and flasks. Needles, L-rods and scalpels are sometimes immersed in
methylated spirit before flaming to prevent spattering.
iii. Incineration This is an excellent method for rapidly destroying materials, such as
soiled dressings, animal carcasses, bedding and pathological material. Plastics, such as
PVC and polythene, can be incinerated but polystyrene materials emit clouds of dense
black smoke and hence, should be autoclaved in appropriate containers.
iv. Hot air oven This is the most widely used method of sterilization by dry heat.
A holding period of 160ºC for one hour or 180ºC for half an hour is used. It is used to
sterilize glassware, forceps, scissors, scalpels, all glass syringes, swabs, some pharmaceutical
products, such as liquid paraffin, sulphonamides, dusting powder, fats, greases, etc.
Hot air is a bad conductor of heat and its penetrating power is low. The oven is usually
heated by electricity, with heating elements in the wall of the chamber and it is fitted
with a fan to ensure even distribution of air and elimination of air pockets. It should not
be overloaded. The material should be arranged in a manner which allows free circulation
of air in between the objects.
Glassware should be perfectly dry before being placed in the oven. Test tubes, flasks, etc.
should be wrapped in kraft paper. Rubber materials (except silicone rubber) cannot withstand
the temperature. At 180°C, cotton wool plugs may get charred. For cutting instruments, such
as those used in ophthalmic surgery, a sterilizing time of two hours at 150°C is recommended.
The oven must be allowed to cool slowly for about two hours before the door is opened, since
the glassware may get cracked by sudden or uneven cooling.
Sterilization control The spores of a non-toxigenic strain of Clostridium tetani are used as a
microbiological test for dry heat efficiency. Paper strips impregnated with 106 spores are placed
in envelopes and inserted into suitable packs. After sterilization is over, the strips are removed
and inoculated into thioglycollate or cooked meat media and incubated for sterility test under
strict anaerobic conditions for five days at 37°C. A Browne’s tube (green spot) is available for
dry heat and is convenient for routine use. After proper sterilization, a green colour is produced
(after 10 minutes at 160°C or 15 minutes at 150°C).
2. Moist heat Microorganisms and their spores are destroyed at lower temperatures by moist
heat than by dry heat. However, moist heat cannot be used to destroy microorganisms from water-
proof materials, such as oils and greases and to dry materials in sealed containers. Moist heat is
used in the sterilization of culture media and other liquids required to retain their water content.
i. Steam under pressure (autoclave) Free-flowing steam under pressure requires the
use of an autoclave, a double-walled metal vessel that allows steam to be pressurized in
the outer jacket. At a designated pressure, the saturated steam is released into the inner
chamber from which all the air has been evacuated. The steam under pressure in the
vacuumed inner chamber is now capable of achieving temperatures in excess of 100°C.
A pressure of 15 lb/inch2 achieves a temperature of 121°C and sterilizes in 15 minutes.
This is the usual procedure, however, depending on the heat sensitivity of the material
to be sterilized, the operating pressure and time conditions can be adjusted.
ii. Boiling Application of free-flowing steam requires exposure of the contaminated
substance to a temperature of 100°C, which is achieved by boiling water. Exposures to
boiling water for 30 minutes will result in disinfection only; all vegetative cells will be
killed, but not necessarily the more heat-resistant spores. Clothing, bed linen, eating
utensils and some equipment may be disinfected by washing in water at 70–80°C for
several minutes. Cystoscopes, specula and other apparatus that may be damaged by
boiling can be disinfected in a bath at 75°C for 10 minutes.
Thermometer
Pressure guage
Safety valve
Steam inlet Steam outlet
Figure 3.1 Steam sterillizer
iii. Tyndallization Tyndallization is also referred to as intermittent or fractional

sterilization. This procedure requires exposure of the material to free-flowing steam
at 100°C for 20minutes for 3 consecutive days with intermittent incubation at 37°C.
The steaming kills all vegetative cells. Any spores that may be present germinate during
the period of incubation and are destroyed during subsequent exposure to a temperature
of 100°C. Repeating this procedure for 3 days ensures germination of all spores and their
destruction in the vegetative form. An apparatus, known as Arnold steamer, is used
for this technique.
Because tyndallization requires so much time, it is used only for sterilization of materials
that are composed of thermolabile materials and that might be subject to decomposition
at higher temperatures.
iv. Pasteurization Pasteurization exposes fairly thermolabile products, such as milk, wine
and beer, for a given period of time to a temperature that is high enough to destroy
pathogens and some spoilage-causing microorganisms that may be present, without
necessarily destroying all vegetative cells. There are two types of pasteurization—the
high-temperature, short-time (flash) procedure, which requires temperature of 71°C
for 15 seconds, and the low-temperature long-time method, which requires 63°C
for 30 minutes.
Sterilization control For determining the efficacy of moist-heat sterilization, the spores of Bacillus
stearothermophilus are used. This is a thermophilic organism with an optimum growth temperature
of 55–60°C and its spores require an exposure of 12 minutes at 121°C to be killed. Paper strips
impregnated with 106 spores are dried at room temperature and placed in paper envelopes.
These envelopes are inserted in different parts of the load and after being sterilized, the strips
are inoculated into a suitable recovering medium and incubated for sterility test at 55°C for
5 days. Chemical indicators, autoclave tapes and thermocouples are also used instead.
3. Sunlight The microbicidal activity of sunlight is mainly due to the presence of ultraviolet
rays in it. It is responsible for spontaneous sterilization in natural conditions. In tropical countries,
the sunlight is more effective in killing germs due to combination of ultraviolet rays and heat. By
killing bacteria suspended in water, sunlight provides a natural method of disinfection of water
bodies such as tanks and lakes. Sunlight is not sporicidal, hence it does not sterilize.
4. Drying Moisture is essential for the growth of bacteria. Four-fifths of the weight of the
bacterial cell is due to water. Drying in air has therefore, a deleterious effect on many bacteria.
However, this method is unreliable and is only of theoretical interest. Spores are unaffected by
drying. One of the oldest methods of food preservation is drying, which reduces water activity
sufficiently to prevent or delay bacterial growth.
5. Sonic and ultrasonic vibrations Sound waves of frequency >20,000 cycles/second kill
bacteria and some viruses on exposing for one hour. Microwaves are not particularly antimicrobial
in themselves, rather the killing effect of microwaves are largely due to the heat that they generate.
High-frequency sound waves disrupt cells. They are used to clean and disinfect instruments as
well as to reduce microbial load. This method is not reliable since many viruses and phages are
not affected by these waves.
II. Osmotic pressure Osmosis is the net movement of water molecules (solvent) across a
semi-permeable membrane (cell membrane) from a solution of higher concentration to a solution
of lower concentration.
The hypertonic solution possesses a higher osmotic pressure and a higher solute concentration,
and therefore, has a lower water concentration therefore, it tends to draw in water. In a hypertonic,
high-pressure environment, all cells lose water by osmosis and become shrivelled. This effect is
called plasmolysis; it inhibits cell reproduction.
The hypotonic solution possesses a lower osmotic pressure and has a lower solute concentration, and
a higher water concentration, therefore, it tends to lose water. In a hypotonic, low-pressure environment,
cells take in water and become swollen and lyse. This phenomenon is called plasmoptysis.
If two solutions separated by a semi-permeable membrane have equal concentrations of solutes
and therefore, equal water concentrations, there is no osmosis and the solutions are isotonic. The
ideal environment of animal cells and microbial cells, which are bounded by fragile cell membranes
are completely or closely isotonic.
III. Radiations Electromagnetic radiations that possess sufficient energy to be microbicidal are the
short-wavelength radiations, i.e., below 300 nm. They include UV, gamma rays and X-rays. The high-
wavelength radiations, those above 300 nm, have insufficient energy to destroy cells.
The gamma radiation, is representative of ionizing form of radiation. Radiations affect cell
molecules through loss of their chemical structures and ionization.
Because of their high energy content and therefore, ability to penetrate matter, X-rays and
gamma radiations can be used as a means of sterilization, particularly for thermolabile materials.
They are not commonly used however, because of the expenses of the equipment and the special
facilities necessary for their safe use. Since there is no appreciable increase in temperature, this
method is referred to as cold sterilization.
Ultraviolet light, which has lower energy content than ionizing radiations, is capable of
producing a lethal effect in cells exposed to the low penetrating wavelengths in the range of
210 nm to 300 nm. Cellular components capable of absorbing UV light are the nucleic acids,
with the DNA acting as the primary site of damage. The major effect is thymine dimerization,
which is the covalent bonding of two adjacent thymine molecules on one nucleic acid strand in
the DNA molecule. This dimer formation, distorts the configuration of DNA and interferes with
replication and transcription. UV radiation is used for disinfecting enclosed areas, such as entry
ways, hospital wards, operation theatres and small virus inoculation rooms and virus laboratories.
IV. Filtration This is the method used to rid heat-labile liquids of microorganisms within the
porous structure of the filter matrix.This is useful for antibiotic solutions, sera and carbohydrate solutions
used in the preparation of culture media. By this technique, we can obtain bacteria-free filtrates of
toxins and bacteriophages.This method is also useful to separate microorganisms, which are scanty in
fluids, and to study them. The filter would retain the organisms and the filter disc could be cultured.
There are two basic types of filters—depth filters and membrane filters. Other type of filters
include syringe filters, pressure filters and air filters.
1. Depth filters Depth filters consist of fibrous or granular materials that are pressed, wound,
fired or bonded into a maze of flow channels. In these, retention of particulates is a matter of
combination of absorption and mechanical entrapment in the filter matrix. The filters are made
of different materials, such as an asbestos pad in Seitz filter, diatomaceous earth in the Berkefield
filter, porcelain in the Chamberland Pasteur filter and sintered glass in other filters.
Seitz filters Seitz filters consist of an asbestos disc supported on a perforated metal disc within
a metal funnel. Before use, the funnel is loosely assembled with the asbestos disc in position and
sterilized in the autoclave and the disc is flushed with sterile saline and the upper part of the
funnel is screwed down tightly onto the softened asbestos. After use, the disc is discarded.
2. Membrane filters Membrane filters are made from polymeric materials, such as cellulose
nitrate, cellulose diacetate, polycarbonate and polyesters. Since it is less absorptive than other
filters, they have a faster rate of filtration for any given porosity. Membrane filters are manufactured
as discs of 13–293 mm diameter with porosities from 0.0150 – 12µm. A filter that is selected for
use must have enough porosity to retain the expected microorganisms. The membrane filters
can be sterilized by autoclaving. The filters are for single use after sterilization. The filter can
be mounted in re-usable holders and fitted to the filtration vessel. The whole assembly may be
packed and autoclaved separately, and with a sterile forcep, the sterilized filter is handled and set
in the filter assembly in a laminar flow chamber or in an inoculation chamber.
Membranes of 0.22 µm pore size are used to retain Pseudomonas diminuta, 0.45 µm filters
are used to retain coliform bacilli and 0.8 µm filters are used to remove air-borne microorganisms
in sterile rooms and to produce bacteria-free gases.
3. Syringe filter Membranes of 13–25 mm diameter can be fitted in syringe-like holders
of stainless steel or polycarbonate. This is used to sterilize small volumes of heat-labile fluids.
The fluid is forced through the filter by pressing down the piston.
Figure 3.2 Vacuum filtration apparatus
Figure 3.3 Syringe filters

4. Pressure filters These are high-performance pressurized pressure-flow pond filters with
integrated UV sterilizer/clarifier. These water filtering systems keep pond water clean. These
all-in-one filters provide mechanical, biological and UV sterilization.
Figure 3.4 Pressure filters
Working of the pressure filters:

1. With the filter connected to the pump, water enters the unit through a water inlet.
2. It then passes through the mechanical filtration stage, consisting of foam filters that intercept
and trap dirt and debris.
3. Next, water enters a biological filtration chamber (except PT1500) where it comes into contact
with Biospheres bio media which harbour beneficial bacteria that keeps the water healthy.
4. Dirt-free water then passes along an integrated UV- lamp, where it is exposed to ultraviolet
rays that inhibit the growth of single-celled algae organisms.
5. Filtered water finally returns to the pond.
5. Air filters Large volumes of air may be freed from pathogens by passing through HEPA
(high efficiency particulate air) filters. HEPA filters are used to decontaminate the air entering
into a laminar flow chamber, and to blow air from an exhaust ventilator for safety in laboratories
where work with dangerous pathogens are carried out.
V. Gaseous chemicals Medical and surgical articles that cannot withstand even heating
at 73°C can be treated with ethylene oxide. This highly lethal gas is an alkylation agent and
kills all microorganisms, including viruses.
Ethylene oxide is toxic and highly explosive. The gas is mutagenic in a variety of animals. The
concentration in the environmental air should not exceed an average of 5 parts per million over
an 8-hour period. Ethylene oxide should be used in purpose-designed pressure vessels capable
of withstanding an explosion. It should be operated at sub-atmospheric pressure in a spark-free
environment.
1. Beta-Propiolactone (BPL) It is a colourless liquid with pungent to slightly sweetish
smell. It is a condensation product of ketone with formaldehyde. It is an alkylating agent and
acts through alkylation of carboxyl and hydroxyl groups. It is an effective sporicidal agent, and
has broad-spectrum activity. 0.2% is used to sterilize biological products. It is more efficient in
fumigation than formaldehyde. It is used to sterilize vaccines, tissue grafts, surgical instruments
and enzymes. Its disadvantages are its poor penetrating power and carcinogenic nature.
2. Low temperature steam sterilization The use of low temperature steam sterilization for
special solutions or special packaging is becoming very common in industry today. Automated
liquid packaging machines using formalin, fill, and seal operations commonly require temperature
of 106°C for sterilization of solutions because this is the maximum temperature that many of
these packages can take. The biological indicator of choice for this process is Bacillus subtilis
ATCC 5230, a spore-forming Bacillus which is much more sensitive to these steam sterilization
temperatures. It is an exciting time in surgical procedure development and instrument design.
In parallel with the many advances in minimum invasive surgical techniques, the instruments
used are becoming more complex, with integrated electronics, robotic control and precise
operating mechanisms. Many of these contain a variety of temperature-sensitive materials
and cannot be exposed to traditional, heat-based disinfection and sterilization techniques.
Low-temperature sterilization is increasingly desired to reprocess these types of devices in a
microbial-effective, device-compatible and safe way. The traditional low-temperature sterilization
techniques based on humidified ethylene oxide and steam formaldehyde are not frequently used in
hospitals, particularly due to longer overall reprocessing times, and safety concerns. More recent
developments in low-temperature sterilization include gas plasma systems, humidified ozone
and liquid chemical peracetic acid. There are a number of new systems recently developed all
of which use hydrogen peroxide gas for low temperature sterilization. These can be considered
as those using plasma as part of the process and those that only use hydrogen peroxide gas.
All these systems expose loads to be sterilized under vacuum to the gas, in the presence or absence
of plasma. A plasma is essentially an excited gas and is produced by adding energy (in the form
of heat or an electromagnetic field).
3. Formaldehyde gas The use of formaldehyde gas for killing microorganisms was practised
before the turn of the century. One of the first uses of formaldehyde gas was to fumigate rooms,
a practice long since shown to be ineffective and unnecessary. There are, however, automatic,
low-temperature steam formaldehyde sterilizers that are effective and can be used to process
heat-sensitive instruments and plastic items. Formaldehyde kills microorganisms by coagulation
of protein in cells. Because formaldehyde vapours are irritating to the skin, eyes and respiratory
tract, the use of formaldehyde in this form should be limited.
DISINFECTION
Disinfectant is an agent, usually a chemical, which kills the growing forms but not necessarily
the resistant spore forms of disease-producing microorganisms. The term is commonly applied to
substances used on inanimate objects. Disinfection is the process of destroying infectious agents.
An antiseptic is a substance that opposes sepsis, i.e., prevents the growth or action of
microorganisms either by destroying microorganisms or by inhibiting their growth and
metabolism. It is usually associated with substances applied to the body.
Characteristics of an Ideal Antimicrobial Chemical Agent

1. The chemical, at a low concentration, should have a broad spectrum of antimicrobial activity.
2. The substance must be soluble in water or other solvents to the extent necessary for effective use.
3. The compound must be stable and should not result in significant loss of germicidal action.
4. Ideally, the compound should be lethal to microorganisms and not injurious to human and
other animals.
5. The preparation must be uniform in composition so that active ingredients are present in each
application. Pure chemicals are uniform, but mixtures of materials may lack homogenicity.
6. It should not combine with extraneous organic materials.
7. It must be toxic to microorganisms at room or body temperature.
8. It should have the capacity to penetrate.
9. It should not rust or disfigure metals, or stain or damage fabrics.
10. Deodorizing and cleansing action while disinfecting, is a desirable attribute.
11. The compound must be available in large quantities at a reasonable price.
In the process of selecting the most appropriate chemical agent for a specific practical
application, the major factors that need to be assessed are:
i. Nature of the material to be treated
ii. Types of microorganisms
iii. Environmental conditions
Common Disinfectants and Antiseptics

1. Phenol and phenolic compounds Phenol and phenolic compounds are very effective
disinfectants. A 5% aqueous solution of phenol rapidly kills the vegetative cells of microorganisms;
spores are much more resistant. Hexylresorcinol, a derivative of phenol, is employed as general
antiseptic.
Aqueous solutions of 2–5% can be employed to disinfect sputum, urine, faeces and
contaminated instruments or utensils.
Lysol is an effective phenolic compound but is pungent and an irritant to the skin. The
effective concentration of lysol is usually 2% for cleaning bench tops.
Exposure of microbial cells to phenolic compounds produces a variety of effects like disruption of
cells, precipitation of cell protein, inactivation of enzymes and leakage of amino acids from the cells.
2. Alcohols Ethyl alcohol, in concentration between 50 and 90% is effective against
vegetative or non-spore forming cells. For practical application, a 70% concentration of alcohol
is generally used. Alcohol is used as a skin antiseptic and for the disinfection of clinical oral
thermometers. Alcohols are protein denaturants, and also solvents for lipids. Hence, they may
damage lipid complexes in the cell membrane. They are also dehydrating agents.
3. Halogens
Iodine Iodine is traditionally used as a germicidal agent in a form referred to as tincture
of iodine. There are several preparations available, such as 2% iodine plus 2% sodium iodide
diluted in alcohol, 7% iodine plus 5% potassium iodide in 83% alcohol, and 5% iodine plus 10%
potassium iodide in aqueous solution. Iodine is also used in the form of iodophores. Iodophores
are mixtures of iodine with surface-active agents, which act as carriers and solubilizers for iodine.
One of these agents is polyvinyl pyrolidone (PVP).
Iodine solutions are chiefly used for skin antisepsis and for other purposes such as disinfection of
water, disinfection of air (iodine vapours) and sanitization of food utensils. Iodine is an oxidizing agent.
Chlorine and chlorine compounds Chlorine, either in the form of gas or in combinations,
represents one of the most widely used disinfectants. Calcium hypochlorite (chlorinated lime)
and sodium hypochlorite are popular compounds. The chloramines represent another category
of chlorine compounds used as disinfectants, sanitizing agents or antiseptics.
Chlorine compounds are widely used in water treatment, in the food industry, for domestic
uses and in medicine. When free chlorine is added to water, hypochlorous acid is formed, which
is further decomposed to nascent oxygen, which is a strong oxidizing agent.
Cl2+H2O → HCl+HClO (hypochlorous acid)
HClO → HCl+[O]
4. Aldehydes Two of the most effective aldehydes are formaldehyde and glutaraldehyde.
Both are microbicidal and both have the ability to kill spores (sporicidal).
Formaldehyde It is marketed in aqueous solution as formalin, which contains 37–40%
formaldehyde. The fumes of formaldehyde are noxious; they are irritating to tissues and eyes.
Formaldehyde in solution is useful for sterilization of certain instruments. In gaseous form, it is used
for disinfection of enclosed area. Only in an emergency, decontamination can be done by adding 10
g of potassium permanganate to 35 ml of 40% formaldehyde solution in a 500 ml beaker placed
in the cabinet. This liberates vapours, but is hazardous. The mixture boils within seconds and there
is a risk of explosion if the potassium permanganate is at any time present in excess in relation to
the formaldehyde. Formaldehyde combines with proteins and nucleic acids and destroys organisms.
Glutaraldehyde A 2% solution of this chemical agent exhibits a wide spectrum of antimicrobial
activity. It is effective against vegetative bacteria, fungi, bacterial and fungal spores and viruses.
It is used in the medical field for sterilizing urological instruments, lenses, instruments and
respiratory therapy equipment.
5. Heavy metals Examples of heavy metals include mercuric chloride, silver nitrate, copper
sulphate and organic mercury salts (e.g., mercurochrome, merthiolate). They act by precipitation
of proteins and oxidation of sulphydryl groups. They are bacteriostatic. 1% silver nitrate solution
can be applied on eyes as treatment for opthalmia neonatorum (Crede’s method). This procedure
is no longer followed. Silver sulphadiazine is used topically to help to prevent colonization and
infection of burn tissues. Mercurials are active against viruses at dilution of 1 : 500 to 1 : 1000.
Merthiolate at a concentration of 1 : 10000 is used in preservation of serum. Copper salts are
used as a fungicide. Bismuth salts have been used to treat stomach ulcers, even long before it
was realised that these are caused by a bacterial infection. Helicobacter pylori is now accepted as
playing a primary role in the pathology of stomach ulcers.
Disadvantages Most heavy metal ion preparations are now considered too toxic for routine use.
Mercuric chloride is highly toxic, are readily inactivated by organic matter.
6. Surface-active agents These are soaps or detergents. Detergents can be anionic
or cationic. Detergents containing negatively charged long-chain hydrocarbons are called
anionic detergents. These include soaps and bile salts. If the fat-soluble part is made to have a
positive charge by combining with a quaternary nitrogen atom, it is called cationic detergents.
Cationic detergents are known as quaternary ammonium compounds (or quat). Cetrimide and
benzalkonium chloride act as cationic detergents. They have the property of concentrating at
interfaces between lipid-containing membrane of bacterial cell and surrounding aqueous medium.
These compounds have long-chain hydrocarbons that are fat-soluble and charged ions that are
water-soluble. Since they contain both of these, they concentrate on the surface of membranes.
They disrupt membrane resulting in leakage of cell constituents. They are active against vegetative
cells, Mycobacteria and enveloped viruses. They are widely used as disinfectants at dilution of
1–2% for domestic use and in hospitals.
Disadvantages Their activity is reduced by hard water, anionic detergents and organic matter.
Pseudomonas can metabolize cetrimide, using them as a carbon, nitrogen and energy source.
7. Dyes Aniline dyes include crystal violet, malachite green and brilliant green. Acridine
dyes include acriflavin and aminacrine. Acriflavine is a mixture of proflavine and euflavine.
Only euflavine has effective antimicrobial properties. A related dye, ethidium bromide, is also
germicidal. It intercalates between base pairs in DNA. They are more effective against gram-
positive bacteria than gram-negative bacteria and are more bacteriostatic in action. They may
be used topically as antiseptics to treat mild burns. They are used as paint on the skin to treat
bacterial skin infections. The dyes are used as selective agents in certain selective media. Acridine
dyes are bactericidal because of their interaction with bacterial nucleic acids.
Testing the Efficiency of the Disinfectant—The In-Use Test

1. Using a sterile pipette, transfer 1 ml of the disinfectant into 9 ml of nutrient broth culture
in a conical flask. Mix well.
2. Using a 0.1 ml pipette, place 0.1 ml drops of this mixture onto ten different areas of two
nutrient agar plates.
3. Incubate one plate for 3 days at 37°C and the other for 7 days at room temperature.
4. Observe the plates following incubation.
Growth in more than 5 areas on either plates, indicates a failure of disinfection.
CULTURE MEDIA PREPARATION

The cultivation of bacteria is necessary for subsequent isolation and identification. The growth
of the organisms is influenced by proper physical and chemical conditions.The physical factors
are provided by proper temperature and pH. The chemical conditions for growth are provided
by components in the growth medium.
Any medium for the cultivation of bacteria requires the following:
1. a carbon source that may also serve as an energy source,
2. water,
3. a nitrogen source,
4. a phosphorous source,
5. a sulphur source and
6. various mineral nutrients, such as iron and magnesium.
The nutrients that are essential for growth vary with microorganisms. If the organisms require
more growth factors, then the organisms are said to be fastidious. For example, the lactic acid
bacteria are very fastidious, which need many growth factors, whereas E.coli are able to grow on
a medium containing simple sugars, ammonium phosphate and a few mineral salts and thus, E.
coli is not considered nutritionally fastidious.
DIFFERENT TYPES OF MEDIA

From a nutritional standpoint the media are divided into two categories: 1) complex media and
2) defined or synthetic media. There are also special media such as enriched media, enrichment
media, selective media, differential media, sugar media and transport media.
1. Simple (Basal) Media

The basic nutrients in these media are provided by plant and animal extracts. For example, nutrient
broth, in which yeast or beef extract and peptones are the basic ingredients, is commonly used
for E. coli.
Nutrient broth composition:
Peptone 5.0g
Beef/yeast extract 3.0g
NaCl 5.0g
Water 1000ml
Peptone, a semi-digested protein, is primarily a nitrogen source. The beef or yeast extract is
a source of organic carbon, nitrogen, vitamins and inorganic salts.
2. Complex Media

They have added ingredients for special purposes or for bringing out certain characteristics of the
bacteria under study. For routine laboratory work, complex media are often employed.
3. Defined or Synthetic Media

These are composed of known quantities of chemically pure, specific organic and/or inorganic
compounds. Their use requires knowledge of the organism’s specific nutritional needs, e.g.,
inorganic synthetic broth.
Inorganic synthetic broth composition:
Sodium chloride (NaCl) 5.0g
Magnesium sulphate (MgSO4) 0.2g
Ammonium dihydrogen phosphate (NH4H2PO4) 1.0g
Water 1000ml
For the growth of microorganisms, liquid, solid and semi-solid media can be used. Liquid
media, also called as broths, contain only nutrients dissolved in water. Liquid media are used
for the preparation of large numbers of organisms, for fermentation studies and for various
other tests. Solid media contain nutrients dissolved in distilled water, plus a solidifying agent,
such as agar, gelatin or silica gel to impart a degree of firmness to the medium. Agar is an ideal
solidifying agent for a microbiological medium because of its melting properties and because it
has no nutritive value for the vast majority of bacteria and fungi. Solid agar melts at 90–100°C;
liquid agar solidifies at about 42°C. Agar is a complex polysaccharide compound of galactose
and galacturonic acid. It is extracted from certain marine red algae, such as Eucheuma, Gelidium,
Gracilaria and Rhodophyta.
Solid media are used for developing surface colony growth of bacteria and moulds.
The development of colonies on the surface of a medium is essential to isolate organism from mixed
cultures. Semi-solid media are similar to solid media except that a lower concentration of solidifying
agent is used, which gives jelly-like consistency. Semi-solid media are used for motility experiments.
4. Special-Purpose Media

Numerous special-purpose media are available for various functions, such as characterization
and identification of bacteria by their abilities to produce chemical changes in different media.
The various special media are outlined as follows:
Numerous special-purpose media are available for the following purposes:
 Isolation of bacterial types from a mixed population of organisms.
 Differentiation among closely related groups of bacteria on the basis of macroscopic
appearance of the colonies and biochemical reactions within the medium.
 Enumeration of bacteria in sanitary microbiology, such as in water and sewage, and also
in food and dairy products.
 Assay of naturally occurring substances such as antibiotics, vitamins and products of
industrial fermentation.
 Characterization and identification of bacteria by their abilities to produce chemical
changes in different media.
i. Selective media These media are used to select (isolate) specific groups of bacteria.
They incorporate chemical substances that inhibit the growth of one type of bacteria while
permitting the growth of another, thus facilitating bacterial isolation.
Mannitol salt agar (MSA) This medium contains a high salt concentration, 7.5% NaCl, which
is inhibitory to the growth of most bacteria other than staphylococci. The medium also performs
a differential function. It contains the carbohydrate mannitol, which some staphylococci are
capable of fermenting and phenol red, a pH indicator for detecting the acid produced by mannitol-
fermenting staphylococci. These staphylococci form yellow colonies and exhibit a yellow zone
surrounding their growth; staphylococci that do not ferment mannitol will not produce any
change in colour.
Salmonella–Shigella agar (SS Agar) SS agar is used to isolate Salmonella and Shigella species.
Its bile salt mixture inhibits many groups of coliforms. Both Salmonella and Shigella sp. produce
colourless colonies because they are unable to ferment lactose. Lactose-fermenting bacteria will
produce pink colonies.
Bismuth sulphite agar (BSA) BSA is used for the isolation of Salmonella typhi, especially from
stool and food specimens. S.typhi reduces the sulphite to sulphide, resulting in black colonies
with a metallic sheen.
ii. Differential media These can distinguish among morphologically and biochemically
related groups of organisms. They incorporate chemical compounds, which following inoculation
and incubation, produce a characteristic change in the appearance of bacterial growth and or on
the medium surrounding the colony which permits differentiation.
MacConkey agar The inhibitory action of crystal violet on the growth of gram-positive organisms
allows the isolation of gram-negative bacteria. Incorporation of the carbohydrate lactose, bile salts
and the pH indicator, neutral red, permits differentiation of the ability to ferment lactose. E. coli
produces acid as a result of lactose fermentation. The acid precipitates the bile salts followed by
absorption of the neutral red, giving a red colouration on the surface of the colony. Salmonella and
Shigella are non-lactose fermenters and therefore, do not produce acid. The colonies appear colourless.
Eosin methylene blue (EMB) Agar This differentiates between lactose fermenters and
non-lactose fermenters. EMB contains lactose, salts and two dyes—eosin and methylene blue.
E.coli, which is a lactose fermenter, will produce a dark colour or one that has a metallic sheen.
S. typhi, a non-lactose fermenter, will appear as colourless colonies.
iii. Enrichment Media Enrichment media contain specific nutrients required for the growth
of particular bacterial pathogens that may be present alone or with other bacterial species in a
patient specimen. This media type is used to enhance the growth of a particular bacterial pathogen
from a mixture of organism by using nutrient specificity. One example of such a medium is
buffered charcoal–yeast extract agar, which provides l-cysteine and other nutrients required for
the growth of Legionella pneumophila, the causative agent of Legionnaire’s disease.
iv. Enriched Media Brain heart infusion (BHI) is a nutritionally rich medium used to
grow various microorganisms, either as a broth or as an agar, with or without added blood.
Key ingredients include infusion from several animal tissue sources, added peptone (protein),
phosphate buffer, and a small concentration of dextrose. The carbohydrate provides a readily
accessible source of energy for many bacteria. BHI broth is often used as a major component of
the media developed for culturing a patients blood for bacteria establishing bacterial susceptibility
to antimicrobial agents.
Chocolate agar Chocolate agar is essentially the same as blood agar except that during
preparation the red blood cells are lysed when added to molten agar base. This lysis releases
intracellular nutrients such as haemoglobin, haemin (‘’ X’’ factor) and the coenzyme nicotinamide
adenine dinucleotide (NAD or ‘’ V’’ factor) into the agar for utilization by fastidious bacteria.
v. Indicator Media These media contain an indicator which changes colour when a
bacterium grows in them, for example incorporation of sulphite in Wilson and Blair medium.
Salmonella typhi reduce sulphite to sulphide in the presence of glucose and the colonies of
Salmonella typhi have a black metallic sheen. Potassium tellurite in McLeod’s medium is reduced
to metallic tellurium by the diphtheria bacillus to produce black colonies.
vi. Sugar Media The usual sugar media consist of 1% of the sugar—pentoses e.g.,
arabinose, xylose, hexoses, e.g., dextrose, mannose; Disaccharides, e.g., saccharose, lactose or
polysaccharides, e.g., starch, inulin,— in peptone water along with an appropriate indicator.
A small tube (Durham’s tube) is kept inverted in the sugar tube to detect gas production. For
organisms which are exacting in their growth requirements (e.g., Pneumococci), Hiss’ serum
sugar is used. They contain 3% serum.
vii. Transport Media In the case of delicate organisms (like gonococci) which may not survive
the time taken for transporting the specimen to the laboratory or may be overgrown by nonpathogens
(such as dysentery or cholera organisms in faeces), special media are devised for transporting the
specimens. These are termed transport media, for example, Stuart’s medium, a non-nutrient soft
agar gel containing a reducing agent to prevent oxidation and charcoal to neutralize certain bacterial
inhibitors used for gonococci and buffered glycerol saline used for enteric bacilli.
viii. Anaerobic Media These media are used to grow anaerobic organisms, for example,
Robertson’s cooked meat medium, and thioglycollate broth. Robertson’s cooked meat medium
is probably the most widely used fluid medium for the culture of anaerobes. It consists of fat-free
minced cooked meat in broth, with a layer of sterile vaseline over it. It permits the growth of
even strict anaerobes and indicates their saccharolytic or proteolytic activities, by the meat being
turned red or black, respectively.
Thioglycollate broth Nonselective for cultivation of anaerobes, facultative anaerobes and aerobes.
Anaerobes grow at the bottom of the tube, facultative anaerobes grow at the middle portion of
the tube and aerobes grow at the top.
STERILIZATION
Sterlization procedures eliminate all viable microorganisms. Petri dishes, test tubes, flasks,
pipettes, transfer loops and media must be free of viable microorganisms before they can be used
for establishing pure cultures.The culture vessels must be capped with sterile plugs to prevent
contamination. The media must be sterilized by autoclaving which permits exposure to high
temperatures for a specific period of time (121°C at 15 1b/in2 for 15 to 20 minutes).
MEDIA INOCULATION AND INCUBATION

After media preparation and sterilization, a culture is inoculated (introduced) into each medium.
Media inoculation can be done using sterile loop, needle, swab or pipette. Inoculated media must
be incubated to allow time for bacterial growth. After incubation, the microbial growth in tubes
and plates becomes visible and thus, can be examined. The presence of a contaminant suggests
the need for better aseptic technique.
Aim
To perform media inoculation procedure.
Materials required
Culture : 24-hours plate culture of E. coli
Media : Nutrient broth, nutrient agar (Appendix III)
Equipment and other materials : Autoclave, balance, incubator, Bunsen burner, inoculating loop
and needle, Petri dish, test tubes, test tube rack, pipette,etc.
Procedure
1. Prepare 10 ml of nutrient broth in a small conical flask. After dissolving, transfer 5 ml to each
of the two tubes with a 10 ml pipette. Cotton plug the tubes and place them in a test tube
rack.
2. Prepare 50 ml of nutrient agar in a 100 ml flask. Cotton plug and place them in the autoclave
for sterilization. Cotton plug the other four test tubes, label and sterilize.
3. After sterilization, dispense 5 ml nutrient agar into each of the other four test tubes. Lean the
two tubes with 5 ml of nutrient agar against a pipette or similar object. When the medium
cools, these tubes will form agar slants. Let the remaining tubes cool upright in the rack.
These tubes will form agar deeps.
4. Let the flask contents cool sufficiently to allow handling without discomfort. After this, pour
the agar (15–20 ml) into the plates. Do not pour hot agar into a Petri dish. This will cause
excess condensation on the lid of the Petri dish and on the agar surface. Excess moisture may
allow the culture to spread across the entire surface of the agar, instead of forming discrete
colonies.
5. When the agar has solidified in tubes and plates, the media are ready to inoculate.
6. Inoculate the broth and slant using inoculation loop. One tube serves as control.
7. Inoculate the agar deep with an inoculating needle.
8. Inoculate the plate by simple streaking.
9. After inoculation, incubate the plates and tubes at 37°C for 24 hours in an incubator.
Observation
Basic Elements in Identifying Colonies are:
 Form The general form of the colony can be determined by looking down at the top of the
colony. For example, circular, filamentous, etc.
 Elevation The nature of the colony elevation is apparent when viewed from the side as the
plate is held at eye level. For example, flat, raised, convex, pulvinate, umbonate
 Margin This is the magnified shape of the edge of the colony. The shape of the edge or margin
can be determined by looking down at the top of the colony, e.g., entire, undulate, etc.
 Surface This is the appearance of colony surface. For example, smooth, glistening, rough,
dull, rugose, etc.
 Opacity For example, transparent (clear), opaque, translucent (almost clear, but distorted vision,
like looking through frosted glass), iridescent (changing colours in reflected light), etc.
 Chromogenesis Some colonies may be pigmented. For example, white, buff, red, purple, etc.
 Consistency Butyrous (butter like), viscous or stringy (a portion of it may come off the agar
surface with the transfer needle), rubbery (whole colony comes off the agar surface with the
transfer needle) and dry, brittle or powdery (colonies that break when touched by a needle)
 Odour Sweet, Putrefactive and Fruity
Agar butt
 Growth only within the line of inoculation (non-motile)
 Growth spread or not within the line of inoculation (motile)
Agar broth
 Amount (scanty, moderate, abundant)
 Distribution and type of growth
 Uniform (even turbid)

 Scum or film (pellicle)
 Sedimentary (granular)
 Ring at the top of the rim.
Identification of bacterial colonies
 Each distinct circular colony should represent an individual bacterial cell or group that
has divided repeatedly.
 Being kept in one place, the resulting cells have accumulated to form a visible patch.
 Most bacterial colonies appear white, cream, or yellow in colour, and fairly circular in shape.
Some of the examples are given as follows:

Bacillus subtilis circular, flat, entire, smooth, opaque, white colony.

Proteus vulgaris medium to large colony, shiny, cream coloured, swarming colony.
Streptococcus pyogenes translucent, domed, greyish, hemolytic colony.
Escherichia coli small, circular, raised, smooth, translucent, entire colony.
Staphylococcus aureus circular, pinhead, entire, white or golden yellow colony.
PURE CULTURE TECHNIQUES

AEROBIC CULTURE TECHNIQUES
Aim
To perform pure culture techniques for the separation of cells of a mixed culture so that discrete
colonies can be isolated.
Principle
The microbial population in our environment—air, soil and water—is large and includes many
species of bacteria, fungi and algae. In nature, microbial populations do not segregate themselves
by species but exist with a mixture of many other cell types. A study of the microorganisms in
these habitats requires knowledge of the specific microbes present.
In the laboratory, these populations can be separated into pure cultures. These cultures
contain only one type of organism and are suitable for the study of their cultural, morphological
and biochemical properties. Pure culture techniques are designed to produce discrete colonies.
Colonies are individual, macroscopically visible masses of microbial growth on a solid medium
surface, each representing the multiplication of a single organism.
The techniques commonly used for isolation of discrete colonies initially require that the number
of organisms in the inoculum be reduced. The resulting diminution of the population size ensures that
following inoculation, individual cells will be sufficiently far apart on the surface of the agar medium
to effect a separation of the different species present. The different techniques used are:
1. Streak-plate method,
2. Spread-plate method ,
3. Pour-plate method and
4. Decimal dilution method
1. The streak-plate method (looping out method) This is a rapid qualitative isolation
method. It is essentially a dilution technique that involves spreading a loopful of culture over
the surface of an agar plate.
2. The spread-plate method This technique requires that a previously diluted culture
be used. During inoculation, the cells are spread over the surface of a solid agar medium with a
sterile, L-shaped bent rod, while the Petri dish is spun on a turn table.
This method has two advantages:
i. By this method, distinct colonies may develop and they can be characterized according
to size, shape, colour, texture and so on. The colonies can be easily transferred to other
media for further study.
ii. This method is satisfactory for growing strict aerobes, since all cells are spread on and
grow at the agar surface.
3. The pour-plate method This technique requires a serially diluted culture. The diluted
inoculum is added with the mixed molten agar medium is a Petri dish, and allowed to solidify.
This technique is satisfactory for growing either the facultative or the microaerophilic forms. The
major disadvantage in this technique is that strict aerobes do not grow below the agar surface and
it is difficult to isolate cells from sub-surface colonies. It is often more difficult to count colonies
embedded in pour plate because they may be stacked on top of one another and some cells may
be damaged or killed when held for even a shorter time at 40–45°C.
Materials Required
Culture : 24–48 hours nutrient broth cultures of mixtures of organisms
Media : Nutrient agar [Appendix III]
Equipment and other materials: Inoculating loop, Bunsen burner, turn table, beaker, 95%
ethyl alcohol, L-rod and other materials
Procedure
Streak plate method Streaking is a rapid method for isolation of cultures. Without decimal
dilution, loopful of the culture is spread over the surface of an agar plate to reduce the number of
colonies and to get isolated colonies. On the area 1, heavy confluent growth will be seen. As the loop
is flamed in between, it reduces the number of colonies in area 2. Again flaming of loop and spreading
to next area reduces the number of colonies and individual colonies will be obtained in area 3 and 4.
Hence it is a simple and rapid method to obtain isolated colonies from a mixed culture.
Four-way streak or quadrant streak
1. Place a loopful of culture on the agar surface in area [1](Figure 3.5 a). Flame and cool the
loop, then drag it rapidy several times across the surface of area [1].
2. Reflame and cool the loop and turn the Petri dish 90°. Then, touch the loop to a corner of
the culture in area [1] and drag it several times across the agar in area [2]. The loop should
never enter area [1] again.
3. Reflame and cool the loop and again turn the dish 90°. Streak area [3] in the same manner
as area [2].
4. Without reflaming the loop, again turn the dish 90° and then drag the culture from a corner
of area [3] across area [4], using a wider streak. Do not let the loop touch any of the previously
streaked areas.
5. Incubate the plates at 37°C for 24 hours.
Apply
flamed Heavy
Heavy loop growth
Apply
confluent flamed
growth loop
[1]
[2]
[3]
[4]
Light
growth
Apply
loopful
of culture
(first step) Discrete Apply
colonies flamed
loop
Figure 3.5 (a) Four-way streak
Observation
Confluent growth will be seen on area 1. The number of colonies will be reduced in area 2, then
in area 3 and 4.
Result
Isolated colonies can be obtained from a mixed culture in areas 3 and 4. Hence this is a better
method for separation of cultures from a mixed culture.
Continuous streak (Simple streak)
i. Spread the culture over the plate in one continuous motion, (Figure 3.5 b) without flaming
the loop.
ii. This technique will work only when few cells are on the loop.
Figure 3.5 (b) Continuous streak
Surface colonies
Sample is pipetted onto Sample is spread evenly Incubation

Typical spread-plate results
surface of agar plate over surface of agar using
(0.1ml or less) sterile glass spreader.
Figure 3.6 Spread plate method

Spread-plate technique
1. Place the bent glass rod into the beaker and add a sufficient amount of 95% ethyl alcohol to
cover the lower, bent portion.
2. Place 0.1 ml of the diluted culture at the centre of the nutrient agar plate that has been
placed on the turn table.
3. Remove the glass rod from the beaker and pass it through the bunsen burner flame with the
bent portion of the rod pointing downward. Allow the alcohol to burn off the rod completely.
Cool the rod for 10–15 seconds.
4. Remove the Petri dish cover and spin the turn table.
5. While the turn table is spinning, lightly touch the sterile bent rod to the surface of the agar
and move it back and forth. This will spread the culture over the agar surface. (Figure 3.6)
6. When the turn table comes to a stop, replace the cover. Immerse the rod in alcohol and reflame.
7. Incubate the plates at 37°C for 24 hours.
iii. Pour-plate technique
1. Serially dilute the culture. Add 1ml of the diluted culture to sterile Petri dishes.
2. Approximately add 15 ml of the nutrient medium, melted and cooled to 45°C to each Petri
dish containing the diluted sample.
3. Mix the contents by rotating the plates.
4. Incubate the plates at 37°C for 24 hrs.
Surface Subsurface
colonies colonies
Sample is pipetted into Sterlie medium is added and Typical pour plate results
sterile plate mixed well with inoculum
Figure 3.7 Pour plate method
Decimal Dilution Method When one intends to determine the number of cells in a bacterial
culture, one way of doing this is by carrying out serial dilution. Since bacterial cell numbers are
usually very high in your original sample, plating out this sample in an undiluted fashion would
just lead to the creation of a bacterial lawn (a smear of many, individual bacterial colonies that
are all growing next to or on top of one another).
Bacterial cell numbers need to be reduced, which is done by repeatedly diluting the amount
of bacteria you have in your sample. A small amount of bacterial sample is mixed with a diluent
solution (such as sterile broth or saline or distilled water), and then successive dilutions are
made. A small amount of each of the diluted bacterial samples is then spread onto an agar plate.
The numbers of bacterial colonies that grow on each plate are counted. By working backwards
using multiplication with the “dilution factor” (the number of times that you have diluted the
bacterial sample with the diluent solution), you will be able to make a determination of the
numbers of bacteria in your original sample. Each colony-forming unit represents a bacterium that
was present in the diluted sample. This method has some drawbacks, however. Injured bacteria
may not always form colonies. Also, since there is no single diluent solution that supports the
growth of all types of bacteria, some bacteria may be left out of any given counting procedure.
If we take one ml of the original sample (say a known weight of soil mixed in known volume
of sterile water to have a soil suspension which contains a microbial mixture) and add it to
9 ml of sterile water, it will give 1 : 10 or 10–1 dilution of original sample, i.e., the original sample
has been diluted to 1/10th. Similarly we may prepare 1 : 100 (10–2), 1 : 1,000 (10–3), 1 : 10,000
(10–4 and so on dilutions of the original sample.
Finally one ml aliquot of any dilution is added to a sterile Petri dish to which are added 15 ml
of sterile, cool, molten agar medium. The plates are incubated at suitable temperature. Within
24 hrs colonies of each kind of microbes grow in the dish. The number of colonies of each kind is
counted. This number is then multiplied by the dilution factor to find the total number of cells
present per ml of the original sample.
1 ml 1 ml 1 ml 1 ml 1 ml
Original 9 ml broth
inoculum in each tube
Dilutions 1:10 1:100 1:1000 1:10

1:10,000 1:100,000
1 ml 1 ml 1 ml 1 ml 1 ml
Plating
1:10 1:100 1:1000 1:10,000 1:100,000
Figure 3.8 Decimal dilution method
A tenfold dilution for each step is called a logarithmic dilution or log-dilution. Dilution is
the mixing of a small volume of accurately measured sample with a large volume of sterile water
or normal saline called diluents or dilution blank.
Laws
Dilution=V of sample
Total V of sample +diluent
Dilution Factor = Total V of sample +diluent

Vof sample (or we can simply say the reciprocal of dilution)
In order to understand how these laws are applied, let us consider the following example.
If we made a dilution of 1ml of bacterial sample in 9ml sterlie water then,
D = 1 / (1+9) = 1 / 10 = 1 × 10–1
in which (1+9) is the total volume of the sample+diluent, 1/10 means 1ml added to 10ml.
D.F = The reciprocal of D = 1 / (1×10–1) = 1 × 101

Observation
For spread-plate method
Observe the number of colonies and record the results.
S.No Dilution plated Number of colonies CFU/ml

1. 10 –3 280 2.8 × 104
2. 10 –4 38
3. 10 –5 TFTC*
* Too few to count
CFU/ mL = CFU/plate × dilution factor × 1/aliquot.

= 280 × 103 × 1/0.1
= 2.8 × 106 cfu/ml.
For pour-plate method
Observe the number of colonies. Count the number of surface and subsurface colonies on each
plate and record the results
S.No Dilution plated Number of colonies CFU/ml

1. 10 –3 TNTC * 2.4 × 106
2. 10 –4 240
3. 10 –5 34
* Too numerous to count
CFU/mL = CFU/plate × dilution factor × 1/aliquot.

= 240 × 104 × 1/1
= 2.4 × 106 cfu/ml.
For decimal dilution method
Count all colonies. A magnifying colony counter can aid in counting small embedded colonies.
Record the data. Calculate CFU/mL or CFU/g.
Calculation Number of colonies on plate × reciprocal of dilution of sample = number of
bacteria/ml (For example, if 32 colonies are on a plate of 1/10,000 dilution, then the count is
32 × 10,000 = 320,000/ml in sample.)
CFU/ mL = CFU/plate × dilution factor × 1/aliquot.
Result
For spread-plate method
The number of bacteria present in 1ml of the given original undiluted sample is 2.8 × 104 cfu/ml.
For pour-plate method
The number of bacteria present in 1ml of the given original undiluted sample is
2.4 × 106 cfu/ml.
For decimal dilution method Results are obtained either by spread plating the decimally diluted
samples or by pour plate method (see also spread and pour plate method)
ANAEROBIC CULTURE TECHNIQUES

An organism, which is able to grow in the presence of atmospheric oxygen is an aerobe, whereas
one that can grow in its absence is an anaerobe. Almost all higher organisms are completely
dependent upon atmospheric oxygen for growth, i.e., they are obligate aerobes. Oxygen serves as
the terminal electron acceptor for the electron transport chain in aerobic respiration. Facultative
anaerobes do not require oxygen for growth, but do grow better in its presence. Aerotolerant
aerobes such as Enterococcus faecalis simply ignore oxygen and grow equally well whether it is
present or not. Strict or obligate anaerobes (Bacteroides, Fusobacterium, Clostridium tetani) do not
tolerate oxygen and die in its presence.
Obligate aerobes and facultative anaerobes usually contain the enzymes superoxide dismutase
and catalase, which catalyse the destruction of superoxide radical and hydrogen peroxide,
respectively. All strict anaerobes lack both the enzymes or have them in very low concentrations
and therefore, cannot tolerate oxygen.
Because anaerobes are killed by oxygen, their cultivation can be accomplished in three ways:
1. Special anaerobic media containing reducing agents, such as thioglycollate or cysteine
or ascorbic acid, may be used. The reducing agents will eliminate any dissolved oxygen
present within the medium so that anaerobes can grow beneath its surface.
2. Oxygen may also be eliminated by removing air with a vacuum pump and flushing out
residual oxygen with nitrogen gas or CO2.
3. One of the most popular ways of culturing small numbers of anaerobes is by the use of
a Gaspak jar. In this procedure, the environment is made anaerobic by using hydrogen
and a palladium catalyst, which removes oxygen through the formation of water.
1. Robertson’s Cooked Meat (RCM) Medium (Clostridium sp.)

Aim
To cultivate anaerobic bacteria using RCM medium.
Principle
This is the most widely used fluid medium for the culture of anaerobes. It consists of fat-free,
minced, cooked meat in broth, with a layer of sterile vaseline over it. It permits the growth of
even strict anaerobes and indicates their saccharolytic or proteolytic activities, by the meat being
turned red or black, respectively.
For fastidious anaerobes, particularly for quantitative cultures, pre-reduced media and an
anaerobic chamber (glove box) may be used. The anaerobic chamber is an airtight, glass-fronted
cabinet filled with inert gas, with an entry lock for the introduction and removal of materials,
and gloves for the hands.
Materials required
Sample : Soil
Media : Robertson’s cooked meat medium (Appendix III)
Equipment and other materials : Incubator, screw-capped tubes, etc.
Procedure
1. Prepare Robertson’s cooked meat medium and dispense into screw-capped tubes and sterilize.
2. After sterilization, allow the medium to cool. Add 0.1 g of soil sample to each screw-capped
tube aseptically.
3. Incubate the tubes in an incubator at 37°C for 24–48 hours.
Result
RCM medium contains unsaturated fatty acids which take up oxygen, the reaction being
catalysed by haematin in the meat and also sulphydryl compounds which bring about a reduced
oxidation-reduction potential. Clostridium grow in the medium, rendering the broth turbid. Most
species produce gas. Saccharolytic species turn the meat pink (Clostridium difficile, Clostridium
tertium); Proteolytic species (Clostridium tetani, Clostridium botulinum) turn the meat black and
produce foul and pervasive odours.
2. Anaerobic Jar (Total Anaerobes)

Aim
To cultivate anaerobic organisms using anaerobic jar.
Principle
A number of methods have been described for achieving anaerobiosis, by exclusion of oxygen
or production of vacuum, displacement of oxygen with other gases, absorption of oxygen by
chemical or biological means and reduction of oxygen.
Mclntosh–Fildes anaerobic jar The most reliable and widely used anaerobic method is the
Mclntosh–Fildes anaerobic jar. It consists of a stout glass or metal jar with a metal lid, which
can be clamped air-tight with a screw. The lid has two tubes with taps, one acting as the gas
inlet and the other as the outlet. The lid also has two terminals, which can be connected to an
electrical supply. Leading from the terminals and suspended by stout wires on the underside of
the lid is a small grooved porcelain spool around which is wrapped a layer of palladinized asbestos.
Inoculated culture plates are placed inside the jar, with the medium in the bottom half of the
plates, and the lid clamped tight.
Mixture of gases
Pressure guage
To vaccum pump
Clamp
Palladium catalyst
Jar
Indicator
Inoculated culture plates
Figure 3.9 McIntosh and Fildes anaerobic jar
The outlet tube is connected to a vacuum pump and the air inside is evacuated. The outlet
tap is then closed and the inlet tube is connected to a hydrogen supply. After the jar is filled
with hydrogen, the electric terminals are connected to a current supply so that the palladinized
asbestos is heated. This acts as a catalyst for the combination of hydrogen with the residual oxygen
present in the jar. This method ensures complete anaerobiosis but carries the risk of explosion,
which may rarely occur. This risk can be eliminated by modification of the catalyst. Alumina
pellets coated with palladium in a gauze sachet suspended from the lid of the jar act as a catalyst
at room temperature, as long as the sachet is kept dry.
Gaspak anaerobic technique The Gaspak is now the method of choice for preparing anaerobic
jars. The Gaspak system is a contemporary method for the exclusion of oxygen from a sealed jar
used for incubation of anaerobic cultures in a non-reducing medium. The Gaspak is commercially
available as a disposable envelope, containing chemicals (sodium borohydride, cobalt chloride,
citric acid and sodium bicarbonate), which generate hydrogen and CO2, on the addition of water.
After the inoculated plates are kept in the jar, the Gaspak envelope, with water added, is placed
inside and the lid is screwed tight. H2 and CO2 are liberated and the presence of a cold catalyst
in the envelope permits the combination of H2 and CO2 to produce an anaerobic environment.
The gaspak is simple and effective, eliminating the need for drawing vaccum and addding
hydrogen.
Figure 3.10 Gaspak anaerobic technique
An indicator should be employed for verifying the anaerobic condition in the jars. Reduced
methylene blue is generally used for this purpose. It remains colourless anaerobically but turns
blue on exposure to oxygen.
Materials required
Culture : E. coli, Bacillus sp., Pseudomonas sp.
Media : Nutrient agar (Appendix III)
Equipment and other materials : Anaerobic jar, Gaspak envelope, Petri plates, etc.
Procedure
1. Inoculate each test organism into two nutrient agar plates.
2. Place one set of plate cultures inside the anaerobic chamber.
3. Tear off the corner of the Gaspak envelope and insert this inside the Gaspak jar.
4. Expose the anaerobic indicator strip and place it inside the anaerobic jar so that it is visible
from outside.
5. With a pipette, add the required 10 ml of water to the gas generator envelope and quickly
seal the chamber with its lid.
6. Place the sealed jar in an incubator at 37°C for 24–48 hours. Observe the indicator strip for
a colour change to colourless, which is indicative of anaerobic conditions.
7. Incubate the duplicate set of plates under aerobic conditions for 24–48 hours at 37°C.
Result
Inoculated test organisms like E. coli, Bacillus and Pseudomonas will not grow on the plates incubated
anaerobically. These organisms are aerobic or facultatively anaerobic and grow in nutrient agar
plates incubated in aerobic conditions. Anaerobic organisms like Clostridium, Bacteroides and
Fusobacterium will grow in anaerobic environment
3. Wright’s Tube Method

Aim
To cultivate anaerobic organisms by Wright’s tube method.
Principle
Microorganisms differ in their abilities to use oxygen for cellular respiration. Respiration involves
the oxidation of substrates for the production of energy necessary to life. A substrate is oxidized
when it loses a hydrogen ion and its electron (H+ e–). Since the H+ e– cannot remain free in the
cell, it must be immediately picked up by an electron acceptor, which becomes reduced. Therefore,
reduction is the gain of the H+ e–. These are termed oxidation–reduction (redox) reactions.
Organisms which use oxygen as an electron acceptor have high oxidation–reduction potentials;
others have low potential and use other substances as electron acceptors.
Wright’s tube method was first introduced by Buchner. This is a specialized method that does
not require the use of sealed jars. The culture tube is made anaerobic by the use of chemicals.
The chemicals absorb oxygen, producing anaerobic environment.
Materials required
Culture : 24-hours culture of E. coli, Bacillus, Pseudomonas, Clostridium
Reagents : Pyrogallic acid, sodium hydroxide

Equipment and other materials : Bunsen burner, test tubes, inoculation loop, glass rod, etc.
Procedure
1. Prepare nutrient agar slants and inoculate it with the test organisms by streaking the surface
of the agar slope.
2. Cut or char the cotton plug right onto the top (mount) of the test tube and push it into the
tube with a glass rod until it nearly touches the slant.
3. Add a pinch of pyrogallic acid crystals.
4. Add 2 ml of 4% sodium hydroxide solution to the pyrogallic acid crystals and immediately
close the mouth of the test tube with a cotton plug tightly.
5. Seal the mouth of the tube by wrapping its mouth with paraffin wax. Invert the tube and
incubate for 24–48 hours at 37°C.
Cotton plug
Addition of pyrogallic
acid and 4% NaOH
Inoculated slant
surface
Cotton plug
Pyrogallic
acid and NaOH
Rubber stopper
Figure 3.11 Wright’s tube method

Result
Agar slants inoculated with E. coli, Bacillus and Pseudomonas will not grow. Clostridium sp. will
grow as it is an anaerobe.
STAINING TECHNIQUES
INTRODUCTION
A stain is a dye consisting of a coloured ion (a chromophore) and a counter ion to balance the
charge. Attachment of the chromophore part of the dye complex to a cellular component represents
the staining reaction. Depending upon the dye, the chromophore can be either positively charged
(cationic/basic) and have an affinity for negative ions, or negatively charged (anionic/acidic) with
an affinity for positive ions. The stains react chemically with cell material and change the contrast
between the cell and the background. Bacteria carry a net negative charge at pH 7. Therefore,
basic stains (methylene blue, basic fuchsin, crystal violet) are more commonly used for bacterial
staining. The presence of a negative charge on the bacterial surface acts to repel most acidic stains
(eosin and nigrosin) and thus prevent their penetration into the cell.
SMEAR PREPARATION
Successful staining of bacteria and other microorganisms requires first of all that a suitable smear be
prepared on a microscope slide. Cells from a culture are spread in a thin film over a small area of the
slide, dried and then fixed by heating or with a chemical fixative to make the cells adhere to the slide.
From solid medium From liquid medium
Place 10 µl of sterile water Place 2 or 3 loopfuls of the liquid culture

on a clean glass slide. on a clean glass slide.
Transfer a small amount of culture with

Spread the bacteria on the slide.
a sterile inoculating loop and mix with
water.
Allow to air dry.
Figure 3.12 Preparation of smear

A good smear preparation will have the following characteristics:

1. It will be of an appropriate thickness to view individual cells;
2. It will withstand repeated washings during staining; and
3. The cells will retain their original morphologies after fixation and staining.
TYPES OF STAINING TECHNIQUES
1. Simple staining Simple staining implies the use of only a single stain, which is usually
sufficient to reveal the basic morphological features of most microbial cells, including the relative
size, shape and characteristic arrangements for groups of cells, e.g., Loeffler’s methylene blue
stain that shows the characteristic morphology of lymphocytes.
2. Differential staining Staining methods that could differntiate the different cell types are
known as differential staining and they have an important role in the identification of taxonomic
groups, e.g., Gram’s stain, acid-fast stain, etc.
1. SIMPLE STAINING

Aim
To study the shapes and arrangements of bacterial cells.
Principle
Simple staining means one dye; a one-step procedure is used to stain microbial cells. The most
commonly used simple stains are cationic (or basic) dyes, such as methylene blue, basic fuchsin
and crystal violet. Dyes impart a colour to cells or cell parts by becoming affixed to them through
a chemical reaction. These basic dyes bind to those cell parts that are negatively charged.
It reveals the shape and arrangement of bacterial cells.
Materials required
Cultures : 24-hour broth or slant cultures of E. coli and Staphylococcus aureus
Reagents : Crystal violet [Appendix I]
Equipment and other materials : Glass slides, microscope, inoculation loop, Bunsen burner,
marker, etc.
Procedure
1. Take a clean, grease-free glass slide. Hold the clean slide by their edges.
2. Label the end of the slide with the initials of the organism.
3. Prepare a smear at the centre, about 2 cm in diameter. A good smear is one that when dried,
appears as a translucent or semi-transparent confluent whitish film.
4. If the cells to be stained are from an agar culture, place a small drop of water on the top of the
slide at the centre. Using a heat-sterilized inoculating loop, aseptically transfer microorganisms
from the agar culture to the slide and mix them with water. Spread the suspension over the
area of the circle. If the cells are from a broth culture, transfer one or several loopful to the
slide without mixing water. Spread the cells over the centre.
5. Allow the smear to air-dry.
6. Heat-fix the preparation by passing the slide through a Bunsen burner flame several times,
with the smear directed away from the flame. During heat fixation, the bacterial proteins
are coagulated and fixed to the glass surface, so that it is not washed away while staining.
7. Place the slide on the staining rack and flood the smear with crystal violet. Leave it for
1 minute.
8. Gently wash the smear with tap water to remove excess stain. Drain and blot-dry with
blotting paper.
9. Air-dry and examine under oil-immersion objective.
Result
Stained cells appear as violet or purple.
2. GRAM STAINING
Aim
To differentiate between gram-positive and gram-negative bacteria.
Principle
The Gram stain reaction is based on the difference in the chemical composition of bacterial cell
walls. Gram-positive cells have a thick peptidoglycan layer, whereas the peptidoglycan layer in
gram-negative cells is much thinner and surrounded by outer lipid containing layers.
Four reagents are used in the Gram staining procedure. They are:
1. A primary stain— Crystal violet All cells stain purple.
2. Mordant—Gram’s iodine Binds to the primary stain, thus forming an insoluble
complex (CV–I).
3. Decolorizing agent—95% ethyl alcohol This reagent serves a dual function as a
protein dehydrating agent and as a lipid solvent. In gram-negative cells, the alcohol
increases the porosity of the cell wall by dissolving the lipids in the outer layers. Thus, the
CV–I complex can be more easily removed from the thinner and less highly cross-linked
peptidoglycan layer. Therefore, the washing out effect of the alcohol facilitates the release
of the unbound CV–I complex, leaving the cells colourless or unstained. The much thicker
peptidoglycan layer in gram-positive cells is responsible for the more stringent retention
of the CV–I complex, as the pores are made smaller due to the dehydrating effect of the
alcohol. Thus, the tightly bound primary stain complex is difficult to remove, and the
cells remain purple.
4. A secondary stain or counterstain—Safranine This reagent stains cells that have
lost the primary stain after alcohol treatment.
Gram-positive
Gram-negative
Fixation
Crystal violet
Iodine treatment
Decolorization
Counterstain
safranine
(a) Cells appear as blue-purple (b) Cells appear as pink-red
Figure 3.13 Gram staining
Materials required
Culture : 24-hour broth or slant cultures
Reagents : Crystal violet, Gram’s iodine, 95% ethyl alcohol, Safranine [Appendix I]
Equipment and other materials : Glass slides, microscope, blotting paper, Bunsen burner,
inoculation loop, marker, etc.
Procedure
1. Take a clean, grease-free glass slide and label it with the organism to be stained.
2. Prepare a smear. Air-dry and heat fix.
3. Flood the smear with crystal violet. Let it stand for 1minute.
4. Wash with tap water.
5. Then, flood the smear with Gram’s iodine and leave it for 1 minute.
6. Wash with tap water. Drain off the excess water.
7. Decolorize with 95% ethyl alcohol (for about 15 seconds) until the alcohol draining from
the slide appears colourless.
9. Counterstain with safranine for about 20–30 seconds.
10. Wash with tap water and blot-dry.
11. Examine under oil-immersion lens.
Result
Stained cells would appear pink-red (gram-negative) or blue-purple (gram-positive).
Modifications of Gram stain
There have been several modifications of Gram’s stain. These are:
1. Kopeloff and Beerman’s modification Primary stain solution consists of freshly
constituted methyl violet with sodium bicarbonate in distilled water. Mordant consists of iodine
dissolved in 4% NaOH solution. Decolorization is by using acetone either alone or as a mixture
with ethanol. Basic fuchsin is used to counterstain the smear. This method may be modified to
stain tissue sections.
2. Jensen’s modification This method involves use of methyl violet as primary stain,
iodine and potassium iodide in water as mordant, absolute alcohol as decolorizer and neutral red
as counterstain. For Neisseria spp., Sandiford’s counterstain is useful.
3. Weigert’s modification This modification is particularly useful for staining tissue
sections. The primary stain carbol gentian violet is prepared using saturated alcoholic solution of
4% gentian violet and 5% phenol solution. Gram’s iodine is used as a mordant and aniline-xylol
is used as a decolorizer. The counterstain carmalum (carminic acid and potassium alum in water),
however is used ahead of primary stain. This method may be used to stain Pneumocystis cysts.
4. Preston and Morrell’s modification The primary stain used in this modification is
ammonium oxalate–crystal violet. The smear is washed in Lugol’s iodine and further treated with
iodine solution. The smear is decolorized using iodine–acetone decolorizer and counterstained
using dilute carbol fuchsin solution. This method has been further modified to overcome the
irritating iodine in aerosols by reducing the iodine concentration to one-tenth and shortening
the duration of decolorization to ten seconds.
5. Hucker’s modification The primary stain used in this modification is ammonium
oxalate–crystal violet. The smear is washed in Lugol’s iodine. The smear is decolorized using
95% ethanol and counterstained using safranine solution.
3. CAPSULE STAINING

Aim
To demonstrate the presence of capsule by differential staining procedure.
Principle
A capsule is a gelatinous outer layer that is secreted by the cell. Chemically, capsule is a
polysaccharide, a glycoprotein or a polypeptide. Capsule staining is difficult than other types of
differential staining because the capsular materials are water-soluble and may be dislodged and
removed with vigorous washing. Smears should not be heated because the resultant cell shrinkage
may create a clear zone around the organism creating an artifact that can be mistaken for capsule.
Capsule staining uses two reagents:
1. Primary stain—1% aqueous crystal violet The cell and the capsular material
will take on the dark purple colour.
2. Decolorizing agent and counterstain 20% copper sulphate Since the capsule is
water-soluble, copper sulphate, rather than water is used to wash the purple primary
stain out of the capsular material without removing the stain that is bound to the cell
wall. At the same time, it acts as a counterstain as it is absorbed into the decolorized
capsular material. The capsule will now appear light blue in contrast to the deep
purple colour of the cell.
Materials required
Culture : 24-hour culture of Enterobacter aerogenes
Reagents : 1% crystal violet, 20% copper sulphate [Appendix I]
Equipment and other materials : Glass slides, microscope, Bunsen burner, inoculation loop,
marker, etc.
Procedure
1. Take a clean glass slide.
2. Place several drops of crystal violet stain on the slide. Using sterile technique, add three
loopful of the culture to the stain and gently mix with the inoculating loop.
3. With a clean glass slide, spread the mixture over the surface of the slide to create a very thin
smear. Let it stand for 5–7 minutes.
4. Allow the smear to air dry. Do not heat fix.
5. Wash the smear with 20% copper sulphate solution.
6. Gently blot dry and examine under oil-immersion objective.
Cell wall Capsule
Nuclear
material
Flagellum Plasmid 70s ribosomes
(a) Schematic diagram showing capsule (b) Microscopic observation of capsule
Figure 3.14
Result
Capsules of Enterobacter aerogenes appear light blue. The cell contents take up crystal violet and
appear purple. Other bacteria that possess capsule are Klebsiella pneumoniae, Streptococcus pneumoniae
and Neisseria meningitidis.
4. SPORE STAINING

(SCHAEFFER AND FULTON METHOD)
Aim
To perform a staining procedure to differentiate bacterial spore and vegetative cell.
Principle
Many microorganisms form spores, but the endospores formed by bacteria are unique because of
their remarkable resistance to a variety of adverse environmental conditions. The most important
endospore-forming bacteria are members of the genera Clostridium, Desulphotomaculum (anaerobic)
and Bacillus (aerobic). Endospores form inside the vegetative cells of these microorganisms, when
conditions are unfavourable for continued cell growth. As the parent cell dies and disintegrates,
the endospore is released as a separate entity. Once formed, endospores can retain viability
indefinitely. Under favourable conditions, the endospore will germinate and give rise to an active
vegetative bacterial cell.
Cortex
Inner spore coat

Germ cell wall
Plasma membrane
Outer spore coat Core
Exosporium
Figure 3.15 Diagram showing endospore
Endospores are dense, multilayered structures. They appear as highly refractile ovoid bodies
under the light microscope. Its highly resistant nature is due to two factors—a multilayer outer
covering containing peptidoglycan and the presence of a protein stabilizing molecule called
dipicolinic acid (DPA). DPA is an organic compound.
The calcium salt of DPA is a major constituent (5–15% of the dry weight) of bacterial
spores. DPA is not detectable in vegetative cells but is rapidly synthesized during the process
of sporulation. Spores become heat-resistant only after the development of DPA, which occurs
approximately 1 hour after the synthesis of the compound. The acid is released into the medium
when spores geminate. Sporulation and germination are not means of reproduction but are
mechanisms that ensure their survival under unfavourable environmental conditions.
Endospores are not stained well by most dyes but once stained, they strongly resist
decolorization. In the Schaeffer and Fulton procedure, endospores are first stained by heating
bacteria with malachite green, which is a very strong stain that can penetrate endospores. After
malachite green treatment, the rest of the cell is washed free of the dye with water and is
counterstained with safranine. This technique yields a green endospore in a pink-red cell.
Materials required
Culture : 48–72 hour nutrient agar slant culture of Bacillus cereus
Reagents : Malachite green, safranin. [Appendix I]
Equipment and other materials : Glass slides, microscope, inoculation loops, Bunsen burner,
marker, etc.
Procedure
1. Prepare a smear on a clean, grease-free glass slide.
2. Air-dry and heat fix.
3. Flood the smear with malachite green. Steam for 2–3 minutes. Do not allow the stain to
evaporate. Replenish the stain as and when needed.
4. Cool the slides and wash with running tap water.
5. Counterstain with safranine for 30 seconds.
6. Wash with tap water, blot dry and examine under oil-immersion objective. Spores will be
stained green and vegetative cells will appear as shades of pink to red.
7. Observe the shape of spores and the position within the cells, whether terminal, subterminal
or in the middle of the cell.
Result
Bacillus cereus is a spore-forming organism. Spores appear green and the vegetative cells are stained
pink. The position of the endospore differs among bacterial species and is useful in identification.
The main types within the cell are terminal, subterminal, and centrally placed endospores. Terminal
endospores are seen at the poles of cells, whereas central endospores are more or less in the middle.
Subterminal endospores are those between these two extremes, usually seen far enough towards
the poles but close enough to the centre so as not to be considered either terminal or central.
Lateral endospores are seen occasionally. Examples of bacteria having terminal endospores include
Clostridium tetani, the pathogen that causes the disease tetanus. Bacillus cereus, has a centrally
placed endospore and Bacillus subtilis has a subterminal endospore. Sometimes the endospore
can be so large the cell can be distended around the endospore, this is typical of Clostridium tetani.
5. ACID-FAST STAINING

(ZIEHL–NEELSEN’S METHOD)
Aim
To differentiate between acid-fast and non-acid-fast bacteria by differential staining.
Principle
Acid-fast bacteria (members of the genus Mycobacteria) contain unusual complex lipids (mycolic
acids) at the surface of the cell. These lipids give the cells waxy properties and makes it difficult
for the cell to take up the stain in ordinary staining procedures. This problem can be overcome
by applying moderate heat or detergents to soften the lipid components of the cell wall in order
to allow the stain to penetrate. Once stained, however, these cells resist decolorization with
acid–alcohol; hence, the name “acid-fast”.
The acid-fast stain uses three different reagents:
1. Primary stain— Carbol fuchsin It is a red phenolic stain that is soluble in the lipoidal
materials that constitute the major portion of the mycobacterial cell wall. Penetration is
further enhanced by the application of heat. A modification of the Ziehl–Neelsen method
circumvents the use of heat by the addition of a wetting agent (turgitol) to this stain,
which reduces surface tension between the cell wall of the Mycobacteria and the stain.
Following the application of primary stain, all the cells will appear red.
2. Decolorizing agent —Acid–alcohol (3% HCl + 95% ethanol) On application of
acid–alcohol, acid-fast cells will be resistant to decolorization since the primary stain is
more soluble in the cellular waxes than in the decolorizing agent. The primary stain is
retained and the Mycobacteria will stay red. This is not the case with non-acid-fast organisms
that lack cellular waxes. The primary stain is more easily removed during decolorization,
leaving these cells colourless or unstained.
3. Counterstain— Methylene blue It is used as the final reagent. As only non-acid-fast cells
undergo decolorization, they may now absorb the counterstain and take on the blue colour.
Materials required
Cultures : 24-hour cultures of Staphylococcus aureus, 72–96 hour trypticase soy broth cultures
of Mycobacterium smegmatis
Reagents : Carbol fuchsin, acid–alcohol and methylene blue [Appendix I]
marker, etc.
Procedure
1. Take clean glass slides.
2. Prepare the bacterial smear, air-dry and heat fix.
3. Flood the smear with carbol fuchsin and heat the preparation in steam for 5 minutes. Prevent
the stain from boiling. Also replenish the stain as needed (for heatless method, flood smear
with carbol fuchsin containing turgitol for 3–5 minutes).
4. Cool the slides. Wash with tap water.
5. Decolorize with acid–alcohol, adding the reagent drop-by-drop until the alcohol runs
almost clear with a slight red tinge.
7. Counterstain with methylene blue for 2 minutes.
8. Wash, blot dry and observe under oil-immersion lens.
Result
Smears of Staphylococcus aureus cells appear blue in colour whereas Mycobacterium smegmatis appears
red as it is an acid-fast bacillus.
6. FLAGELLAR STAINING

Aim
To identify the presence of flagella in the given organism through staining method.
Principle
Motility is a characteristic feature of most vibrios, spirilla and spirochetes, some bacilli and a
few non-pathogenic cocci. Motile bacteria produce one or more flagella, which can be visualized
directly by dark-field microscope and in stained preparation by light and electron microscopy.
Flagella can also be detected serologically. Flagellated cells react with specific antisera to give a
typical loose, flocculant agglutination.
Flagella are thin, proteinaceous structures, which originate in the cytoplasm and project out
from the cell wall. Bacteria show four types of flagellation patterns. They are:
i. Monotrichous, possessing a single flagellum at one end of the cell, e.g., Vibrio
cholerae.
ii. Lophotrichous, having many flagella in tufts or clusters at one end, e.g., Bacillus brevis.
iii. Amphitrichous, possessing flagella at both ends, either singly or in tufts, e.g., Spirillum
serpens
iv. Peritrichous, possessing flagella all over the surface, e.g., Bacillus cereus.
To make the flagella visible, it is necessary to increase flagellar diameter by precipitating a
coat of dye over their entire length. Leifson’s method accomplishes this by using a single staining
reagent that utilizes pararosaniline as a staining agent and tannic acid as a mordant. Staining
takes place within 5–15 minutes.
Materials required
Culture : E. coli and Staphylococous aureus
Reagents : BHI broth, normal saline, leifson’s stain, (Appendix I)

Equipment and other materials : Glass slide, microscope, inoculation loop, test sample,
staining solutions
Structure
Flagella Type Example
Monotrichous Vibrio cholerae
Lophotrichous Bartibella bacilliformis
Amphitrochous Sporillum serpens
Peritrichous Escherichia coli
Procedure
1. Inoculate a tube of brain–heart infusion broth with the organism and incubate at room
temperature for 18–20 hours.
2. Add 0.25 ml of formalin to the culture, mix by shaking and allow it to stand for 15 minutes.
3. Fill the tube upto 1 cm on top with distilled water, mix by shaking and allow it to stand
for 15 minutes.
4. Discard the supernatant without disturbing the pellet.
5. Add fresh normal saline, mix and centrifuge again.
6. Remove the supernatant and resuspend the pellet in 2 ml of normal saline.
7. Dilute the suspension with normal saline, until the suspension is barely turbid.
Staining procedure
 Clean a slide without grease. Place several loopful of organisms at right end of the slide.
 Tilt the slide to allow organisms to flow down over the surface of the slide.
 Allow the smear to air-dry completely. Do not apply any heat.

 Flood the smear with Leifson’s stain and leave it on the slide until all the alcohol has evaporated.
Wash gently to remove the stain from the slide.
 Allow the stained organisms to air-dry and examine under oil-immersion lens. Basic fuchsin
is a strong basic dye and most bacteria are stained red by the basic fuschin in Leifson’s stain.
The precipitate that builds up around the flagella will also appear red.
Result
E. coli is motile by peritrichous flagella. Flagella appears thicker and red due to the precipitation
of basic fuchsin around it. Staphylococcus aureus, Klebsiella pneumoniae, Shigella and Streptococcus are
non-motile.
7. NEGATIVE STAINING

Aim
 To observe the natural size and shape of the cells by negative staining.
 To demonstrate the presence of capsule by negative staining.
Principle
The purpose of negative staining is to stain everything in the background, but not the cells
themselves. Negative staining has been a useful method to demonstrate the presence of capsule,
surrounding the cell. Capsule is made up of polysaccharides or proteins (polymer of d-glutamic
acid in Bacillus anthracis). Because the capsule is a highly hydrated polymer, it will shrink during
heat fixation. In negative staining, since heat fixation is not required and the cells are not subjected
to the distorting effects of chemicals and heat, their natural size and shape can be seen.
Negative staining requires the use of an acidic stain, such as India ink or nigrosin.
The acidic stain, with its negatively charged chromogen, will not penetrate the cells because of
the negative charge on the surface of bacteria. Therefore, unstained cells are clearly seen as halos
against the dark background.
Materials required
Cultures : 24-hour cultures of Enterobacter aerogenes and Klebsiella sp.
Reagent : India ink or nigrosin [Appendix I]
marker, etc.
Procedure
1. Place a small drop of nigrosin close to one end of a clean slide.
2. Using sterile technique, place a loopful of inoculum from the culture into the drop of nigrosin
and mix.
3. With the edge of a second slide held at a 45º angle and placed in front of the bacterial
suspension, push the mixture to form a thin smear.
4. Air-dry. Do not heat fix the slide.
5. Examine under oil-immersion lens.
Result
Enterobacter aerogenes and Klebsiella are capsulated bacilli. Since the dyes, India ink and nigrosin,
are acidic, bacterial cells will not take up these dyes. Hence unstained bacilli can be seen in a
dark background.
8. GRANULE STAINING

Ponder’s stain
Metachromatic granules stain pink and the bacilli light blue.
1. Flood the smear with Ponder’s stain (toluidine blue). [Appendix. I]
2. Let it stand for 8–10 minutes. Do not wash. Blot-dry gently
Albert’s Stain
To demonstrate metachromatic granules present in Corynebacterium diphtheriae. They appear
bluish- black whereas bacillary body appears green.
1. Flood the smear with stain no. 1 (toluidine blue and malachite green). [Appendix I]
2. Let it stand for 3–5 minutes. Wash with tap water.
3. Flood the smear with stain no. 2 (Gram’s lodine).
4. Let it stand for 3 minutes. Wash. Blot gently.
9. GIEMSA STAIN FOR THIN FILMS (BLOOD)
This stain differentiates various parts of a cell and so can be used to identify mammalian cells
and also for demonstrating parasites in tissues and blood.
1. Air-dry thin films.
2. Fix in methanol for 1 minute.
3. Wash in tap water and flood the slide with Giemsa diluted 1 in 10 with buffered distilled
water (pH 7.2). The diluted stain must be freshly prepared each time.
4. Stain for 25–30 minutes.
5. Run tap water on the slide to float off the stain and to prevent deposition of precipitate on
the film. Drain, dry vertically.
6. Examine the film using the 100× objective.
Notes on the Stained Film

1. Examine the tail end of the slide where the red cells are separated into a one-cell-layer thick.
2. An alkaline buffer of pH 7.2 is vital for clear differentiation of nuclear and cytoplasmic
material and to visualize inclusions such as Schuffner’s/James’s dots in the red cells. Acidic
buffer is unsuitable.
3. The red cells are fixed in the thin film so the morphology of the parasitized cells and the
parasites can be seen.
4. On a well stained film the chromatin stains red/purple and the cytoplasm blue. Leucocytes
have purple nuclei, the red stippling if present should be clearly visible.
10. FONTANA’S STAIN FOR LEPTOSPIRES
Leptospires stain brownish black and the background will stain yellow.
1. Make a film and allow it to dry in air.
2. Flood the smear with fixative and leave for 1 min.
3. Wash in running tap water for 10 seconds.
4. Flood smear with mordant, heat gently until it steams and leave for 30 seconds.
5. Wash in running tap water for 20 seconds.
6. Rinse in distilled water.
7. Flood the slide with 0.5% silver nitrate; add one drop of concentrated ammonia; heat to
steam and leave for 20 seconds.
9. Blot-dry and examine.
11. PERIODIC ACID—SCHIFF (PAS) STAIN
Periodic acid - Schiff stain is used to detect yeast cells and fungal hyphae in tissues. Periodic acid
(5%) hydrolyses the cell wall aldehydes, which then combine with the modified Schiff reagent
and stains the cell wall carbohydrates pink-magenta against a light green background. This is
a stain useful for many things. It stains glycogen, mucin, mucoprotein and glycoprotein. The
stain is time-consuming and also requires the specimens to be digested. Because this staining
procedure is complex, most laboratories have replaced it with the calcofluor white stain. PAS is
useful for outlining tissue structures—basement membranes, capsules, blood vessels, etc. It does
stain a lot of things and, therefore, can have a high background. It is very sensitive, but specificity
depends upon interpretation.
Specimen
Standard paraffin section fixed in 10% neutral buffered formalin. For blood smears, the
recommended fixative is methanol.
Notes
Schiff reagent
Test of activity The Schiff reagent may be tested by adding a few drops to 3 to 5 mL of
40% formaldehyde on a Petri dish—active Schiff reagent will lead to the rapid development of
a reddish purple colour; delayed development of a deep bluish purple indicates that the reagent
is going off.
Procedure
1. Deparaffinize and hydrate to distilled water.
2. Oxidize with periodic acid for 5 minutes.
4. Treat with Schiff’s reagent for 5 minutes. [Appendix I]
5. Wash in running water for 10 minutes; this intensifies the colour reaction.
6. Stain the nuclei with Mayer’s haematoxylin for 1 minute (Do not keep for more than 1minute).
7. Wash in water.
8. Dehydrate, clear and mount.
Result
PAS-positive material stains magenta.
12. FUNGAL WET MOUNT—LACTOPHENOL

COTTON BLUE STAINING
Aim
To study the fungal morphology using lactophenol cotton blue staining.
Principle
The identification of moulds is based on surface colour, or colour on the reverse of the Petri plate,
hyphal structure and types of spores. However, the mould colony may change appreciably as it
gets older. Conclusive identification can be made only with microscopic observation of fungal
wet mount. The commonly used staining reagent is lactophenol cotton blue.
Lactophenol cotton blue is formulated with lactophenol, which serves as a mounting fluid.
Organisms suspended in the stain are killed due to the presence of phenol. The high concentration
of the phenol deactivates lytic celluar enzymes, thus the cells do not lyse. Cotton blue is an acidic
dye that stains the chitin present in the cell wall of fungi.
Materials required
Culture : Given fungal culture
Reagent : Lactophenol cotton blue [Appendix I}
Equipment and other materials : Inoculation needle, burner, cover glass, glass slide, etc.
Procedure
1. Take a clean glass slide.
2. Place a drop of lactophenol cotton blue at the centre of the slide.
3. Transfer a small amount of fungal culture with an inoculation needle from the culture plate
and place it over the stain on the slide.
4. Cover the preparation with a cover glass and examine under high-power objective lens.
Result
Lactophenol cotton blue stain is useful in the recognition and presumptive identification of fungi.
Additional characteristics including colony morphology and biochemical tests should be used
where appropriate for final identification.
In case of Aspergillus niger, delicate blue hyphae and fruiting structures with a pale blue background
are observed.
SLIDE CULTURE METHOD

Aim
To study the fungal morphology using slide culture technique.
Principle
The isolation, culture and microscopic examination of moulds require the use of suitable selective
media and special microscopic slide techniques. The arrangement of spores in the fungal hyphae
is necessary in the identification of fungi. In slide culture method, stained slides of moulds are
prepared and the method is superior to wet mounts in that the hyphae, sporangiophores and
spores remain more or less intact when stained.
For slide culture, Sabouraud’s agar can be used. It is a simple medium consisting of 1% peptone,
4% glucose and 2% agar. The pH of the medium is adjusted to 5.6 to inhibit the bacterial growth.
For some moulds, the pH of Sabouraud’s agar is too low and the glucose content is too high.
A better medium for such organisms is the one suggested by C.W. Emmons that contains only
2% glucose with 1% neopeptone and an adjusted pH of 6.8 to 7.0. To inhibit bacterial growth,
40 mg of chloramphenicol is added to the medium. In addition to these media, corn meal agar,
Czapek–Dox agar can also be used..
Materials required
Culture : Given fungal culture
Media : Sabouraud’s dextrose agar [Appendix III]
Reagent : Lactophenol cotton blue [Appendix I]
Equipment and other materials : Petri plates, burner, inoculation needle, glass slide, cover glass
Procedure
Slide culture preparation
1. Aseptically place a sheet of filter paper into the bottom of a Petri dish.
2. Place a sterile U-shaped glass rod on the filter paper.
3. Moisten the filter paper with sterile water (about 5 ml).
4. Place a sterile slide on the U-shaped rod.
5. Flame a scalpel to sterilize, cool and cut a 5 mm square block of the medium from the plate
of Sabouraud’s agar or Emmons’ medium. [Appendix III]
6. Pick up the block of agar by inserting the scalpel into one side.
7. Inoculate both top and bottom surfaces of the agar block with spores from the mould colony.
8. Place the inoculated block of agar in the centre of the slide with one of the inoculated surface
down.
9. Place a sterile cover glass on the upper inoculated surface of the agar block.
10. Place the lid of the Petri dish and incubate at room temperature for 48 hours.
11. After 48 hours of incubation, examine the slide under low-power for hyphae and spores.
Application of stain
 Place a drop of lactophenol cotton blue stain on a clean microscopic slide.
 Remove the cover glass from the slide culture. Add a drop of 95% ethanol to the hyphae on
the cover glass. (95% ethanol is a wetting agent).
 After evaporation of the alcohol, place the cover glass, mould side down on the drop of
lactophenol cotton blue stain on the slide.
 Remove the slide from the Petri dish, add a drop of 95% ethanol to the hyphae and follow
this up with a drop of lactophenol cotton blue stain.
 Cover the entire preparation with a clean cover glass.
 Observe both the stained slides under the microscope.
Result
Species of Cladosporium, Monilia, and Alternaria have spores connected in very fragile chains that
can fall apart at the slightest movement of air. Mounts of these fungi invariably reveal only loose
spores and a network of hyphae. To overcome this problem it is useful to set up slide cultures.
When you do slide cultures, you are growing the fungi directly on the slide on a thin film of agar.
By doing this, you do not have to remove a portion of the fungus from a culture and transfer
it to the slide, so there is less chance for the features that are key to identification, notably the
spore-bearing structures, to be damaged. In order to accurately identify many fungi it is essential
to observe the precise arrangement of the conidiophores and the way the spores are produced.
Slide culturing permits fungi to be studied virtually in situ with as little disturbance as possible.
MICROMETRY
MICROMETRY AND MEASUREMENT
OF MICROORGANISMS
Aim
To perform an experiment to measure microorganisms and to become familiar with the calibration
of an ocular micrometer.
Principle
The length and diameter of a bacterium can be measured with the help of optic devices. Before
an accurate measurement of cells can be made, the diameter of the microscopic field must be
established by means of an ocular micrometer and a stage micrometer.
The ocular micrometer has a scale divided into 100 divisions of arbitrary length and there
are markings such as 10, 20 and so on. It is placed on a circular shelf inside the eyepiece. The
distance between these graduations will vary depending on the size of the field. This distance is
determined by using a stage micrometer.
Stage micrometer is a special glass slide with etched graduations that are 0.01 mm (10 µm)
apart. Five consecutive small divisions are marked by slightly longer lines, 10 consecutive small
divisions are indicated by still longer graduations.
The calibration procedure for the ocular micrometer requires that the graduations on both
micrometers be superimposed on each other. This is accomplished by rotating the ocular lens.
A determination is then made of the number of ocular divisions per known distance on the stage
micrometer. Finally, the calibration factor for one ocular division is calculated as:
0 10 20 30 40 50 60 70 80 90 100
Ocular scale
Stage micrometer scale
0.0
1
2
0.1 3
4
5
6
0.2
7
8
9
0.3
Alignment of the stage micrometer and the ocular micrometer
0 10 20 30
Figure 3.16 (b) Ocular micrometer superimposed over a view of bacterial chains of cocci
under the microscope.
Known distance between two lines

on stage micrometer
One division on ocular micrometer in µm =
Number of divisions on ocular
micrometer
For example:
1. If one space of stage micrometer is occupied (0.01 mm) by six spaces of ocular micrometer,
then one ocular division = 0.01/6 = 0.006 mm or (1.6 µm).
2. If 12 ocular divisions coincide with two stage divisions (2×0.01)
Then, one ocular division
2×0.01
= = 0.0016mm(or)1.6µm
12
Calibration factor varies with objectives and can be calculated for low-power, high-power
and oil-immersion objectives.
Once the ocular micrometer is calibrated, the size of a microorganism can easily be determined
by counting the number of spaces occupied by the organism.
The stage micrometer slide is then removed from the microscope; the slide containing the
bacterial smear is focused and observed under the oil-immersion objective.
If an organism occupies five spaces on the ocular micrometer, then,
Length of organism = Number of ocular divisions occupied × Calibration factor (oil-
immersion objective)
=5×1.6 mm =8.0 mm
Materials required
Slides : Gram-stained slides of bacteria, lactophenol cotton blue-stained yeast cells
Equipment and other materials : Ocular micrometer, stage micrometer, microscope, immersion
oil, lens paper, etc.
Procedure
1. Place the ocular micrometer into the eyepiece.
2. Place the stage micrometer on the microscope stage.
3. With the stage micrometer in clear focus under the low-power objective, slowly rotate the
eyepiece to superimpose the ocular micrometer graduations over those of the stage micrometer.
4. Add a drop of immersion oil to the stage micrometer; bring the oil-immersion objective into
position and focus.
5. Move the mechanical stage so that a line on the stage micrometer coincides with a line on the
ocular micrometer at one end. Find another line on the ocular micrometer that coincides with
a line on the stage micrometer. Determine the distance on the stage micrometer (number of
divisions × 0.01 mm) and the corresponding number of divisions on the ocular micrometer.
6. Determine the value of the calibration factor for the oil-immersion objective.
7. Remove the stage micrometer from the stage. Focus the prepared slides under oil-immersion objective.
8. Calculate the number of ocular divisions occupied by each three separate bacilli or yeast.
9. Determine the size (length and width) by multiplying the average with the calibration factor.
Calculation and Result
Example 1
S.No. Length of the bacterial cell Width of the bacterial cell

Number of Average Average × Number of Average Average ×
ocular divisions Calibration ocular Calibration
occupied factor divisions factor
occupied
1. 2 2.3 1.6 × 2.3= 1 1.3 1.3 ×1.6=
2. 2 3.68 μm 1 2.13μm
3. 3 2
Length of the bacterial cell =3.68 μm Width of the bacterial cell= 2.13 μm
Example 2
A hypha was measured using an ocular micrometer in the eye piece of a phase-contrast micro
scope and its 40× darkfield objective. The hypha was 3 ocular micrometer units wide.
The calibration factor for that specific micrometer used in the phase-contrast micro scope with
the 40× darkfield objective is 2µm.
3 ocular micrometer units × 2 µm = 6 µm ocular micrometer

The hypha is 6 µm wide.
MOTILITY DETERMINATION
Aim
To determine the motility of bacterial cells and to become familiar with different types of motility
exhibited by bacteria.
Principle
1. Hanging drop method Hanging drop preparation is useful for microscopic examination
of living microorganisms, especially bacteria, without staining them and to see their motility
due to flagella. It is essential to differentiate between actual motility and Brownian movement, a
vibratory movement of the cells due to their bombardment by water molecules in the suspension.
Most motile bacteria move by the use of flagella, threadlike locomotory appendages, extending
outward from the plasma membrane and cell wall. They are slender, rigid structures about
20 nm across and up to 15 or 20µm long. Flagella are so thin that they cannot be observed
directly with a bright-field microscope, but must be stained with special techniques designed to
increase their thickness.
Bacterial species often differ distinctively in their patterns of flagella distribution. Flagellation
patterns are very useful in identifying bacteria. They may be monotrichous, amphitrichous,
lophotrichous or peritrichous. Bacteria can move by mechanisms other than flagella rotation.
Spirochetes are helical bacteria that travel through viscous substances, such as mucous or mud, by
flexing and spinning movements caused by a special axial filament composed of periplasmic flagella.
A very different type of motility, called gliding motility, is employed by many bacteria like
cyanobacteria, myxobacteria, cytophages and some mycoplasmas. Although there are no visible
external structures associated with gliding motility, these bacteria can coast along solid surfaces
at rates up to 3µm/s.
For observing motility of pathogenic organisms, it is safer to inoculate them in semi-solid
medium and observe their growth and motility.
2. Semi-solid culture technique The motility of bacteria can be indirectly observed by using
soft agar deeps. Agar deeps with media that contain 0.5% agar are inoculated by stabbing. If
the organism is actively motile, the turbidity radiates outward from the stabbed line.
3. Craigie’s tube method The motility of bacteria can also be observed by placing a piece
of open-end glass tubing in the semi-solid medium before sterilization. Culture is inoculated
inside the glass tubing. Motile bacteria will migrate downwards inside the tubing, emerge from
the bottom of the tube and then migrate upwards to the surface of the medium outside the
glass tubing. This method is helpful for selecting highly motile organisms for use in preparing
H-antigens for immunization.
4. Motility observation on plates The motility of bacteria can be observed indirectly by observing
the mass movement of cells in a culture growing on plates. Very actively motile bacterial culture
may rapidly spread itself across the surface of agar plates. This type of culture is called a spreader.
Species of the genus Proteus show this characteristic swarming motility.
Examples of motile organisms with flagella.
1. Monotrichous, e.g., Vibrio cholerae 2. Lophotrichous, e.g., Bacillus brevis

3. Amphitrichous, e.g., Spirillum serpens 4. Peritrichous,e.g., Salmonella typhimurium
Materials required
Cultures : Pseudomonas aeruginosa, Bacillus cereus, Proteus sp., Staphylococcus aureus
Media : Nutrient agar, soft agar [Appendix III]
Equipment and other materials : Inoculating loop, cavity slide, microscope, petroleum jelly,
coverslip, Petri plates, Craigie’s tube, etc.
Procedure
I. Using hanging drop method
1. With a matchstick, apply petroleum jelly at the four corners of a clean coverslip.
2. Using sterile technique, place a loopful of the mixed culture at the centre of the cover slip.
3. Place the depression slide, with the concave surface facing down, over the coverslip so that
the depression covers the drop of culture. Press the slide gently to form a seal between the
slide and the coverslip.
4. Quickly turn the slide right side up so that the drop continues to adhere to the inner surface
of the coverslip.
5. For microscopic examination, first focus the drop of culture under low-power objective with
reduced light and focus the boundary of the drop. Place a drop of oil on the coverslip and
use the oil-immersion objective for detailed observation.
II. Using agar deeps
1. Prepare soft nutrient agar deeps.
2. Inoculate the agar deeps by stab inoculation with Proteus sp. and Staphylococcus aureus.
3. Inoculation of agar deeps with sterile needle serves as the control.
4. Incubate the tubes at 37°C for 24 hours.
III. Using Craigie’s tube method
1. Place a piece of open-end glass tubing in the semi-solid nutrient medium before sterilization.
2. Inoculate the culture inside the glass tubing.
3. Incubate at 37°C for 24 hours.
Cavity slide
Sample placed
on coverslip
with loop
Vaseline
Oil drop
Figure 3.17 Hanging drop method
IV. Using plates

1. Prepare nutrient agar plates.
2. Divide the agar plates into two equal parts by drawing a line at the bottom of the plate with
a glass marking pen.
3. Inoculate Proteus sp. in a spot about 5 mm in diameter near the centre of one half of the plate.
4. Inoculate Staphylococcus aureus in a spot about 5 mm in diameter near the centre of other half
of the plate. Incubate the plates at 37°C for 48 hours.
Result
I. Using hanging drop method Bacillus cereus and Proteus sp. are motile rods. Moving cells
clinging to the sides can be easily observed at the edge of the water droplet. Staphylococcus aureus
is non-motile.
Figure 3.18 Hanging drop technique
To the right a properly illuminated hanging drop can be observed. The arrows indicate various
organims present.
II. Using agar deeps Motility can be seen in Proteus culture tubes which is shown by spreading
growth away from the line of inoculation. S. aureus, which is non-motile grows only at the line
of inoculation.
Figure 3.19 Using agar deeps

The tube on the left stabbed with Proteus vulgaris is positive for motility, while the tube on
the right was inoculated with Staphylococcus aureus, a species lacking motility.
III. Using Craigie’s tube method This method is used particularly to select motile strains
of Salmonella. Motile bacteria can be isolated from the surface of the medium outside the glass
tubing. Motile organisms move away from the line of inoculation and migrate upwards to the
surface of the medium outside the glass tubing. Non-motile organisms grow along the line of
inoculation inside the glass tubing.
Figure 3.20 A Craigie’s tube
IV. Using Plates Proteus shows swarming motility and spreads on the entire half of the plate,
whereas S. aureus is non-motile and grows only at the inoculated spot.
ENUMERATION OF BACTERIA,
FUNGI AND ACTINOMYCETES FROM SOIL
Aim
 To become familiar with the microbial soil flora.
 To determine the number of bacteria, fungi and actinomycetes present in a sample of soil.
Principle
Fertile soil is inhabited by the root systems of higher plants, by many animal forms (e.g., rodents,
insects and worms), and by tremendous numbers of microorganisms. The vast differences in the
composition of soils, together with differences in their physical characteristics and the agricultural
practices by which they are cultivated, result in corresponding large differences in the microbial
population, both in total numbers and in kinds.
Soil is inhabited by a great variety of microorganisms. Bacteria, fungi, algae, protozoa
and viruses make up this microbial flora, which may reach a total of billions of organisms per
gram of soil. The great diversity of the microbial flora makes it extremely difficult to determine
accurately the total number of microorganisms present. Cultural methods will reveal only those
physiological and nutritional types compatible with the cultural environment.
The bacterial population of soil exceeds the population of all other groups of microorganisms,
both in number and variety. Predominantly, members of the orders Pseudomonadales and
Eubacteriales are present in soil. Large numbers of actinomycetes, as many as millions per
gram, are present in dry warm soils. The most predominant genera of this group are Nocardia,
Streptomyces and Micromonospora. These organisms are responsible for the characteristic musty or
earthy odour of freshly ploughed field. Hundreds of different species of fungi inhabit the soil.
Predominatly, members of the classes Phycomycetes (Rhizopus, Mucor) and Deuteromycetes
(Penicillium, Aspergillus, Alternaria) are present in the soil.
The soil environment differs from one location to another and from one period of time
to another. Therefore, factors such as moisture, pH, temperature, gaseous oxygen content,
organic and inorganic composition of soil are crucial in determining the specific microbial flora
of a particular sample. Only the relative number of bacteria, actinomycetes and fungi can be
determined by serial dilution–agar plate procedure.
Different media are employed to support the growth of these three types of microorganisms:
glycerol yeast agar for the isolation of actinomycetes, Sabouraud’s agar for the isolation of fungi
and nutrient agar for the isolation of bacteria. The glycerol yeast agar and Sabouraud’s agar are
supplemented with 10 mg of chlorotetracycline (aureomycin) per ml of medium to inhibit the
growth of bacteria.
Materials required
Sample : 1 g of garden soil
Media : Glycerol yeast extract agar, Sabouraud’s dextrose agar, nutrient agar [AppendixIII]
Equipment and other materials : Bunsen burner, pipettes, conical flasks, marker, etc.
Procedure
1. Respective nutrient plates are prepared.
2. 1 g of soil is suspended in 100 ml of sterile distilled water.
3. The sample is diluted decimally after vigorous shaking (10–1 to 10–7).
4. From the appropriate dilutions, 1 ml is transferred with sterile pipettes to sterile Petri dishes.
Three Petri dishes are used for each dilution.
5. Approximately, 15 ml of the nutrient medium, melted and cooled to 45°C is added to each
Petri plate containing the diluted sample. The contents of each plate are mixed by rotating
gently to distribute the cells throughout the medium.
6. For bacterial enumeration, 10–4, 10–5 and 10–6 dilutions are plated and incubated at 37°C for
24 hours.
7. For fungal enumeration, 10–2, 10–3 and 10–4 dilutions are plated and incubated at 25°C for
3 days.
8. For the enumeration of actinomycetes, 10–3, 10–4 and 10–5 dilutions are plated and incubated
at 37°C for 3–7 days.
9. After incubation, the number of bacteria, fungi and actinomycetes per gram of soil is calculated.
Observation
BACTERIA FUNGI ACTINOMYCETES

Average of Dilution CFU/g Average Dilution PFU/g Average Dilution CFU/g
colonies of of
from 3 colonies colonies
plates from 3 from 3
(CFU/Plate) plates plates
TNTC 10 –4 1.8× 7 10–2 7× 4 10– 3 4×103
180 10 –5 107 TFTC 10–3 102 TFTC 10–4
TFTC 10 –6 TFTC 10–4 TFTC 10– 5

TNTC—Too Numerous To Count
TFTC—Too Few To Count
Result
Calculate CFU/mL or CFU/g (Colony forming units)
CFU/ mL = CFU/plate × dilution factor × 1/aliquot

Number of bacteria per gram of soil sample: 180 × 105 × 1/ 1(Volume of sample plated)
: 1.8×107 cfu/g
Number of fungi per gram of soil sample: 7 × 102 × 1/ 1(Volume of sample plated)
: 7 × 102 pfu/g(propagule forming units)
Number of Actinomycetes per gram of soil sample: 4 × 103 × 1/ 1(Volume of sample
plated)
: 4 × 103 cfu/g
PHENOL COEFFICIENT TEST
Aim
To compare the effectiveness of disinfectants.
Principle
The phenol coefficient test compares the antimicrobial activity of a chemical compound to that
of phenol under standardized experimental conditions. This specific test method is called the
AOAC phenol coefficient method (Association of Official Agricultural Chemists) or the FDA
method (Food and Drug Administration).
This procedure is suitable for testing disinfectants miscible with water and exerting their
antimicrobial action in a manner similar to that of phenol. The test organism employed in this
procedure is a specific strain of either Salmonella typhi or Staphylococcus aureus.
To a series of dilutions of the disinfectant being tested (5 ml per tube), 0.5 ml of 24 hours broth
culture of the test organism is added. At the same time, similar additions of the same amounts are
made to a series of dilutions of phenol and organisms and the tubes are placed in a 20°C water bath. At
intervals of 5, 10 and 15 minutes, subcultures are made with an inoculation loop into sterile nutrient
broth (5 ml). The inoculated subculture tubes are incubated and subsequently examined for growth.
The phenol coefficient is determined by dividing the highest dilution of the chemical being
tested that destroyed the microorganisms in 10 minutes but not in 5 minutes by the highest
dilution of phenol that destroyed the microorganisms in 10 minutes but not in 5 minutes. Suppose
that the phenol dilution was 1/90 and the maximum effective dilution for disinfectant X was
1/450. Then, the phenol coefficient of X would be 5. The higher the phenol coefficient value,
the more effective the disinfectant under these test conditions. A phenol coefficient greater than
1 indicates that this agent is as effective as or less effective than phenol. A phenol coefficient of
5 indicates that the chemical agent under evaluation is five times as effective as phenol.
Materials required
Culture : 24-hour broth culture of Staphylococcus aureus
Media : Nutrient broth (Appendix III)
Disinfectants : Phenol, dettol (test disinfectant)
Equipment and other materials : Test tubes, Bunsen burner, inoculating loop, etc.
Procedure
1. Prepare serial dilutions of phenol (1 : 20, 1 : 40, 1 : 80, 1 : 160) in sterile nutrient broth
tubes.
2. Similarly, prepare dilutions of the test disinfectant (1 : 20, 1 : 40, 1 : 80, 1 : 160, 1 : 320 and
1 : 640) in sterile nutrient broth tubes.
3. Inoculate 0.5 ml of the Staphylococcus aureus culture into each of the test tubes of disinfectant
dilutions. Mix and incubate at 37°C.
4. Using sterile technique, at intervals of 5, 10 and 15 minutes, transfer one loopful from each of
the test tube containing the disinfectant and microorganisms into the appropriately labelled
sterile tube of nutrient broth (5 ml).
5. Incubate all nutrient broth cultures for 48 hours at 37°C and observe the results.
Observation
S.No. Dilution Turbidity in 10 minutes tubes
Phenol Dettol
1. 1 : 20 – –
2. 1 : 40 + –
3. 1 : 80 + –
4. 1 : 160 + +
5. 1 : 320 + +
+ Organism live
– Organism destroyed
Result
Highest dilution of dettol that destroyed organisms in10 minutes
Phenol coefficient=
Highest dilution of phenol that destroyed organisms in10 minutes
=80 / 20= 4
Hence dettol is 4 times as effective as phenol.
MAINTENANCE AND PRESERVATION OF CULTURES

INTRODUCTION
Once a pure culture of a microorganism has been established, it is important to preserve that
culture, i.e., to prepare a stock culture that can be used for further studies. Methods for culture
preservation must permit the survival of the microorganisms; it is desirable to achieve maximal
survival of the population. Culture preservation also must prevent genetic and physiological
changes from occurring in the cultures. There are several techniques for preserving cultures.
These include:
1. Repeated subculturing,
2. Mineral oil slant cultures,
3. Using minimal medium, distilled water or water agar;
4. Freezing in growth media,
5. Drying,
6. Freeze-drying (lyophilization) and
7. Ultrafreezing.
1. Repeated subculturing This is the simplest method for culture preservation. Aerobes
are maintained on agar slants. Anaerobes are maintained by growing the bacteria deep in the
agar where air is excluded. Variables of periodic transfer to new media include transfer frequency,
medium used and holding temperature. This can lead to increased mutation rates and production
of variants. Repeated subculturing is done for certain bacteria that do not readily withstand
freezing/drying.
2. Mineral oil slant cultures A stock culture is grown on a slant and covered with sterilized
mineral oil. The slant can be stored at refrigerator temperature. This method is time-consuming,
especially if large numbers of cultures are involved.
3. Storage in minimal medium, distilled water or water agar Washed cultures are
stored under refrigeration. Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium and several
other common bacteria have been found to survive in water agar and simple nutrient media
for upto 30 years. In comparison, with more complex procedures used for culture preservation,
such as lyophilization and storage in liquid nitrogen, this preservation approach is only slowly
being recognized and accepted by microbiologists. These cultures remain viable for three to five
months or longer.
4. Freezing in growth media This is not a reliable method. This can result in damage to
microbial structures. However, with some microorganisms, this can be a useful means of culture
maintenance.
5. Drying In the absence of water, a condition known as desiccation, microorganisms cannot
grow or reproduce but can remain viable for years. Then, when water is made available to them,
they can resume their growth and division. This ability is used in the laboratory when microbes
are preserved.
Cultures are dried on sterile soil (soil stocks), on sterile filter paper disks, or in gelatin drops.
These can be stored in a desciccator at refrigeration temperature or frozen to improve viability. The
resistance of vegetative cells to desiccation varies with the species and the organism’s environment.
For example, the gonorrhea bacterium can withstand dryness for only about an hour, but tuberculosis
bacterium can remain viable for months. Viruses are generally resistant to desiccation, but they are
not as resistant as bacterial endospores, some of which have survived for centuries.
6. Freeze-drying (lyophilization) One of the best ways to store a bacterial, fungal, yeast
or other microorganism culture for long periods of time is to use a process called lyophilization,
or freeze-drying, In this method, the culture is rapidly frozen at a very low temperature (–70°C)
and then dehydrated by vacuum. Under these conditions, the microbial cells are dehydrated and
their metabolic activities are stopped; as a result, the microbes go into dormant state and retain
viability for years. Lyophilized or freeze-dried pure cultures are then sealed and stored in the
dark at 4°C in refrigerators. Freeze-drying method is the technique used most frequently by
culture collection centres. Viability even upto 30 years has been reported.
7. Ultrafreezing Cryopreservation Liquid nitrogen at ×196ºC is used, and cultures of fastidious
microorganisms have been preserved for more than 15 years by this method. Cryopreservation
(i.e., freezing in liquid nitrogen at ×196°C) helps survival of pure cultures for long storage times.
In this method, the microorganisms of culture are rapidly frozen in liquid nitrogen at – 196°C
in the presence of stabilizing agents such as glycerol, that prevent the formation of ice crystals
and promote cell survival.
4
MICROBIAL PHYSIOLOGY
GROWTH CURVE
1. DIRECT COUNT
Aim
 To measure microbial growth in a population of cells.
 To determine the generation time of bacterial cultures.
Principle
Growth may be defined as an increase in cellular constituents. Population growth is studied
by analysing the growth curve of a microbial culture. When microorganisms are cultivated
in liquid medium, they are usually grown in a batch culture or closed system, i.e., they are
incubated in a closed culture vessel with a single batch of medium. Because no fresh medium is
provided during incubation, nutrient concentrations decline and concentration of wastes increases.
The growth of microorganisms reproducing by binary fission can be plotted as the logarithm
of cell number versus the incubation time. The resulting curve has four distinct phases (Figure
4.1). There is an initial period of what appears to be no growth (the lag phase), followed by
rapid growth (the exponential or logarithmic phase), then a levelling off (stationary phase)
and finally a decline in the viable population (death or decline phase). Between each of these
phases, there is a transitional period (curved portion). This represents the time required before
all cells enter the new phase.
During the exponential phase, each microorganism divides at constant intervals. Thus, the
population will double in number during a specific length of time called the generation time
or doubling time.
Stationary
Logarithm Death
of cell
number
Exponential
(logarithmic)
Lag
Time
Figure 4.1 Phases of microbial growth (Batch culture)
Not all bacteria have the same generation time; for some, such as E. coli, it is 15–20 minutes;
for others, it may be many hours (for Mycobacterium tuberculosis, it is approximately 12 hours).
Similarly, the generation time is not the same for a particular species under all conditions. It is
strongly dependent upon the nutrients in the medium and on prevailing physical conditions.
Measurement of microbial growth

There are many ways to measure microbial growth, and to determine the growth rates and
generation times. Either population mass or number may be followed because growth leads to
increases in both. The most commonly employed techniques for growth measurement are:
1. Cell count: Directly by microscopy (Haemocytometer or Petroff–Hausser chamber) or
by using an electronic particle counter or indirectly by a colony count.
2. Cell mass: Directly by weighing or by measuring the cell nitrogen or indirectly by
turbidity.
3. Cell activity: Indirectly by relating the degree of biochemical activity to the size of the
population.
Direct microscopic count (Petroff–Hausser counting chamber)
The most commonly used method to determine microbial numbers is through direct counting.
The use of a counting chamber is easy, inexpensive and relatively quick. It also gives information
about the size and morphology of microorganisms. Petroff–Hausser counting chambers can
be used for counting bacteria (Figure 4.2). Haemocytometers (similar in principle to Petroff–
Hausser chamber, but the depth is 0.01mm) can be used for larger eukaryotic microorganisms.
Microbial physiology 119
These specially designed slides have chamber of known depth (0.02 mm in Petroff–Hausser
chamber) with an etched grid at the chamber bottom.
The Petroff–Hausser chamber has ruling in 9 mm2 (9 large squares) of which one square can be
seen under 100 × objective of microscope. Each of the large square (divided into 25 small squares)
is 1 mm on each side or 1 mm × 1 mm =1 mm2 in area. The depth of the chamber is 0.02 mm3.
Figure 4.2 Petroff-Hausser counting chamber
Disadvantages of direct counting are:

i. The microbial population must be fairly large for accuracy because such a small volume
is sampled.
ii. It is also difficult to distinguish between living and dead cells.
Materials required
Culture : 24 hour broth culture of E. coli
Equipment and other materials : Petroff–Hausser chamber, micropipettes, conical flask, Bunsen
burner, etc.
Procedure
1. Wash, drain and dry the counting chamber and coverslip.
2. Inoculate a sterile 50 ml nutrient broth with 0.5 ml of E. coli.
3. From this inoculated medium, place a small drop at the centre of the chamber platform and
place the coverslip over it.
4. Observe under a high-power microscope. Start counting the cells on the large square (one
large square). Start counting the cells from the top row and continue to the bottom row.
Count only the cells that are on the line forming top and right side of the square. This will
avoid the chances of counting the cells twice.
5. For quick counting, four corner squares and one centre square can be counted. Thus, obtained
cell number could be multiplied with 5 to get the total number of cells present in all 25
squares.
6. For every 30 minutes, repeat this and note the observations to determine the generation time.
Calculations
The number of microorganisms in a sample can be calculated by taking into account, the
chamber’s volume and any sample dilutions required.
Since there are 25 squares covering an area of 1 mm2, the total number of bacteria in 1 mm2
of the chamber is equal to the number/square × 25 squares.
The chamber is 0.02 mm deep, and therefore,
Bacteria/mm3 = Bacteria/square × 25 squares × 0.02 (volume).
The number of bacteria/cm3 is 103 times this value.
Example 1
Suppose the average count per square is 28 bacteria,
Bacteria/cm3 (or ml) = 28 bacteria × 25 squares × 0.02(vol.)×103 =1.4×104.
Calculation of generation time The direct method uses the log of cell number scale on the
growth curve and the formula used is
t log 2
GT =
log b – log B
Where,
GT = Generation time
B = Number of bacterial cells at some point during the log phase
b = Number of bacterial cells at a second point of the log phase
t = Time in hours or minutes between B and b
Result
Example 1
When a bacterial cell multiplies from 106 cells to 108 cells in 3.5 hrs., then
t log 2
Generation time =
log b – log B
Where,
b = Number of bacterial cells at a second point during the log phase
3.5×0.302
GT =
log(10 ) – log(10 )
8 6
1.057
=
8–6
1.057
=
2
= 0.52 hrs
= 30 min
The generation time of the given bacterial culture is 30 minutes.
Example 2
When a bacterial cell multiplies from 103 cells to 109 cells in 10 hrs., then
t log 2
Generation time =
log b – log B
where,
10 × 0.302
GT =
log(109 ) – log(103 )
3.02
=
9–3
3.02
=
6
= 0.50 hrs.
= 30 min.
The generation time of the given bacterial culture is 30 min.
2. VIABLE COUNT

Aim
To determine quantitatively the number of viable cells and to study the generation time.
Principle
Analysis of food, water and milk, requires quantitative enumeration of microorganisms in the
substances. Many methods have been devised to accomplish this, including direct microscopic
counts. The disadvantage in the direct method is that the total count includes dead as well as
living cells. Sanitary and medical microbiology at times require determination of viable cells. To
accomplish this, the serial dilution–agar plate technique is used.
This method involves serial dilution of a bacterial suspension in sterile water blanks. Once
diluted, the suspensions are placed on suitable nutrient media. The spread-plate method involves
spreading a small volume (0.1 ml) of cell suspension onto the surface of a pre-poured agar plate.
The inoculum is spread evenly over the agar surface and the colonies develop after incubation.
In pour-plate technique, the molten agar, cooled to 45°C, is poured into a Petri dish
containing 1 ml of the diluted sample. The plate is gently rotated in a circular motion, to achieve
uniform distribution of microorganisms. Dilutions should be plated in duplicates for greater
accuracy, incubated overnight and counted.
Advantages of the serial dilution–agar plate technique are:

i. Only viable cells are counted.
ii. It allows isolation of discrete colonies that can be subcultured into pure cultures, which
may then be easily studied and identified.
Disadvantages of this method are:
i. Overnight incubation is necessary before colonies develop on the agar surface.
ii. It is necessary to use more glassware.
iii. The need for greater manipulation may result in enormous counts due to errors in dilution
or plating.
iv. It works well for cells that separate shortly after division but not for cells that stick
together after division.
Materials required
Culture : 24 hour broth culture of E. coli
Equipment and other materials: Conical flask, test tubes, pipettes, etc.
Procedure
1. Inoculate a 50 ml sterile nutrient broth with 5 ml of overnight culture.
2. Determine the viable count from this broth by plating 10–1 dilution, by pour-plate method.
3. Incubate the broth at 37ºC.
4. Every half an hour, serially dilute the culture by transferring 1 ml from the broth at regular
intervals and plate by pour plate method.
5. Incubate the plates at 37ºC for 24 hours.
6. The number of organisms per ml of original culture is calculated by multiplying the number
of colonies counted by the dilution factor.
Calculations
Number of cells per ml = Number of colonies × Dilution factor
Examples
a. Colonies per plate = 50
Dilution factor = 1 × 106
Volume of dilution added to plate = 1 ml
No. of cells/ml = 50 × 1,000,000
= 5 × 107 cfu/ml (colony-forming units).
b. Colonies per plate = 50

Dilution factor = 1 × 106
Volume of dilution added to plate = 0.1 ml
No. of cells/0.1 ml = No. of colonies × Dilution factor × Volume
= 50 × 106 × 0.1
= 50,000,000 × 0.1
= 5 × 106
Therefore, no. of cells/ml = 5 × 106 × 10
= 5 × 107 cfu/ml
Calculation of generation time The direct method uses the log of cell number scale on the
growth curve and the formula used is:
t log 2
GT =
log b – log B
Where,
GT = Generation time
b = Number of bacterial cells at a second point of the log phase
Result
Example 1
The initial population of bacteria is 104 cells. If it reaches 108 cells in 7 hrs, then
t log 2
Generation time =
log b – log B
Where,
7 × 0.302
GT =
log(108 ) – log(104 )
2.114
=
8–4
2.114
=
4
= 0.52 hrs
= 30 min.

Example 2
When a bacterial cell multiplies from 106 cells to 109 cells in 5 hrs, then
t log 2
Generation time =
log b – log B
Where,
5 × 0.302
GT =
log(109 ) – log(106 )
1.51
=
9–6
1.51
=
3
= 0.50 hrs
= 30 min.
3. TURBIDITY METHOD

Aim
To determine the generation time by turbidity method.
Principle
Microorganisms do not settle in suspension and they scatter light, creating turbidity.
The spectrophotometer measures the amount of light transmitted (T) or absorbed (A). It
transmits a beam of light at a single wavelength (monochromatic light) through a liquid culture.
The cells suspended in the culture interrupt the passage of light, and the amount of light energy
transmitted through the suspension is measured on a photoelectric cell and converted into electrical
energy. The electrical energy is then recorded on a galvanometer as 0% to 100%T.
The density of a cell suspension is expressed as absorbance or optical density (O.D.) rather than %T,
since O.D. is directly proportional to the concentration of cells, whereas %T is inversely proportional
to the concentration of suspended cells. Therefore, as the turbidity of a culture increases, the O.D.
increases and %T decreases, indicating growth of the cell population in the culture.
The major disadvantage is that the turbidity includes dead as well as living cells.
While measuring the increase in turbidity (growth) with conventional cuvette, samples must
be transferred from the culture flask to the cuvette. If many measurements are made, it will
deplete the culture volume, which can alter the microorganisms, growth characteristics. This
can be avoided by using a side-arm growth flask. Instead of transferring samples from the flask
to a cuvette, the culture is simply tipped into the attached cuvette and the cuvette is inserted
into the spectrophotometer. A black cloth bag placed over the flask prevents stray light from
interfering with measurements.
To determine the generation time, the O.D. values are plotted on the semi-log paper.
Two points on the optical density scale, such as 0.04 and 0.08, are selected that represents a
doubling of turbidity. Using a ruler, extrapolation is done by drawing a line between each of
the selected optical densities on the Y-axis and the plotted line of the growth curve. Then,
perpendicular lines are drawn from these end-points on the plotted line of the growth curve to their
respective time intervals on the X-axis. With this information, generation time is calculated as:
GT = t (O.D. 0.08) – t (O.D. 0.04)

Materials required
Culture : 24 hour broth culture of E. coli or yeast
Media : Nutrient broth for E. coli or Sabouraud’s broth for yeasts (Appendix III)
Equipment and other materials: Spectrophotometer, side-arm flask, inoculation loop, Bunsen
burner, etc.
Procedure
1. Switch on the spectrophotometer. Adjust the wavelength to 620 nm.
2. Allow 10 minutes for the unit to warm up before taking the readings.
Measurement of Growth–Turbidity Method

3. Inoculate a fresh, sterile nutrient broth with 5 ml of overnight culture.

4. Adjust the galvanometer to zero per cent transmittance (%T). This is dark current adjustment.
The galvanometer now indicates that no light is being transmitted to the phototube.
5. Fill one cuvette with sterile broth (same broth used for the culture growth). This is called the
blank. Wipe the outside of this cuvette and insert into the sample holder. Adjust to 100%T.
6. Place the cuvette with the T0 sample (the sample taken immediately after inoculation) into
the sample holder of the adjusted spectrophotometer. Read the O.D.
7. Measure the O.D. for every 30 minutes and plot the values on a semi-log paper.
Observation
Sl.No Time of Incubation O D Value
(in minutes)
1 30 0.2
2 60 0.2
3 90 0.3
4 120 0.6
5 150 0.8
6 180 0.8
Generation time = t(0.4 OD) – t(0.2OD)

The time difference between any two doubling OD values in log phase.
GT = t (0.6) – t (0.3)
= 120 – 90
= 30 minutes.
Result
The generation time of the given culture is 30 minutes.
BIOCHEMICAL TESTS
I. IMViC REACTIONS
1. Indole Production Test
Aim
To determine the ability of microorganisms to degrade the amino acid, tryptophan.
Principle
Tryptophan is an essential amino acid that can undergo oxidation by way of enzymatic activities
of some bacteria. Tryptophan is converted to indole by the enzyme tryptophanase. Indole test
is performed by inoculating a bacterium into peptone broth. The presence of indole is detected
by adding Kovac’s reagent (p-dimethyl amino benzaldehyde, butanol and hydrochloric acid).
Indole is extracted from the medium into the reagent layer by the acidified butyl alcohol
component and forms a complex with the p-dimethyl amino benzaldehyde, yielding the cherry
red colour.
Examples of indole-positive organisms are Escherichia coli and Proteus vulgaris and that of
indole-negative organisms are Klebsiella pneumoniae.
Indole Reaction
— CH2— CH — COOH
—
Tryptophanase
—
— NH2
N
Tryptophan
CH3
—
—
—
+ C =O+NH3
—
—
N Ammonia
COOH
—
H Pyruvic Acid
Indole
+
Kovac’s Reagent
Cherry Red colour
Materials required
Cultures : 24–48 hour old bacterial broth cultures
Media : Peptone broth (Appendix III)
Reagent : Kovac’s reagent (Appendix I)
Equipment and other materials : Bunsen burner, test tubes, inoculation loop, etc.
Procedure
1. Prepare peptone broth, sterilize and dispense into sterile test tubes (5 ml each).
2. Inoculate peptone broth with a loopful of the test culture.
3. Incubate at 37ºC for 24 hours.
4. Add 0.2 ml of Kovac’s reagent.
Interpretation
Cultures that produce a red reagent layer or ring are indole-positive, while the cultures in which
there is no colour change are indole-negative.
2. Methyl Red–Voges Proskauer Test

Aim
 To differentiate between organisms that produce large amounts of acid from glucose and
those that produce the neutral (non-acidic) product, acetoin.
 To differentiate between Escherichia coli and Enterobacter aerogenes.
Principle
Glucose is the major substrate oxidized by all enteric organisms for energy production.
The end products vary depending on the substrate, the incubation time and the organism.
Both E. coli and Enterobacter aerogenes initially produce organic acids during the early incubation
period. The acidicity (pH 4) is stabilized and maintained by E. coli at the end of incubation.
During the later incubation period, E. aerogenes enzymatically converts these acids to non-acidic
end products, such as 2,3-butanediol and acetoin (acetyl methyl carbinol), resulting in an elevated
pH of approximately 6.
Glucose + H2O E.coli Lactic acid, acetic acid, formic acid +CO2 +H2
(pH 4.0)
Glucose + O2→ Acetic acid Enterobacter aerogenes 2, 3 –butanediol (acetyl
methyl carbinol) + CO2 + H2 (pH 6.0)
MR–VP medium is a glucose-supplemented nutrient broth used for the methyl red (MR) test
and the Voges Proskauer (VP) test. If an organism produces a large amount of organic acid from
glucose, the medium will remain red when methyl red is added, indicating that the pH is below
4.4. If neutral products are produced, methyl red will turn yellow, indicating a pH above 6.0.
The production of acetoin is detected by the addition of potassium hydroxide and α -napthol.
If acetoin is present, the upper part of the medium will turn red; a negative VP test will turn
the medium light brown.
Shigella dysentriae, Proteus vulgaris and E. coli are MR-positive and Klebsiella pneumoniae
and Enterobacter aerogenes are MR -negative. Klebsiella pneumoniae and Enterobacter aerogenes are
VP-positve while E. coli and Shigella dysentriae are VP-negative.
Materials required
Cultures : 24–48 hour nutrient broth cultures
Media : MR–VP broth (Appendix III)
Reagents : Barritt’s reagents A and B, methyl red solution (Appendix I)
Equipment and other materials: Bunsen burner, inoculation loop, test tubes, etc.
Procedure
1. Prepare MP–VP broth, dispense into test tubes and sterilize.
2. Inoculate a loopful of the test culture into appropriately labelled tubes.
3. Incubate for 24–48 hours at 37ºC.
4. For methyl red test, add 5 drops of methyl red.
5. For VP test, add 0.6 ml (12 drops) of Barritt’s reagent A and 0.2 ml (2–3 drops) of Barritt’s
reagent B. Shake and allow the tubes to stand for 15 minutes.
Interpretation
Development of red colour is positive for methyl red test. A positive VP test will develop pink
to red colour.
3. Citrate Utilization Test

Aim
To differentiate between enteric organisms on the basis of their ability to ferment citrate as a
sole carbon source.
Principle
In the absence of glucose or lactose, some microorganisms are capable of using citrate as a carbon
source. This ability depends on the presence of citrate permease that facilitates the transport of
citrate inside the cell. Citrate is acted on by the enzyme citrase, which produces oxaloacetic acid
and acetate. These products are then enzymatically converted to pyruvic acid and carbon dioxide.
Carbon dioxide combines with sodium and water to form sodium carbonate, an alkaline product.
This changes the bromothymol blue indicator in the medium from green to deep Prussian blue.
Klebsiella pneumoniae and Enterobacter aerogenes are citrate-positive. E. coli and Shigella dysentriae
are citrate-negative.
Citrate Reaction
Citrate Citrate Permease Oxaloacetic acid

Citrase
Pyruvate
+
Co2
+
Excess sodium
Na2CO3
Alkaline pH
Medium turns blue
Materials required
Cultures : 24–48 hr nutrient broth cultures
Media : Simmons citrate agar (Appendix III)
Procedure
1. Prepare Simmons citrate agar, sterilize and dispense into sterile test tubes (5 ml). Keep it in
a slanting position for solidification.
2. Using sterile technique, inoculate each of the organisms into its appropriately labelled tubes.
3. Incubate all cultures for 24–48 hours at 37ºC.
4. Observe for the presence or absence of growth and colouration of the medium.
Interpretation
Citrate positive cultures are identified by the presence of growth on the surface of the slant, which
is accompanied by blue colouration.
2. OXIDASE TEST

Aim
To differentiate bacteria based on cytochrome oxidase activity.
Principle
Oxidase enzymes play a vital role in electron transport chain during aerobic respiration.
Cytochrome oxidase catalyses the oxidation of a reduced cytochrome by molecular oxygen
(O2), resulting in the formation of H2O or H2O2. Four general classes of bacterial cytochromes
have been identified and the oxidase test is used to determine the presence of one of these,
cytochrome c. The oxidase test aids in the differentiation among members of the genera, Neisseria
and Pseudomonas, which are oxidase positive and Enterobacteriacaeae, which are oxidase negative.
The ability of bacteria to produce cytochrome oxidase can be determined by the addition
of the test reagent, p-aminodimethyl aniline oxalate to the colonies grown on a plate medium.
This light pink reagent serves as an artificial substrate, donating electrons and thereby becoming
oxidized to a black-coloured compound in the presence of the oxidase and free oxygen. Following
the addition of the test reagent, the development of pink, then maroon and finally black colouration
on the surface of the colonies is indicative of cytochrome oxidase production.
Alcaligenes faecalis and Pseudomonas aeruginosa are examples of oxidase-positive organisms and
E.coli and Klebsiella pneumoniae are examples of oxidase-negative organisms.
Materials required
Cultures : Nutrient agar plate cultures of the test organisms
Reagent: p-aminodimethyl aniline oxalate (Appendix I) or oxidase discs
Equipment and other materials : Bunsen burner, marker pen, etc.
Procedure
1. Add 2 or 3 drops of p-aminodimethyl aniline oxalate to the surface of 24 hours old plate
cultures of the test organisms.
2. Observe for a colour change to pink within 1 minute, then blue and then to black. This is
a positive reaction.
or
3. Moisten an oxidase disc and place it on colonies. Incubate the plates for 15 seconds at 35ºC.
Interpretation
Oxidase-negative colonies will not change colour. Oxidase-positive bacterial colonies will turn black.
3. CATALASE TEST

Aim
To determine the ability of some microorganisms to degrade hydrogen peroxide by producing
the enzyme catalase.
Principle
During aerobic respiration, microorganisms produce hydrogen peroxide and in some cases, an
extremely toxic superoxide. Accumulation of these substances will result in death of the organism.
Most aerobic, facultative anaerobic and microaerophilic bacteria produce the enzyme catalase,
which breaks down hydrogen peroxide to water and oxygen.
2H2O2 catalase 2H2O+O2
Hydrogen Water Oxygen
Peroxide
Aerobic organisms that lack catalase can degrade toxic superoxides using the enzyme
superoxide dismutase. The inability of strict anaerobes to synthesize catalase, peroxidase or
superoxide dismutase may explain why oxygen is poisonous to these microorganisms. Catalase
production can be determined by adding the substrate H2O2 to the culture. If catalase is present,
the culture shows bubbling or foaming.
Staphylococcus aureus, Pseudomonas aeruginosa, etc., are catalase-positive while Streptococcus is
catalase-negative.
Materials required
Cultures : 24 hour slant cultures of the test organisms
Reagent: 3% hydrogen peroxide (Appendix I)
Equipment and other materials: Glass slides, glass rod, Bunsen burner, etc.
Procedure
1. With a glass rod, transfer the culture from the slant to a clean glass slide.
2. Add hydrogen peroxide in drops to the organisms on the slide and observe for bubbles.
(or)
3. Allow three or four drops of hydrogen peroxide to flow over the entire surface of each slant
culture. Examine for the presence of bubbles.
Interpretation
Catalase-positive test is indicated by the formation of bubbles, while the absence of bubble
formation indicates a negative catalase test.
4. UREASE TEST

Aim
To determine the ability of microorganisms to degrade urea by the enzyme urease.
Principle
Proteins are organic molecules that contain carbon, hydrogen, oxygen and nitrogen.
The subunits that make up a protein are called amino acids. Amino acids bond together by
peptide bonds forming a small chain (a peptide) or a larger molecule (a polypeptide). Urea is a
waste product of protein digestion in most vertebrates. Presence of the enzyme urease, which
liberates ammonia from urea is a useful diagnostic test for identifying bacteria. Urea agar or broth
contains peptone, glucose, urea and phenol red. As the substrate urea is split into its products,
the presence of ammonia creates an alkaline environment that causes the phenol red to turn to
a deep pink colour. This is a positive reaction for the presence of urease.
Urease
Urea + Water Carbon dioxide + Water + Ammonia
Urease
H2N NH2 + 2H2O CO2 + H2O + 2NH3
—
—
C
—
—
O
Klebsiella pneumoniae and Proteus vulgaris are examples of urease-positive organisms where as
E. coli and Enterobacter aerogenes are examples of urease-negative organisms.
Materials required
Cultures: 24 hour broth cultures of test organisms
Media: Urea broth or Christensen’s urea agar (Appendix III)
Procedure
1. Prepare urea broth, dispense into test tubes and sterilize (if urea agar is used, prepare slants).
2. Inoculate each test culture into its appropriately labelled tube.
3. Incubate at 37ºC for 24–48 hours.
4. Observe for the colour change in the medium.
Interpretation
Deep pink colour shows a positive reaction for the presence of urease.
5. HYDROGEN SULPHIDE TEST

Aim
To determine the ability of microorganisms to produce hydrogen sulphide from substrates, such
as sulphur-containing amino acids or inorganic sulphur compounds.
Principle
Some bacteria liberate hydrogen sulphide (H2S) from the sulphur-containing amino acids, cystine,
cysteine and methionine.
Cystine
Cysteine H2S + NH3 + Pyruvic acid
desulphydrase
H2S can also be produced from the reduction of inorganic compounds, such as thiosulphate.
Thiosulphate
3S2O32– + 4H+ + 4e– 2SO32– + 2H2S
reductase
Thiosulphate Sulphite Hydrogen
sulphide
H2S is commonly called rotten egg gas because of the copious amounts liberated when eggs
decompose. To detect H2S production, a heavy metal salt containing ferrous ion (Fe2+) is added
to a nutrient culture medium. When H2S is produced, the sulphide (S2–) reacts with the metal
salt to produce a visible black precipitate.
H2 S + FeSO4 FeS + H SO
2 4
Hydrogen Ferrous Ferrous Sulphuric acid

Sulphide sulphate sulphide
(Black precipitate)
Salmonella typhimurium and Proteus vulgaris are H2S positive where as E. coli and Klebsiella pneumoniae
are H2S negative.
Materials required
Media: SIM agar or peptone iron agar (Appendix III)
Equipment and other materials: Bunsen burner, inoculation needle, test tubes, marker pen, etc.
Procedure
1. Prepare SIM agar or peptone iron agar, (Appendix III) sterilize and dispense into sterile test
tubes in an upright position for solidification.
2. Inoculate the test cultures into its appropriately labelled tubes by means of stab inoculation.
3. Incubate the tubes for 24–48 hours at 37ºC.
4. Observe for the presence of growth.
Interpretation
Blackening in the butt of the tube indicates a positive test. Growth along the line of stab
inoculation shows the absence of motility. Spreading growth shows the presence of motility.
6. TRIPLE SUGAR IRON AGAR TEST

Aim
 To study the bacteria commonly found in the gastrointestinal tract.
 To differentiate enteric bacteria and other groups of intestinal bacilli.
Principle
Gram-negative, facultatively anaerobic rods are a large and diverse group of bacteria that includes
the enteric family (Enterobacteriaceae). The triple sugar iron (TSI) agar test is designed to
differentiate between the genera of Enterobacteriaceae. To facilitate observation of carbohydrate
utilization patterns, the TSI agar slants contain 0.l% glucose, 1.0% lactose and 1.0% sucrose.
To detect carbohydrate fermentation, the acid–base indicator, phenol red, is incorporated in the
medium. 0.02% ferrous sulphate present in the medium facilitates the detection of hydrogen
sulphide (H2S). Organisms capable of producing H2S will show an extensive blackening in the butt.
Materials required
Cultures : 24 hour broth cultures of test organisms
Media: Triple sugar iron (TSI) agar (Appendix III)
Equipment and other materials : Bunsen burner, inoculation needle, test tubes, etc.
Procedure
1. Prepare TSI agar, sterilize and dispense into sterile test tubes. Keep it in a slanting position
to prepare an agar slant with a butt.
2. Inoculate each organism into its appropriately labelled tube by means of a stab and streak
inoculation (Insert a sterile, straight needle from the base of the slant into the butt.
Upon withdrawal of the needle, the slant surface of the medium is streaked).
3. Incubate for 24 hours at 37ºC.
4. Examine the colour of both butt and slant of all cultures. Examine all cultures for the presence
or absence of blackening within the medium. Breaks or bubbles in the medium show gas
production.
Interpretation
Alkaline slant (red) and acid butt (yellow) with or without gas production (breaks in the
agar): Only glucose fermentation has occurred. Since glucose is present in minimal concentration,
the small amount of acid produced on the slant is oxidized rapidly. The peptones in the medium
are also used in the production of alkali. In the butt, the acid reaction is maintained because of
reduced oxygen tension and slower growth of the organisms.
Acid slant (yellow) and acid butt (yellow) with or without gas production Lactose
and/or sucrose fermentation has occurred.
Alkaline slant (red) and alkaline butt (red) or no change (orange-red butt) Peptones
are catabolized resulting in an alkaline pH due to the production of ammonia. No carbohydrate
fermentation has occurred,e.g., Alkaligenes, Pseudomonas, Acinetobacter.
Alkaline slant, acid butt Acid butt, acid slant Alkaline slant,
alkaline butt,
without gas
H 2S+Ve H 2S–Ve H 2S+Ve H 2S–Ve
Gas+Ve Gas–Ve Gas+Ve Gas+Ve
Salmonella Shigella Citrobacter,Proteus sp. Escherichia coli, Alcaligenes,
Arizona Klebsiella pneumoniae Pseudomonas,
Citrobacter Enterobacter Acinetobacter
7. NITRATE REDUCTION TEST

Aim
To determine the ability of some microorganisms to reduce nitrates (NO3–) to nitrites (NO2–) or
beyond the nitrite stage.
Principle
In the process of anaerobic respiration, inorganic compounds other than oxygen act as final
electron acceptors. During anaerobic respiration, some bacteria reduce nitrates to nitrites; others
further reduce nitrites to nitrous oxide or nitrogen gas. Other bacteria reduce nitrates to nitrites
and then to ammonia.
Nitrate broth (nutrient broth plus 0.1% potassium nitrate) is used to determine a bacteria’s
ability to reduce nitrates. Nitrites are detected by the addition of sulphanilic acid (solution A)
and dimethyl α-naphthylamine (solution B) to nitrate broth. A red colour, indicating that nitrite
is present, is a positive test for nitrate reduction. A negative test (no nitrites) is further checked
for the presence of nitrate in the broth by the addition of zinc. Zinc will reduce nitrate to nitrite
and a red colour will appear. If nitrates are present, it means reduction has not taken place. If the
addition of zinc does not produce a colour change, then the nitrates in the medium were reduced
beyond nitrites to ammonia or nitrogen gas. This is a positive reaction.
Nitrate-positive organisms include E. coli and Enterobacter aerogenes. Alcaligenes faecalis is a
nitrate-negative organism.
– +
NO3 + 2H + 2e– Nitrate –
NO 2+H2O
Nitrate Hydrogen electrons Reductase Nitrite Water
+
NO 2
–
NH3
Nitrite Ammonia
(or)
2NO3 +12H +10e–

– +
N2+6H2O
Molecular Nitrogen
– –
NO3 NO2 (Red colour on addition of Solution A and B)
Nitrate Reductase
NH2 NH2
—
—
— — —
—
—
+ + HNO2
— — — Nitrous Acid
—
—
SO3H α – napthylamine
Sulfanilic Acid (Colourless)
(Colourless) NH2
N=N
—
— —
— — —
—
+ H2O (Water)
—
— — —
—
SO2H
Sulfobenzene azo– α – napthylamine (red)
Materials required
Media : Nitrate broth (Appendix III)
Reagents: Solution A (sulphanilic acid), solution B (a-naphthylamine) (Appendix I)
Equipment and other materials : Bunsen burner, inoculation loop, test tubes, etc.
Procedure
1. Prepare nitrate broth, dispense into test tubes and sterilize.
2. Inoculate each of the test organisms into appropriately labelled tubes.
3. Incubate at 37ºC for 24–48 hours.

4. Add five drops of solution A and then five drops of solution B to all nitrate broth cultures.
Shake gently.
Interpretation
A red colour within 30 seconds is a positive test. If it does not turn red, then add a small pinch
of zinc powder; if it turns red now, the test is negative. If not, it is positive for nitrate reduction.
8. POLYMER (STARCH, CASEIN, GELATIN, LIPID) DEGRADATION

Aim
To determine the ability of microorganisms to excrete hydrolytic exoenzymes.
Principle
Starch hydrolysis Exoenzymes are hydrolytic enzymes that leave the cell and break down large
substrates into smaller components that can then be transported into the cell. The exoenzymic
amylase hydrolyses the polysaccharide starch into smaller carbohydrates. Starch agar is used to
demonstrate the hydrolytic activity of these exoenzymes. The medium is composed of nutrient
agar supplemented with starch, which serves as the polysaccharide substrate. The detection of
the hydrolytic activity is made by performing the starch test. Starch, in the presence of iodine will
impart a blue-black colour to the medium, indicating the absence of starch-splitting enzymes and
represent a negative result, e.g., E. coli, Klebsiella pneumoniae. If the starch has been hydrolysed, a
clear zone of hydrolysis will surround the growth of the organism. This is a positive result, e.g.,
Bacillus cereus, Bacillus subtilis.
Casein hydrolysis Bacteria can hydrolyse peptides or polypeptides to release amino acids.
They use the amino acids as carbon and energy sources when carbohydrates are not available.
Some bacteria can hydrolyse the protein in milk, called casein. Casein hydrolysis can be detected
in milk agar. The medium is composed of nutrient agar supplemented with casein. Casein in the
medium, gives the colour and opacity. Following incubation of milk agar plate cultures, organisms
secreting proteases, e.g., Bacillus cereus, Bacillus subtilis will exhibit a zone of proteolysis, which is
demonstrated by a clear area surrounding the bacterial growth. This loss of opacity is the result
of a hydrolytic reaction yielding soluble, non-colloidal amino acid. In the absence of protease
activity, the medium surrounding the growth of the organisms, e.g., E. coli, Klebsiella pneumomiae
remains opaque, which is a negative reaction.
Gelatin hydrolysis Gelatin is a protein produced by the hydrolysis of collagen, a major
component of connective tissue and tendons in humans and other animals. Hydrolysis of gelatin
can be demonstrated by growing bacteria in nutrient gelatin. Nutrient gelatin dissolves in warm
water (50ºC), solidifies (gels) when cooled below 25ºC and liquefies when heated to about 25ºC.
When an exoenzyme (gelatinase) hydrolyses gelatin, it liquefies and does not solidify even when
cooled below 20°C.
Examples for organisms which hydrolyse gelatin are Pseudomonas aeruginosa, Proteus vulgaris.
Examples for organisms which do not hydrolyse gelatin are E. coli, Klebsiella pneumoniae.
Lipid hydrolysis The degradation of lipids such as triglycerides is accomplished by hydrolytic
enzymes called lipases that cleave the ester bonds to form glycerol and fatty acids. Tributyrin
agar is used to demonstrate the hydrolytic activity of lipase. The medium is composed of nutrient
agar supplemented with the triglyceride, tributyrin, as the lipid substrate. Tributyrin forms
an emulsion when dispersed in the agar, producing an opaque medium. Following incubation,
organisms excreting lipase, e.g., Pseudomonas aeruginosa, Bacillus cereus will show a zone of lipolysis,
which is demonstrated by a clear area surrounding the bacterial growth. In the absence of lipolytic
enzymes, the medium retains its opacity. This is a negative reaction in organisms like E. coli and
Klebsiella pneumoniae.
Materials required
Cultures : 24 hour broth cultures of test organisms
Media : Starch agar, milk agar, nutrient gelatin deeps, tributyrin agar (Appendix III)
Reagent : Gram’s iodine solution [Appendix I]
Equipment and other materials : Bunsen burner, inoculation loop and needle, test tubes, Petri
plates, refrigerator, etc.
Procedure
1. Prepare starch agar, milk agar, gelatin deep tubes and tributyrin agar plates.
2. Make a single line streak inoculation of the test organisms in appropriately labelled plates
and gelatin deep tubes (stab inoculation).
3. Incubate all plates in an inverted position for 24–48 hours at 37ºC. Incubate the gelatin deep
tubes for 48 hours.
Interpretation
Starch hydrolysis Flood the starch agar plates with Gram’s iodine. Areas of starch hydrolysis
will appear clear, while unchanged starch will stain dark blue.
Casein hydrolysis The presence of clear area of zone of proteolysis surrounding the growth of
each of the bacterial test organisms, on the milk agar plate, shows casein hydrolysis while the
absence of zone indicates no hydrolysis.
Lipid hydrolysis The presence of clear area or zone of lipolysis surrounding the growth of
organisms on tributyrin agar is indicative of lipid hydrolysis while the absence of zone indicates
no hydolysis.
Gelatin hydrolysis Place all gelatin deep culture tubes in refrigerator at 4ºC for 30 minutes. The
tubes, if even after placing in the refrigerator, remains liquid, then they are positive for gelatin
hydrolysis. If they solidify, then it shows negative result.
9. CARBOHYDRATE FERMENTATION TEST
Aim
To determine the ability of microorganisms to degrade and ferment carbohydrates with the
production of an acid, or acid and gas.
Principle
Fermentation is a bio-oxidative process not requiring oxygen in which an organic substrate serves
as the final electron acceptor. Most microorganisms obtain their energy through bioxidation of
carbohydrates. Many carbohydrates, including monosaccharides, such as glucose, disaccharides
like sucrose and polysaccharides, such as cellulose can be fermented.
A fermentation tube is used to detect acid and gas production from carbohydrates.
The fermentation broth contains peptone, an acid–base indicator (phenol red), an inverted tube to
trap gas and 0.5–1.0% of the desired carbohydrate. The phenol red indicator is red (neutral pH)
in an uninoculated fermentation tube and fermentation results in acid production, which will turn
the indicator yellow; when gas is produced, some will be trapped in the inverted Durham’s tube.
Organism Fermentation of sugars
Lactose Dextrose
Escherichia coli AG AG
Enterobacter aerogenes AG AG
Klebsiella pneumoniae AG AG
Alcaligenes faecalis – –
Staphylococcus aureus A A
Bacillus cereus – A
AG—acid and gas production
A—acid production
“–” —Negative
The nature of the fermentation reaction and the activity of the indicator make it imperative
that all cultures should be observed within 48 hours. Extended incubation may mask
acid-producing reactions by production of alkali because of enzymatic action on substrates other
than the carbohydrates.
Materials required
Media: Fermentation broth with the desired carbohydrates (Appendix III)

Equipment and other materials: Bunsen burner, inoculation loop, test tubes, Durham’s tubes, etc.
Procedure
1. Prepare fermentation broth, dispense into test tubes. Sterilize with Durham’s tubes inverted
in the medium, without air bubbles.
2. Inoculate the test cultures into the appropriately labelled medium. Do not shake the
fermentation tube, as they may force air bubbles into Durham’s tubes.
3. Incubate at 37ºC for 24 hours.
Interpretation
Carbohydrates that have been fermented with the production of acid will cause the phenol red
to turn yellow, indicating a positive reaction. Evolution of gas will be visible as a bubble in
the inverted tube. Negative culture tubes appear red without bubbles in the Durham’s tubes.
10. AMINO ACID DECARBOXYLASE TEST

Aim
To find out the ability of bacteria to produce enzymes that decarboxylate certain amino acids.
Principle
Determining the ability of bacteria to produce enzymes that either deaminate, hydrolyse, or
decarboxylate certain amino acids is often used in identification schemes. The amino acid substrates
most often tested include lysine, ornithine, arginine, and phenylalanine. Decarboxylases cleave the
carboxy group from amino acids so that amino acids are converted into amines; lysine is converted
to cadaverine, and ornithine is converted to putrescine. Because amines increase medium pH, they
are readily detected by colour changes in a pH indicator medium. The most common medium
used for this test is Moeller decarboxylase base, whose components include glucose, the amino
acid substrate of interest (i.e., lysine, ornithine, or arginine), and a pH indicator.
Organisms are inoculated into the tube medium that is then overlaid with mineral oil to ensure
anaerobic conditions. Early during incubation, bacteria utilize the glucose and produce acid, resulting
in a yellow colouration of the pH indicator. Organisms that can decarboxylate the amino acid then
begin to attack that substrate and produce the amine product, which increases the pH and changes
the indicator back from yellow to purple (if bromocresol purple is the pH indicator used.) Therefore,
after overnight incubation a positive test is indicated by a purple colour and a negative test (i.e., lack
of decarboxylase activity ) is indicated by a yellow colour. With each amino acid tested, a control
tube of the glucose-containing broth base without amino acid is inoculated. This standard’s colour
is compared with that of the tube containing the amino acid following incubation.
Materials required
Culture: 18 to 24 hour culture
Media: Moeller decarboxylase broth or agar (Appendix III)
Equipment and other material: Test tubes, inoculation loop, mineral oil, etc.
Procedure
Method of Use, Broth
1. Prior to inoculation, the media should be brought to room temperature.
2. Lightly inoculate each culture media with organisms taken from a pure 18 to 24 hour culture.
3. A control tube without an amino acid should always be inoculated with each test run.
4. Overlay all broth tubes, including the control, with 2–3 ml of sterile mineral oil. Incubate
at 35°C for 18 hours to 4 days.
5. Examine daily. Prolonged incubation from 6–10 days or longer may be required to demonstrate
weak reactions due to an organism’s delayed decarboxylation activity.
Method of Use, Agar
1. Touch a well-isolated colony or broth culture with a straight wire and stab the media to the
bottom of the tube. Do not overlay with mineral oil.
2. Incubate aerobically at 35°C for 18–24 hours.
Interpretation
With Moeller broth or agar plates, all amino acids give the same colours in their reactions:
Positive test Purple to a faded out yellow-purple colour.
Negative test Bright yellow colour for dextrose fermenters, little or no colour change in
comparison to uninoculated tube for nonfermenters.
Test organism Arginine Lysine Ornithine
Klebsiella pneumoniae – + –
Enterobacter cloacae + – +
11. PHENYLALANINE DEAMINASE TEST

Aim
To test the ability of organisms to deaminate the aminoacid phenylalanine.
Principle
This test is used to determine the ability of an organism to oxidatively deaminate phenylalanine
to phenylpyruvic acid. The phenylpyruvic acid is detected by adding a few drops of 10% ferric
chloride; Ferric chloride reacts with phenyl pyruvie acid to form a green-coloured compound.
Materials required
Culture: 24 hour brain-heart infusion broth culture.
Reagents: 10% ferric chloride (Appendix III)
Media: Phenylalanine agar slants
Equipment and other materials: Test tubes, inoculation loop, etc.
Procedure
1. Inoculate phenylalanine slant with 1 drop of a 24-hour brain-heart infusion broth culture.
2. Incubate 18 to 24 hours (or until good growth is apparent) at 35oC in ambient air with cap
loose.
3. After incubation, add 4 to 5 drops of 10% aqueous ferric chloride to the slant.
4. Observe for colour change.
Interpretation
Positive Green colour develops on slant after ferric chloride is added.
Negative Slant remains in original colour after the addition of ferric chloride.
Quality Control
Positive Proteus vulgaris
Negative Escherichia coli
Phenylalanine Agar
Approximate formula per litre purified water.
dl-Phenylalanine 2.0 g
Yeast extract 3.0 g
Sodium chloride 5.0 g
Sodium phosphate 1.0 g
Agar 12.0 g
12. COAGULASE TEST

Aim
To perform coagulase test to differentiate coagulase positive and negative staphylococci.
Principle
This test is used to differentiate Staphylococcus aureus (positive) from coagulase-negative
staphylococci. S. aureus produces two forms of coagulase: bound and free. Bound coagulase,
or “clumping factor” is bound to the bacterial cell wall and reacts directly with fibrinogen.
This results in an alteration of fibrinogen so that it precipitates on the staphylococcal cell, causing
the cells to clump when bacterial suspension is mixed with plasma. The presence of bound
coagulase correlates well with free coagulase, an extracellular protein enzyme that causes the
formation of a clot when S. aureus colonies are incubated with plasma. The clotting mechanism
involves activation of a plasma coagulase-reacting factor (CRF), which is a modified or derived
thrombin molecule, to form a coagulase CRF complex. This complex in turn reacts with fibrinogen
to produce the fibrin clot.
Materials Required
Culture: Staphylococcus aureus
Reagents: Rabbit plasma
Equipment and other material: Glass slides, test tubes, inoculation loop, wooden sticks, etc.
Procedure
A. Slide test
1. Place a drop of plasma (preferably rabbit plasma with EDTA) on a clean, dry glass slide.
2. Place a drop of distilled water or saline next to the drop of plasma as a control.
3. With a loop, straight wire, or wooden stick, emulsify a portion of the isolated colony being
tested to each drop, inoculating the water or saline first. Try to create a smooth suspension.
4. Mix well with a wooden applicator stick.
5. Rock the slide gently for 5 to 10 seconds.
Results and interpretation
Positive Macroscopic clumping in 10 seconds or less in coagulated plasma drop and no clumping
in saline or water drop (Staphylococcus aureus)
Negative No clumping in either drop. Note: All negative slide tests must be confirmed using
the tube test (Staphylococcus epidermidis)
Equivocal Clumping in both drops indicates that the organism autoagglutinates and is
unsuitable for the slide coagulase test.
B. Tube test
1. Emulsify several colonies in 0.5 ml of rabbit plasma (with EDTA) to give a milky suspension.
2. Incubate tube at 35oC in ambient air for 4 hours.
3. Check for clot formation.
Note: Tests can be positive at 4 hours and then revert to negative after 24 hours.
Result
Positive Staphylococcus aureus
Negative Staphylococcus epidermidis
13. ESCULIN HYDROLYSIS

Aim
To determine the ability of an organism to hydrolyse esculin.
Principle
This test is used to determine whether an organism is able to hydrolyse the glycoside esculin.
Bile esculin agar is a selective and differential medium which is used to presumptively identify
enterococci and group D streptococci based on the ability of an organism to hydrolyse esculin.
Bile esculin agar contains oxgall (bile salts) to inhibit the growth of gram-positive organisms
other than enterococci and group D streptococci. It also contains nutrients, esculin, and ferric
citrate. When an organism hydrolyses the glycoside esculin to form esculetin and dextrose, the
esculetin reacts with the ferric citrate to produce a dark brown or black phenolic iron complex.
If an organism can hydrolyse esculin, the media will turn dark brown or black. However, the
test is interpreted as a positive result only if more than half the medium is dark brown or black
after incubation.
Materials required
Culture: Enterococcus, Listeria monocytogenes
Media: Bile esculin agar (Appendix III)
Procedure
1. Inoculate the medium with 1 drop of a 24 hour broth culture.
2. Incubate at 35oC for up to 7 days.
3. Examine the slants for blackening and under the ultraviolet rays of a Wood’s
lamp for esculin hydrolysis.

Positive Blackened medium which would also show a loss of fluorescence under the Wood’s
lamps.
Negative No blackening and no loss of fluorescence under Wood’s lamps, or slight blackening
with no loss of fluorescence under Wood’s lamp. Uninoculated plate is shown in the following
Figure.
Uninoculated plate Positive culture (blackening)
Quality control
Positive Klebsiella pneumoniae
Negative Shigella flexneri.
14. ONPG ( O-NITROPHENYL β-d-GALACTOPYRANOSIDE) TEST
Aim
This test is used to determine the ability of an organism to produce b-galactosidase, an enzyme
that hydrolyses the substrate ONPG to form a visible (yellow) product, orthonitrophenol.
Principle
Lactose is a disaccharide composed of molecules of galactose and glucose. The ability of bacteria
to ferment lactose depends on two enzymes; permease and beta-galactosidase. Permease regulates
the movement of lactose across the bacterial cell wall. Once lactose is inside the cell, it is broken
down into the individual components, glucose and galactose, by beta-galactosidase.
However, some organisms lack permease and consequently appear as late or non-lactose-
fermenters. The ONPG test is valuable for the detection of beta-galactosidase activity in late
lactose-fermenting organism like Shigella sonnei and some strains of Escherichia coli. The ONPG test
detects the enzyme beta-galactosidase with greater speed and sensitivity than lactose-fermentation
tests. o-nitrophenyl-beta-d-galactopyranoside (ONPG) is an artificial substrate structurally similar
to lactose with the exception that glucose is substituted with an o-nitrophenyl group. ONPG
is able to enter the bacterial cell more easily than lactose as it is not dependent on the presence
of the permease enzyme. If the organism possesses beta-galactosidase, the enzyme will split the
beta-galactoside bond, releasing o-nitrophenol, which is a yellow-coloured compound. The activity
of the galactosidase enzyme is increased in the presence of sodium ions.
The organism to be tested is taken from a medium containing a high concentration of
lactose. A dense suspension is prepared. An ONPG disc is added to 0.5 ml of the suspension.
If the organism possesses beta-galactosidase, the enzyme will split the beta-galactoside bond,
creating a yellow colour change in the suspension. Organisms with strong beta-galactosidase
activity can produce a positive reaction a few minutes after inoculation of the ONPG medium;
other organisms may take up to 24 hours.
Materials required
Culture: 18 to 24 hour culture of E.coli, Salmonella typhimurium
Reagents: ONPG Disc (Sterile filter paper discs (diameter 6mm) impregnated with
o-nitrophenyl-β-d-galactopyranoside)
Equipment and other materials: Saline, test tubes, Inoculation loop, etc.
Procedure
1. Use a loop to transfer bacteria from pure 18–24 hour culture to a test tube containing 0.5
ml of 0.85% sterile saline. The resulting suspension should be approximately equivalent in
density to a McFarland 3 opacity standard.
2. Add a single ONPG disc to the dense bacterial suspension. Upon the addition of the disc the
bacterial suspension will be clear.
3. Incubate at 37oC and check hourly, for up to 4 hours, for the development of a yellow colour
change.
4. Incubate any negative reactions (colourless) for 24 hours. Observe after 24 hours for possible
delayed reactions of late lactose-fermenters.
Results and Interpretation

A yellow colour change is a positive reaction; indicating beta-galactosidase activity. No colour
change is a negative reaction; indicating the absence of beta-galactosidase activity.
Quality Control
Positive Escherichia coli
Negative Salmonella typhimurium
15. OF TEST FOR CARBOHYDRATE UTILIZATION

Aim
To differentiate the oxidative and fermentative metabolism of carbohydrates by gram-negative
rods on the basis of acid reaction in either the open or closed system.
Principle
OF medium was developed by Hugh and Leifson who described the taxonomic significance of
fermentative versus oxidative metabolism of carbohydrates by gram-negative bacteria.
1. They showed that when an organism is inoculated into two tubes of OF basal medium
containing a carbohydrate and the medium in one of the tubes is covered with melted
petroleum prior to incubation, the patterns of metabolism are of differential significance.
Oxidative organisms only produce an acid reaction in the open tube with little or no
growth and no acid formation in the covered tube. Fermentative organisms will produce
an acid reaction in both types of tubes. Changes in the covered agar are considered to be
due to true fermentation, while changes in the open tubes are due to oxidative utilization
of the carbohydrate present. If the carbohydrate is not utilized by either method, there is
no acid production in either tube. The medium contains a high concentration of added
carbohydrates relative to the peptone concentration to avoid the utilization of peptone
by an aerobic organism and the resultant production of an alkaline reaction which would
neutralize slight acidity produced by an oxidative organism.
2. The dipotassium phosphate adds buffering capacity to the medium. The agar permits
the determination of motility and aids in the even distribution of any acid produced at
the surface of the medium.
3. Dextrose is the most important carbohydrate for use in OF basal medium; however, certain
organisms may metabolize other carbohydrates even if they are unable to utilize dextrose.
Tubes containing media of arabinose, dextrose, dulcitol, fructose, galactose, lactose, maltose,
mannose, raffinose, rhamnose, salicin, sorbitol, sucrose and xylose are prepared and used.
Materials Required
Culture: Escherichia coli, Klebsiella pneumoniae
Media: OF basal medium (Appendix III)
Equipment and other material: Vaselin, test tubes, inoculation loop, etc.
Procedure
1. Heat two tubes of medium in boiling water for 10 minutes to drive off the oxygen, cool and
inoculate by inserting a straight wire vertically.
2. Incubate one tube aerobically and either incubate the second tube anaerobically or seal the
surface with a layer of sterile liquid paraffin oil to create anaerobic conditions.
3. Incubate at 35–37°C for 72 hours. Longer incubation may be required for slowly growing
species.
4. Examine the tubes daily for colour change.
Interpretation
If the organism is an oxidizer it will produce acid only in the open tube (without vaseline), if
it is a fermenter it will produce acid in the vaseline-covered tube and in the open tube. Some
aerobic bacteria may use the peptone in the medium, producing ammonia, with resulting
alkalinity (blue) in the top part of the open tube. The indicator used is bromothymol blue.
Sometimes the Hugh and Leifson’s medium can be used to determine whether an organism is
motile. Motile bacteria will show diffuse growth away from the stab line. Non-motile bacteria
will show growth confined only to the stab line. In the examples given here E. coli is motile and
Klebsiella is non-motile.
Oxidation Acid in aerobic tube only (yellow colour in aerobic tube, green in anaerobic)
Fermentation Acid in both tubes (yellow colour)
Neither fermentation nor oxidation No acid production (green colour in aerobic tube, purple in
anaerobic)
Results
When glucose is used as an example
Fermenter: Escherichia coli
Oxidizer: Pseudomonas aeruginosa
Nonutilizer: Alcaligenes faecalis
16. MALONATE UTILIZATION TEST

Aim
To differentiate Enterobacter from Escherichia based on malonate utilization.
Principle
Malonate broth contains ammonium sulphate, which is the sole source of nitrogen in the
medium; sodium malonate is the sole source of carbon. Malonate is an enzyme inhibitor and
inhibits utilization of succinic acid by bacteria, shutting down the Krebs and glyoxylic cycles.
Growth is indicative of malonate utilization as a carbon source. Dipotassium phosphate and
monopotassium phosphate provide buffering capability. Sodium chloride maintains the osmotic
balance of the medium. Increased alkalinity resulting from malonate utilization causes the
indicator, bromothymol blue, to change colour from green to blue. Enterobacter group utilizes
malonate whereas the Escherichia group is unable to grow on the medium. Malonate broth is
further described for differentiating Enterobacteriaceae in food and dairy products.
Materials required
Culture: Enterobacter, Escherichia coli
Media: Malonate broth (Appendix III)
Procedure
1. Inoculate tubes with a loopful of test organism.
2. Incubate at 35oC for 18–48 hours.
3. Examine tubes for a change in the colour of the medium from green to blue.
Malonate utilization is indicated by a change in the colour of the medium from green to blue:
Positive: Blue
Negative: Green
Quality control
Enterobacter aerogenes—Blue
Enterobacter cloacae—Blue
Escherichia coli—Green
Klebsiella pneumoniae—Blue
Salmonella choleraesuis subsp. arizonae—Blue

Salmonella choleraesuis subsp. choleraesuis serotype Typhimurium—Green
17. LECITHINASE TEST

Aim
To detect the production of lecithinase enzyme and in the presumptive identification of various
Clostridium.
Principle
Microorganisms that possess the enzyme lecithinase break down lecithin to insoluble diglyceride
and phosphorylcholine. The insoluble diglyceride produces a white opaque zone of precipitation
that spreads beyond the edge of the colony. If clostridia are suspected clinically or from the Gram
stain of clinical material, an egg yolk agar plate should be inoculated to check for the production
of lecithinase. Egg yolk agar, originally formulated by McClung and Toabe, is a non-selective
medium supplemented with a suspension of egg yolk and enriched with haemin and vitamin
K. Egg yolk supplies lecithin and free fats, substrates needed to detect lecithinase and lipase
production and proteolytic activity. Haemin and vitamin K are incorporated into the medium
to enhance the growth of obligate anaerobic microorganisms.
Material Required
Culture: 24–72 hour culture of Clostridium welchii
Media: Egg yolk agar (Appendix III)
Equipment and other material: Petri plates, inoculation loop, etc.
Procedure
Note: If clostridia are suspected clinically or upon gram stain, a primary egg yolk agar plate can
be inoculated to check for lipase and lecithinase production. If bacillus with parallel sides and
rounded or truncated ends are observed after gram staining, a direct Nagler test can be performed.
Method of Use
1. Inoculate using a pure 24–72 hour culture. Streak the medium to obtain isolated colonies.
2. Immediately following inoculation, place the medium in an anaerobic atmosphere and incubate
at 35–37oC for up to one week.
3. Observe for the appearance of lecithinase activity.
Nagler Test
1. Prior to inoculation, allow medium to equilibrate to room temperature.
2. Swab one-half of the medium with C. perfringens type A antitoxin and allow it to dry.
3. Starting from the side of the plate that does not contain antitoxin, make a single streak of
the test organism.
4. Incubate the inoculated medium for 24–48 hour at 35oC in an anaerobic atmosphere.
Interpretation of results
Lecithinase A positive lecithinase test is noted by the appearance of a white, opaque, diffuse zone
that extends into the medium surrounding the colonies. A negative lecithinase test is indicated
by the absence of a white, opaque zone extending from the edge of the colony.
Nagler Test A positive lecithinase reaction that occurs on the half of the medium without
antitoxin and inhibition of lecithinase reaction on the half containing the antitoxin is indicative
of a positive Nagler test. A negative Nagler test is noted by a positive lecithinase reaction on
both sides of the plate or no reaction on the agar.
PIGMENT EXTRACTION FROM ALGAE

Aim
To extract pigments from algae.
Principle
Three major classes of photosynthetic pigments occur among the algae: chlorophylls, carotenoids
(carotenes and xanthophylls) and phycobilins. Chlorophylls and carotenes are generally fat-soluble
molecules and can be extracted from thylakoid membranes with organic solvents such as acetone,
methanol or DMSO. The phycobilins and peridinin, in contrast, are water-soluble and can be
extracted from algal tissues after the organic solvent extraction of chlorophyll in those tissues.
The rationale behind the extraction techniques is to distrupt cell integrity as much as possible,
thereby, removing pigment molecules from intrinsic membrane proteins. Freezing the tissue with liquid
nitrogen, and grinding the still frozen tissue with a mortar and pestle or blender, overcomes some of
the problems of working with material that produces large amounts of viscous polysaccharides.
“Freeze-thawing’’ the tissue also breaks down cellular membranes, but may liberate more
polysaccharides. Finely ground tissue can then be homogenized in organic solvent to further disrupt
cellular membranes, and to liberate pigment molecules from the light-harvesting pigment–protein
complexes. Once the pigments are extracted into appropriate solvents, they can be separated
chromatographically by thin-layer chromatography and then identified.
Table 4.1 Pigment composition of several algal groups (after Dring 1982)
Division Common name Major accessory pigment

Chlorophyta Green algae Chlorophyll b
Charophyta Charophytes Chlorophyll b
Euglenophyta Euglenoids Chlorophyll b
Phaeophyta Brown algae Chlorophyll c1 + c2, fucoxanthin
Chrysophyta Yellow-brown or golden-brown algae Chlorophyll c1 + c2, fucoxanthin
Pyrrhophyta Dinoflagellates Chlorophyll c2 +peridinin
Cryptophyta Cryptomonads Chlorophyll c2 , phycobilins
Rhodophyta Red algae Phycoerythrin, phycocyanin
Cyanophyta Blue-green algae Phycocyanin, phycoerythrin
Materials required
Sample: Fresh weight of blotted tissue of algae
Reagents : Liquid nitrogen, petroleum ether, ice-cold 100% acetone
Equipment and other materials: Mortar and pestle, capillary tube, gel plate, Eppendorf tubes,
cuvette, etc.
Procedure
Method 1: Acetone extraction This technique can be used for green, brown and red algae as well
as for sea grasses. Some of the green algae and sea grasses may be extractable without grinding
in liquid nitrogen; for brown and red algae, extreme care should be taken while grinding.
Note: Field-collected tissue should be cleaned of epiphytes prior to the extraction.
1. Grind approximately 2.0 g (fresh weight) of blotted tissue in a chilled mortar with liquid
nitrogen. The mortar and pestle can be placed in the freezer prior to use, and chilled
even further by adding a small amount of liquid nitrogen to it prior to adding the tissue.
It may help to chop tough blades into workable pieces with a sharp razor blade before grinding.
2. Quickly transfer powdered tissues to a ground-glass homogenizer. Add 2 to 3 ml of ice-cold
100% acetone. Grind over ice until the remaining debris is colourless.
3. Transfer pigmented solution to centrifuge tube and spin at 1400 g for 2 minutes. Decant the
supernatant onto a fresh tube and measure the OD at 660 and 670 nm.
Note: If there is colour still remaining in the pellet, repeat steps 2 and 3 until the pellet is
colourless. (Samples can be stored in the dark at 4ºC at this point for a limited period of time)
Observation
Total chloroform per gram tissue = 57.6 (A670) + 54.2 (A660) × V/ 1000 × W
where
V— Final volume of chloroform extract in acetone.
W—Fresh weight of tissue extract
A—Absorbance at specific wavelength.
Example
Total chloroform per gram tissue = 57.6 (A670) + 54.2 (A660) × V/ 1000 ×W
= 57.6 (0.229) + 54.2 (0.270) × 10/ 1000 ×2
= 0.5 g.
Result
The amount of chlorophyll present in the given sample was 0.5g.
Method 2 : Thin-layer chromatography
1. On a silica gel plate, draw a light pencil line above the solvent level in the developing tank,
approximately 2 cm in width and 2 cm from the bottom.
2. Draw sample into capillary tube and carefully spot the pigment extract in a solid line along
the pencil line until a thin, dark line appears. Each layer should be dry before the next is
added.
3. Place a small amount of petroleum ether : acetone (7:3 v/v) into the bottom of the developing
tank. Dip filter paper in solvent and put it on side of the tank to saturate the atmosphere
with solvent.
Note: This should be done well in advance to the introdution of thin-layer chromatography
plates into the development tank.
4. Develop the thin-layer chromatography plate in a tank containing petroleum ether : acetone
(7:3 v/v) for approximately 20–30 minutes, or how much ever long is necessary to move the
solvent front near, but not off, the top of the plate.
5. Mark the solvent front as soon as the strip is removed from the tank.
6. Mark the location of the pigment bands and measure the distance moved by the pigments
as well as the distance moved by the solvent. The Rf values for the individual pigments can
be determined from the following formula:
Rf = Distance moved by the pigment/Distance moved by the solvent

7. Slight difference in the dryness of the plates and in the polarity of solvent system will affect
the Rf values. To aid in an approximate identification of the different pigmented bands, the
following values are reasonable:
Table 4.2 Rf Values
Pigment Rf
Chrolophyll a 0.68
Chrolophyll b 0.54
Chrolophyll c 0.03
β-carotene 0.94
Fucoxanthin 0.51
Lutein 0.43
Violaxanthin 0.22
Neoxanthin 0.08
Table 4.3 Wavelength maxima for pigments in various solvents
Pigment Wavelength maxima Solvent

β-carotene 452, 470 Ethanol
Lutein 446, 474 Ethanol
Violaxanthin 442, 470 Ethanol
Neoxanthin 437, 466 Ethanol
Myxoxanthophyll 445, 471, 503 Ethanol
Siphonoxanthin 455 Ethanol
Peridinin 455 Ethanol
Chlorophyll a 428.5, 660.5 Diethyl ether
Chlorophyll b 452.5, 642 Diethyl ether
Chlorophyll c1 629.1 100% acetone
Chlorophyll c 447, 533 or 449, 635 90% acetone
8. After measuring the Rf values, scrape the pigments off the plate with a spatula or razor blade.
Collect the residue and transfer to an Eppendorf tube. Resuspend in the appropriate solvent
(acetone for chlorophyll and ethanol for carotenoids). The amount of the solvent will depend
upon the amount of pigment in the chromatographic band.
9. Centrifuge for 1–2 minutes and decant the supernatant into the appropriate cuvette.
10. Determine the absorption spectrum for that pigment and note the absorption maxima
(wavelength maxima). The absorption maxima for several pigments are listed in Table 4.3.
11.The absorption data and the chromatographic data can be used together to identify specific
pigments for each algae.
Observation and results
The Rf value is calculated as follows.
Distance moved by pigment
Rf value =
Distance moved by solvent
Distance moved by pigment = 8.2

Distance moved by solvent = 12
8.2
Rf value =
12
= 0.68
The Rf value of the pigment separated was found to be equal to the standard Rf values of
chlorophyll a.
Confirmation using absorption maxima: The absorption maxima was 660 nm. This shows
the presence of chlorophyll a in the given sample.
EFFECT OF TEMPERATURE ON THE GROWTH OF BACTERIA

AND FUNGI (TDP AND TDT)
Aim
 To reveal the diverse growth temperature requirements of bacteria.
 To determine the thermal death point (TDP) and thermal death time (TDT) of
bacterial cultures.
Principle
Environmental temperature profoundly affects microorganisms, like all other organisms.
Microorganisms are particularly susceptible because they are usually unicellular and their
temperature varies with that of the external environment.
A most important factor influencing the effect of temperature on growth is the temperature
sensitivity of enzyme-catalysed reactions. High temperatures damage microorganisms by
denaturing enzymes, transport carriers and other proteins. Microbial membranes are also disrupted
by temperature extremes; the lipid bilayer simply melts and disintegrates. Thus, although
functional enzymes operate more rapidly at higher temperatures, the microorganisms may be
damaged to such an extent that growth is inhibited because the damage cannot be repaired. At
very low temperatures, membranes solidify and enzymes do not work rapidly. Thus, it affects
function but not necessarily all chemical composition and structures.
The optimal temperatures vary greatly between microorganisms. Optimal temperature
usually range from 0ºC to as high as 75ºC, whereas microbial growth occurs at temperatures
extending from 20ºC to over 120ºC. Specific temperature ranges consists of the following
significant temperature points:
1. Minimum growth temperature The lowest temperature at which growth will occur.
Below this temperature, enzyme activity is inhibited and the cells are metabolically
inactive so that growth is negligible or absent.
2. Maximum growth temperature The highest temperature at which growth will occur.
Above this temperature, most cell enzymes are destroyed and the organism dies.
3. Optimum growth temperature The temperature at which the rate of reproduction
is most rapid; however, it is not necessarily optimum or ideal for all enzymatic activities
of the cell.
All bacteria can be classified into one of the three groups, depending on their temperature
requirements:
i. Psychrophiles These include bacterial species that will grow within a temperature
range of –5ºC to 20ºC. The distinguishing characteristics of all psychrophiles is that
they will grow between 0 and 5ºC (e.g., Bacillus psychrophilus, Chlamydomonas nivalis).
ii. Mesophiles These include bacterial species that will grow within a temperature range
of 20ºC to 45ºC (e.g., E. coli, Neisseria gonorrhoea, Trichomonas vaginalis). The distinguishing
characteristics of all mesophiles are their ability to grow at human body temperature
(37ºC) and their inability to grow at temperatures above 45ºC. Included in the mesophiles
are two distinct groups:
 Those whose optimum growth temperature is in the range of 20ºC to 30ºC, called
plant saprophytes.
 Those whose optimum growth temperature is in the range of 35ºC to 40ºC, i.e.,
organisms that prefer to grow in the bodies of warm-blooded hosts.
iii. Thermophiles These include bacterial species that will grow at 35º C and above
(e.g., Bacillus stearothermophilus, Thermus aquatius Chaetomium thermophile.) Two groups of
themophiles exist:
 Facultative thermophiles Organisms that will grow at 37ºC, with an optimum growth
temperature of 45ºC to 60ºC.
 Obligate thermophiles Organisms that will grow only at temperature above 50ºC,
with optimum temperatures above 60ºC.
iv. Psychrotrophs Bacterial species that can grow at 0–7oC even though they have optimum
between 20 and 30oC and the maxima at about 35oC. These are called psychrotrophs or
facultative psychrophiles, e.g., Listeria monocytogenes, Pseudomonas fluorescens.
Determining the Optimum Temperature for Growth
Cultures can be grown in nutrient broth at various temperatures (5, 25, 38 and 42ºC) to determine
approximately their optimum growth temperatures. Turbidity determination can be made to
evaluate the amount of growth.
Thermal Death Point (TDP) and Thermal Death Time (TDT)

The time of exposure to temperature is a vital factor in assessing the lethal effect of high
temperatures on bacterial cells. The susceptibility of various microorganisms to elevated
temperatures can be compared by two methods:
1. The thermal death point (TDP), the temperature at which an organism is killed in
10 minutes of exposure.
2. The thermal death time (TDT), the time required to kill a suspension of bacterial cells
or spores at a given temperature.
Materials required
Cultures: 24 hour broth cultures of E. coli and Saccharomyces cerevisiae
Media: Nutrient broth, yeast broth, nutrient agar, Sabouraud’s agar [Appendix III]
Equipment and other materials: Water bath, incubator, test tubes, Bunsen burner, etc.
Procedure
1. Prepare nutrient broth tubes and inoculate with E. coli culture.
2. Prepare yeast broth and inoculate with Saccharomyces cerevisiae.
3. Incubate different tubes at different temperatures of 5, 25, 38 and 42ºC for 24–48 hours.
4. Following incubation, compare the growth using the differences in turbidity.
5. Use a spectrophotometer at 620 nm with sterile broth as blanks to measure turbidity.
Interpretation
Optimum temperature is the temperature at which the rate of reproduction is most rapid.
Hence the tube with maximum turbidity shows the optimum temperature for growth.
The optimum temperature for E. coli is 42oC and Saccharomyces cerevisiae is 25oC.
Determination of TDP
1. Prepare a series of tubes of bacterial and yeast suspension with the test cultures (E. coli and
Saccharomyces cerevisiae separately).
2. Set up a water bath. Set the temperature, say 40ºC for 10 minutes.
3. Place a tube of bacterial suspension and another tube of yeast suspension in the water bath
for 10 minutes at 40ºC.
4. After 10 minutes, remove the culture from the water bath and inoculate on nutrient agar for
bacteria and Sabouraud’s agar for yeast as a single line inoculation. Discard the 40ºC broth
tubes.
5. Place the second set of tubes in water bath. Set the temperature at 50ºC for 10 minutes.
6. Remove the tubes and inoculate the culture on the respective agar plates. Discard the 50ºC
tubes.
7. Continue to raise the temperature, expose the culture for 10 minutes and make inoculations
as before, for 60, 70, 80 and 90ºC.
8. Incubate the plates at 37ºC for 24 hours for bacteria and 25ºC for 24–48 hours for yeast.
Observation
S.No Organism Growth in tubes exposed to different temperatures
for 10 minutes
40°C 50°C 60°C 70°C
1. E. coli + + – –
2. Saccharomyces + – – –
cerevisiae
Interpretation
From this observation, TDP (i.e., the temperature at which an organism is killed in 10 minutes
exposure) for E. coli is 60oC and for Saccharomyces cerevisiae it is 50oC.
Determination of TDT
1. Prepare a series of tubes of bacterial and yeast suspension.
2. Place the cultures at constant temperature (40ºC) in a water bath.
3. At different time intervals (5, 10, 15, 20, 25 minutes), withdraw a culture tube from water
bath and inoculate a loopful onto nutrient agar/Sabouraud’s agar plates.
4. Observe the thermal death time of the test cultures and interpret the results.
5. Perform the test at elevated temperatures (50, 60 and 70ºC) with different time intervals.
Observation
Growth of organism
Temperature Time E. coli Saccharomyces
interval (mts) cerevisiae
40°C 5° + +
10° + +
15° + +
20° + +
25° + +
50°C 5° + +
10° + –
15° + –
20° + –
25° + –
60°C 5° + –
10° – –
15° – –
20° – –
25° – –
70°C 5° – –
10° – –
15° – –
20° – –
25° – –
Result and interpretation

From the above observation, it can be concluded that E.coli can be killed at 60 o C in 10 minutes
and Saccharomyces cerevisiae can be killed at 50oC in 10 minutes.
EFFECT OF OSMOTIC PRESSURE ON THE GROWTH OF

BACTERIA AND YEAST
Aim
To study the effect of osmotic pressure on the growth of bacteria and yeast.
Principle
A selectively permeable plasma membrane separates microorganisms from their environment.
If a microorganism is placed in a hypotonic solution, water will enter the cell and cause it to
burst. Many microorganisms keep the osmotic concentration of their protoplasm above that of
the habitat, by the use of compatible solutes (solutes that are compatible with metabolism
and growth, when at high intracellular concentrations). Most bacteria increase their internal
osmotic concentration through the synthesis or uptake of choline, betaine, proline, glutamic
acid and other amino acids; elevated levels of potassium ions are also involved to some extent.
Algae and fungi employ sucrose and polyols, for example, arabitol, glycerol and mannitol in
plasma membrane for the control of osmotic pressure. Polyols and amino acids are ideal solutes
for this function because they normally do not disrupt enzyme structure and function. When
microorganisms with rigid cell walls are placed in a hypertonic environment, water leaves and the
plasma membrane shrinks away from the wall, a process known as plasmolysis. Water activity
(aw) is the quantitative expression of the degree of water availability. Water activity is inversely
related to osmotic pressure. If a solution has high osmotic pressure, its aw is low.
A microorganism must spend extra effort to grow in a habitat with a low aw value, because it
must maintain a high internal solute concentration to retain water. Some microorganisms can do
this and are osmotolerant. For example, Staphylococcus aureus can be cultured in media containing
any sodium chloride concentration up to about 3 M. The yeast Saccharomyces rouxii will grow in
sugar solutions with aw values as low as 0.6.
Halophiles have adapted so completely to saline conditions that they require high levels
of sodium chloride to grow, for example concentrations between about 2.8 M and saturation
(6.2 M) for extreme halophilic bacteria (Halobacterium). These extreme halophiles accumulate
enormous quantities of potassium in order to remain hypertonic in their environment; the internal
potassium concentration may reach 4 to 7 M.
Materials required
Cultures: Staphylococcus aureus, E. coli, Saccharomyces cerevisiae and Bacillus sp.
Media: Nutrient agar plates with NaCl and sucrose respectively (Appendix III)
Equipment and other materials: Petri plates, test tubes, inoculation loop, incubator, etc.
Procedure
1. Prepare nutrient agar plates containing sodium chloride concentrations in 0.5, 5.0 and
10.0% (w/v).
2. Prepare nutrient agar plates containing sucrose in 0.5, 15.0 and 60.0% (w/v) concentrations.
3. Inoculate the test organisms onto the plates containing NaCl and sucrose.
4. Incubate all plates at 37°C for 48 hours.
5. Following incubation, measure the average width of each inoculated streak and record the
relative density of growth of that microorganism. Let the E. coli streak on the 0.5% sucrose
plate be the standard for very dense (++) growth. Record the growth as zero (0) if no growth
is detected.
Observation and interpretation

S.No Organism Salt concentration Sugar concentration
0.5% 5% 10% 0.5% 5% 10%
1. E. coli + – – + – –
2. Saccharomyces cerevisiae + – – + + +
3. Bacillus + – – + – –
4. Staphylococcus aureus + + + + – –
From the above observation, S.aureus can tolerate high concentration of salt and Saccharomyces
cerevisiae can tolerate up to 15% sugar. This shows the difference in tolerance of osmotic pressure
in different organisms.
EFFECT OF pH ON THE GROWTH OF BACTERIA AND FUNGI

Aim
To become familiar with the pH requirements of microorganisms.
Principle
pH is a measure of the hydrogen ion activity of a solution and is defined as the negative logarithm
of the hydrogen ion concentration.
pH = – log [H+] = log (1/[H+])
The pH scale extends from pH 0.0 (1.0 M [H+]) to pH 14.0 (1.0 × 10–14 M [H+]), and each
pH unit represents a ten-fold change in hydrogen ion concentration.
Bacteria are sensitive to variations in pH. Each species has a pH range, above or below which
it does not survive and an optimum pH at which it grows best. The majority of pathogenic
bacteria grow best at neutral (neutrophiles) or slightly alkaline pH (7.2–7.6). Some acidophilic
bacteria, such as lactobacilli grow under acidic conditions. Others, such as the Vibrio cholerae, are
very sensitive to acid, but tolerate high degrees of alkalinity (alkalophiles).
Drastic variations in pH can harm microorganisms by disrupting the plasma membrane or
inhibiting the activity of enzymes and membrane transport proteins. Despite the diversity in pH,
the specific range for bacteria is between 4 and 9, with the optimum being 6.5 to 7.5. Fungi,
moulds and yeasts prefer an acidic environment with optimum activities at a pH of 4 to 6.
Microorganisms frequently change the pH of their habitat by producing acidic or basic
metabolic waste products. Fermentative microorganisms form organic acids from carbohydrates.
Other microorganisms make their environment more alkaline by generating ammonia through
amino acid degradation. Buffers are often included in media to prevent growth inhibition by
large pH changes.
A commonly used buffering system involves the addition of equimolar concentrations of

K2HPO4, a salt of a weak base, and KH2PO4, a salt of a weak acid. In a medium that has become
acidic, the K2HPO4 absorbs excess H+ to form a weakly acidic salt and a potassium salt with the
anion of the strong acid.
K2HPO4 + HCl KH2PO4 + KCl

(Salt of a (Strong (Salt of a (Potassium
Weak base) acid) weak acid) chloride salt)
In a medium that has become alkaline, KH2PO4 releases H+ to form water by combining
with the excess OH–, and the remaining anionic portion of the weakly acidic salt combines with
the cation of the alkali.
KH2PO4 + KOH K2HPO4 + H2O

(Salt of a (Strong (Salt of a (Water)
weak acid) base) weak base)
Most media contain amino acids, peptones and proteins, which because of their amphoteric
nature, can act as natural buffers.
Materials required
Cultures : 24 hour nutrient broth cultures of E. coli and Saccharomyces cerevisiae
Media : Nutrient broth, yeast broth [Appendix III]
Reagents : 1N NaOH, 1N HCl
Equipment and other materials: Bunsen burner, spectrophotometer, test tube, conical flask, etc.
Procedure
1. Using a sterile pipette, inoculate (0.1 ml) a series of nutrient broth tubes (5 ml) of pH values
3, 6, 7 and 9 with E. coli.
2. Inoculate a series of yeast broth tubes (pH 3, 6, 7 and 9) with 0.1 ml of Saccharomyces cerevisiae
culture.
3. Incubate the E. coli cultures for 24–48 hours at 37ºC and the S. cerevisiae cultures for 48–72
hours at 25ºC.
4. After incubation, measure the growth in a spectrophotometer at 600 nm using sterile nutrient
broth for E. coli and yeast broth for S. cerevisiae as blanks.
Observation and Interpretation
S.No. Organism Turbidity

pH 3 pH 6 pH 7 pH 9
1. E. coli – + ++ –
2. Saccharomyces cerevisiae + + ++ –
E. coli is sensitive to acid and alkaline pH. Hence turbidity was observed in nutrient broth
tubes with pH 6 and 7. Saccharomyces cerevisiae shows turbidity at pH 3, 6 and 7. Heavy growth is
observed at pH 7 for both organisms. Thus pH 7 is the optimum pH for E. coli and Saccharomyces
cerevisiae.
5
INDUSTRIAL MICROBIOLOGY
WINE PRODUCTION
Aim
To become acquainted with wine production by fermentative activities of yeast cells.
Principle
Wine is a product of natural alcoholic fermentation of grape juice and other fruit juices, such as
peaches, pears, plums and apples, by the action of yeast cells. This biochemical conversion of juices
to wine occurs when the yeast cells enzymatically degrade the fruit sugars, fructose and glucose,
first to acetaldehyde, and then to alcohol. Grapes containing 20–30% sugar concentration will
yield wine with alcohol content of approximately 10–15%. Grapes also contain acids, minerals,
tannin, pigments, vitamins, enzymes and other aroma compounds whose concentration in the
final product gives its characteristic taste.
The commercial production of wine is a long process. First, the grapes are washed and pressed to
extract the juice, which is called the must. Potassium metabisulphite is added to the must to retard
the growth of acetic acid bacteria, moulds and wild yeast that are endogenous to grapes in the
wineyard. A wine-producing strain of yeast, Saccharomyces cerevisiae, is used to inoculate the must
and incubated for 3–5 days at 21°C–31°C. Then, the wine is allowed to settle down, clarified and
stored for maturation. The chemical changes that occur during ageing are responsible for the aroma.
The clarified product is taken, filtered, pasteurized at 62.5°C for 30 minutes and stored.
Materials required
Culture : Conical flask cultures of S. cerevisiae
Substrate : One kg of grapes
Reagents : 1% phenolphthalein solution, 0.1N sodium hydroxide, glucose solution, Anthrone

reagent, potassium dichromate, conc. H2SO4.
Equipment and other materials : Pipette, burette, test tubes, waterbath, spectrophotometer,
fermented wine, distilled water etc.
Procedure
1. One kg of grapes is washed thoroughly and crushed to obtain the juice.
2. The juice is filtered and 750 ml of filtrate is taken. This is separated at 62.5°C for
30 min.
3. The flask containing 50 ml is inoculated with yeast and placed in a shaker for 48 hours.
4. The 50 ml of the developed inoculum is added to 700 ml of the filtrate. Immediately,
50 ml is withdrawn from the mixture for the evaluation of sugar content, total acidity, taste
and flavours.
5. The inoculated extract is allowed to undergo fermentation for a few days.
6. On the 7th, 14th and 21st day, 50 ml is withdrawn to check acidity, taste and flavour.
7. A standard chart for estimating sugar is plotted using the concentration of glucose from
100–1000 mg/ml by Anthrone method.
8. The sugar content on the 7th, 14th and 21st day is estimated.
Estimation of sugar content
1. About 1 ml of the extract is diluted to about 1000 times with distilled water and 1 ml from
each dilution is taken and 4 ml of Anthrone reagent is added to it under cold condition.
2. The content is then kept in a boiling water bath for 10 min.
3. Then, the optical density values are recorded using a spectrophotometer at 620 nm.
4. Sugar content is estimated using a standard graph obtained using glucose
(100–1000 mg/ml)
Test for acidity 20 ml of the sample is taken and 5 drops of phenolphthalein is added to it. It
is then titrated against 0.1N NaOH .The end point is a pale pink colour.
Xml of alkali×Normality of alkali

Total acidity (Expressed asacetic acid)=
Amount of sample taken

Estimation of alcohol
Preparation of chromic acid About 34 g of potassium dichromate is dissolved in 350 ml of
distilled water. Then, 325 ml of concentrated sulphuric acid is added slowly by placing the flask
on an ice-bed. The volume is made up to 1000 ml with distilled water. This gives 3.4% chromic acid.
Industrial Microbiology 169
Preparation of standard curve for alcohol estimation

1. Absolute alcohol (1–10% v/v) is prepared in various test tubes.
2. To 1 ml of various dilutions in a conical flask, 25ml of distilled water is added and kept at
room temperature for 15 min.
3. From this, 15 ml is taken separately and 25 ml of chromic acid solution is added.
The solution is made upto 50 ml.
4. The reaction mixture is kept in water bath at 60ºC for 30 min.
5. The optical density (O.D.) is recorded at 600 nm.
6. A standard graph is plotted with concentration of alcohol along the X-axis and O.D. values
along the Y-axis.
7. With 1 ml of wine sample, the same procedure is carried out. The O.D. values are extrapolated
with the standard graph to find the alcohol content of the wine sample.
Observation
Conc. of standard Optical
sugar solution density
100 0.22
200 0.30
300 0.45
400 0.68
500 0.74
600 0.76
700 0.79
800 0.81
900 0.83
1000 0.85
Result
The optical density of the test sample is found to be 0.74. Hence the sugar content of the wine
is estimated from the graph, which is 500 mg/ml.
CITRIC ACID PRODUCTION

Aim
To produce citric acid by solid-state fermentation.
Principle
Citric acid, a tricarboxylic acid is widely used as an acidifying agent and antioxidant in food, beverages
and pharmaceutical industries. Before the development of fermentation technology, citric acid
was obtained by extraction from the juices of certain fruits (e.g., lemon), and later, from pineapple
wastes. Today, most of the commercial citric acid is obtained by microbial fermentation process.
Conventionally, it is a submerged fermentation using molasses as raw material. In recent
years, considerable interest has been shown in solid-state production of citric acid by Aspergillus
niger using agro residues like bagasse, corncob, carob pod and wastes from food processing
industries, like apple and grape pomace and fruit peel, due to its several advantages like solid
waste management, biomass energy conservation, production of high-value products and little
risk of bacterial contamination.
Materials required
Culture : Aspergillus niger
Media : Citric acid production media, Potato dextrose agar (PDA), SDA slant (Appendix III)
Reagents : Tween 80, acetic anhydride, 2N HCl, pyridine
Substrate : Oven-dried, ground sugar cane bagasse with the particle size of 1.2–1.6 mm is
used as a carrier. The carrier is soaked overnight in 2N HCl at room temperature, washed
thoroughly with distilled water, dried and used for fermentation medium.
Equipment and other materials : Centrifuge, spectrophotometer, hot air oven, test tubes, flasks, etc.
Procedure
Inoculum
1. Add 25 ml of sterile distilled water and Tween 80 (0.1%) on PDA or SDA slant and shake
vigorously for a minute.
2. Suitably dilute to get a spore concentration of 2 × 107 spores/ml.
Fermentation
1. Transfer the inoculum to flasks (5 flasks) of treated substrate (100 g of sugar cane bagasse
moistened in sucrose medium).
2. Incubate at 30°C for 2–8 days.
3. Harvest one flask, every alternate day for the estimation of residual sugar and citric acid
content.
4. Dry the harvested fermented mass in an oven at 90°C, to get dry fermented mass (DFM).
5. Extract the DFM, three times with 100 ml of distilled water at 40°C and centrifuge at
3000 rpm for 30 min. to separate the mycelium from the support.
6. Filter through a muslin cloth and centrifuge the filtrate at 6000 rpm for 15 min.
7. Estimate the citric acid content from the supernatant.
Estimation of citric acid
1. To 1 ml of the supernatant, add 1.3 ml of pyridine.
2. Shake the tubes and add 5.7 ml of acetic anhydride under ice-cold condition.
3. Bring the test tubes to a constant temperature by keeping it in a water bath at 32°C for
30 min.
4. Measure the intensity of colour developed in the tube at 420 nm in a spectrophotometer.
5. Plot a standard graph for citric acid at various concentrations. Estimate the concentration of
citric acid produced by Aspergillus niger from the standard graph.
Observation
Conc. of citric acid O.D.Value
100 0.24
200 0.51
300 0.72
400 0.99
500 1.28
600 1.48
700 1.58
800 1.82
900 1.92
1000 1.98
Culture assay
O.D.Value Conc.of citric

acid mg/ml
Aspergillus 0.99 400
niger
Result
The amount of citric acid produced by Aspergillus niger is 400 mg/ml.
PRODUCTION OF GLUTAMIC ACID

Aim
To produce glutamic acid using Corynebacterium glutamicum and/or Micrococcus glutamicus.
Principle
Amino acids, such as lysine and glutamic acid, are used in the food industry as nutritional supplements
in bread products and as flavour-enhancing compounds, such as monosodium glutamate (MSG).
Amino acid production is typically carried out by means of regulatory mutants, which have a reduced
ability to limit synthesis of an end product. Mutants of Corynebacterium glutamicum that have only
a limited ability to convert the TCA cycle intermediate, alpha-keto glutarate to succinyl-CoA, is
used. A controlled low biotin level and the addition of fatty acid derivatives results in increased
membrane permeability and excretion of high concentrations of glutamic acid. The impaired
bacteria use the glyoxalate pathway, to meet their needs for essential biochemical intermediates,
especially during the growth phase. After growth becomes limited because of changed nutrient
availability, an almost molar conversion of isocitrate to glutamate occurs.
Glucose and starch hydrolysates are the principal raw materials for the fermentative production
of amino acids. More efforts have been made to replace these materials with such cheaper and more
easily available ones as molasses, ethanol, pentose, acetic acid and other petrochemicals. Urea or
ammonia may be used as nitrogen sources. Also, phosphates and other ordinary salts are added to
the production medium in the reaction. The optimum temperature is 30°C and duration is 40 hrs.
Materials required
Culture : Corynebacterium glutamicum, Micrococcus glutamicus
Media : Inoculum medium, Production medium (Appendix III)
Reagents : DNS reagent, 1% ninhydrin, solvent system
Equipment and other materials : Shaker, incubator, test tubes, spectrophotometer, waterbath,
centrifuge, hot air oven, chromatogram
Procedure
1. Grow Micrococcus glutamicus in 50 ml of inoculum medium for 24 hours.
2. Transfer aseptically 10% of inoculum to 125 ml of production medium.
3. Incubate in a shaker at 30°C. Withdraw samples after 24, 36, 48, 60 and 72 hours and
estimate the presence of glucose, total amino acids and glutamic acid content.
Estimation of Glucose content The estimation can be performed by DNS method. Sugar solution
in the range of 0.1 to 2.0 mg/ml is used.
1. To each sample, add 1ml of distilled water and 3 ml of DNS reagent.

2. Shake the tubes and heat in the boiling water bath for 3 to 10 minutes.
3. Immediately cool to room temperature and add 3ml of distilled water to each of the tubes.
4. Measure the optical density of the samples at 600 nm against the blank, which is prepared
by taking 10 ml of distilled water instead of sugar solution. Construct a standard graph with
known concentration of glucose and compare the results of samples.
Preparation of DNS Reagent
1. Suspend 1.6 g of sodium hydroxide in 50 ml of distilled water.
2. Add 0.9 g of 3, 5-dinitrosalicylic acid and mix until it is dissolved.
3. Add 28.22 g of potassium sodium tartarate and mix until it is dissolved.
4. Make up the volume to 100 ml.
5. Store in a dark bottle.
Estimation of total amino acids by colorimetry
1. Adjust the pH of the centrifuged broth to 7.0.
2. To 1.0 ml of this broth, add 0.25 ml of 1% ninhydrin reagent.
3. Plug the tubes and heat in water bath for 10 minutes.
4. Make up the volume to 10 ml with distilled water.
5. Read the developed colour at 550 nm against the blank solution.
6. Estimate the amounts from the standard graph.
Estimation of glutamic acid by Descending Paper chromatography
1. The solvent system used is butanol : acetic acid : water (4:1:3).
2. Spot the sample on the paper as dots.
3. After drying, place the paper in the chromatographic tank, previously saturated with the
solvent system.
4. Allow the chromatogram to run for 16 hrs. Then dry in the atmosphere.
5. After drying, spray the paper with 1% ninhydrin reagent in acetone solution.
6. Dry the sprayed paper in an oven at 80°C for 10 min. and cool.
7. Observe the spots.
8. Cut these spots and extract with 50% alcohol.
9. Measure the optical density at 550nm against a blank solution and with an authentic sample
of glutamic acid (1 mg/ml).
10. Extrapolate the optical density readings on a standard graph and determine the glutamic
acid content of each sample.
Observation
Estimation of Glutamic Acid Using DNSA method

Conc.of glutamic acid O.D.value

100 0.01
200 0.02
300 0.04
400 0.29
500 0.36
600 0.45
700 0.51
800 0.80
900 0.92
1000 0.98
Culture assay
O.D.Value Con.of glutamic

acid µg/ml
Corynebacterium 0.48 650
glutamicum
Result
The amount of glutamic acid produced by Corynebacterium glutamicum was found to be
700 mg/ml.
PROTEASE ESTIMATION
Aim
To produce and estimate the amount of alkaline protease produced by Bacillus subtilis.
Principle
Complex mixtures of true proteinases and peptidases are usually called proteases. Proteases
are produced, both by bacteria (Bacillus subtilis, Bacillus licheniformis) and fungi (Aspergillus niger,
Aspergillus oryzae). There are two types of proteases: (i) alkaline serine proteases and (ii) acid
proteases.
i. Alkaline serine proteases: This is the most widely used detergent protease. It is obtained
from Bacillus licheniformis by submerged culture method. The temperature of fermentation
is in the range of 30–40ºC and the pH is 7.0.
ii. Acid proteases: These enzymes are mostly produced by fungi. Acid proteases may be produced
by either semi-solid culture or submerged culture depending upon the fungal species employed.
The optimum temperature for fermentation is 30ºC and the process is complete within 3–7
days.
Materials required
Culture : 24-hour-old slant culture of Bacillus subtilis
Media : Production medium (Bacillus subtilis) and inoculum medium (Bacillus subtilis) peptone,
beef extract agar medium (Appendix III)
Equipment and other materials : Erlenmeyer flasks, shaker incubator, centrifuge, test tubes,
pipettes, inoculation loop, autoclave, etc.
Procedure
1. Add 5 ml of sterile distilled water to a 24 hrs old slant of Bacillus subtilis. Scrap the culture
from the slant into the sterile distilled water and transfer 10% (5 ml) from the resulted cell
suspension to 250 ml Erlenmeyer flasks containing 45 ml of sterile inoculum medium.
2. Incubate the flask in a shaker incubator at 220 rpm at 37ºC for 24 hrs.
3. Transfer 10% (5 ml) of inoculum into a 250 ml Erlenmeyer flask containing 45 ml of
production medium.
4. Incubate the flasks at 37ºC for 48 hrs.
5. Centrifuge the culture broth (5 ml) at 3000 rpm for 20 minutes. The supernatant contains
the crude enzyme source.
PROTEASE ASSAY
5 ml of culture medium
Centrifuge at 3000rpm for 20 minutes
Get the supernatant DNS method (reducing sugar)
For protease assay (diluted or undiluted)
Protease determination
Reagents
1. A solution of 0.6% casein in 0.5 M boric acid–NaOH buffer (pH 9.0): Suspend 3.09 g of
boric acid in 700 ml of distilled water, adjust to pH 9 using 1N NaOH, and then add 6 g of
casein. Make the volume to 1000 ml, keep at 4ºC.
2. 0.4 M trichloroacetic acid: Take 65.57 ml of 100% TCA and then make the volume to
1000 ml. The 100% TCA is prepared from 500 g TCA in 227 ml of distilled water; keep
the solution in a dark bottle.
3. 0.4 M sodium carbonate: Suspend 42.40 g in 1000 ml of distilled water.
4. Folin–Ciocalteau reagent: Mix folin in distilled water in the ratio 1 : 1.
Method to Determine Protease in the Sample
Sample
1. Create a tyrosine standard curve (30 mg in 30 ml of distilled water).
2. Pipette 1 ml of sample (diluted or undiluted) into a test tube.
3. Add 1 ml of caesin solution (mix the two solutions together) and incubate at 45ºC for
10 min.
4. Add 2 ml of trichloroacetic acid (TCA) to stop the reaction and then incubate at 45ºC for
10 min. (along with control).
5. Centrifuge at 4,800 rpm for 5 minutes.
6. Pipette 0.5 ml of supernatant into a new test tube.
7. Add 2.5 ml of sodium carbonate solution and mix together.
8. Add 0.5 ml of Folin–Ciocaltaeu reagent solution and mix again.
9. Incubate at room temperature for 30 min.
10. Read the optical density at 660 nm and calculate the obtained tyrosine from standard curve
of tyrosine (0–60 µg/ml).
11. Calculate the alkaline protease activity (unit/ml).
Control
1. Pipette 1 ml of sample into a test tube.
2. Add 2 ml of TCA to stop the reaction.
3. Add 1 ml of caesin (mix together), incubate at 45ºC for 10 minutes (with the sample).
4. Centrifuge at 4,800 rpm for 5 minutes (with the sample). A white pellet is seen at the base
of the test tube when completed.
5. Pipette 0.5 ml of supernatant into a test tube and complete steps 7–11 as given above.
Observation
Unit calculation of enzyme
One unit of protease activity is defined as the amount that liberates 1mg of tyrosine in a reaction
mixture per minute at 45ºC and at pH 9.0.
Alkaline protease (unit/ml) = A×B (1/C) × (1/D) × dilution factor (if diluted)
where,
Protein Estimation-Lowry’s Method

A = Tyrosine concentration (µg/ml)

B = Total volume (ml), (4 ml)
C = Incubation time (min.), (10 min.)
D = Sample volume (ml), (1 ml)
Protein Estimation–Lowry’s Method
B S1 S2 S3 S4 S5 T1 T2
1. Volume of Working std - 0.2 0.4 0.6 0.8 1.0 - -
2. Concentration of working - 20 40 60 80 100 - -
standard (ml)
3. Volume of Unknown (ml) - - - - - - 0.4 0.4
4. Volume of Distilled water 1.0 0.8 0.6 0.4 0.2 0 0.6 0.6
5. Volume of Alkaline reagent 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5
(ml)
10 minutes at room temperature
6. Volume of Folin-ciocalteau 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
reagent (ml)
20 minutes at room temperature, colour developed was read at 640 nm
7. Optical density at 640 nm 0.00 0.05 0.0 0.1 0.14 0.20 0.1 0.10
9 4 0
Determination of Reducing Sugar by Dinitrosalicylic Acid (DNS)

Reagents DNS solution.
Method to determine sugar in the sample
1. Create a standard curve for glucose.
2. Pipette 0.5 ml of the supernatant (sample) into a test tube.
3. Add 0.5 ml of DNS. Then mix the sample and DNS together. Once mixed, boil for
10 minutes at 110ºC, place a lid on the test tubes.
4. Stop the reaction by placing the test tubes in an ice bath. Take the lids off to allow the
cooling process to happen quickly. Allow the test tubes to remain in the ice bath for
5 to 10 min.
5. Read the optical density at 570nm.
6. Calculate the sugar concentration from the standard curve of glucose.
Result
In the protease estimation the enzyme activity was 42 units/ml/min. i.e., 42 units/ml/minute of
protease enzyme was used for the liberation of tyrosine.
PRODUCTION OF EXTRACELLULAR CELLULASE BY

SOLID-STATE FERMENTATION
Aim
To determine the ability of microorganisms to produce the extracellular enzyme, cellulase, for
degradation of cellulose.
Principle
Cellulose is the most important structural component of the cell wall of plants. Cellulose
is utilized as a carbon source and energy source by numerous bacteria, actinomycetes and
fungi. e.g., Trichoderma sp., Aspergillus sp, etc.
The production of reducing sugar (glucose) due to the cellulolytic activity is measured by
dinitrosalisylic acid (DNSA) method. The enzyme activity is expressed as milligram of glucose
released per minute.
Materials required
Culture : Trichoderma sp
Media : Carboxymethyl cellulose medium (Appendix III).
Reagents :
1. Sodium citrate buffer (0.1 M) (pH 5.0): Carboxy methyl cellulose (CMC) is dissolved in
sodium citrate buffer (1g of CMC in 100 ml of sodium citrate buffer).
2. Dinitrosalicylic acid (DNSA) reagent.
3. 40% Rochelle salt solution (potassium sodium tartarate)
Equipment and other materials : Pipette, test tubes, conical flask.
Procedure
Isolation and confirmation of cellulolytic activity of microorganisms Carboxymethyl cellulose
medium is prepared and dispensed into Petri plates. The test organism is inoculated into the
medium. The plates are incubated at room temperature for 3 days. Cellulolytic activity is confirmed
for Trichoderma sp. with solid-state fermentation for the production of extracellular cellulase, as
given below:
Wheat straw is chopped into 2 cm fibrils and subjected to chemical pretreatment with
NaOH and water until neutrality. Wheat straw is then weighed and dispensed into conical flask.
The straw is wetted with sterile distilled water. The medium is supplemented with minerals.
The spores of Trichoderma are inoculated into the flask. The set-up is kept at room temperature
for 4 days. After incubation, the straw is filtered to filter out the enzyme present in the medium.
The filtrate is taken as the source of the enzyme. The standard graph for glucose is plotted by
DNSA method.
A standard graph is prepared with glucose concentration ranging from 100–1000 mg
per ml. About 0.45 ml of 1% carboxymethyl cellulose solution is pipetted out into 0.05 ml of
enzyme extract. The mixture is incubated at 55ºC for 15 min. Immediately after removing the
enzyme–substrate mixture from the broth, 0.5 ml of DNSA reagent is added.
The mixture is heated in a boiling water bath for 5 min. While the test tube is warm,
0.1 ml of Rochelle salt solution is added and cooled to room temperature. Water is added to
make up the volume to 50 ml. The absorbance is measured at 540 nm. The activity is measured
by extrapolating the values in the standard graph. The cellulase activity of the given organism
is expressed in units/min/ml.
Observation
Concentration of glucose Optical Density value
µg/ml
100 0.27
200 0.52
300 0.80
400 1.04
500 1.32
600 1.58
700 1.84
800 2.10
900 2.46
1000 2.60
Calculations
Enzyme activity = Concentration of cellulase
80 µg
Aspergillus sp =
60
=1.99unit/ml.
Result
The cellulase activity of the given organism is observed 1.5 units.
Estimation of cellulase
MUSHROOM CULTIVATION
Aim
To cultivate oyster mushrooms (Pleurotus).
Principle
Mushrooms are fleshly fungi, which constitute the lower group of the Plant Kingdom.
The mushroom is a common fungal fruiting body that forms basidiospore at the tip of the
club-like structure, called basidia, which are arranged along the gills of mushrooms. So, these
are placed under the family Basidiomycetes.
Mushrooms can be grouped as:
 Edible mushrooms: Agaricus, Pleurotus, A. campestris, A. bisporus, P. sojariaji, P. sapidus,
P. florida, P. ostreatus, Volveriella, Lentinus, Flammulina, V. volvacia, L. edodus.
 Non-edible mushrooms: Amanita (poisonous mushroom), A. phailorides, A. virosa
The fungi are incapable of causing infections but produce toxic substances. These poisonous
substances are collectively called as mycotoxins and result in mycastimus (mushroom poisoning),
following this ingestion.
The oyster mushrooms are rich in proteins and minerals, and devoid of starch and low in
calories and carbohydrates. These are ideal for diabetic and heart patients and those do not want
to put on weight.
The various substrates used for the cultivation of Pleurotus sp., are banana pseudostems, wheat
straw, paddy straw, raggi straw, saw dust, sunflower stakes, rice husks and karad hay. Rice straw
gives highest yields. These can be grown in any container—earthen pot, cane gusket, polyethylene
bags, iron baskets, wooden trays, etc.
Materials required
1. Thatched bud or polythene chamber
2. Dry paddy straw (chopped)
3. Horse gram powder
4. Spawn bottles of Pleurotus sp.
5. Water sprayers, etc.
Procedure
Spawn preparation
1. The grains are boiled for 15–20 minutes in water.
2. The excess water is drained.
3. The grains are mixed with 2% CaSO4 or 0.5% of CaCO3.

4. This chemical mixture is placed in 500 ml container.
5. This is kept at 25ºC for 2–3 weeks.
6. It is observed for the growth of white mycelium spawn. Spawn is the true mycelia grown on
a special media; it is also known as basidiocarp.
Preparation of mushroom bed
1. Dried paddy straw is taken and chopped into small bits of 1–2 cm length.
2. They are soaked in clear water for an hour.
3. The excess water is drained off and the paddy straw is sterilized.
4. Polyethylene bags are made into cylindrical shapes.
5. The bags are filled up to some height with sterilized paddy straw.
6. The paddy is spread with the spawn.
7. Again, paddy straw substrate is filled up to some height.
8. It is repeated for 5 times and the bag is tied with the help of a thread.
9. Small holes are made on the mushroom bed for aeration.
10. The bag is kept away from ventilation in a dark room for 15 days.
11. The polyethylene cover is removed and water is sprinkled 2 times a day.
12. Young fruiting bodies develop.
13. The fruiting bodies are harvested every 8 days after 20–22 days of spawning.
Harvesting
It has three-stage harvesting process:
First harvest: 22–24 days
Second harvest: 24–30 days
Third harvest: 34–36 days
Discussion
Mushroom provides a rich addition to the diet in the form of carbohydrates, minerals and vitamins.
They have a high percentage of all essential amino acids. They are rich in vitamins B1 and B2,
niacin, pantothenic acid, vitamin B12 and vitamin C. They are also a good source of mineral salts,
like phosphorous, potassium ion, copper and almost free of fat.
Mushrooms are known as “poor man’s food” as it is grown on dead organic matter under
suitable environmental conditions. It derives the carbonaceous food by decomposing lignin,
cellulose, hemicelluose, with the help of extracellular enzymes secreted by the mycelium. Microbial
protein present in compost is the chief source of organic N2.—
Pleurotus sp. or Oyster mushroom or dhingai mushroom or wood fungus is a most important
mushroom. The leading countries producing it are Japan, Taiwan and Italy. The most common
species are P. ostreatus, P. fiabellatus, P. sojariajii, P. sapide, P. sapthulatis and P. floride.
General moulds like Sclerotium rollie, Trichoderma sp., Fusarium sp., etc., and weeds like Copanus
sp., and Penziza sp., are the common ones which damage the oyster mushrooms. Among insects,
mushroom flies and mites are common. After using the full crop, the substrate may be used as
compost. This can be used for a wide range of crops and it is as good as farmyard manure.
EXTRACELLULAR ENZYME PRODUCTION—AMYLASE

The starch components have glycosidic bonds, which are split by the α-amylase enzyme which
converts starch into simpler sugars like dextrins, disaccharides and monosaccharides. The product
of hydrolysis differs according to the source of the enzyme.
Bacillus, Pseudomonas, Clostridium produce bacterial α-amylases, but for industrial production
Bacillus subtilis is preferred. Bacillus is used for commercial fungal amylases production. α-amylases
are mainly used for designing of textile fabrics, manufacture of glucose and in brewing process.
Aim
To produce and measure the activity of amylase enzyme.
Principle
Amylase are the enzymes which carry out the hydrolysis of polysaccharide. The amylases are
of three types namely alpha amylase, amylo glucosidase and beta amylase. Alpha amylase is an
exoenzyme which hydrolyse the internal 1,4-glucosidic which links both of the amylase and
amylopectin constituent present in starch. The main source for the production of bacterial amylase
enzymes are Bacillus subtilis , B. licheniformis and fungal enzyme is obtained from Aspergillus oryzae
and Aspergillus niger.
Amyloglucosidase or gluco amylase hydrolyses the alpha-1,4 glucan link in
polysaccharide and removes the glucose units from the non-reducing end of the chain and also
hydrolyses 1,6-glucosidic link of amylopectin.
Beta amylase hydrolyses an alternate alpha-1,4 glucan link in starch and removes maltose
units from the non-reducing end of the chain. This enzyme is utilized in the production of beer,
spirits and also in syrup production.
Materials Required
Media : Starch agar medium, fermentation medium (starch broth)
Reagents : Dinitrosalicylic acid, 0.1 M sodium acetate buffer,1N HCl.
Equipment and other materials: Conical flasks, test tubes, pipette
Procedure
1. The soil sample is inoculated onto starch agar plates and amylase-producing organism is
isolated.
2. The culture is purified by plating onto starch agar plate. It is incubated at room temperature.
3. The plate is then flooded with 0.15% of iodine solution. Starch–iodine complex gives a purple
colour on starch agar plates.
4. The amylase producing organisms produce a clear zone around them. These colonies are
selected and inoculated into fermentation medium.
5. The flasks are incubated for 5 days at room temperature.
6. The amount of amylase produced is estimated by dinitrosalicylic acid (DNSA method).
DNSA method of amylase estimation
1. About 5 ml of 1% starch in 0.1 M sodium acetate buffer (pH 5.8) is taken.
2. To it, 3 ml of buffer and 1 ml of the enzyme preparation is added.
3. The contents are mixed and incubated at 60ºC for 10 min. in a water bath.
4. The reaction is then arrested by adding 2 ml of 1N HCl.
5. From the above mixture, 1 ml is taken and 2 ml of DNSA reagent is added.
6. This is incubated at 60ºC for 10 min. in a water bath. It is then cooled and 10 ml of distilled
water is added to dilute the mixture.
7. The intensity of the colour developed is read at 520 nm in a spectrophotometer.
8. The result is then expressed in units/ml/min.
IMMOBILIZATION OF CELLS
Immobilization is a physical or chemical attachment of a cell or an enzyme, to a support material,
which can be hydrophilic organic polymers or certain other metal oxides. Alginate exists in brown
algae as a most abundant polysaccharide comprising up to 40% of dry weight. It is loaded in the
intracellular matrix as a gel containing sodium, calcium, magnesium, bromium and strontium
ions; its main function is believed to give strength and flexibility to the algal tissues. Because
of its gelling, viscosifying and stabilizing properties, alginate is widely used industrially and
in technical applications. Alginate belongs to the family of unbranched polymer of 1-4 linked
D-mannuronic acid and L-gluconic acid.
Aim
To immobilize the whole cells using sodium alginate beads.
Principle
Immobilization of cells by entrapping into the hydrogel is generally carried out by mixing the
cells with water-soluble polymers and subsequent gelling of the polymers by adding cations,
such as calcium or strontium, by dipping the alginate cell mixture into a solution containing the
multivalent cations. The droplets will instantly form gel spheres, by ionotrophic gelatin, the gel
with a three-dimensional lattice of chemically cross-linked polymers.
Materials required
Culture : Yeast cell culture
Reagents : Sodium alginate (3.6%) : 3.6g of sodium alginate is dissolved in 100 ml of distilled
water
Equipment and other materials : Mortar and pestle, syringe, centrifuge, beaker, etc.
Procedure
1. Yeast cell culture of stationary growth phase is harvested by centrifugation.
2. The harvested yeast cells are broken into small segments using mortar and pestle.
3. After homogenization, 3.6% sodium alginate solution is added into homogenized yeast cells.
4. The contents are mixed well with continuous shaking.
5. The yeast cell suspension containing sodium alginate is extracted dropwise through a sterile
syringe into the calcium chloride solution in beaker. Sodium is replaced by calcium ions and
fine beads of calcium alginate spheres are formed.
6. The polymerization reaction between sodium alginate and calcium chloride results in the
formation of calcium alginate which are strong enough to hold the contents inside stably.
Result
The yeast cells are immobilized in the calcium alginate beads and this can be confirmed by
comparing the weight of the calcium alginate beads to that of the beads which are formed by
mixing of the yeast culture.
6
ENVIRONMENTAL MICROBIOLOGY
ISOLATION OF NITROGEN FIXERS

The vast majority of the nitrogen in the environment exists as nitrogen gas (N2). It constitutes
about 80% of the atmospheric gases. Nitrogen, in its gaseous form, is not a suitable source of
nitrogen for most microorganisms, which require it for various cellular constituents, especially
proteins. Biological nitrogen fixation in the environment occurs primarily as a result of the activities
of microorganisms that are free living or participate in mutualistic relationship with plants.
These bacteria convert nitrogen gas into ammonia, which is assimilated into biological systems.
Non-symbiotic nitrogen fixation is carried out by free-living bacteria in the soil and surface waters.
Examples are Azotobacter, Bacillus, Clostridium and Cyanobacteria. Symbiotic nitrogen fixation occurs
when bacteria, such as Rhizobium, enter into symbiotic relationships with leguminous plants,
such as clover and alfalfa; the combined plant and bacterial relationship effects nitrogen fixation.
ISOLATION OF SYMBIOTIC NITROGEN FIXERS (RHIZOBIUM SP.)
Aim
To isolate Rhizobium sp. from the root nodules of leguminous plants.
Principle
It is known that legumes enrich the soil by contributing nitrogen through symbiotic nitrogen
fixation by Rhizobium through centuries. However, scientific demonstration of value of legumes
in contributing nitrogen nutrition of plants was only done in 19th Century. This was established
by the facts that nodules on legume roots are responsible for fixing atmospheric nitrogen through
bacterium Rhizobium. Due to new technological development a substantial contribution in
increasing production of legumes besides improving microbial fertility of soil, is made.
Different types of legumes form various sizes and shapes of nodules on their roots. Differences
have been even found within the same species of legumes. Hence, nodules are collected when
plants are in flowering stage and one can make out an effective nodule which is large in size and
red in colour. Such nodules are used for separation of Rhizobium in the laboratory. Yeast mannitol
agar is the special medium used to grow Rhizobium.
Materials required
Sample : Root nodules
Media : Yeast extract mannitol agar (YEMA) medium (Appendix III)
Reagents : 0.1% mercuric chloride, 75% ethyl alcohol
Equipments and other materials : Sterile Petri plates, scalpel, forceps, Bunsen burner, etc.
Procedure
1. Select well-formed, healthy pinkish nodule from the tap root of leguminous plants.
2. Surface sterilize by immersing in 0.1% mercuric chloride for five minutes and wash repeatedly
with sterile water to remove the adhering chemicals.
3. Again sterilize with 75% ethanol for 3 minutes. Wash repeatedly in sterile water.
4. Cut the sterile nodule into 2 halves.
5. Rub the exposed, pinkish-brown portion on the surface of yeast extract mannitol agar medium
using sterile forceps. Incubate the plates at 37°C for 24–72 hours.
Interpretation
White, mucoid colonies are formed by Rhizobium sp.
ISOLATION OF FREE-LIVING
NITROGEN FIXERS (AZOTOBACTER SP.)
Aim
To isolate Azotobacter sp. from soil.
Principle
Azotobacter is a gram-negative bacteria, polymorphic, i.e., they are of different sizes and shapes. Their
size ranges from 2–10×1–2.5 m. Young cells possess peritrichous flagella and are used as locomotive
organs. Old population of bacteria includes encapsulated forms and have enhanced resistant to
heat, desiccation and adverse conditions. The cyst germinates under favourable conditions to give
vegetative cells. They also produce polysachharides. Azotobacter spp., are sensitive to acidic pH, high
salts, and temperature above 350C. There are four important species of Azotobacter, viz., A. chroococcum,
A. agilis, A. paspali and A. vinelandii of which A. chroococcum is most commonly found in our soils.
Environmental Microbiology 191
These are free-living bacteria which grow well on a nitrogen free medium. These bacteria
utilize atmospheric nitrogen gas for their cell protein synthesis. This cell protein is then mineralised
in soil after the death of Azotobacter cells thereby contributing towards the nitrogen availability
of the crop plants.
Organisms in the genus Azotobacter are chemoorganotrophs, capable of using sugars, alcohols
and salts of organic acids for growth. During growth, many species will produce water soluble
and water insoluble pigments, causing cultures and colonies of the organism to appear in shades
of yellow, green, red and brown. While growing on sugars, some Azotobacter will produce copious
amounts of an extracellular polysaccharide (EPS). Often in this laboratory the microorganisms
isolated will produce so much EPS that the culture will have the appearance of cream pudding.
Under nutrient limiting conditions, the organism forms resting structures called cysts. Cysts
can be described as vegetative cells encapsulated in a desiccation resistant coat. They are very
resistant to drying and the encysted bacteria can survive for many years in this state.
Materials required
Sample : Soil sample
Media : Nitrogen-free mannitol broth, nitrogen-free mannitol agar (Appendix III)
Equipment and other materials : Bunsen burner, inoculation loop, conical flask, marker, etc.
Procedure
1. Add 1 g of soil sample to 50 ml of sterile nitrogen-free mannitol broth. Shake vigorously.
2. Incubate the culture for 4–7 days at room temperature (25°C).
3. Examine the surface of the culture for the presence of a film. Do not shake or disturb the film.
4. Using sterile inoculating technique, transfer a loopful of the surface film to an appropriately
labelled nitrogen-free mannitol agar plate. Perform quadrant streaking for isolation of colonies.
5. Incubate at 25°C for 4–6 days.
6. Observe for pigmentation of colonies.
Interpretation
Azotobacter chroococcum produce brown to black-coloured colonies. Azotobacter vinelandii fluoresces
green under UV light.
ISOLATION OF PHOSPHATE SOLUBILIZERS

Aim
To isolate phosphate solubilizers from the given soil sample.
Principle
Phosphorous is a vital nutrient for plants and organisms, next to nitrogen. Several soil bacteria,
particularly those that belong to the genera Pseudomonas and Bacillus, and fungi belonging to the
genera Aspergillus and Penicillium, possess the ability of converting the insoluble phosphate in the
soil to soluble form by secreting organic acids, such as lactic acid, acetoproponate and succinate.
This acid lowers the pH and brings about the dissolution of bound forms of phosphate with
some of the calcium and iron, resulting in effective solubilization and utilization of phosphates.
Materials required
Sample: Rhizosphere soil sample
Media: Pikovskaya’s medium (Appendix III)
Equipments and other materials: Sterile distilled water, sterilized Petri plates, pipettes, conical
flasks, water bath, L-rod, etc.
Procedure
1. Prepare Pikovskaya’s media.
2. Dispense the medium in 100 ml quantities in 250 ml conical flasks.
3. Maintain the sterilized medium at 50°C in a water bath.
4. Collect rhizosphere soil sample and prepare dilution (10–1, 10–2, 10–3, 10–4, 10–5) in sterile
distilled water.
5. Dispense 15 ml of the medium into each plate and add 0.1 ml of dilution and spread it with L-rod.
6. Rotate the plate for uniform distribution and allow the media to solidify.
7. Incubate the plate at 37ºC for 24 hours.
Interpretation
Following incubation, clear zones are observed around the bacterial colonies surrounded by a
turbid background, which denotes the presence of phosphate solubilizers.
STANDARD QUALITATIVE ANALYSIS OF WATER BY MPN METHOD

Aim
To determine the presence of coliform bacteria in water by most probable number (MPN) method.
Presumptive Test
Principle
The presumptive test is specific for the detection of coliform bacteria present in the water sample.
Measured aliquots of water to be tested are added to a lactose fermentation broth containing an
inverted glass vial. Because these bacteria are capable of using lactose, their detection is facilitated
by the use of this medium. The lactose fermentation medium also contains a surface tension
depressant, bile salt, which is used to suppress the growth of organisms other than coliform
bacteria. Development of gas in any of the tubes is a presumptive evidence for the presence of
coliform bacteria in the sample.
Materials required
Sample: Samples included sewage, lake water, river water, well water, domestic usage water
Media: Lactose fermentation broth (Appendix III)
Equipments and other materials: Test tubes, test tubes rack, pipettes, etc.
Procedure
1. Set up three series consisting of three groups in a test tube rack. In each tube, label the water
sample and volume of sample inoculated.
2. Mix the water sample by shaking thoroughly.
3. Transfer 10 ml aliquots of water sample to the tubes labelled LB 2×, 10 ml.
4. Transfer 1 ml aliquots of water sample to the tubes labelled LB 1×, 1 ml.
5. Transfer 0.1 ml aliquots of water sample to the tubes labelled LB 1×, 0.1 ml.
6. Repeat the steps 2–5 for the other water samples.
7. Incubate all the tubes at 37°C for 48 hours.
Intrepretation and result
In the presumptive test a series of nine or twelve tubes of lactose broth are inoculated with
measured amount of water to see if the water contains any lactose-fermenting bacteria that produce
gas. If after incubation, gas is seen in any of the lactose broth, it is presumed that coliform are
present in water sample.
Confirmed Test
Principle
The presence of positive or doubtful presumptive test suggest that the water sample is not potable.
It requires that selective and differential media, such as, EMB or Endo agar be streaked from
a positive lactose broth tube obtained from the presumptive test. Eosin methylene blue (EMB)
contains the dye, methylene blue, which inhibits the growth of gram-positive organisms. In the
presence of an acid environment, EMB forms a complex that precipitates out onto the coliform
colonies producing dark centres and green metallic sheen. Endo agar is a nutrient media containing
the dye, fuchsin, which is present in decolorized state. In the presence of acid produced by the
coliform bacteria, fuchsin forms a dark pink complex that turns the E. coli colonies red and the
surrounding medium pink.
Materials required
Culture: 24-hour-old positive lactose broth cultures
Media: EMB agar plates, Endo agar plates (Appendix III)
Equipments and other materials: Inoculating loop, Bunsen burner, glasswares, marking pencil, etc.
Procedure
1. Label the covers of EMB plates or Endo agar plates with the source of the water sample.
2. Using a positive 24-hour lactose broth culture from the water series from the presumptive
test, streak the surface of EMB or Endo agar plates.
3. Repeat the above step, using lactose broth culture for different water series. Incubate all the
plate cultures in inverted position for 24 hours at 37°C.
If Endo agar is used, the colonies will be

are red in color and the surrounding medium
will also be red. This indicates the organism is
a fermenter of lactose which may be a coliform.
A result of a metallic sheen on the plate
If the organism is a non-fermenter of lactose
may indicate the presence of E.coli. If the
then the colonies will be colourless and will not
organism is a gram-negative lactose fer-
affect the colour of the surrounding medium.
menter (coliform) then “nucleated colonies”
(dark centres) will appear.
Figure 6.1 Results of the Endo agar or Levine’s EMB plate for the confirmed test
Interpretation
To confirm the presence of gram-negative lactose fermenters the next step is to inoculate media
such as Eosin methylene blue agar and Endo agar from positive presumptive tubes.
EMB agar contain metallic sheen bacteria which inhibits gram-positive bacteria, gram-
negative lactose fermenter which grow on this medium will produce nucleated colonies (dark
centre). Colonies of E. coli and E. aerogenes can be differentiated on the basis of size and presence
of a greenish metallic sheen. E. coli colonies on this medium are small and have this metallic sheen
whereas E. aerogenes colonies usually lack the sheen and are large.
Completed Test
Principle
The completed test is the final analysis of the water sample. It is used to examine the coliform
colonies which are isolated in the confirmed test. Isolated colonies, picked from confirmatory
test plate, are inoculated into a tube of lactose broth and streaked on nutrient agar slant to
perform Gram staining. Presence of gram-negative bacilli on microscope examination is further
confirmation of presence of E. coli and they are indicative of the positive completed test.
Materials required
Culture: 24-hour coliform positive EMB or Endo agar culture
Media : Lactose fermentation broth, nutrient agar slant (Appendix III)
Reagents : Crystal violet, Gram’s iodine, 95% ethanol, safranine (Appendix I)
Equipment and other materials : Bunsen burner, staining tray, inoculating loop, microscope,
glasswares and marking pencil, etc.
Procedure
1. Inoculate one lactose broth and one nutrient agar slant, from the same isolated E. coli colony
obtained from the EMB or Endo agar plates, from each of the experimental water sample.
2. Incubate all the tubes for 24 hours at 37ºC.
3. Using nutrient agar slant, prepare a Gram stain that shows positive result.
Interpretation
When the slide is observed microscopically, it shows the presence of Gram-negative short bacilli,
indicative of E. coli.
QUANTITATIVE ANALYSIS OF WATER BY MEMBRANE

FILTER TECHNIQUE
Aim
To determine the quality of water samples using the membrane filter technique.
Principle
Membrane filters capable of retaining microorganisms larger than 0.4 µm are frequently used
for analysis of water. These filters offer several advantages over the conventional multiple tube
method of water analysis. These include:
 Results are available in short period of time.
 Larger volume of sample can be processed.
 Because of high accuracy of the method, the results are readily reproducible.
A disadvantage of this method is the processing of turbid specimen that contains larger
quantities of suspended material. Particulate matter clogs the pores and inhibits the passage of
specific volume of water.
A water sample is passed through sterile membrane filter, i.e., it is forced into a special
filter apparatus connected to a suction flask. Following filtration, the filter disc containing the
trapped microorganisms is aseptically transferred to a sterile Petri dish containing an absorbent
pad saturated with selective, differential liquid medium. Following incubation, colonies present
on the filter are counted with the aid of microscope.
A total count of coliform bacteria determines the potability of water sources. Also the type
of faecal pollution, if any, is established by means of faecal coliform count, indicative of human
pollution, and faecal streptococcal count, indicative of pollution from other animal origins.
The ratio of faecal streptococci per ml of the sample is interpreted as follows: between 2 and
4 indicates human and animal pollution; less than 4 indicates human pollution; greater than
7 indicates poultry and live-stock pollution.
Materials required
Sample : Water samples, collected from sewage treatment plant.
Media : 20 ml of M-endo broth (tube), 20 ml tube of K–F broth, M-FC broth.
Equipment and other materials : Sterile membrane filtration apparatus, suction flask, sterile
membrane filter, absorbent pad, Petri dish, pipettes, beaker, forceps, water-proof tape, water-tight
plastic bags, water bath, microscope and marking pencil, sterile water blank, etc.
Procedure
1. Label the four water samples with their source and dilution (10–1, 10–2, 10–3, 10–4).
2. Using a pipette, aseptically perform serial dilution of the water sample.
3. Arrange the Petri dish into 3 sets of 5 plates. Label each set as follows:
a. For total coliform count: TCC (undiluted, 10–1, 10–2, 10–3, 10–4)
b. For faecal coliform count: FCC (undiluted, 10–1, 10–2, 10–3, 10–4)
c. For faecal streptococcal count: FSC (undiluted, 10–1, 10–2, 10–3, 10–4)
4. Use sterile forceps to place the absorbent pad in a Petri dish.
5. Use sterile pipettes to add:
a. 2 ml of M-endo broth to each pad in plates labelled TCC.
b. 2 ml of M-FC broth to each pad in plates labelled FCC.
c. 2 ml of K–F broth to each pad in plates labelled FSC.
6. Assemble the sterile paper-wrapped membrane filter unit aseptically.
a. Unwrap and insert the sintered glass filter into the neck of the 1 litre side arm of the
suction flask.
b. With sterile forceps, place a sterile membrane filter disc, grid side upon the sintered
glass platform.
c. Unwrap and carefully place the funnel suction on top of the filter disc. Using filter clamp,
place the funnel to the filter apparatus.
d. Attach a rubber hose on the side arm of the vacuum flask to a vacuum source.
7. Use the highest sample dilution (10–4) and place 20 ml of the dilution into the funnel and
start the vacuum. The entire sample gets filtered; wash the inner surface of funnel with
20 ml of sterile water.
8. Disconnect the vacuum. Unclamp the filter assembly.
9. With sterile forceps, remove the membrane filter and place on the media saturated pad in
Petri dish labelled TCC 10–4.
10. Aseptically place a new membrane on the platform and reassemble the filtration apparatus.
Repeat the steps 7 to 9 twice, add the filter disc to the plates labelled FCC and FSC.
11. Repeat steps 8 to 10 using 20 ml of the 10–3, 10–2, 10–1 dilutions and the undiluted sample.
Incubate the plates at 37ºC for 24 hours.
1. Collect the sample and make any necessary dilutions.

2. Select the appropriate nutrient or culture medium. Dispense the broth into a sterile Petri
dish, evenly saturating the absorbent pad.
3. Flame the forceps, and remove the membrane from the sterile package.
4. Place the membrane filter into the funnel assembly.
5. Flame the pouring lip of the sample container and pour the sample into the funnel.
6. Turn on the vacuum and allow the sample to draw completely through the filter.
7. Rinse funnel with sterile buffered water. Turn on vacuum and allow the liquid to draw
completely through the filter.
8. Flame the forceps and remove the membrane filter from the funnel.
9. Place the membrane filter into the prepared Petri dish.

10. Incubate at the proper temperature and for the appropriate time period.
11. Count the colonies under 10–15 X magnification. Confirm the colonies and report the results.
Result
Colonies produced by faecal coliform bacteria on M-FC medium are various shades of blue.
Nonfaecal coliform colonies are grey to cream-coloured. Normally, few nonfaecal coliform colonies
will be observed on M-FC medium because of selective action of the elevated temperature and
addition of rosolic acid salt reagent. Count colonies with a low-power (10 to 15 magnifications)
binocular wide-field dissecting microscope or other optical device.
DISSOLVED OXYGEN (DO)

Aim
To determine the dissolved oxygen (DO) of water samples.
Principle
Dissolved oxygen of water is of paramount importance to all living organisms and is considered
to be the lone factor, which to a great extent can reveal the nature of the whole aquatic system
at a glance, even when information on other chemical, physical and biological parameters is not
available. The presence of DO in water may be mainly attributed to two distinct phenomena:
a. Direct diffusion from the air.
b. Photosynthetic evolution by aquatic autotrophs.
The first one is purely a physical process and depends on the solubility of oxygen under
the influence of temperature, salinity, water movements, etc., whereas the latter is a biological
process and depends on the availability of light and the rate of metabolic process resulting in
diurnal fluctuation.
The estimation of DO is done by titrimetric method. The oxygen of the water combines with
manganous hydroxide, which on acidification liberates iodine equivalent to that of oxygen fixed.
This iodine is titrated by standard sodium thiosulphate solution using starch as an indicator.
MnSO4 + 2KOH Mn(OH)2 + K2SO4
Mn(OH)2 + O MnO(OH)2
MnO(OH)2 + 2H2SO4 + 2KI MnSO4 + K2SO4 + 3H2O + I2
Materials required
Sample: Water samples obtained from different sources
Reagents:
1. Sodium thiosulphate (0.025N) Dissolve 6.240g of sodium thiosulphate in freshly boiled
and cooled distilled water and dilute to 100 ml; add one pellet of NaOH as preservative.
2. Manganous sulphate solution Dissolve 3.46g of manganous sulphate in distilled water,
filter and dilute it to one litre.
3. Alkaline iodide azide solution Dissolve separately 700g of KOH and 15g of KI in distilled
water, mix them and make up to one litre. Dissolve separately by adding 10g of sodium
azide in 40 ml of distilled water. Add the solution to 960 ml of alkaline iodide reagent.
4. Starch indicator Filtered extract of boiled potato (or) dissolve one gram of starch (soluble)
in 200 ml of hot distilled water and add few drops of toluene as preservative.
5. Sulphuric acid (concentrated)
Equipments and other materials: Narrow-mouthed 250 ml BOD bottle, titration assembly,
three 2 ml pipettes, etc.
Procedure
1. Collect water samples without bubbling in the 250 ml BOD bottle.
2. Add 2 ml each of manganous sulphate and alkaline iodide azide solution in succession, right
at the bottom of the bottle with separate pipettes and replace the stopper.
3. Shake the bottle in the upside down direction at least six times.
4. Allow the brown precipitates to settle.
5. Add 2 ml of concentrated sulphuric acid and shake the stoppered bottle to dissolve the brown
precipitates.
6. Take 50 ml of the sample in a flask and titrate with thiosulphate solution (taken in the burette)
till the colour changes to pale straw.
7. Add two drops of starch solution to the above flask which changes the colour of the contents
to blue.
8. Titrate again with thiosulphate solution till the blue colour disappears.
S. No. Sample Burette reading Volume of sodium

thiosulphate solution
Intial Final
1 50 ml 0 2.5 ml
Calculations
The dissolved oxygen content of water (mg/L) is calculated by applying the equation:
(8* 1000 N )
DO (mg / L) v
V
where,
V = Volume of sample taken (ml)
v = Volume of titrant used
N = Normality of the titrant
* = 8 is the constant, since 1 ml of 0.025 N sodium thiosulphate solution is
equivalent to 0.2 mg of oxygen.
BIOLOGICAL OXYGEN DEMAND (BOD)
Aim
To determine the biological oxygen demand (BOD) of water samples.
Principle
Biological oxygen demand is the measure of oxygen used by microorganisms to decompose the
waste present in water. The word “waste”, used here, includes, any organic matter, such as dead
plants, leaves, grass clipping, manure, sewage or food waste. The more waste in the water, the
more bacteria and microorganisms will be found breaking it down. With an increased number of
organisms in the water, there will be a greater need for oxygen and thus a high biological oxygen
demand. As the waste is decomposed and dispersed, the biological oxygen demand will go down.
Nitrate and phosphate levels in water may increase biological oxygen demand levels. Nitrates
and phosphates are plant nutrients which may cause plant life and algae to grow quickly.
The increase in plant and algae growth is accompanied by an increase in the levels of waste in
the water, which in turn increases the bacterial population.
High biological oxygen demand levels negatively impact dissolved oxygen levels and may
cause aquatic animals, such as fish, to die. A biological oxygen demand level of 1–2 ppm is
very good, signifying very little waste in water. A level between 3 to 5 is considered moderately
clean. Once levels reach 6 to 9, the water is considered somewhat polluted and levels over
10 ppm indicate heavy pollution.
The BOD of water sample is generally measured by incubating the samples at 20°C for
5 days in the dark under aerobic conditions. In tropical and subtropical belts, where the
temperature and rate of metabolic activities are higher, incubation should preferably be done at
27°C for 3 days.
Materials required
Sample: Water samples obtained from different sources.
Reagents: 1N acid or 1N alkali solution, conc. sulphuric acid, sodium thiosulphate, starch solution.
Equipments and other materials: BOD bottles
Procedure
1. Water samples are filled in BOD bottles of known volume (250 ml) without air bubbles in
bottle after placing the stopper.
2. The pH of the water sample is adjusted to neutral using 1N acid or 1N alkali solution.
3. From each sample, a bottle is taken and it is incubated in BOD incubator at 27°C for three days.
4. For determination of initial dissolved oxygen (DO), the remaining set of bottles are taken
and 2 ml of manganous sulphate and alkaline azide solution are added to each bottle.
5. The bottle is then shaken so that the brown precipitate is formed.
6. To this bottle, 2 ml of concentrated sulphuric acid is added and stoppered, and the bottle
is shaken to dissolve the brown precipitate.
7. 50 ml of water from each sample is taken and titrated against sodium thiosulphate solution
taken in the burette, till the colour changes to pale yellow.
8. Two drops of starch solution are added to the above flask in which the colour changed from
pale yellow to blue.
9. This is titrated against sodium thiosulphate solution till the blue colour disappears. This is
the initial DO.
10. After 3 days of incubation, the bottles are taken from the BOD incubator and the final DO
is determined as that of the initial DO.
Calculations
The BOD value is calculated using the formula:
BOD (mg/L) =D1 – D2
where,
D1 = Initial DO in the sample

D2 = Final DO (after 3 days of incubation)
For determining the dissolved oxygen content, the following formula is used:
(8* ×1000 × N )
DO (mg / L) = ×v
V
where,
V = Volume of sample taken
v = Volume of titrant used
N = Normality of titrant
S. No. Sample Burette reading Volume of DO µg/ml

sodium
thiosulphate (ml)
1 Tap water 0 2.5 2.5 10
2.5 5
8×1000×0.025
×2.5=10
50
CHEMICAL OXYGEN DEMAND (COD)

Aim
To determine the COD of the given water sample.
Principle
Chemical oxygen demand is the oxygen required by organic substances in water to get oxidized
by chemical oxidation, thereby leading to the increase of pollution by the discharge of large
amounts of various chemically oxidizable organic substances of different nature entering the
aquatic systems; BOD alone does not give a clear picture of the organic matter content of the
water sample. In addition, the presence of various toxicants in the sample may severely affect
the validity of the BOD test. Hence, chemical oxygen demand (COD) is a better estimate of
the organic matter, which needs no sophistication and is time-saving. However, COD, i.e., the
oxygen consumed (OC) does not differentiate the stable organic matter from the unstable form.
Therefore, the COD values are not directly comparable to that of BOD.
The amount of organic matter in water is estimated by their oxidizability by chemical oxidants,
such as potassium permanganate or potassium dichromate (the constituents, carbon and hydrogen
are oxidized and but not nitrogen). In the permanganate method, the organic matter is first
oxidized with a known value of KMnO4 and then the excess of oxygen is allowed to react with
potassium iodide to liberate iodine in amounts equal to the excess oxygen, which is estimated
titrimetrically with sodium thiosulphate solution using starch as an indicator.
Materials required
Sample : Water samples obtained from different sources
Reagents:
1. Potassium dichromate solution (0.1N) 3.676 g of K2Cr2O7 in one litre of distilled water.
2. Sodium thiosulphate (0.1M) 15.811 g of sodium thiosulphate in two litres of distilled water.
3. Sulphuric acid (2M) 10.8 ml of conc. H2SO4 in 100 ml of distilled water.
4. Potassium iodide solution (10%)
5. Starch solution (1%)
Equipment and other materials: Water bath, titration assembly, 100 ml conical flasks, water blanks
Procedure
1. Take three 100 ml conical flasks and pour 50 ml of water sample in each (i.e., in triplicate).
2. Simultaneously run distilled water blank standards (also in triplicate).
3. Add 5 ml of K2Cr2O7 solution in each of the six flasks.
4. Keep the flasks in water bath at 100°C (boiling temperature) for one hour.
5. Allow the samples to cool for ten minutes.
6. Add 5 ml of potassium iodide in each flask.
7. Add 10 ml of H2SO4 in each flask.
8. Titrate the contents of each flask with 0.1 M sodium thiosulphate until the appearance of
pale yellow colour.
9. Add 1 ml of starch solution to each flask (the solution turns blue).
10. Titrate it again with 0.1 M sodium thiosulphate until the blue colour disappears completely.
Calculations
COD (mg/L) per litre of the water sample is calculated by applying the formula:
COD of water sample (mg/L) = 8 × C × (B – A) / S

where,
C = Concentration of titrant (mmol/litre)
A = Volume of titrant used for blank (ml)
B = Volume of titrant used for sample (ml)
S = Volume of water sample taken (ml)
S.No Sample Burette readings Volume of
thiosulphate
1. Blank 0 5.4 5.3
5.4 10.9
2 Tap water 0 5.8 5.7
5.8 11.7
8×0.1×(5.7 – 5.3)×1000
= 64 µg ml
50
TOTAL SUSPENDED SOLIDS

Total suspended solids (TSS) is a water quality measurement, usually abbreviated as TSS. This
parameter was at one time called non-filterable residue (NFR), a term that refers to the identical
measurement—the dry-weight of particles trapped by a filter, typically of a specified pore size.
However, the term “non-filterable” suffered from an odd (for science) condition of usage—in
Oceanography, for example, “filterable” means the material retained on a filter, so non-filterable
would be the water and particulates that passed through the filter. In dictionary definition,
“filterable” means just the opposite—the material passed by a filter, usually called total dissolved
solids or TDS. Thus, in chemistry, the non-filterable solids are the retained material called the
residue.
Aim
To determine the total suspended solids in water sample.
Principle
TSS of a water sample is determined by pouring a carefully measured volume of water (typically
one litre; but less if the particulate density is high, or as much as two or three litres for very
clean water) through a pre-weighed filter of a specified pore size, then weighing the filter again
after drying to remove all water. Filters for TSS measurements are typically composed of glass fibres.
The gain in weight is a dry weight measure of the particulates present in the water sample expressed
in units derived or calculated from the volume of water filtered (typically milligrams per litre or mg/L).
If the water contains an appreciable amount of dissolved substances (as certainly would be
the case when measuring TSS in sea water), these will add to the weight of the filter as it is dried.
Therefore, it is necessary to “wash” the filter and sample with deionized water after filtering the
sample and before drying the filter. Failure to do this step is a fairly common mistake made by
inexperienced laboratory technicians working with sea water samples, and this will completely
invalidate the results as the weight of salts left on the filter during drying can easily exceed that
of the suspended particulate matter.
Materials required
Sample: Water sample obtained from different sources.
Equipment and other materials: Glass fibre filters, filtering flask, sample bottle.
Procedure
1. Before sampling, prepare glass fibre filters by first soaking them in distilled water, drying
them at 103°C, and weighing and recording their weights (Figure 6.2).
2. Place the dried, weighed glass fibre filter onto a filtering flask—wrinkled side up. Shake the
sample bottle first, then pour in the water and turn on the pump. (The amount of water you
need to filter may change according to water conditions. Start with 100 ml. Use less volume
if the filter gets clogged too quickly and more if the water filters through very fast). Record
the volume of water filtered.
3. Dry the filter at 103–105°C. Let it cool to room temperature, and weigh it. Dry it, cool it and
weigh it again. Continue until the fibre reaches a constant weight. Record the end weight.
4. The increase in weight represents TSS. Calculate TSS by using the following formula:
TSS (mg/L) = ([A–B]×1000)/C
where,
A = End weight of the filter
B = Initial weight of the filter
C = Volume of water filtered
Sample volume Final weight (A) Initial weight (B) Concentration

(ml) (g) (mg/ml)
100
Dry at 103°C.
Read and record
weights.
Soak glass fibre

filters in distilled H2O.
Place fibre filter on

Shake sample bottle, filtering flask,
pour into funnel and wrinkled side up.
turn on pump.
Start with 100 mL H2O.

Record volume.
Calculate TSS.
Dry filter at 103–105°C.

Let it cool to room
temperature and weigh again.
Continue drying and

weighing until filter
reaches a constant
weight.
Figure 6.2 TSS analysis

DECOLORIZATION OF DYE
AND DYE-CONTAINING EFFLUENTS
With the advent of industrialization and urbanization, exploitation and utilization of resources
has reached the maximum level. Current population explosion urges enlargement of industrial
sectors resulting in pollution of water, air and soil. As prevention of pollution is not possible,
control of pollution can be practised effectively.
The discharge of pollutants into the environment from various industries is posing a threat
to life, resulting in great environmental stress.
Currently, large textile dyeing industries utilize a considerable amount of water in its
production process that eventually results in waste water with large amount of dye particulates or
molecules. This waste water, discarded to large water sources, causes a drastic decrease in oxygen
concentration due to the presence of hydrosulphides in certain dyes that can react with oxygen.
It can also block the passage of light into the water body by increasing the turbidity, which is
detrimental to water ecosystem.
Several methods are used in the treatment of textile effluents to achieve decolorization,
including physiochemical methods like filtration, coagulation and flocculation. It is thus important
to explore the possibilities of isolating efficient aerobic degraders for use in decolorization and
biotreatment of textile effluents.
Microbial populations have an amazing and extensive capacity to degrade a variety of
organic compounds. Versatile metabolic activities of microorganisms have played a key role in
biodegradation of various toxic organic compounds.
Aim
To isolate and screen microbial strains for their ability to decolorize dyes aerobically and
dye-containing effluents.
Principle
Azo dyes (N = N group) form the largest class of synthetic dyes with a variety of colour and
structure. These dyes account for approximately 60–70% of all dyes used in food and textile
manufacture. The worldwide production of these dyes is currently estimated at 4,50,000 tons/
year with almost 50,000 tons/year lost in effluent during application and manufacture. At the
time of production and application about 250% of these dyes are lost as waste effluents. Congo
red (sodium salt of benzidinediazobis-1naphthyl-amine-4-sulphonic acid) has been reported to be
a carcinogenic direct diazo dye used for colouration of paper products. The dye infested soils are
detrimental to the growth of plants also. Extensive work has been carried out on the pollution
problems associated with the discharge of dye effluent from industries. It has been documented
that the safe method for azo dye biodegradation is combined aerobic treatment . Many organisms
such as Bacillus, E. coli, Klebsiella, Enterobacter, Pseudomonas and a group of fungi, yeast have been
studied for their decolorization of congo red dye.
Materials required
Culture: Pseudomonas aeruginosa culture
Media: Nutrient broth (Appendix III)
Equipment and other materials: colorimeter, cuvette, eppendorf tubes, conical flasks, etc.
Procedure
1. Sterile nutrient broth is prepared, sterilized and inoculated with the bacterial culture and
incubated at 37°C for 24 hours.
2. About 50 ml of raw effluent is taken in a clean conical flask and to it, 5 ml of the 24-hour
bacterial culture is added and incubated for about 14 days.
3. The colour change can be checked with the colorimeter at 650–720 nm and the percentage
of decolorization is calculated using the formula:
Percentage decolorization = [(A0 – At)/ A0] × 100
where,
A0 = Absorbance of raw effluent
At = Absorbance of treated effluent (14 days after microbial inoculation)
Absorbance value of raw effluent = 0.40
OD value of effluent treated with Pseudomonas = 0.26
NITROGEN CYCLE
The fixation, interconversions and turnover of nitrogen form a global biogeochemical cycle
(Figure 6.3). All the critical steps in this nitrogen cycle are carried out almost exclusively by
microorganisms. The microbial conversions of nitrogen are essential for soil fertility and the
growth of plants. The four distinct phases of this cycle are:
1. Ammonification Sequential degradation of nitrogenous organic compounds with the
release of ammonia.
2. Nitrification Oxidation of ammonia to nitrite (NO ) and then to nitrate (NO ) a form
–
2
–
3
that is assimilated by plants.

3. Denitrification Reduction of nitrates to gaseous nitrogen (N ↑). 2
4. Nitrogen fixation Chemical combination of free nitrogen (N ↑) with other elements to

2
form fixed nitrogen.

Figure 6.3 Nitrogen cycle
AMMONIFICATION
Aim
To demonstrate the liberation of ammonia from nitrogenous organic compounds by soil
microorganisms.
Principle
Most nitrogen fixed into biological material, such as plants and animals, is not released until their
death. Ammonification occurs during decomposition and putrefaction of proteinaceous organic
matter. Hydrolysis of proteins and nucleic acids releases simpler compounds, such as amino acids
and nitrogenous bases, which can be utilized by microorganisms for fermentation or respiration.
Bacteria belonging to the genera, Bacillus, Pseudomonas and Clostridium, are soil inhabitants that
carry out ammonification. These bacteria excrete proteolytic enzymes that hydrolyse the proteins
of plant and animal origin into their constituent amino acids. The amino acids are subsequently
enzymatically deaminated, with the release of ammonia.
Materials required
Sample : 0.1g of garden soil (different sample)
Media: Peptone broth tubes (Appendix III)
Reagent: Nessler’s reagent (Appendix I)
Equipment and other materials : Bunsen burner, inoculating loop, spot plates, Pasteur pipette, etc.
Procedure
1. Label each of the peptone broth tubes with the soil sample to be inoculated.
2. Add 0.1 g of soil sample to the peptone broth.
3. Incubate the tubes for 7 days at 25°C.
4. Test each culture for the presence of ammonia on days 3, 5 and 7, following inoculation.
5. Using a Pasteur pipette, place one drop of Nessler’s reagent in the depressions on the spot
plate. Add a loopful of culture from each tube into separate depressions on the spot plate.
Mix and observe for colour change.
Interpretation
Development of pale yellow colour shows the presence of small amount of ammonia (1+), deep
yellow (2+) and a brown precipitate (3+) show the presence of large amount of ammonia.
NITRIFICATION
Aim
To demonstrate the enzymatic conversion of ammonia to nitrates by soil microorganisms.
Principle
The conversion of ammonia to nitrate is an oxidative process brought about by two highly specialized
chemolithotrophic bacteria in the soil. Nitrosomonas oxidizes ammonia to nitrite, which is further
oxidized to nitrate by Nitrobacter. These bacteria are of prime importance in soil fertilization because
their combined activities convert ammonia to nitrate, the principal nitrogenous source for plants.
NH+4+→ O2 Nitrosomonas NO2+→ H2O
No–2+→ O2 Nitrobacter NO3
Materials required
Sample : 0.1 g of soil samples from alkaline and acidic garden soil
Media : Ammonium sulphate broth (25 ml) (Appendix III)
Reagents : Nessler’s reagent, Trommsdorf’s reagent (Appendix I), conc. sulphuric acid
Equipment and other materials : Bunsen burner, spot plate, Pasteur pipette, marker, etc.
Procedure
1. Label each of the flasks of ammonium sulphate broth with the soil sample to be inoculated.
2. Add 0.1 g of soil samples to the labelled flasks. Shake vigorously.
3. Incubate all the flasks for 3 weeks at 25ºC.

4. Test for the presence of ammonia by using Nessler’s reagent. A yellow colour indicates that
ammonia was not oxidized to nitrite. No colour change indicates the absence of ammonia
and therefore, nitrite should be present.
5. Test for the presence of nitrite by using Trommsdorf’s reagent and sulphuric acid.
Interpretation
The presence of a blue-black colour is indicative of the presence of nitrite. No colour change is
indicative of the absence of nitrite.
DENITRIFICATION
Aim
To demonstrate the reduction of nitrates to nitrogen gas by soil microorganisms.
Principle
Nitrate can serve as an electron acceptor for many aerobic bacteria when aerobic conditions are
present. Some bacteria, such as Escherichia coli, only reduce nitrate to nitrite ions, whereas others,
such as Paracoccus reduce nitrates and nitrites to nitrogen gas. Denitrification is of major ecological
importance in the soil. It represents a net loss of nitrogen back to the atmosphere, depleting the
soil of needed nutrients essential for plant growth.
NO3– → NO2– → NO → N2
In this experiment, nitrate broth is used in which nitrate is the substrate, and the evolution
of nitrogen gas is identified by the presence of air bubbles in the Durham’s tubes.
Materials required
Sample: 0.1 g samples of rich and poor soil
Media: Nitrate broth tubes (5 ml) containing Durham’s tubes (Appendix III)
Equipment and other materials: Bunsen burner, test tube rack, marker, etc.
Procedure
1. Add aseptically 0.1 g of soil sample to the sterilized and cooked nitrate broth tubes with
Durham’s tubes . Do not shake the tubes during inoculation.
2. Label each tube appropriately with the inoculated soil sample.
3. Incubate for 2 weeks at 25oC.
4. Observe for the presence of air bubbles (N ↑) in Durham’s tubes.
2
Result
Presence of air bubbles indicates the presence of nitrogen gas.
ENUMERATION OF MICROORGANISMS FROM

WOOD AND PAINT
Aim
To isolate and characterize bacteria and fungi from wood and paint.
Principle
Paints are generally used to protect wood surface. Wood is a natural raw material and fully
biodegradable. Biodegradation of natual components is catalysed by enzymes. The biological
degration of wood in nature is based on enzymes produced by various wood degrading
microorganisms. These enzymes are typically inducible, i.e., only produced upon contact with
available substrate. The main enzymes are cellulose, hemicellulose, lignases.
Materials required
Sample: Wood pieces, paint
Media: Nutrient agar, potato dextrose agar, peptone broth (Appendix III)
Equipment and other materials: Petri plates, pipettes, test tubes, etc.
Procedure
1. To 20 ml of peptone broth prepared, 1 g of wood pieces is added.
2. To another 20 ml of peptone broth, 1 ml of paint is added.
3. From both the broths, 1 ml is taken and added to 99 ml of sterile distilled water in a separate
conical flask.
4. 1 ml of the above sample is taken from the conical flask and serially diluted (up to 10–7
dilution) with 9 ml of sterile distilled water. This is done for both the samples.
5. The dilutions are plated onto nutrient agar and potato dextrose agar.
6. The nutrient agar plates are incubated at 37ºC for 24 hours, and the potato dextrose agar
plates are incubated at room temperature and observed for growth.
Result
In nutrient agar plates colonies were found. In PDA fungi will be seen.
ASSAY OF MICROORGANISMS FROM BIOMEDICAL WASTE

In hospital and clinics, wounded patients are cleaned with cotton and injected with medicine
intravenously. After the treatment, the cotton and the needles are thrown off; these are called
the biomedical wastes. It is hazardous and harbour dangerous pathogens. The biomedical waste
should be decontaminated and must be destroyed properly, if not, they will spread contagious
diseases. Hence, assaying the biomedical waste in the laboratory becomes important.
Aim
To assay microorganisms from biomedical waste.
Principle
“Bio-medical waste” means any waste, which is generated during the diagnosis, treatment or
immunisation of human beings or animals or in research activities pertaining thereto or in the
production or testing of biologicals and including human tissues, organs, body parts.
Materials required
Sample: Biomedical waste
Media: Nutrient agar, Saboraud’s dextrose agar, blood agar, MacConkey agar, IMViC media
(Appendix III)
Reagents: Crystal violet, safranine, Gram’s iodine
Equipment and other materials: Petri plates, test tubes, Bunsen burner, inoculating loop,
incubator, etc.
Procedure
1. The biomedical wastes such as needles and cotton are collected.
2. The samples are dipped into sterile broth and the broth is incubated at 37ºC for 24 hours.
3. The samples are streaked on fungal media (SDA) and incubated at room temperature for
3–5 days.
4. The incubated broth is observed for turbidity; if there is turbidity it is further processed to
identify the bacterial species.
Processing: A loopful of culture is taken and Gram’s staining is performed. A loopful of
the same culture is streaked on nutrient agar, blood agar and MacConkey agar. IMViC tests
are also performed.
Result
Based on gram staining we can find whether it is gram-positive or gram-negative, further it is
confirmed by biochemical test.
ISOLATION AND CULTURE OF ALGAE

Algal cultures are essential when conducting competition studies, bioassays, assessment of
zooplankton food preferences, and determination of algal life histories. They are also necessary for
molecular systematic work. Algal cultures may be unialgal, which means they contain only one
kind of algae, usually a clonal, or axenic, meaning that they contain only one algae and no bacteria,
fungi or protozoa. There are four major techniques for obtaining unialgal isolates—streaking,
spraying, serial dilution and single-cell isolations. Streaking and spraying are useful for single-
celled, colonial, or filamentous algae that will grow on an agar surface; cultures of some flagellates,
such as Chlamydomonas and Cryptomonas may also be obtained by these procedures. However, many
flagellates, as well as other types of algae, must be isolated by single-organism isolations or serial-
dilution techniques. Spraying and single-organism isolation techniques are given is this section.
SPRAYING
Aim
To isolate unialgal isolates by using spraying techniques.
Principle
A fine or atomized spray of cells can be used to inoculate agar plate, in general liquid cell
suspension is atomized with forced sterile air so that cells are scattered onto the plate. A stream
of compressed air is used to disperse algal cells froma mixture onto the surface of a petriplate
containing growth medium solidified with agar.
Materials required
Petri plate, compressed air, Pasteur pipettes, growth medium
Procedure
In this technique, a stream of compressed air is used to disperse algal cells from a mixture onto the
surface of a Petri plate containing growth medium solidified with agar. A Petri plate is held about
18 inches from the touching tips of two Pasteur pipettes. One of these is attached to an airline
via a hose, and mounted onto a ring stand. The other pipette is suspended tip-up into a container
holding the algal mixture. The air flow from the first pipette creates a vacuum that draws a stream
of algae-containing liquid up from the container through the second pipette. The airflow also sprays
the suspended algae through the air, where they can be intercepted by the agar plate.
Result
Plates are kept for incubation. After colony formation, cells are selected and further inoculated.
SINGLE CELL/COLONY/FILAMENT ISOLATIONS

Aim
To isolate single cell and filamentous algal isolates.
Materials required
Sample: Water sample is collected from ponds and microalgae.
Media: Bold’s Basal medium (BBM), SD11, DYIII, (Appendix III)
BBM is widely used artificial freshwater medium, especially for growing green algae.
The medium lacks vitamins and some of the trace metal concentrations are relatively high, making
the medium the medium unacceptable for growth of many non-green algae. Six macronutrient stock
solutions, an alkaline EDTA solution, an acidified iron solution, a boron solution and a trace metals
solution are individually prepared. The usual recipe is based upon 400 ml stock solutions, but for
comparison with other media, we have included 1 litre stock solutions.
To prepare the final medium, begin with 940 ml of dH2O and add 10 ml of the first six stock
solutions. Add 1 ml each of the alkaline EDTA, acidified iron, boron and trace metals solutions.
Autoclave. The final pH should be 6.6.
Equipment and other materials: Petri dish, dissecting microscope, micropipette tip.
Freshwater Growth Media
1. BBM or Bold’s basal medium a chemically defined medium is good for many green algae.
2. Soil water is undefined and used for algae whose nutritional requirements are unknown, or
which will not grow on simple inorganic media. The soil should be from a site where herbicides
have not recently been used. Sometimes, it is advisable to add a dried pea to the medium
before autoclaving.
3. SD11 is a defined medium that is somewhat more complex than BBM; it contains a vitamin
mixture. This medium is suitable for many green algae.
4. DYIII is a defined medium to which vitamins are often added; it is used for the culture of
chrysophytes and cryptomonads as well as some dinoflagellates.
Procedure
1. Preparation of micropipettes The first step in this procedure is to prepare a number
of micropipettes from glass Pasteur pipettes. A pipette is held in both hands; the tip end
is held with a forceps so that the glass near the tip is within the flame of a Bunsen burner.
The pipette is held in the flame only until the glass becomes slightly soft. This is determined
by testing for flexibility by moving the tip with the forceps. Then the pipette is removed
from the flame and pulled out straight or at an angle so that there is a bend. The pipette
must always be removed before pulling it! The forceps is used to break the tip. The diameter
of the finely pulled tip can be varied by changing the speed of pulling; the diameter of a slowly
pulled tip will be greater than that of a rapidly pulled tip. A narrow diameter tip is used
when trying to isolate very small algae, but a larger diameter tip is required for large cells.
The diameter of the pipette tip must be matched to the size of the algal cells to be isolated.
2. Prepare a multiwell plate with sterilized media in each well.
3. Place multiple drops of sterilized media or water onto the inside surface of a sterile Petri plate.
4. Attach a micropipette to a length of rubber tubing; attach a ethanol-sterilized mouthpiece
to the other end of the tubing, and put the mouthpiece in your mouth.
5. Place a Petri dish of algae on the stage of a dissecting microscope and locate the single
cell/colony/filament to be isolated.
6. Then find the tip of the micropipette and move it to the vicinity of the algae, then suck it
up into the pipette tip, then stop the suction. Try to avoid sucking up any other algae.
7. Now, remove the pipette from the dish, then blow the liquid and algae into one of the drops
of water on a Petri plate.
8. Break off and dispose off the portion of the micropipette tip that contained liquid; this has
been contaminated. The micropipette tipils used till all the algae sucked in the pipette is
inoculated onto the plate.
9. Now, use the micropipette to transfer the isolated algae from the first drop into a series of
fresh drops. This is a washing procedure that helps remove contaminants.
10. After transfer through 5–10 drops, transfer the algae into a well of the multiwell plate
holding liquid growth medium suitable for that particular species.
11. Repeat the procedure. Usually several attempts are made because not all isolated algae will
continue to grow, or some may be contaminated with other algal cells.
NOTE A particularly effective means of obtaining unialgal cultures is isolation of zoospores

immediately after they have been released from parental cell walls, but before they stop swimming
and attach to a surface. Recently released zoospores are devoid of contaminants, unlike the surfaces
of most algal cells. But catching zoospores requires a steady hand and experience. Filaments can
be grabbed with a slightly curved pipette tip and dragged through soft agar (less than 1%) to
remove contaminants. It is best to begin with young branches or filament tips which have not
yet been extensively epiphytized.
Antibiotics can be added to the growth medium to discourage growth of contaminating
cyanobacteria and other bacteria. Addition of germanium dioxide will inhibit the growth of diatoms.
Result
Single cell or filament isolates will be grown on the suitable medium.
7
FOOD MICROBIOLOGY
METHYLENE BLUE REDUCTION TEST

Aim
To determine the quality of milk sample by methylene blue reduction test.
Principle
Methylene blue reduction test is an enzymatic test to determine the quality of milk sample. Milk is
an excellent medium for the growth of many bacteria. Milk is contaminated with microorganisms
right from the time it is drawn from the cow and further contamination occurs during handling
and processing.
Milk if contaminated, will contain a markedly decreased concentration of dissolved oxygen
because of the vigorous growth of organisms. In other words, the oxidation–reduction potential
of the milk is greatly lowered. The rate of oxidation–reduction potential shift to negative value is
directly proportional to the bacterial population. To determine the oxidation–reduction potential
and thereby, the concentration of microorganisms in milk, an oxido-reduction dye (methylene
blue, resazurin) can be employed. Methylene blue loses its colour in an anaerobic environment
and is said to be reduced. The blue colour of methylene blue is transformed to colourless
leuco-methylene blue due to reduction in the anaerobic environment. This colour change forms
the basis of this test.
Raw milk contains a large population of enteric bacteria and Streptococcus lactis, which are the
potent reducers of the dye. The speed at which reduction occurs following addition of methylene
blue in the milk sample indicates the milk quality. This determination is made as follows:
Materials required
The necessary equipment consists of test tubes with rubber stoppers, a pipette or dipper graduated
to deliver 10 ml of milk and a water bath for maintaining the samples at 35 to 37ºC. The bath
should contain a volume of water sufficient to heat the samples to 35ºC within 10 minutes after
the tubes enter the water and should have some means of protecting the samples from light
during the incubation period. If a hot-air chamber is used, the samples should be heated to 35ºC
in a water bath since warm air would heat the milk too slowly.
The dry tablets contain methylene blue thiocyanate and may be obtained from any of the
usual laboratory supply houses. They should be certified by the Commission on Standardization
of Biological Stains. The solution is prepared by autoclaving or momentarily boiling 200 ml of
distilled water in a light resistant (amber) stoppered flask and then adding one methylene blue
tablet to the flask of hot water. The tablet should be completely dissolved before the solution is
cooled. The solution may be stored in the stoppered, amber flask or an amber bottle in the dark.
Fresh solution should be prepared weekly.
Reduction time Quality of milk

Less than 30 minutes Very poor
30 minutes to 2 hours Poor
2–6 hours Fair
6–8 hours Good
More than 8 hours Excellent
Procedure
1. The test tubes are labelled as raw milk and pasteurized milk.
2. Using different pipettes, 10 ml of different milk samples are transferred into appropriately
labelled test tubes.
3. About 1 ml of methylene blue (1: 25,000) dye is then added to each test tube.
4. The test tubes are closed air-tight and are gently inverted few times and placed in a water
bath at 37°C.
5. The time of incubation is recorded. Decolorization of methylene blue is checked at every
15 minutes for the first three hours. If it is not decolorized, then hourly readings, for
6 hours, are taken.
Observation
The methylene blue in the milk sample got reduced in 6 to 8 hours.
Result
The time taken (6 to 8 hours) for the dye reduction indicates that the milk is of good quality.
Food Microbiology 221
PHOSPHATASE TEST FOR PASTEURISATION IN LIQUID MILK

Aim
Milk testing and quality control of any milk processing industry.
Principle
Phosphatase, an enzyme in milk is destroyed during pasteurization. The test is based on the above
principle to judge the efficiency of pasteurization. Any phosphatase present in the milk splits the
substrate, p-nitrophenyl phosphate to give p-nitrophenol, which is highly coloured in alkaline
solution. The test does not apply to sour milk and milk preserved with chemical preservatives.
Materials required
Sample:
i. At the farm
Quality control and assurance must begin at the farm. This is achieved through farmers
using approved practices of milk production and handing, and observation of laid down
regulations against adulterations of milk etc.
ii. At milk Collection Centres
All milk from different farmers or bulked milk from various collecting centres must be
checked for ‘wholesomeness’ bacteriological and chemical quality.
iii. At the Dairy factories
Milk from individual farmers or bulked milk from various collecting centres.
iv. Within the Dairy factory
Once the dairy factor has accepted the farmers’s milk it has the responsibility of ensuring
that the milk is handled hygienically during processing. It must carry out quality assurance
test to ensure that the products produced confirm to specified standards as to the adequacy
of effect of processes applied and the keeping quality of manufactured products. A good
example is the phosphatase test used on pasteurised milk and the acidity.
Reagents: All reagents should be of analytical grade.
1. Buffer solution 1.5 g of sodium bicarbonate and 3.5 g of anhydrous sodium carbonate
dissolved in water and made up to one litre. Store in a refrigerator and discard after 1
month.
2. Disodium p-nitrophenylphosphate. The solid substrate must be kept in the refrigerator.
3. Buffer–substrate solution—Weigh accurately 0.15 g of substrate (disodium p-nitrophenyl
phosphate) into a 100 ml measuring cylinder and make up to 100 ml with buffer solution.
The solution should be stored in refrigerator and protected from light. The solution should
give a reading of less than the standard marked 10 on comparator disc APTW or APTW
7 when viewed through a 25 mm cell (distilled water is used as a blank). The solution
must be discarded after one week.
Equipment and other materials: A Lovibond comparator with stand for work in reflected
light, a Lovibond comparator disc APTW or APTW 7, two fused glass cells of 25 mm depth,
a water bath or incubator capable of being maintained at 37.5ºC 0.5 ºC, 1 ml pipette and
5 ml pipette, 1 litre graduated flask, 100 ml measuring cylinder, test tubes, nominal size
150/16 mm with rubber stoppers, etc.
Precautions
a. All glassware must be cleaned before use. Cleaning should be done by soaking in chromic
acid solution prepared by slowly adding 4 volumes of conc. H2SO4 to 5 volumes of 8% pot
as. dichromate. After cleaning in chromic acid glassware must be rinsed in warm water and
distilled water and finally dried. Glassware used for the test must not be used for any other
purpose and must be kept apart from other apparatus in the laboratory.
b. Test must not be carried out in direct sunlight.
c. A fresh pipette must be used for each sample of milk. Pipettes must not be contaminated
with saliva.
d. The sample of milk should be examined as soon as possible after arrival at the laboratory.
If not examined immediately it must be kept at a temperature between 3ºC and 5ºC until
examined. The sample must be brought to room temperature immediately before being tested.
Procedure
Into a test tube pipette 5 ml of buffer substrate solution, stopper and bring the temperature to
37ºC. Add 1 ml of test milk to it shake and replace stopper, incubate at 37ºC for 2 hrs. Incubate
one blank prepared from boiled milk of the same type as that undergoing the test with each series
of sample. Remove the tubes after 2 h and the content should be well mixed. Place the boiled
milk blank on left hand side of the comparator stand and test sample on the right. Take reading
in reflected light by revolving the disc until the test sample is matched. Record readings falling
between two standards by affixing a plus or minus sign to the figure for the nearest standard.
Interpretation
The test is considered satisfactory if it gives a reading of 10 µg or less of p-nitrophenyl per ml of
milk. Properly pasteurized milk gives no discernible colour.
TURBIDITY, COLONY AND COLIFORM TESTS

FOR PASTEURIZED MILK
Aim
The turbidity test is designed to scientifically and objectively judge the solubility of a sample to
the solvent specified in clarity of solution in purity in the individual monograph.
Turbidity test
Principle
The turbidity test depends upon the denaturation of proteins of milk especially albumin after
sterilization. When solutions of inorganic salts or acids are added, albumin separates with the
casein. The sample on treatment with ammonium sulphate, filtration and heating of the filtrate
shows turbidity due to presence of albumin on account of insufficient heat treatment. If milk has
been sterilized properly all albumin will have been precipitated and no turbidity will be produced.
The test is not suitable for UHT milk.
Materials required
Reagents: Ammonium Sulphate AR
Equipments and other materials: Conical flask, 50 ml, graduated cylinder, 25 ml, test tubes
150 /16 mm, Funnels, (6 cm diameter), beaker, 400 ml, whatman No. 12 or equivalent| (12.5
cm folded filter paper) pipette, 20 ml, etc.
Procedure
Pipette 20 ml of milk in a 50 ml conical flask, add 4.0 ml ammonium sulphate. Shake the flask
till the ammonium sulphate is completely dissolved. Allow the mixture to settle for 5 min., filter
through a folded filter paper in a test tube. Keep about 5 ml of the above filtrate in a boiling water
bath for 5 min. Cool the tube in a beaker of cold water and examine the contents for turbidity
by moving the tube in front of an electric light shaded from the eyes of the observer.
Interperetation
The milk is considered sterilized when the filtrate shows no turbidity.
Standard plate count (SPC)

The total bacterial forming units per ml of milk was calculated using the count made by adding
1 ml of milk sample into the following formula (IDF 1991). Sterile test tube having 9 ml
peptone water. After thoroughly mixing, the sample was serially diluted up to 10–7 to 1:10 and
duplicate samples (1 ml) were pour plated using 15–20 ml standard plate count agar solution
and mixed thoroughly. The plated sample was allowed to solidify and then incubated at 30ºC
for 48 hours. Colony counts were made using colony counter.
Coliform count (CC)

One ml of milk sample was added into sterile test tube having 9 ml peptone water. After mixing,
the sample was serially diluted up to 1: 10-5 and duplicate samples (1 ml) were pour plated using
15–20 ml Violet Red Bile Agar solution (VRBA). After thoroughly mixing, the plated sample was
allowed to solidify and then incubated at 30ºC for 24 hours. Finally, colony counts were made
using colony counter. Typical dark red colonies were considered as coliform colonies.
ISOLATION OF FOOD SPOILERS

Aim
To isolate food spoilers from meat, milk, cereals and bread.
Principle
The presence of microorganisms in food is considered harmful in some cases, while in others, it
is definitely beneficial. Certain microorganisms are necessary in the preparation of foods, such as
cheese, pickles, sauerkraut, yoghurt and sausage. The presence of other microorganisms, however,
is responsible for serious and sometimes fatal food poisoning and toxicity as well as spoilage.
As with milk or water, the presence and number of coliform bacteria and other enteric organisms
in food is indicative of faecal contamination and may suggest the presence of pathogens.
Materials required
Samples : Spoiled meat, milk, cereals and bread
Equipment and other materials : Test tubes, Petri plates, Bunsen burner, pipette, conical flask,
inoculation loop, balance, colony counter, glassware marking pencil, incubator, etc.
Media: Nutrient agar, potato dextrose agar, Sabouraud’s dextrose agar and buffered peptone
water (Appendix III)
Procedure
1. About 25 ml of liquid food sample or 25 g of solid food sample is added to 225 ml or 250
ml of buffered peptone water.
2. It is homogenized, in case of solid food sample.
3. Serial dilutions from 10–1 to 10–7 are made.
4. Then, about 1 ml of each dilution is inoculated using pour plate technique on nutrient agar
for bacteria, potato dextrose agar for moulds and Sabouraud’s dextrose agar for yeast.
5. These plates are then incubated at 37°C for 24 hours, in case of bacteria, at room temperature
for 3 days, in case of fungi and yeast.
6. The colonies formed are then counted.
7. The bacterial colonies are further subjected to Gram staining for identification.
8. To observe moulds, a wet mount is prepared by suspending some of the culture in a few
drops of lactophenol cotton blue solution. Care should be taken not to dampen the fungal
structures. The preparation is examined under low-power and high-power magnifications
with the aid of a dissecting microscope.
9. The yeast colonies are identified with their characteristic shape (generally, egg-shaped,
elongated or even spherical, and usually longer than bacteria).
Figure 7.1
Note
1. The organisms involved in spoilage of milk are Alcaligenes, Pseudomonas, Proteus and
Flavobacterium.
2. Moulds involved in the spoilage of bread are called bread moulds.These moulds include
Rhizopus that formes white colonies and have black dots of sporangia, and green-spored
Penicillium expansum, Aspergillus niger and Mucor.
3. Organisms involved in spoilage of meat are Pseudomonas, Sporotrichum and Candida.
4. Organisms involved in spoilage of cereals are Erwinia, Pseudomonas, Alcaligenes, Fusarium,
Penicillium, Rhizobium, Alternaria and Saccharomyces.
Observation
The plates were incubated and growth of bacterial and fungal species were observed.
Result
The bacterial colonies were counted to be 120. The fungal colnies were stained and identified as
Penicillium sp. and Aspergillus sp. Thus the given food sample was found to be contaminated and
the spoilage-causing organisms were isolated.
ANALYSIS OF FOOD SAMPLE FOR MYCOTOXIN (AFLATOXIN)

Aim
To analyse the mycotoxin in food sample by thin layer chromatography (TLC).
Principle
Aflatoxins are present in foods due to mould growth. These foods are consumed by animals.
The toxin is present in milk, meat and egg of the ingested animal. Aflatoxins are potent
carcinogens and their control needs qualitative method of analyses of microgram (µg) or picogram
(pg) per kg of food. TLC helps in detecting, quantitating and purifying aflatoxin at high levels.
The release of toxin from the mycelium is due to polar solvent breaking the protein–lipid bonds
in membranes by denaturing the protein.
Materials required
Media: Yeast extract glucose agar (Appendix III)
Developing system: TEF–Toluene, acetate, 90% formic acid (5 : 4 : 1)
Extraction liquid: Methanol, chloroform (1 : 2)
Equipment and other materials: Thin layer chromatography plate, scalpel, syringe, etc.
Procedure
1. The toxigenic culture from food and feed is grown in yeast extract glucose agar.
2. One or more agar plugs are cut out from a mould colony near the centre with a flame-
sterilized stainless steel tube (inner diameter 0.4 cm).
3. The plug is removed by using a flame-sterilized needle or scalpel.
4. By means of a syringe, a drop of extraction liquid is placed directly on the mycelium.
5. The mycelium side of the plug is gently pressed against the application line on the TLC
plate and then removed immediately.
6. After drying the application spot, the TLC plates are activated for 2 hours at 110°C.
7. The TLC plate is then developed in TEF (5 : 4 : 1) developing system.
8. The plates are removed after 3/4th of development and are air-dried.
9. The TLC is sprayed with 20% aluminium chloride (AlCl3) solution in 96% ethanol and
heated for 5 minutes at 120°C.
10. The Rf values are calculated and compared against the standard to identify the toxin.
1 1. The toxin can also be visualized in the UV at 366 and 254 nm. It appears bluish-green in
day light and reddish-brown in UV.
Table showing Rf values of different aflatoxin
Type of aflatoxin Rf value in Ethyl acetate 2- Propanol

water system
Aflatoxin B1 0.86
Aflatoxin B2 0.82
Averugin 0.97
Versicoral acetate 0.93
Versicolorin 0.95
Observation
The aflatoxin were separated and were visualized as reddish-brown bands under UV.
Result
The Rf value of the separated band was 0.86. It was identified as aflatoxin B1 by comparing the
Rf value against the standard Rf values.

8
GENETICS
ISOLATION OF CHROMOSOMAL DNA FROM BACTERIA

Aim
To isolate chromosomal DNA from bacterial culture.
Principle
Nucleic acid Deoxyribonucleic acid (DNA) is the single most important molecule of
living cells and contains all the information that specifies cellular properties. Isolation of
DNA is an essential step in many experiments. Although the methods are straightforward,
a particular method must be tailored depending upon the organism from which the DNA is
to be obtained, because the structure and composition of the organisms vary. The difference
between the techniques follows how the DNA is enclosed and the total dry weight of the
DNA (which varies from about 1% in complex mammalian cells to 50% in bacteriophages).
The common feature in all procedures is that the cell or virus is first broken and the DNA is
separated from other components like proteins, RNA, lipids and carbohydrates.
Bacteria The contents of a bacterial cell are enclosed in a multilayered cell wall and a cell
membrane. These structures cannot simply be removed by exposure to phenol but should be
removed by successive treatment of the cell suspension with lysozyme (an enzyme isolated
from chicken egg white) or with any one of the several different detergents, the most common
one being sodium dodecyl sulphate (SDS). The result of this treatment is the release of the
contents of the cell. Detergent SDS can disrupt cell membrane. The DNA will be protected
from the attack of endogenous nucleases by another detergent EDTA, which acts as a chelating
agent that binds the Mg2+ ions that is generally considered as cofactor for most nucleases.
RNA is then removed by digestion with specific enzymes. Protein is removed both
enzymatically and with phenol. The standard to remove proteins from nucleic acid solution is to
extract first with phenol–chloroform and then with chloroform. This procedure takes the advantage
of the fact that deproteinization is more efficient when two different organic solvents are used
instead of one. Although the phenol denatures the proteins efficiently, it does not completely
inhibit RNAase activity and it is a solvent for RNA molecules that contains long tracts of poly
(-A) tail. Both the problems can be circumvented by using a mixture of phenol : chloroform :
isoamyl alcohol (25 : 24 : 1). The subsequent extraction with chloroform removes any lingering
traces of phenol from precipitated nucleic acids.
The DNA is collected by precipitation with ethanol. Precipitation with ethanol is one of the
standard methods to recover nucleic acid from aqueous solution. It is very rapid and efficient,
even nanogram levels of DNA and RNA can be quantitatively precipitated with ethanol. Ethanol
depletes the hydration shell from the nucleic acid and exposes negatively charged phosphate
groups. Counter ions, such as Na+ ions, bind to charged groups and reduce repulsive force between
polynucleotide chains to the point where the precipitation can form. Ethanol precipitation can,
therefore, occur only if cations are available in sufficient quantity to neutralize the charge on
exposed phosphate residues. Examples for most commonly used cations are ammonium acetate,
lithium chloride, sodium chloride and sodium acetate.
Method I: Using STE Solution

Materials required
Sample: Bacterial culture
Reagents: STE solutions I and II (Appendix II), SDS phenol : chloroform:isoamyl alcohol
(25 : 24 : 1), 95% ethanol, sodium acetate solution (Appendix II), TE buffer (Appendix II),
TAE buffer (Appendix II), Ethidium bromide (Appendix II)
Media: Agarose
Procedure
1. Harvest cells from actively growing culture and wash them in 750µl STE I and suspend in
500µl STE II.
2. Add 50µl SDS to a final concentration of 0.5%; mix well and incubate the sample at 70°C
for 20 min.
3. Add equal volume of phenol:chloroform and mix well. Centrifuge for 10 min. and save the
top phase.
4. Extract the aqueous phase using chloroform : isoamyl alcohol.
5. Precipitate DNA by adding 3 M sodium acetate (pH 7.0) and 95% ethanol.
6. Suspend the pellet in minimum volume of TE buffer and store it at 4°C.
Result
Fine bands were visible under UV illumination
Genetics 231
Method II: Using STET buffer

Materials required
Reagents: STET buffer, lysozyme, 80% ethanol, 4 M Lithium chloride, isopropanol, STE saturated
phenol
Preparation of STE saturated phenol
1. Melt the solid phenol in a 50°C water bath.
2. Add 20% of the above STE buffer (v/w).
3. Shake until the two become an emulsion.
4. Allow the phases to separate.
5. Decant off the aqueous phase.
6. Add hydroxyquinolin to 0.1% (w/v) and beta-mercaptoethanol (BME) to 0.2%
(v/v).
7. Add the same amount of STE as you did in step 2 and store at 4°C ( It may take more than
one time of mixing to an emulsion to saturate the phenol)
Procedure
1. Grow well-isolated bacterial colonies in 15 ml of the medium overnight.
2. Transfer the culture into a single Eppendorf tube or into a 15 ml disposable tube depending
on the volume and harvest the cells.
3. Resuspend the pellet with 300µl of STET buffer (900µl) Filter and sterilize. Store at 4°C.
4. After resuspending, add 30µl of RNase/lysozyme mixture. (10mg/ml lysozyme,
1 mg/ml RNase).
5. Boil the pellet for 1–2 min.
6. Spin in microfuge for 15 min.
7. Take the supernatant and add phenol extract saturated with STET- phenol.
8. Spin and take the supernatant. Add 1/10 volume of 4 M lithium chloride (autoclaved). Keep
it in ice for 5–10 min.
9. Spin and take the supernatant. Add equal volume of isopropanol and incubate in room
temperature for 5 min.
10. Centrifuge the mixture and collect the pellet. Wash with 80% ethanol (95% will cause the
residual Triton to precipitate). Resuspend pellet in 50 – 200µl of buffer and store at 20°C.
Result
Method III: Using lysozyme

Materials required
Sample: 24-hour broth culture of E. coli
Reagents: Lysozyme (5%), phosphate buffer solution (PBS), SDS (10%), chloroform: N-amyl
alcohol (24:1), double distilled water, 3 M Sodium acetate solution, ice-cold ethanol,
Equipments and other materials : Glassware, micropipettes, water bath and centrifuge
Procedure
1. Centrifuge 2 ml of 24-hour culture broth at 10,000 rpm for 5 min. to obtain a pellet.
2. Discard the supernatant. Add 2 ml of phosphate buffer solution and 0.1 ml of lysozyme to
the pellet, mix and incubate in the water bath at 37ºC for 15 min.
3. After incubation, transfer the reaction mixture into a sterile test tube, and add 1 ml of sodium
dodecyl sulphate and agitate the mixture.
4. Incubate this in the water bath at 40ºC for 30 min. Mix the contents of the tube gently at
regular intervals.
5. Add 1 ml of double distilled water and 2 ml of choloroform : N-amyl alcohol in the ratio
24 : 1 to the mixture.
6. Mix the contents thoroughly and centrifuge at 10,000 rpm for 5 minutes.
7. After centrifugation, transfer the supernatant to a sterile test tube and add 0.2 ml of sodium
acetate and twice the volume of ice-cold ethanol.
8. Shake the tube and observe for results.
Results
ISOLATION OF GENOMIC DNA FROM CAULIFLOWER BY

SUPAQUICK METHOD
Aim
To isolate the genomic DNA from cauliflower by employing the supaquick method.
Principle
Extraction of high molecular weight DNA from protein and RNA is essential for all molecular
biology investigations. The cell wall as well as the cell membrane must be broken by grinding
the tissue along with the extracting buffer in a mortar and pestle.
Genetics 233
The extracting buffer contains the detergent, SDS, which disrupts the cell membrane.
The DNA will be protected from the attack of endogenous nucleases by another detergent,
EDTA (which is also the component of extraction buffer), which acts as the chelating agent and
binds magnesium ions, generally considered as a cofactor for most nucleases. The tissue mixture is
emulsified with chloroform and isoamyl alcohol to denature proteins. Incubation at 60°C degrades
DNA-degrading enzymes but not the DNA, since DNA can withstand that temperature.
Materials required
Reagents: Supaquick buffer (Appendix II), chloroform : isoamyl alcohol (24 : 1), isopropanol,
95% ethanol, 70% ethanol, TE buffer (Appendix II), acid-washed sand or glass wool, TAE buffer
(Appendix II),
Equipment and other materials: Centrifuge, microfuge tube, mortar and pestle
Reagents for agarose gel electrophoresis:
i. Electrophoretic agarose gel tank
ii. Agarose
iii. TE buffer
iv. TAE buffer (Tank Buffer)
v. 1µl of ethidium bromide
vi. Gel loading Buffer
vii. UV electrophoresis
Procedure
1. Collect 100 mg of experimental tissue (cauliflower).
2. Add a pinch of acid-washed sand or glass wool and grind thoroughly in a mortar and pestle.
3. Add 800µl of pre-warmed (60°C) supaquick buffer and mix gently. Incubate at 60°C in water
bath for 30 min.
4. Add an equal volume of chloroform : isoamyl alcohol (24 : 1) and mix well for 5 min. by
inverting the tubes several times.
5. Centrifuge at 10,000 rpm for 10 min. Carefully transfer the supernatant (aqueous phase, i.e.,
top of viscous layer) into a sterilized Eppendorf tube.
6. Add equal volume of ice-cold isopropanol (500 µl approximately). Mix gently by inverting
and place at –20°C for 30 min. Centrifuge at 10,000 rpm for 10 min. and decant to drain
the isopropanol.
7. Add 500 µl of 70% ethanol and invert the tubes and drain the ethanol. (Care should be taken
so that the pellets do not slip down along the tube wall).
8. Resuspend the pellets in 100 µl of TE buffer and gel loading dye is added to it.
9. The agarose gel electrophoresis is run and observed under UV.
Result
DNA bands were viewed under UV Transilluminator.
ISOLATION OF PLASMID DNA

Introduction
Bacterial plasmids are frequently used in cloning protocols. Plasmids are extrachromosomal
covalently closed (CCC) self-replicating genetic material found in bacteria. These are responsible
for various functions, such as drug resistance, degradative fertility, virulence, tumour induction
and for the production of restriction enzymes. They are 1–200 kb in size. Plasmid DNA can be
purified and isolated using caesium chloride/ethidium bromide protocol.
Aim
To isolate plasmid DNA from microorganisms.
Principle
This protocol involves three steps: growth of bacterial culture, harvesting and lysis of bacterial
cells and purification of plasmid DNA.
Materials required
Media: Luria–Bertani medium (Appendix III)
Equipments and other materials: Laminar air flow chamber, incubator cum environmental shaker,
refrigerated centrifuge, agarose gel electrophoresis unit, vortex mixture, micropipette, Eppendorf
tube and tips, inoculation loop and test tube, sterile tooth picks and spreader
Reagents: Solutions I, II and III (Appendix II), isopropanol, 70% ethanol, TE buffer (Appendix
II), loading dye, (Appendix II), TAE buffer (Appendix II), Reagents for agarose gel electrophoresis
(refer next expt.)
Procedure
Culturing of bacteria Incubate 2 ml of sterile LB broth with a single bacterial colony of Escherichia
coli. Grow the culture to saturation (O.D. 600 nm) overnight at 37°C in an environmental shaker
(150–200 rpm).
Genetics 235
Harvesting and lysis of bacterial cells

1. Fill a micro centrifuge with the grown culture (1.5 ml).
2. Centrifuge for one minute at 10,000 rpm at 4°C.
3. Drain the supernatant and take the pellet.
4. Add 0.2 ml of ice-cold solution I to the cell pellet, mix well and keep at room temperature
for 5 minutes.
5. Add 0.4 ml of solution II, invert 5 times, and gently place in ice for 5 minutes.
6. Add 0.3 ml of ice-cold solution III, invert 5 times gently. Incubate in ice for 3–5 minutes.
7. Centrifuge for 5 minutes at 10,000 rpm at 4°C. Take the supernatant for plasmid DNA
isolation.
Purification of plasmid DNA
8. To the pellet, add isopropanol and allow it to stand at room temperature for 2 minutes.
Repeat the same procedure with supernatant for plasmid DNA.
9. Centrifuge the tubes for 5 minutes at 10, 000 rpm and discard the supernatant.
10. Add 1ml of ice-cold 70% ethanol. Mix by inverting several times. Centrifuge for 1 minute
and discard the supernatant.
11. Dry, add 50µl of TE buffer to the tube and store.
AGAROSE GEL ELECTROPHORESIS

Introduction
The most popular method to separate, identify and purify nucleic acids is done by agarose gel
electrophoresis. This technique is simple, rapid and capable of resolving fragments that cannot be
done by other methods like density-gradient centrifugation. Above all, DNA or RNA fragments
that get separated can be located in the gel density by ethidium bromide staining. Ethidium
bromide, as an intercalating fluorescent dye binds with RNA and DNA. When ethidium bromide
DNA complexes are examined in UV light, they give fluorescent bands. By this method, DNA
or RNA fragments, whose concentration in gel is as little as few nanograms, can be detected.
The mobility of DNA in agarose gel depends on several parameters:
 Size of the DNA molecules
 Concentration of gel
 Conformation of the molecule
 Electrical parameters like voltage/current
 Temperature
Electrophoresis of DNA is generally carried out at room temperature. However, low

concentration gels are run at 4°C as they gain source rigidity at low temperature.
Electrophoresis of DNA is generally carried out in horizontal apparatus. Agarose gel is
prepared in desirable thickness in a special tray called gel platform. While preparing the gel,
suitable comb is placed in the gel platform to make the sample wells. Gel platform with gel is
then placed on an elevated gel bed in the apparatus and suitable buffer is poured to submerge
the gel. Current passes through the gel between the cathode and anode. This kind of separation
in submerged gel is popularly known as submarine gel electrophoresis.
Submarine gel electrophoresis has got several advantages:
 Unlike vertical gel electrophoresis apparatus, the submarine electrophoresis apparatus
is somewhat compartmentalized. There is no separate anode and cathode reservoir.
The gel is placed in between cathode and anode under submerged condition in buffer.
Considerable amount of current flows through the gel uniformly. During electrophoresis,
buffer acts as a heat link to minimize heat in the gel and it also homogenizes pH changes
taking place at cathode and anode.
 Thick agarose gel can be casted easily by using horizontal tray like gel platform. Especially
when low percentage gel is required, the only safe way of handling thick gel is in horizontal
gel platforms.
 Gel platform can be made by using UV transparent materials. When such UV transparent
platforms are used, gel need not be taken out for viewing bands; also other gel handling
risks are avoided.
Aim
To separate and extract DNA by agarose gel electrophoresis.
Materials required
Sample: 24 hr bacterial culture
Media: Agarose
Reagents: running buffer (5X), gel loading buffer or sample buffer (6X), ethidium bromide.
Equipments and other materials: horizontal electrophoresis apparatus, agarose,
Procedure
Preparation of agarose gel
1. The volume of gel required is found out by multiplying the size of gel platform with that
of the gel thickness:
Gel platform = 7 × 5
Genetics 237
Gel thickness = 0.6

7 × 5 × 0.6 = 21 ml
About 21 ml of 1% agarose gel is prepared by weighing 210 mg of agarose powder and
dissolved in 21 ml of buffer.
2. About 300 ml of 1X buffer is prepared by mixing 60 ml of 5X buffer with 240 ml of deionized
water.
3. Agarose powder of 210 mg is weighed and placed in a dry flask to which 2 ml of 1X
electrophoresis buffer is added.
4. The flask is placed in the boiling water bath for heating. After the agarose particles get
dissolved on boiling and the solution becomes transparent, it is allowed to cool. In the
meanwhile, the gel platform and comb is kept ready.
5. The gel platform is washed thoroughly and wiped with tissue paper. The open glass slides
are sealed with an adhesive tape. A clean comb is placed on the platform and it is checked
to verify whether 0.5 mm gap is left. The whole assembly is placed on a levelled table.
6. 0.1% of stock ethidium bromide solution is added, when the agarose solution is at about
56°C. The entire solution is poured onto the gel platform slowly without trapping any air
bubbles. The solution becomes translucent after 10–15 minutes, which is indicated by the
formation of gel.
7. The assembly is left undisturbed for 30–45 minutes.
8. The comb is lifted carefully after gel formation and the adhesive tape is removed.
9. The gel platform is placed on the bed of the tank.
10. Enough tank buffer is poured to cover about 1–2 mm buffer layer above the gel surface.
Sample preparation
1. The sample is prepared after knowing the maximum or minimum volume of sample that can
be loaded to each well. The volume of the well is found out by using the following formula:
Well width × comb thickness × gel thickness gap
0.6 × 0.1 × (0.6 – 0.2) = 0.024 ml
Maximum capacity of the well ~ 25µl
2. Samples are prepared in 6X gel loading buffer. The quality of DNA loaded is based on the
number of fragments (0.2 to 0.5 mg per slot is sufficient). Different concentrations of the
sample DNA is taken in separate Eppendorff tubes and their final volume is made to 25µl.
Volume of sample taken is 20µl
To this, 5µl of gel loading buffer is mixed.
Therefore, total sample volume is 25µl.
3. Each sample is injected into different wells using a micropipette. The sample settles down at
the bottom of the well because of the density of gel loading buffer.
4. The lid is closed.
5. The apparatus is connected to a power pack.
6. The power is turned on and it is set at the constant volume mode C.
7. The run is continued till the marker dye is about 0.5 cm from the end of the gel.
8. The voltage is reduced, the power pack is switched off and the power is disconnected.
9. The gel is taken out and examined under UV transilluminator.
Examination
CAUTION: Since UV rays are harmful to eyes and skin, the entire viewing window is covered
with UV blocking cover.
The agarose gel is taken out after electrophoresis and placed on the transilluminator viewing
window. It is then covered with UV blocking cover. It is switched on to observe orange fluorescent
bands (if UV transparent gel platform is used, the gel need not to be taken out of it and the gel
with the platform can be placed in the transilluminator). Photograph is taken for permanent
recording of the banding pattern.
DNA extraction from agarose gel
1. Freeze the gel slice at –20°C for 30 min. so that gel can be easily handled.
2. Cut a 5cm length of dialysis tubing and rinse it in and out with distilled water.
Then, rinse with the same buffer used for the gel (0.5X TBE) and leave it submerged in a
small beaker of this buffer. Seal one end with a dialysis clip.
3. Insert the frozen gel slice into the tubing and add 200 – 400µl of the buffer, 0.5X TBE.
Seal the other end of the tubing with a second dialysis clip. The buffer around the gel slice
must be the same as the buffer inside the gel.
4. Immerse the sealed tubing in an electrophoresis tank so that the DNA band is parallel to
the electrodes and apply 5 V/cm electric field. The DNA will migrate out of the gel towards
the positive electrode. It will be retained by the dialysis tubing. It takes about 10–15 min.
5. Remove the buffer from the tubing and place into a 1.5 ml microfuge tube.
6. Using phenol/chloroform extract and ethanol, precipitate the DNA. Re-dissolve the DNA
pellet in an appropriate volume of water or TE buffer (e.g., 10µl). The pellet is often so small
that it is invisible.
Result
Fine bands were visible under UV illumination.
Genetics 239
ISOLATION OF RNA
Introduction
Ribonucleic acid (RNA) is a nucleic acid polymer consisting of covalently bound ribonucleotides,
which contains ribose sugar, a phosphate and a base (uracil, adenine, guanine or cytosine). It is
transcribed from DNA and it serves as a template for translation of genes into proteins (mRNA),
transferring amino acids to the ribosome to form proteins (tRNA) and also for translating the
transcript into proteins (rRNA).
Living cell, whether prokaryotes or eukaryotes, contain three major types of RNA (ribosomal
RNA, transfer RNA and messenger RNA).
Aim
To isolate RNA.
Principle
The basic steps involved in the isolation of RNA are: disruption of cells or tissue, denaturation
of the nucleic acid–protein complexes, inactivation of endogenous ribonucleases and finally,
purification of RNA from contaminating DNA and proteins.
RNA is generally isolated by using strong denaturants, such as guanidine thiocyanate or
phenol–chloroform with a reducing agent, and beta-mercapto ethanol, to inhibit RNAase activity.
Successful isolation of RNA depends on the suppression of endogenous RNA ases during
the isolation procedure. Since RNAses are found almost everywhere, all surfaces, glassware, gel
equipment, etc. are decontaminated (e.g., by using DEPC [diethyl pyrocarbonate], to prevent
degradation of RNA.
Quality of RNA purified can be analysed by agarose gel electrophoresis (native or denaturing).
Denaturing agarose gel electrophoresis involves the use of toxic chemicals like formaldehyde and
this is used if RNA isolated has to be used for further downstream applications like northern
blotting, nuclease protection analysis, etc. Otherwise, native agarose gel electrophoresis can be
used to visualize and assess the quality of RNA isolated.
Materials required
Sample: E.coli
Media: LB broth, LB Agar
Reagents: Extraction buffer, proteinase K,3M sodium acetate, gel loading buffer, alchohol, beta-
mercaptoethanol, nuclease free-water, agarose, TAE buffer
Equipments and other materials: Microfuge, conical flask, measuring cylinder, Petri dish, UV
transilluminator, 70% ice-cold ethanol, sterile water, choloroform, isopropanol, ethidium bromide,
sterile microtips, vials,
Procedure
1. Pick a single colony from the overnight culture on LB plate and inoculate into 5 ml of broth.
2. Incubate in a shaker set at 37°C overnight.
RNA extraction
3. Pipette 1.0 ml of culture into a sterile 1.5 ml microfuge tube.
4. Spin at 6000 rpm for 8 to 10 min.
5. Discard the supernatant and invert the vial on blotting paper to drain out the leftover
supernatant.
6. Pipette 600µl of the extraction buffer into a microfuge tube and add 10µl of
beta-mercarptoethanol and 50µl of proteinase K.
7. Mix well and add 300µl of the buffer into each of the microfuge tube containing the bacterial
pellet.
8. Resuspend the bacterial cell pellet in the extraction buffer.
9. Incubate the microfuge tube at 37°C for 20 min.
10. Add 1/10th volume of 3 M sodium acetate and equal volume of chloroform.
11. Mix well and spin at 10, 000 rpm for 10 min. at 4ºC.
12. Carefully aspirate the aqueous layer into another microfuge tube and add an equal volume
of chloroform and spin at 10,000 rpm for 10 minutes at 4ºC.
13. Collect the supernatant in a new sterile microfuge tube and add two volumes of alcohol and
mix well.
14. Incubate at –20ºC for 2 hrs.
15. Centrifuge for 30 min. at 10, 000 rpm at 4ºC.
16. Decant the supernatant and add 100µl of ice-cold 70% ethanol and centrifuge for 10 min.
at 10,000 rpm at 4ºC.
17. Decant the supernatant and allow the pellet to dry at room temperature.
18. Add 20µl of RNAase-free water to the bacterial RNA pellet.
19. Add 4µl of bacterial RNA to the microfuge tube.
20. Load in the 1% agarose gel and run at 50 V current.
Result
RNA bands under UV illumination were visualized.
Genetics 241
ISOLATION OF MUTANTS
I. INDUCED MUTATION—ISOLATION
OF ANTIBIOTIC-RESISTANT MUTANTS
Aim
To increase the frequency of mutation with the help of UV radiation and to study the mutants
for its induced mutation frequency.
Principle
Radiation is the process of emitting radiant energy in the form of waves or particles. The effect
of radiant energy on microbes depends on its wavelength. UV radiation (100–400 nm) is of
special interest because it kills the microbes. The germicidal region of UV spectrum occurs in the
240–300 nm region. The purine and pyrimidine bases in the nucleic acid absorb radiation
strongly at 260 nm where the greater lethal effects occur. Several effects are known but the most
thoroughly studied effect is the formation of thymine dimers. UV radiation causes both base
substitution and deletion in bacterial genes. Weak induction of frameshift has also been reported.
Most of the microorganisms have enzymes that can repair the UV-damaged host. There are two
types of repair systems:
 Photoreactivation
 Dark repair
Photoreactivation in bacteria was observed by Albert Kelner in 1949. Such repair mechanisms
are also reported in fungi, algae, higher plants and animals, including humans. Photoreactivation
is catalysed by an enzyme called photolyase. In dark repair, enzymes like nuclease and ligase play
a role in the re-formation of the DNA to its original state. In this experiment, the chosen bacterial
culture is exposed to UV light at fixed density and the time of exposure is valued.
Materials required
Sample: 24 hours E.coli culture
Media: LB broth, LB agar, LB agar with streptomycin,
Equipments and other materials: Petri plates, pipettes, Eppendorf tubes, Erlenmeyer flasks,
centrifuge, saline, UV lamp, conical flasks, test tubes, etc.
Procedure
1. Overnight culture of E. coli is centrifuged at 10,000 rpm for 10 minutes.
2. The pellet is suspended in 1 ml of sterile saline.
3. The bacterial suspension is serially diluted with 9 ml of sterile saline blanks up to 10–8.
4. From 10–6 to 10–8 dilutions, 0.1ml is spread onto LB agar plates for determining the total
viable count.
5. From the dilutions 10–2 to 10–5, 0.1 ml is taken and spread plated on the LB agar plate with
streptomycin.
6. First set of LB–streptomycin-inoculated (10–2, 10–3, 10–4 dilutions) plates are exposed to
UV for a fixed period of 10 minutes by keeping one half of the Petri plate lids open during
exposure.
7. The procedure is repeated with different sites of LB–streptomycin plate with change in time
of exposure.
8. The LB plates exposed to UV are covered with dark sheet to prevent photoreactivation. All
LB agar and LB–streptomycin plates are incubated at 37ºC for 24 hours.
Result
Antibiotic resistant mutants were isolated.
II. ISOLATION OF AUXOTROPHIC MUTANTS

(REPLICA-PLATING TECHNIQUE)
Aim
To isolate auxotrophic mutants by physical mutagen (UV radiation).
Principle
The physical and chemical properties of each protein are determined by its amino acid sequence.
Any change in the amino acid sequence is capable of altering the activity and inactivating a
protein. Change in amino acid sequence is influenced by alterations in the base sequence. The
change in the base sequence is brought about by mutation. Mutation is a random sequence
event, spontaneous or induced. Spontaneous mutation occurs at low frequency in the bacterial
population. The induced mutation is obtained by two methods, namely physical and chemical
methods. These agents are called mutagens.
In this experiment, mutations will be induced in Escherichia coli by using UV radiation.
Materials required
Sample : Escherichia coli culture
Media : Nutrient agar, Luria–Bertani (LB) agar/broth
Reagents: Magnesium sulphate (0.1 M)
Genetics 243
Equipment and other materials : Test tubes, graduated pipettes, graduated Erlenmeyer flasks,
conical flasks, beaker, tissue paper, cotton, incubator, hot air oven, autoclave, centrifuge, UV
lamp, colony counter, replicator.
Procedure
1. LB broth is prepared and inoculated with E.coli culture and incubated overnight at 37°C.
2. After overnight growth, subculturing is done into four tubes containing 5 ml of LB broth.
3. The tubes are then incubated for about 4–6 hours.
4. The cells are centrifuged at 5000 rpm for 5 minutes.
5. After that, the cells are re-suspended in 10 ml of 0.1 M magnesium sulphate.
6. The mixture is placed in ice for about 5 minutes to prevent growth.
7. Then, 5 ml of cultures are transferred to open Petri plates and the culture is exposed to UV
for 10, 20, 30, 40 and 50 minutes.
8. The UV lamp must be set up at a distance of 24–30 cm from the culture.
9. About 0.1 ml of UV exposed sample is inoculated onto LB agar by spread-plate technique.
10. The plates are incubated overnight in dark.
11. Following incubation, the colonies on the agar are counted.
12. Later, the colonies are transferred from the medium to the minimal medium by replica-plate
technique.
Principle for replica-plate technique Replica-plating technique was first accomplished by
J. Laderberg and E. M. Laderberg. The bacterial colonies are transferred from the master plate
(containing both the wild type and mutant) by means of a replicator. The replicator is a wooden
plank covered tightly with a velvette cloth whose hundreds of threads serve as inoculation needles.
Non-selective plates contain complete nutrition and so all organisms tend to grow. In selective
plates, which lack complete nutrition, only wild-type organisms that synthesize their own nutrition
can grow. Therefore, the organism which appears on non-selective plates but not on the minimal
media are isolated as the auxotrophic mutants. This is an indirect method of selection.
Procedure
The mutagenized culture plate is selected from the previous experiment as the master plate.
Selective, minimal and non-selective LB agar and wooden plank are sterilized. Before the transfer
of colonies, marks are made to ensure the position of colonies. The wooden plank is gently pressed
onto the master plate, removed and then impressed on LB agar plate (non-selective media) and
minimal agar plates (selective media). The inoculated plates are incubated at 37ºC for 24 hours
and checked for the presence of mutants.
RESTRICTION DIGESTION OF LAMBDA DNA

Introduction
Most parts of DNA will have recognition sites for various restriction enzymes and it is often
beneficial to know the relative locations of some of these sites. The technique used to obtain
this information is known as restriction mapping. This involves cutting a DNA fragment with
selective restriction enzymes, singly or in combination. Restriction enzymes are enzymes that cut
DNA in a specific way (HindIII, BamHI, EcoRI). For the digestion of the DNA, it is incubated
with the enzymes under appropriate conditions. Each restriction enzyme requires a particular
environment in which one can digest the DNA. The buffer of each enzyme is supplied along
with the enzyme and the DNA is incubated. When the fragments produced are run in agarose
gel, their sizes are determined.
Aim
To determine the location of some recognition sites of DNA.
Principle
Most pieces of DNA will have recognition sites for various restriction enzymes and it is often
beneficial to know the relative locations of some of these sites. The technique used to obtain
this information is known as restriction mapping. This involves cutting a DNA fragment with
selective restriction enzymes, singly or in combination. Restriction enzymes are enzymes that cut
DNA in a specific way (HindIII, BamHI, EcoRI). For the digestion of the DNA it is incubated
with the enzymes under appropriate working conditions. Each restriction enzyme requires a
particular environment in which one can digest the DNA. The buffer of each enzyme is supplied
along with the enzyme and the DNA is incubated. When the fragments produced are run with
agarose gel, their sizes are determined.
Materials required
Sample: DNA sample,
Reagents: Restriction enzymes, restriction buffer, sterile distilled water,
Equipment and other materials: Eppendorf tubes, water bath, agarose gel electrophoresis apparatus
Procedure
1. In a clean Eppendorf tube, 5µl of lambda DNA sample is taken and kept in ice.
2. To this, 4µl of XbaI restriction enzyme and 2µl of restriction buffer is added. The final volume
of the mixture is made up to 20µl with sterile distilled water.
3. This procedure is also carried out for XhoI restriction enzyme.
Genetics 245
4. For double digestion with restriction enzymes XbaI and XhoI, 2µl of each of these enzymes
are added to 5µl of DNA sample.
5. The mixtures are then incubated in water bath at 37°C for 2–3 hours.
6. Finally, the digested sample is run in 0.8% agarose gel and the banding pattern is studied.
Result
The lambda DNA is known to carry only one restriction site for enzymes XbaI and XhoI and
this can be confirmed based on the number of bands or fragments formed after restriction on
agarose gel electrophoresis. Two bands are formed due to digestion at a single site. The size of
the bands are 20.4 kb and 25 kb for digestion with XhoI, and 15 kb and 34.5 kb for digestion
with XbaI. For double digestion, 3 distinctive bands of sizes 9 kb, 5 kb and 24.5 kb are formed.
BACTERIAL CONJUGATION
Introduction
Conjugation is a gene transfer process in which the donor makes physical contact with the
recipient and transfers genetic material. A recipient that requires donor genetic information is
called a transconjugant. Bacterial conjugation was discovered in 1946 by Joshua Lederberg and
E.Tatum. In this experiment, the donors are wild-type E. coli Hfr strains, 12200 and 12202, and
the recipient is SSH 57 (an E. coli auxotropic mutant). Strains 12200 and 12202 have different
origins of transfer as F-plasmid is integrated at different sites of bacterial chromosomes. Thus,
during conjugation, the genes from both the strains are transferred in different orders. CSH 57
is a mutant for the markers His, Trp, Pur, Leu, Met, Ile and Val and Arg. The best markers for
selection of the conjugant are His, Pur and Leu.
Aim
To determine the efficiency of bacteria to uptake the DNA from the medium through conjugation
Material required
E.coli strains, Minimal Agar, Aminoacids, LB medium
Principle
Conjugation is a gene transfer process in which the donor makes physical contact with and
transfers genetic materials to the recipient . A recipient that requires donor genetic information
is called a transconjugant.
Bacterial conjugation was discovered in 1946 by Joshua Lederberg and E.Tatum. In this
experiment the donors are wild type E.coli Hfr strains 12200 and 12202 and the recipient is
SSH 57 (An E.coli auxotropic mutant) 12200 and 12202 have different origins of transfer as F
plasmid is integrated at different sites of bacterial chromosomes. Thus during conjugation the
genes from both the strains are transferred in different orders. CSH 57 is a mutant for the markers
His, Trp, Pur, leu, Met, Ile and Val and Arg. The best markers for selection in the conjugant are
His, Pur and Leu.
Procedure
1. Each of the three strains, 12200, 12202 and CSH 57, are inoculated into 10 ml of sterile
LB broth. It is incubated to reach the mid log phase.
2. CSH 57 is kept in a shaker, and 12200 and 12202 strains are incubated at 37ºC.
3. Three flasks, each containing 200 ml of minimal agar is prepared and then sterilized.
4. To select for His marker, all 6 amino acids (Trp, Leu, Met, Ile, Val, Arg and Pur) are added
to the minimal agar.
5. This is poured into eight sterile Petri plates. Four of the plates are used to select the conjugants
from the 12200 donor and the other four are used to select the conjugants from 12202 donor.
6. Similarly, plates are made for Leu and Pur selection.
7. Once the cells reach the log phase, 1200 and CSH 57 are mixed in the ratio
1 : 9 (0.2 + 1.8 ml) under aseptic conditions. Similarly, 12202 and CSH 57 are mixed.
8. The mating is allowed to take place for 2 to 2.5 hours.
9. The two test tubes containing the mating pairs are incubated at 37ºC in a slanted position
for better aeration.
10. Two different mating pairs are serially diluted in saline separately up to a dilution of
10–3. Spread-plate technique is performed for each of the dilutions, on His–, Pur– and
Leu– plates. The undiluted sample is also plated.
11. The undiluted sample is centrifuged and the pellets are resuspended in equal amounts of
saline. Then, 0.1 ml is plated.
12. Each of the cells, 12200, 12202 and CSH 57, are centrifuged and the pellet is resuspended
in about 1 ml of the saline.
13. These cells are then plated separately (control plating).
14. The washing with saline is done to remove any residual LB medium, that might allow the
cells to grow on minimal agar.
15. All plates are incubated overnight at 37ºC.
Genetics 247
Result
Gradient transfer of the markers is observed.
Discussion
Shortly after Hfr and F– cultures are mixed, transfer of the chromosome from the Hfr begins
during the transfer of Hfr DNA to a recipient cell. The mating pair usually breaks apart before
the entire chromosome is transferred owing to Brownian motion. The number of cells that remain
paired decreases with time and therefore, the number of transformants of an early marker is higher
than that of a late marker. An early marker is a marker close to the origin and the late marker
is further away from the origin. As the number of recombinants decreases, the distance of the
marker from the origin increases. Thus, the order of the genes can be determined.
The donor is prototrophic strs, kans and the recipient is autotrophic strr, kanr. A plate
containing kan and str, and lacking the amino acid, can select only the recombinants.
The transferred allele that is selected by means of the medium composition is called as selective
marker and the allele used to prevent the growth of the donor is called as counter selective
marker (strr).
BACTERIAL TRANSFORMATION
Introduction
Transformation is a process in which a recipient cell acquires genes from free DNA molecules in
the surrounding medium. In the laboratory, bacterial transformation or transfer is accomplished
by adding plasmid DNA from donor cells. It is then added to a suspension of recipient cells,
where transformation occurs. It was first observed in 1928, by Fred Griffith. It was only in 1944
that Oswald Avery, Colin Macleod and McCarty, carried out the critical three experiments, that
led to our current understanding of the process.
Aim
Transformation of free DNA molecules into recipient cells.
Principle
In this experiment, E.coli (strain CSH 57) is the recipient cell used. The cells of E.coli are
transformed with two different plasmids, pAC and pSRJ. The pAC plasmid carries genes for
tetracycline resistance, while pSRJ has genes for ampicillin resistance. The recipient CSH 57 strain
is sensitive for both ampicillin and tetracycline. Transformed cells (transformants) are checked
by plating the cells in a medium incorporated with ampicillin and a separate one containing
tetracycline.
Materials required
Sample: E.coli, plasmid DNA
Reagents: 50 MM Cacl2, ampicillium, tetracycline
Media: LB broth
Procedure
1. About 10 ml is taken from a mid-log culture of E. coli (CSH 57).
2. It is then centrifuged at 5000 rpm for 10 min. The supernatant is then removed.
3. 5ml of 50mM CaCl2 is added to the test tube containing the pelleted cells. (CaCl2 is added
in such a way that it makes up half the volume of cells). The tube is shaken gently.
4. It is then incubated in ice for about half an hour. The cells are then centrifuged.
5. The supernatant is removed and 1ml of 50mM CaCl2 is added (CaCl2 volume is now
1/10th the volume of cells).
6. The cells in the test tube are again incubated in ice for around half an hour.
7. 100 µl of it is pipetted out, using a micropipette, into two test tubes. Transformation is
carried out separately using two different plasmid DNA, in each test tube.
8. 5µl of the pSRJ is added to one of the test tubes. 3µl of the pAC is added to the second tube.
Both the tubes are incubated in ice for half an hour.
9. The tubes are then placed in a water bath kept at 42ºC for 2 min. Thus, the cells are given
heat shock. The tubes are immediately incubated in ice for 20 min.
10. About 3ml of LB broth is added to each of the test tubes. The tubes are then placed in a
shaker for 30 min. to one hr. This is carried out for the gene expression.
11. Serial dilutions are carried out, using each of the test tubes (up to 10–2) .
12. The cultures transformed with pSRJ are plated onto ampicillin containing LB agar plates.
The final concentration of ampicillin in the agar is 50 µl/ml.
13. About 0.1 ml is taken from each of the dilutions, including 0 dilution and used for plating.
A control plate is also made using non-transformed cells.
14. The cells transformed with pAC are similarly plated in tetracycline LB agar, whose final
concentration is 10µg of tetracycline per ml.
15. All the plates are then incubated at 37ºC for 24 hrs. The plates are observed for the presence
of transformants.
Genetics 249
Result
Few colonies of ampr and tetr transformants are observed.
Discussion
Transformation begins with uptake of DNA fragment from the surrounding medium by recipient
cell and terminates with recombinational exchange of part of the donor DNA with the homologous
segment of the recipient chromosome. Probably most species are capable of the recombination
step, but the ability of most bacteria to take up DNA efficiently is limited. Even in a species
capable of transformation, DNA can penetrate only a very small fraction of the cells in a growing
population. Such cells are said to be competent. They give rise to the transformant colonies.
SOUTHERN BLOTTING
Introduction
Southern blotting is named after M. Southern, who developed this procedure at Edinburgh
University in the 1970s. Southern hybridization is extensively used for the detection of specific
DNA that is blot transferred onto nitrocellulose membrane. This technique uses the specificity
and quantitative nature of annealing of complementary strands of nucleic acid (probes) for the
detection of DNA, which is done by either staining or autoradiography.
Aim
To check for a match between DNA molecules.
Principle
Southern hybridization follows the principle of blotting except that a labelled DNA probe is used to
detect the fragment of interest. The DNA fragment of interest is subjected to a series of denaturation,
depurination and neutralization steps after its separation and are blot transferred to membrane
by capillary action. The pre-hybridized membrane is then hybridized with biotinylated probes.
The biotin-labelled DNA of interest is then detected by using specific conjugate and substrate.
Materials required
Sample: DNA sample mixture (5µl)
Media: Agarose (500 mg)
Reagents: Acid solution 10X (125 ml), denaturation solution 2X (250 ml), neutralization
solution 2X (250 ml), transfer buffer, TEB 10X (60 ml), Probe (50 µl), blocker (50 ml),
conjugate (25ml), substrate (10ml), pre-hybridization buffer (100ml), washing buffer

5X (100ml)
Equipment and other materials: Electrophoresis unit, micropipettes and tips, UV transilluminator,
blotting tray, support, glassware, whatman filter paper, paper towels, nitrocellulose membrane
(5nos)
Preparation of working solutions

Acid solution (1X) Take one volume of 10X solution and add nine volumes of double distilled
water; it gives 1X acid solution. This is ready to use for depurination step.
Denaturation solution (1X) Take one volume of 2X solution and add equal volume of double
distilled water; it gives 1X denaturation solution. This is ready to use for denaturation step.
Neutralization solution (1X) Take one volume of 2X solution and add equal volume of double
distilled water; it gives 1X denaturation solution. This is ready to use for denaturation step.
Transfer buffer(20X SSC) Dissolve the provided transfer buffer powder in 2 litres of double
distilled water to prepare transfer buffer solution (20X SSC). This has to be used as such, there
is for no need for any further dilution. This transfer buffer can be reused for 3–4 times.
TEB (0.5 X) Take one volume of 10X TEB and add 19 volumes of double distilled water, it
gives 0.5 X TEB solution. This is used to prepare agarose gel and tank buffer for electrophoresis.
Washing buffer (5X) Take one volume of 5X solution and add 4 volumes of double distilled
water. This solution is ready to use for washing step.
Procedure
Agarose gel electrophoresis
1. Prepare 0.8% agarose solution in the buffer provided (0.5X TEB).
2. Boil the agarose until it is completely dissolved and make sure no obvious particles of agarose
remain in the suspension.
3. Seal the gel casting tray on both sides and place the comb on the gel tray in appropriate place.
4. When the gel temperature is around 40°C, pour the agarose mixture into the tray containing
the comb. Do not add ethidium bromide.
5. After complete solidification of agarose, remove the seal from either side of the tray without
disturbing the gel.
6. Keep the gel tray in a tank containing 0.5X TEB buffer with the wells at the cathode
(negative) side. The buffer level in the tank should be maintained above the gel tray.
7. Gently lift the comb without damaging the wells, and now the gel is ready for loading.
Genetics 251
8. Connect the power cords between the electrophoresis tank and the power pack before loading
the sample.
9. Load 5µl of sample in the third well.
10. After loading, switch on the power pack and adjust the voltage to 50V or 100V.
11. Continue the electrophoresis until the dye reaches to 3/4th of the gel or above.
Blotting procedure
1. After electrophoresis, place the gel in a small tray containing 100 ml of acid solution (acid
purination). Rock the tray gently for 10 min. (completely cover the gel with acid solution).
2. Decant the acid solution and rinse the gel twice in distilled water.
3. Add 100 ml of denaturation solution to the tray (denaturation step). Gently rock for 15
min.
4. Add 100 ml of neutralization solution. Rock the tray gently for 30 minutes and decant the
solution. Completely cover the gel with neutralization solution (neutralization).
5. Place a support larger than the gels in a tray containing the transfer buffer. Place the paper
wicks on the support to reach to the bottom of the dish on either side.
6. Place the gel upside down on the platform. Remove any air bubbles trapped between gel
and platform by rolling a pipette several times.
7. Place the transfer buffer soaked nitrocellulose (NC) membrane on top of the gel. Remove
air bubbles by using clean test tube.
8. Place three pre-wetted Whatman filter papers on the top of the nitrocellulose membrane.
Remove air bubbles by using a clean test tube.
9. Place a pile of dry filter papers on the membrane (atleast 10–15 cm height) and place a glass
plate on the top of the filter paper and place weight, approximately 1 kg.
10. Leave the arrangement undisturbed overnight. Add additional transfer buffer, if required.
11. Remove the wet paper towels and replace them with dry paper atleast one time during
transfer to improve efficiency of transfer.
12. After the transfer period, carefully remove weight, filter papers and gel. Air dry the NC
membrane for sometime.
Southern hybridization
1. After Southern blotting, bake the filter for one hour at 80º C under vaccum (keep the NC membrane
in between the Whatman filter papers and wrap with aluminium foil and then bake).
2. Rehydrate the baked NC membrane with 2X SSC uniformly.
3. Add 10 ml of prehybridization buffer in a hybridization tube or polypropylene bag along
with NC membrane (Use sealed polypropylene bag).
4. Incubate the filter at 42ºC for 2 hours in a hybridization oven.
5. Take 10µl of biotinylated probe, boil it for 10 minutes in water bath and keep it ice.
6. Add 10µl of denatured probe in 10 ml of prehybridization buffer just before the step.
7. Pour off the prehybridization buffer and add 10 ml of prehybridization buffer containing
probe in a hybridization tube or a polypropylene bag along with NC membrane and incubate
the filter at 42ºC overnight to achieve maximum sensitivity.
8. Wash the NC membrane in 50 ml of 2X SSC solution for 3 min. at room temperature (for
effective washing, add 0.1% SDS to the SSC solution).
9. Repeat as mentioned above (Step 8).
Hybridized probe detection
1. Wash the hybridized membrane using washing buffer for 1 min.
2. Add 10 ml of blocker in a flat tray or propylene bag along with filter. Seal the bag and
incubate for 1 hour at 65ºC.
3. Wash the filter using washing buffer for 5 min.
4. Add 5 ml of conjugate in a propylene bag along with NC membrane.
5. Seal the bag and incubate for 1 hour at 37ºC in a shaking incubator.
6. Wash the filter using washing buffer for 5 minutes.
7. Repeat the above-mentioned step.
8. Add 2 ml of substrate solution in polypropylene bag along with NC membrane. Seal the bag
and incubate for 1 hour at 37ºC in a shaking incubator.
9. For result development, keep the NC membrane in dark or low light till the colour develops.
Colour gets developed within 30 min.
Weight
Stack of paper
towels
3MM paper
NC
Gel
3MM paper
Figure 8.1 Southern blotting
Result
Hybridisation of the probe to a specific DNA fragment on the filter membrane indicates that
this fragment contains DNA sequence that is complementary to the probe.
Genetics 253
WESTERN BLOTTING
Introduction
Western blot analysis can detect a protein in a mixture of any number of proteins while giving
information about the size of the protein. It does not matter whether the protein has been
synthesized in vivo or in vitro. This method is, however, dependent on the use of a high-quality
antibody, directed against a desired protein. Immunoblotting procedures combine the resolution
of gel electrophoresis with the specific antibody detection. Blotting can be used to ascertain a
number of important characteristics of protein antigens, including the presence and quantity of
an antigen, the molecular weight of the antigen, and the efficiency of antigen extraction. This
method is especially helpful when dealing with antigens that are insoluble, difficult to label, or
easily degraded and thus, not amenable to procedures, such as immunoprecipitation.
Aim
To immunoblot the protein bands frone SDS–PAGE gel to the NC membrane.
Principle
Proteins fractionated by SDS–PAGE is transferred to a solid support by electroblotting
(semi-dry and tank transfer systems). The more efficient and most widely used method of transfer is
electro blotting. In this procedure, a sandwich of gel and solid support membrane (nitrocellulose
or PVDF) is compressed in a cassette and immersed in buffer between two parallel electrodes.
A current is passed at right angles to the gel, which causes the separated proteins to electrophorese
out of the gel and onto the solid support membrane. Once the proteins have been transferred to
the solid support membrane, the membrane is referred to as a blot. The efficiency with which
a particular antigen will be transferred to the blot is dependent on the protein-binding capacity
of the membrane used, the transfer method and conditions employed as well as the nature of
the antigen.
Materials required
Sample: Protein sample: Whole serum
Reagents:
1. Labelled antibody: Rabbit anti-human IgG whole serum conjugated with ALP
2. Substrate: BCIP/NBT (1X)
3. Other reagents: bSA (1mg/ml), Tween 20, 2X sample buffer [130 mm Tris HCL (pH 8.0),
20% (v/v) glycerol; 4.6% SDS; 0.2% bromophenol blue; 2% DTT], solutions for stacking
and separating gels, 10X blotting buffer [30.3 g Trizma base (0.25 M), 144 g glycine (1.92
M0)], 1X blotting buffer (400 ml methanol, 200 ml of 10X Z blotting buffer, 1400 ml sterile
water), Blocking buffer (PBS with 1 mg/ml BSA and 0.2% Tween 20, Tris-buffered saline,
Washing buffer (PBS with 0.2% Tween 20)
Equipments and other materials: SDS–PAGE apparatus, semi-dry electroblotter, staining trays,
clean boxes, sterile tips, micropipettes, pipettes, NC membrane, Whatman filter paper,
Procedure
1. Set up the PAGE apparatus and separate the given mixture of proteins on a 10% separating
gel.
2. After the separation is over, transfer the gel into a sterile container containing 1X blotting
buffer and incubate at room temperature for 15 min.
3. Simultaneously, cut the NC membrane to the required size and wet the membrane with 1X
blotting buffer for 30 min.
4. Set up the transfer assembly in the following order.
Cathode
Sponge
Blotting paper
PA gel
Nitrocellulose
Blotting paper
Sponge
Anode
5. Transfer the protein for 1 hour at 0.8 mA/sq.m of the gel.

6. After transfer, carefully remove the NC membrane and block the membrane overnight at
4°C in blocking buffer.
7. Wash the membrane thrice with washing buffer; each wash takes 5 minutes.
Genetics 255
8. Add the labelled antibody (1 : 200 times diluted) and incubate at 37°C for 2 hours with
intermittent shaking.
9. Add 5 ml of substrate solution and incubate in dark for the development of bands.
10. After the bands develop, stop the reaction by adding 0.1% EDTA and wash the blot with
distilled water and air-dry the blot.
CATHODE (–)
Filter paper/
Cathode Buffer
SDS-PAGE gel
Transfer Membrane
Filter Paper/
Anode Buffer II
Filter Paper/
Anode Buffer I
ANODE (+)
Result
SupH2O 355µl
RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD)

Introduction
RAPD (pronounced “rapid”) stands for Random Amplification of Polymorphic DNA. It is
a type of PCR reaction, but the segments of DNA that are amplified are random. The primers
will bind somewhere in the sequence, but the position is not certain. This method is popular
for comparing the DNA of biological systems that have not had the attention of the scientific
community or in a system in which relatively few DNA sequences are compared (it is not suitable
for forming a DNA databank).
Materials required
Sample: Genomic DNA (that is to be amplified)
Media : Agarose
Reagents: Specific primers (both forward and reverse), dNTPS, NH 4 reaction buffer, Taq
polymerase enzyme, sterile water, TBE buffer, 1 kb DNA ladder, Ethidium Bromide
Equipment and other materials: Gel electrophoresis unit, microfuge tubes, thermal cycler
Principle
RAPD does not require any specific knowledge of the DNA sequence of the target organism.
The identical 10-mer primers will or will not amplify a segment of DNA, depending on positions
that are complementary to the primers’ sequence. For example, no fragment is produced if primers
annealed too far apart or 3´ ends of the primers are not facing each other. Therefore, if a mutation
has occurred in the template DNA at the site that was previously complementary to the primer,
a PCR product will not be produced, resulting in a different pattern of amplified DNA segments
on the gel. Polymorphism of amplified fragments are caused by: 1) base substitutions or deletions
in the priming sites, 2) insertions that render priming sites too distant to support amplification
or 3) insertions or deletions that change the size of the amplified fragment.
Separation
Cleavage of DNA by of DNA
restriction enzyme fragments by
electrophoresis
Filter paper
Nitrocellulose
Binding of Transfer to
radioactive DNA a membrane Gal
probe to specific (Southern blot) Southern blotting
DNA fragments Plant A SamplePlant B
Membrane X-ray film

washed free of used to detect
excess probe radioactive DNA comparison
pattern
Figure 8.2 RAPD
Procedure
Preparation of reaction mixture
1. Prepare the core buffer in a 1.5 ml microtube (enough for 100 reactions):
dNTPs, 100 mM 20µl
NH4 reaction buffer, 10X 250µl
MgCl2, 50 mM 125µl
SupH2O 355µl
Genetics 257
Total volume 750µl

Mix by inversion and spin to collect solution.
2. Prepare the cocktail in a 1.5 ml microtube (enough for 10 reactions, adjust amount according
to need). Cocktail should be prepared just before use.
Taq polymerase, 5u/µl 2 µl
Primer, 10 µm 10 µl
SupH2O 138 µl
Total volume 150 µl
Mix by inversion and spin to collect solution.
3. Prepare the reaction mixture by mixing the following components in a PCR tube:
Core buffer 7.5 µl
Cocktail 15.0 µl
DNA sample 2.5 µl
Total volume 25.0 µl
Flick the bottom of PCR tubes and spin to collect the mixture. Overlay the mixture with a
drop of mineral oil.
4. Stock and final concentrations per 25 µl of reaction mixture:
Components Stock concentration Final concentration Volume/reaction

dNTPs 100 mM 0.8 mM 0.2 µl
NH4 reaction buffer 10X 1X 2.5 µl
MgCl2 50 mM 2.5 mM 1.25 µl
Taq 5u/µl 1 u/rxn 0.2 µl
Primer 10 µM 0.4 µM 1.0 µl
SupH2 O 17.35 µl
DNA 2 ng/µl 5 ng/µl 2.5 µl
DNA amplification
1. Place PCR tubes in a thermal cycler. Amplify using the following temperature profile:
Temperature (°C) Time No. of cycles

94 2 minutes 1
94 30 seconds
5 1 minute 2
72 2 minutes
94 30 seconds
35 1 minute 2
72 2 minutes
93 30 seconds
35 1 minute 41
72 2 minutes
72 5 minutes 1
Hold temperature: 25°C
2. After amplification, remove the PCR tubes from the thermal cycler. Add 3 µl of 10X loading
buffer to each tube. Mix by flicking the bottom of the tube and spin to collect the mixture.
The mixture is now ready for loading in the agarose gel.
Electrophoresis
1. Get a gel mould and seal both edges with 1-inch masking tape. Place in a level platform and
attach combs.
2. Prepare 1.4% agarose by weighing 3.5 g agarose. Transfer this to a 500 ml flask and add
250 ml of 0.5X TBE buffer.
3. Boil for 6 minutes in a microwave. Allow the solution to cool to 60°C.
4. Pour agarose into the gel mould and allow it to solidify.
5. Fill the electrode tank with 0.5X TBE buffer.
6. Remove masking tape from both ends of the gel mould. Mount the gel mould onto the
electrode tank making sure no bubbles form beneath the mould.
7. Gently remove the comb.
8. Load 10 µl of 1 kb DNA ladder on the first well and 10 µl of each reaction mixture in the
succeeding wells (a gel can accommodate 54 samples in 2 comb positions).
9. Close the tank and attach electrode wires to the power supply. Run for 3 hours at 150 V.
Genetics 259
Staining and documentation

1. After electrophoresis, switch off the power supply and remove the tank cover.
2. Remove the gel from the moulder and transfer it to a tray with EtBr staining solution in
1000 ml of H2O. Stain for 20 minutes. (EtBr staining solution can be reused but staining
time should be for an hour).
3. After staining, rinse with distilled water.
4. Photograph the gel under UV light.
Scoring and analysis Designate a name or a number for each RAPD marker based on the molecular
size and primer used and carry out the comparison of the two individuals.
RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP)

Introduction
Restriction fragment length polymorphism or RFLP refers to the difference between two or more
samples of homologous DNA molecules arising from differing locations of restriction sites. In
RFLP analysis, the DNA sample is broken into pieces (digested) by restriction enzymes and the
resulting restriction fragments are separated according to their lengths by gel electrophoresis.
RFLP is an important tool in genome mapping, localization of genes for genetic disorders,
determination of risk for disease and paternity testing.
Aim
To perform restriction digestion of the given DNA using restriction enzymes and analyse the
restriction pattern by Agarose gel electrophoresis.
To map and find the similarity of the given two different DNA samples using RFLP (DNA
fingerprinting)
Principle
The basic technique for detecting RFLPs involves fragmenting a sample of DNA by a restriction
enzyme, which can recognize and cut DNA wherever a specific short sequence occurs, in a process
known as restriction digestion. The resulting DNA fragments are then separated by length by a
process known as agarose gel electrophoresis and transferred to a membrane by the Southern
blot procedure. Hybridization of the membrane to a labelled DNA probe then determines the
length of the fragments which are complementary to the probe. A RFLP occurs when the length
of a detected fragment varies between individuals. Each fragment length is considered an allele,
and can be used in genetic analysis.
DNA samples, Restriction enzymes, Gel loading dye (Bromophenol blue), Lambda DNA
marker/ruler, Nylon membrane, Blotting apparatus, Hybridization buffer, Probes.
DNA extracted from Restriction enzyme cleavage
blood cells of DNA
Bloodstain
Radioactive Transfer of DNA fragments Fragments of DNA are

DNA probe to a membrane separated by electrophoresis
binds to (Southern blot)
specific DNA
fragments
Membrane is
washed free of X-ray film, sandwiched DNA pattern is compared
excess probe to the membrane to detect with patterns from known
radioactive pattern
subjects
Figure 8.3 RFLP
Analysis of RFLP variation in genomes is a vital tool in genome mapping and genetic disease
analysis. For a particular chromosomal location of a particular disease gene, we would analyse
the DNA of members of a family afflicted by the disease, and look for RFLP alleles that show a
similar pattern of gene inheritance as that of the disease gene. Once a disease gene is localized,
RFLP analysis of other families could reveal who is at risk for the disease, or who is likely to be
the carrier of the mutant gene.
RFLP analysis is also the basis for early methods of genetic fingerprinting, useful in the
identification of samples retrieved from crime scenes, in the determination of paternity and in
the characterization of genetic diversity or breeding patterns in animal populations.
Materials required
Sample: Genomic DNA
Reagents: Restriction enzymes, HindIII digested Lambda DNA ladder (Marker), Ethidium
Bromide, 0.25 M HCl, 0.4N NaOH, labelling reaction mixture (if probe is used)
Media: Agarose
Equipment and other materials: Gel electrophoresis unit, Southern blotting apparatus, nylon
membrane, filter papers,
Genetics 261
Procedure
Digestion, run and gel blotting
1. Digest 3 – 5 µg of genomic DNA with 10 units of enzyme per µg of DNA (try to do
reactions in a final volume of 15µl).
2. Incubate samples at a suitable temperature for a minimum of 5 hours.
3. At least 30 min. before use, prepare a 0.6–0.8% agarose gel in 1X TBE, without EtBr. Use
wide but thin combs. Do not load the two most external lanes.
4. Add gel loading dye to each sample 3 µl and load 18 µl of the sample onto the well.
5. You can use 5µl of HindIII-digested Lambda DNA (250 ng) as size marker.
6. Run the gel overnight at 70V (about 16 hours) in 1X TBE running buffer. No cooling is
needed.
7. Stain the gel in EtBr for 20 min. wash with tap water and take a picture by placing a ruler
beside the gel in order to estimate the distance run by the size marker.
8. Put the gel in 0.25 M HCl for 20 min.
9. Meanwhile, set up the vacuum blot apparatus. Wash the porous support and wet the sealing
rubber. Connect the vacuum pump to the vacuum trap and then to the apparatus.
1 0. Wash the gel with millipore water.
11. Put the nylon membrane onto the porous support, then the plastic mask (the mask must
be slightly smaller than the gel). Carefully, slide the gel onto the mask.
12. Close the apparatus and start the pump. Close the screws tightly and pour 1 litre of
0.4N NaOH. Apply a vacuum pressure of 50 mM/H2O.
13. Leave for 1 hour, checking from time to time that there is no leakage. If there is a small
leakage add more NaOH to maintain the gel always submerged.
14. Suck out the remaining NaOH and mark with a pencil, the slots onto the nylon membrane.
15. Remove the gel, and wash the membrane with 2X SSC to clean free from agarose. Air-dry
the filter. Note with a pencil the date of the blot on the filter in an area covered by the mask.
Cut to reduce the size of the membrane.
Labelling of the probe
1. Boil for 5 minutes, 25 to 50 ng of the DNA fragment to be labelled.
2. Place on ice for 1min.
3. Set up the following labelling reaction (Pharmacia oligolabelling kit):
 25 to 50 ng denatured DNA (max 36 ml).
 10µl provided reagent mix.

 3µl [alpha-32P]dCTP
 1µl Klenow enzyme
 Distilled water to 50 ml
4. Incubate at 37°C for 60 minutes.

5. If the probe is not needed immediately, store it in the freezer for few days.
6. Before use, boil the probe for 5 minutes.
Pre-hybridization and hybridization
1. If the membrane to be used is employed for the first time, then an overnight pre-hybridization
is needed, otherwise 4–5 hours should be enough.
2. Wet the membrane with either distilled water or 2X SSC. Drain off the excess of water or SSC and
roll the membrane into the hybridization tube. Add 20 ml of hybridization solution and 600mg
of sonicated and denatured (boil for 5 minutes) salmon sperm DNA (usually stock is 10 µg/µl).
3. Incubate in the rotating hybridization oven at 65°C.
4. After pre-hybridization, start hybridization by adding the boiled, labelled probe to the
pre-hybridized membrane.
5. Incubate overnight.
Hybridization solution
1% SDS
1M NaCl
10% dextrane sulphate. (Make 1 litre with 50 mM Tris–Cl, pH 7.5. Resuspend in water bath at
65°C. You can store the solution in the freezer).
Washing of the membranes
1. Add just a little (50–100 ml) of 500 ml of 2X SSC to the hybridization solution.
2. Mix for a couple of minutes, then pour the solution into the radioactive waste tank.
3. Put the hybridized membrane in a large box and pour the rest of the 500 ml of 2X SSC.
4. Shake at room temperature for 10 min. (be careful since the radioactivity is still high).
5. Pour off the solution and replace with 500 ml of pre-heated (65°C) 1X SSC + 0.1% SDS.
Incubate for 20 minutes at 65°C.
6. Substitute the solution with pre-heated (65°C) 0.2X SSC + 0.1% SDS. Incubate while
shaking for 20 minutes at 65°C.
7. Now measure the radioactivity level of the filter using the hand counter. The filter should
not count more than 5 cpm. If it counts too much, wash at 65°C with 0.1X SSC +0.1%
SDS for 20 min.
8. Wrap the membrane with Saran Wrap and expose. Leave at – 80°C for 1 to 3 days.
Genetics 263
Removal of the probe

1. Put the membrane into boiling water +0.1% SDS, let it to slowly cool down at room
temperature.
2. Put the membrane into a plastic bag or wrap it with Saran wrap and store in the fridge (do
not let the membrane to dry out).
Result and Interpretation

The band patterns obtained were observed. By comparing the migration distance with that of
the marker, we can determine the approximate sizes of DNA fragments.
9
IMMUNOLOGY
BLOOD GROUPING
Introduction
Blood has been held as a mysterious fascination by human beings from the dawn of time. Blood
and blood transfusion became scientifically feasible only after the discovery of blood groups by
Karl Landsteiner (1900). Grouping of blood is based on agglutination reaction between antigen
and antibody present in blood cells (RBC). When particulate antigen is mixed with its specific
antibody in the presence of electrolytes at a suitable temperature and pH, the particles are clumped
or agglutinated. This is known as agglutination.
Aim
To perform blood grouping technique.
Principle
The ABO System and Rh Factor Human red blood cells (RBCs) have glycolipid and
glycoprotein components on their cell membrane surface that have antigenic properties.
One example of these glycoproteins is substance H, which gives rise to the ABO system of
blood types.
The ABO system contains four blood groups and is determined based on the presence
or absence of two antigens, A and B, and the isoantibodies for the antigen that are absent.
The presence or absence of antigens and their isoantibodies are under genetic control. The blood
groups, antigens and their isoantibodies are presented in Table 9.1.
H-antigen Red blood cells of the ABO group possess a common antigen—the H-antigen
or the H substance, which is the precursor for the formation of A-and B-antigens. A-,B- and
H-antigens are glycoproteins. A-antigen is formed by the addition of N-acetylgalactosamine to
H substance. B-antigen is formed by the addition of galactose to H substance. Blood group A
is subdivided into A1 and A2. A1 blood group has two molecules of N-acetylgalactosamine with
H substance and A2 blood group contains one molecule of N-acetylgalactosamine with H
substance. Thus, there are six blood group types, namely A1, A2, B, A1B, A2B and O.
Table 9.1 Types of blood groups
Blood group Antigen on RBC Isoantibodies

A A Anti-B
B B Anti-A
AB A and B Nil
O Nil Anti-A and
anti-B
Bombay blood group Blunde et al., from Bombay reported a rare instance in which antigens
A, B and H were absent on the RBCs. This is known as Bombay blood group or OH blood.
They have anti-A, anti-B and anti-H antibodies.
Rh blood group system Levine and Stetson (1939) demonstrated a new type of antibody in
the serum of a woman who developed serious reactions following blood transfusions from her
husband’s ABO compatible blood. She had delivered a still born infant with haemolytic disease
known as erythroblastosis foetalis. Landsteiner and Weiner (1940) identified that majority of
persons have the antigen in their RBC, which was similar to the antigen present on the surface
of Rhesus monkey erythrocytes. These antigens reacted with rabbit antiserum. This was named
as Rhesus or Rh factor. The antibody identified by Levin and Stetson was the anti-Rh factor
antibody. The typing of a person as Rh-positive or Rh-negative depends on the presence or absence
of the Rh-antigen on the erythrocytes.
Erythroblastosis foetalis If the mother is Rh-negative and the unborn is Rh-positive,
(inherited from the Rh-positive father) then the mother’s immune system will produce anti
Rh-antibodies. These may attack and destroy the baby’s blood cells. This is a rare problem in
first pregnancy. Without treatment, it becomes a serious problem in subsequent pregnancies as
the mother’s immune system gets sensitized after the first pregnancy.
Materials required
Reagents : 70% alcohol
Equipment and other materials: Glass slide, porcelain tile, lancet needle, applicator stick, cotton,
marker, etc.
Procedure
1. A clean glass slide or porcelain tile is taken and three circles are drawn and marked as A, B
and D.
Immunology 267
2. The left middle finger is wiped with 70% alcohol and punctured at the tip with a lancet
needle.
3. The first drop blood is wiped off and subsequent drops are placed onto the circles marked
A, B and D.
4. A drop of anti-A, anti-B and anti-D are placed on circles A, B and D, respectively.
5. The drops are mixed well with separate applicator sticks and agglutination pattern is observed.
Interpretation
The serum of group A individual has group B antibody. Group B has anti-A antibody and group
O has both anti-A and anti-B, while in group AB both anti-A and anti-B were absent.
RBC’ Ss agglutinate if they posses the antigen which react with the corresponding antibody
present in the serum .With the help of known sera unknown blood group can be found.
Blood type
A B AB O
Anti-A
Anti-A
Anti-B
Anti-B
Figure 9.1 ABO Blood Reactions
ANTISTREPTOLYSIN O
Aim
To determine the presence or absence of antistreptolysin O.
Principle
Antistreptolysin O (ASO) is a latex agglutination slide test performed to determine the presence
or absence of antistreptolysin O. Both qualitative and quantitative tests are performed. Most of
the strains of Streptococcus, which are pathogenic for human beings belong to group A. All these
organisms produce an exotoxin, known as streptolysin O (SLO).
SLO is a soluble oxygen-labile haemolysin. This has a lethal toxic effect on heart muscles.
In heart muscles, it produces vascular endocarditis, myocarditis and pericarditis. This is usually
seen in the pediatric age group. In the joint, it produces migratory non-purulent arthritis, which
is observed in adults.
SLO, when compared to other toxins and enzymes, is a patient antigen. SLO induces the
formation of antibodies when Streptococcus sp. infection occurs. The antibodies directed against
SLO are known as ASO or anti-streptolysin O. About 80% of patients with rheumatoid fever,
scarlet fever, tonsilitis, glomerulonephritis show ASO titre in their serum.
Polystyrene latex particles coated with streptolysin O (purified protein preparation from
b-haemolytic streptococci), reacts with ASO antibodies resulting in agglutination of latex particles.
Materials required
Sample: Serum
Reagents : Latex reagent coated with streptolysin O, positive control, negative control
Equipment and other materials: Glass slide with 3 reaction circles, plastic disposable sample
dispensing pipettes with rubber teat
Procedure
Qualitative slide test
1. A clean microscopic slide is taken and three circles are drawn and marked as test, positive
control and negative control.
2. A drop of specimen (patient serum) is placed in the test circle marked on a clean microscopic
slide. Positive and negative controls are included.
3. To this, a drop of ASO latex antigen is added.
4. It is mixed well with an applicator stick and the slide is rotated gently for 2 min.
5. Agglutination pattern is observed for test serum, positive and negative control.
6. If agglutination occurs, the test reaction is said to be positive and absence of agglutination
indicates negative results.
Note: The test result is not reliable after 2 min.
Semi-quantitative test
1. The serum showing positive agglutination in slide test, is taken for semi-quantitative test.
2. The serum is diluted with saline (0.85% NaCl). The dilutions made are 1 : 2, 1 : 4, 1 : 8,
1 : 16, 1 : 32.
Immunology 269
3. A clean slide is taken and circles are drawn corresponding to each saline dilution and the
dilutions are marked.
4. A drop of the diluted saline is placed on each circle marked on the slide.
5. To this, one drop of ASO latex antigen is added and mixed well for agglutination.
6. The highest serum dilution showing agglutination gives the approximate ASO concentration
in IU/ml of serum.
Figure 9.2 ASO slide test
Calculations
ASO in IU/ml = Titre value × ASO sensitivity
ASO sensitivity = 200 IU/ml (Standard)
Result
If agglutination is observed till 1 in 8 dilution, the highest titre value is 8 and the ASO in IU/
ml is 8×200= 1600 IU/ml.
WIDAL TEST
Introduction
Enteric fevers, such as typhoid and paratyphoid fever, are caused by Salmonella typhi and S. paratyphi,
respectively. These are gram-negative rods, motile with peritrichous flagella. In 1986, Grandaum
and Widal described the method of determining the presence or absence of antibodies in the
serum of patients infected with either of the organisms. Salmonella antibodies start appearing in
the serum at the first week and rises sharply during the third day of the fever. So, the specimens
should be tested at an interval of 7 to10 days to confirm infection. These antibodies are detected
using Widal test.
Widal is an agglutination reaction involving the antibodies from patient’s serum and Salmonella
antigens. The important antigens that are used for the test are:
1. flagellar (H) antigens and
2. somatic (O) antigens. The H antigen, present in the flagella is called flagellin protein. This is
strongly immunogenic inducing the formation of antibodies. This on combining with its antibody
forms large loose, fluffy clumps resembling wisps of cotton wool. This antigen is different for
S. typhi, S. paratyphi A and B.
The O antigen is a phospholipid–protein–polysaccharide complex. It is an antigen extending
from the outer membrane of most of the gram-negative organisms. They are less immunogenic
than the H antigen. This antigen forms tight compact deposits resembling chalk powder.
This antigen is closely related in all species.
SLIDE AGGLUTINATION METHOD

This is a qualitative test performed for the diagnosis of typhoid and paratyphoid fever.
Aim
To perform Widal test by slide agglutination method.
Principle
Agglutination is the clumping formed when a particulate antigen reacts with its specific antibodies
at optimal conditions. The patient’s serum is allowed to react with S. typhi and S. paratyphi A and
B antigens purified from the organisms. The O antigen, common for both the species and the
H antigen individually for the two species are used to find agglutination. So, the test procedure
is carried out for four different antigens to find their specific antibodies in the patient’s serum.
The antibodies are detected against the following:
1. Antigen for S. typhi, S. paratyphi A and B
2. H antigen for S. typhi
3. AH antigen (H antigen of S. paratyphi A)
4. BH antigen (H antigen of S. paratyphi B)
Materials required
Sample: Serum
Reagents : Suspensions of O, H, AH, and BH antigens, normal saline,
Immunology 271
Equipment and other materials : Applicator stick, disposable plastic dropper, rubber teat, glass
slides, pipettes, disposable gloves for specimen handling, etc.
Procedure
1. A glass slide is cleaned and wiped free of water.
2. Circles are drawn and marked as 1, 2, 3, 4, 5 and 6.
3. One drop of antiserum is placed in the circles 1, 2, 3 and 4, respectively.
4. One drop of positive and negative control are placed in circles 5 and 6 respectively.
5. One drop of O, H, AH and BH antigens are placed in circles 1, 2, 3 and 4 respectively.
To circles 5 and 6, O and H antigens are added.
6. The contents of each circle are mixed with separate applicator sticks. The slides are rocked
for 2 minutes and observed for agglutination.
O H
Figure 9.3 Slide agglutination method
Interpretation
Agglutination was seen in the positive control circle and similar agglutination was observed in the
circle containing O and H antigen (Figure 9.3) which shows the infection is with Salmonella typhi.
TUBE AGGLUTINATION METHOD

This is a quantitative test performed to diagnose and determine the antibody titre of typhoid
and paratyphoid fever.
Aim
To perform Widal test by tube agglutination method.
Principle
The patient’s serum is serially diluted and the highest dilution at which agglutination is seen is
designated as antibody titre. This is expressed as 1 : 20, 1 : 40, 1 : 80, 1 : 160, etc. The dilution
of 1 : 80 is the significant titre. A daily increase of antibody titre is indicative of the disease.
Materials required
Sample: Serum
Reagents : Normal saline, suspensions of O,H, AH, and BH antigens
Equipment and other materials: Applicator stick, disposable plastic dropper, rubber teat, test
tube, pipettes, disposable gloves for specimen handling
Procedure
1. Cleaned serological test tubes are set up in a test tube rack and numbered from 1 to 10.
2. 1.9 ml of saline is taken in the first test tube and 1ml of saline in remaining tubes.
3. To the first tube, 0.1ml of patient’s serum is added, which gives a 1 : 20 dilution.
4. Serial dilution is performed using 1ml of 1 : 20 dilution serum sample to give
1 : 40,1 : 80,1 : 160,1 : 320,1 : 640 and 1 : 1280 dilutions.
5. The last tube serves as negative control that has only the saline in it.
6. Four sets of similar dilutions are made for the four antigens and are labelled appropriately.
7. One drop of O, H, AH and BH antigens are added to the appropriately labelled tubes.
The tubes are shaken well and incubated overnight at 37°C.
8. The agglutination pattern is observed after incubation.
Agglutination in tube
Interpretation
Reading should be taken at least 30 min.–1 hour after removing the assay tube from the incubator.
O antigen shows disc like pattern whereas H antigen shows the characteristic floccular appearance
In negative reaction uncharged with a minute button of deposit at the bottom of the tube which
shows a typical swirl when the tube is flicked.
In addition to pattern of sedimented organism, reduction in opacity of supernatant as compared
to saline control tube must be observed and considered, to measure the degree of agglutination.
Agglutinin titre greater than 1 : 80 is considered significant and suggests infection, where
as low titre are found in normal individuals.
Immunology 273
SEROLOGICAL TESTS FOR DIAGNOSIS OF SYPHILIS

Introduction
Syphilis is a well known and much dreaded venereal disease. It is a sexually transmitted disease
caused by the Spirochaete, Treponema pallidum. The organism is a tightly coiled, highly motile,
delicate spirochaete that can be cultivated only in rabbit tissue culture. It quickly loses viability
outside the human body and no reservoir other than human population is known.
Syphilis is a systemic infection that if untreated progresses through three clinical stages:
1. The first stage or primary syphilis is characterized by the formation of painless papule
called as chancre, at the site of infection.
2. The secondary syphilis represents the systemic extension of the infection and presents
itself in the form of a maculopapular rash, malaise and lymphadenopathy. Following this
stage, the disease becomes self-limiting and the patients appear asymptomatic until the
development of tertiary syphilis.
3. In the final stage or tertiary syphilis, life-threatening complications may develop as a
result of extensive cardiovascular and nervous tissue damage that has ensued.
After Treponema pallidum infection, two main types of antibodies are found in the human serum:
1. reagin antibodies,
2. treponemal antibodies. Reagin antibodies are produced more rapidly than treponemal
antibodies. Serological testing for the diagnosis of syphilis is based on the detection of
reagin type of antibodies using cardiolipin antigens.
The organism is resistant to common staining procedures and is observed under dark or
phase contrast microscopy. The disease is diagnosed by various serological tests, which include
agglutination, precipitation and complement fixation. The two commonly used tests to diagnose
syphilis are VDRL (veneral disease research laboratory) agglutination test and RPR card
(Rapid Plasma Reagin) test. Both the tests are based on agglutination between serum reagin
(a non-specific antibody found in serum of syphilis patients) and cardiolipin (a soluble antigen
extracted from beef heart). This antigen is a phospholipid that reacts with reagin in patient’s
serum to form flocculation or agglutination.
I. VDRL (VENEREAL DISEASE RESEARCH LABORATORY) TEST
Aim
To perform VDRL agglutination test for diagnosis of syphilis.
Principle
VDRL test is done for the diagnosis of syphilis, based on the detection of reagin by using the
antigen prepared from normal tissues. The antigen preparation contains cardiolipin (0.3%),
lecithin (0.24%) and cholesterol (0.9%). VDRL test is performed by mixing the VDRL antigen
with heat-inactivated patient’s serum on a glass slide. The letters “VDRL” is a designation for
the laboratory in which it was developed.
Materials Required
Sample : Patient’s serum
Reagents : Antigen
Equipment and other materials: Plastic dropper, VDRL slide, applicator stick
Procedure
1. Using disposable plastic dropper, one drop of patient’s serum is placed onto the circles on
the VDRL slide and spread over the entire surface, using applicator stick.
2. To this, one drop of antigen is added and the slide is rotated vigorously for about
4 min.
3. The slide is then examined under low-power objective, immediately after rotating.
4. Formation of large clumps shows the strongly reactive result, small clumps indicate weakly
reactive result and absence of clumps shows non-reactive result.
5. Results are confirmed with positive and negative controls.
Non reactive Weakly reactive Strongly reactive
Figure 9.4 VDRL slide test

Immunology 275
II. RPR (RAPID PLASMA REAGIN) TEST

Aim
To perform RPR test for diagnosis of syphilis.
Principle
The rapid plasma reagin test is based on the agglutination reaction that occurs between
cardiolipin–lecithin–cholesterol antigen and the non-specific antibody called reagin that is found
in the serum of syphilis patients. In RPR test, the serum of patient is mixed with the soluble
antigen bound to carbon particles. The carbon particles help in visualizing the agglutination
reaction without the aid of a microscope unlike the VDRL test.
Materials required
Sample : Patient’s serum
Reagent : RPR antigen suspension, positive control serum, negative control serum
Equipment and other materials: Disposable plastic card, antigen delivery dropper for
delivering a drop of approximately 15–20µl, rubber teats, etc.
Procedure
1. About 0.5 ml of the test serum (taken from syphilis suspected patients) is placed on one of
the circles on a clean test slide.
2. The serum is spread around the circle with an applicator stick.
3. One drop of antigen is added to the serum, mixed with applicator sticks and it is rotated
back and forth for 8 min.
4. Agglutination is seen as presence or absence of black clumping in the serum–antigen mixture.
5. Black agglutination reaction shows a positive result, grey colour suspension indicates a
negative result.
6. The same procedure is carried out for positive and negative controls.
Result
Results are interpreted as follows.
1. Reactive
2. Nonreactive
3. Weakly reactive
Negative
Sample Antigen
Rotate 100 rpm/8 minutes
Sample and Positive

antigen are mixed
Figure 9.5 RPR test
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)

Aim
To perform ELISA test.
Principle
Enzyme-linked immunosorbent assay (ELISA) is being extensively used as a tool in research
as well as in analytical and diagnostic tests. The specificity, sensitivity and ease to perform this
technique have made this method popular. This method can be used for estimating any type of
multivalent antigen using the appropriate antibodies.
ELISA is so named because the technique involves the use of an immunosorbent, which is an
absorbent material specific for one of the components of the reaction, the antigen or the antibody.
This may be particulate, such as cellulose or agarose, or a solid phase such as polystyrene or
microwells. ELISA is usually done using 96-well microtitre plates suitable for automation.
The method requires two antibodies that can react with two different epitopes or antigens.
One of the antibody is immobilized on a solid support and the other one is linked to an enzyme.
Antigen containing sample is first added to the immobilized antibody and allowed to react.
The wells are washed and a suitable antibody linked to an enzyme is then added. The wells are
then washed and a suitable substrate is added. Untreated enzyme–antibody conjugate is washed
out and the enzyme bound to the solid support is estimated by colorimetry. The enzyme activity
is directly proportional to the antigen concentration. Also, the positive reaction can be identified
by means of colour development.
Materials required
Sample : Test serum
Reagents : Positive and negative control serum, phosphate-buffered saline, Tween 20, bovine serum
albumin, conjugate (anti-IgM linked with horse radish peroxidase), tetramethyl benzoate (TMB)
Equipment and other materials : ELISA plate coated with antigen, incubator, sterile distilled
water, etc.
Immunology 277
Procedure
1. The 96-well polyvinyl microtitre plates are washed with sterile distilled water and 3 to 4
times with phosphate-buffered saline–Tween 20.
2. After washing, the wells are coated with 100 µl of bacterial antigen and are kept for overnight
incubation.
3. After overnight incubation, the antigen-coated wells are washed with phosphate buffer
saline–Tween 20 for 4–5 times.
4. Then, 80 µl of dilution fluid (phosphate-buffered saline–Tween 20–bovine serum albumin
solution) are added to each well. To this, 20µl of sample is added (positive control serum,
negative control and the test serum). A blank is also kept using sterile distilled water.
5. Then the plate is covered with aluminium foil and incubated at room temperature for one hour.
6. After incubation, washing is carried out again for 4 times by adding phosphate buffer
saline–Tween 20.
7. After washing, 100 µl of conjugate (1 : 2000 dilutions) is added and incubated at room
temperature for another one hour.
8. Then the contents are discarded and washing is repeated as described earlier. Finally, 100 µl
of TMB is added and formation of blue colour is observed.
9. Formation of colour in the well after the addition of TMB indicates the presence of antibodies
in the given serum.
ELISA
Antibody Target Analyte Labeling Reagents

(Antigen) Enzyme
Binding Substrate
site
Second Antibody Product
or
Target Analyte
Sample
Bind Wash Label Read
Figure 9.6 ELISA Test

Result
Based on the colour observed, positive or negative result is obtained.
C-REACTIVE PROTEIN (CRP)

Aim
To detect the presence of infections that cause inflammatory response.
Principle
C-reactive protein (CRP) is a alpha-globulin formed during inflammatory response, necrotic
and neoplastic disease. The CRP serves as a sensitive test to detect the disease in the
acute phase of infection, as inflammation takes place at a higher rate and its rate increases
1000- fold within 10 hours of infection. Increased CRP levels is seen in acute myocardial infection,
bacterial and viral infections, RA, etc. CRP is a more reliable and sensitive method to detect
inflammatory responses than other methods.
Figure 9.7 C-reactive Protein test
The test is based on agglutination of latex particles coated with specific anti-human CRP
antibody, when mixed with CRP in serum.
Immunology 279
Materials required
Sample: Serum
Reagent : CRP latex reagent, positive control serum, negative control serum
Equipment and other materials : Glass slide, disposable applicator stick, disposable plastic
dropper, rubber teats
Procedure
Qualitative test
1. About 50 µl of serum (sample to be tested) is placed on a circle marked on a clean glass slide.
2. A drop of CRP latex reagent is added and mixed well with an applicator stick.
3. The slide is rotated gently for at least 2 minutes to observe for agglutination.
4. Presence of agglutination shows the presence of CRP in serum, indicating infection.
5. A semi-quantitative test is performed for samples showing positive result.
1. The positive serum is taken and diluted serially with normal saline.
2. The dilutions are double-fold, 1 : 2, 1 : 4, 1 : 8, 1 : 16 and so on, until the test shows negative.
3. An appropriate CRP level is calculated using the formula:
CRP in mg/ml = S × D
where,
S = Sensitivity limit = 6 mg/ml
D = Highest dilution showing agglutination
RHEUMATOID ARTHRITIS (RA) FACTOR

Aim
To determine the presence of RA factor in patient serum.
Principle
Rheumatoid arthritis is a systemic disease characterized by a chronic proliferative and inflammatory
reaction in the synovial membrane, which eventually results in erosion and destruction of joint
cartilage. RA factors are a group of autoantibodies directed against the IgG Fc fragment. These
autoantibodies can be of any of the groups, i.e., IgG, IgM, IgA and IgE. RA factor is found in
serum of patients with rheumatoid arthritis.
To determine the presence of RA factor, the RF autoantibodies in patient’s serum is made to

react with polystyrene latex particles coated with purified human IgG. This forms agglutination,
if the autoantibody (RA factor) is present in the patient’s serum.
Materials required
Sample: Serum
Reagents: Latex gammaglobulin reagent, positive control serum, negative control serum
Equipment and other materials : Disposable plastic dropper, disposable applicator stick, rubber
teats, glass slide
Procedure
Qualitative test
1. A clean microscope slide is taken. A circle is marked on it.
2. About 50 µl of patient’s serum is placed in the circle. To this, a drop of the reagent
(at room temperature) is added.
3. The reagent is mixed thoroughly with an applicator stick and the slide is gently rotated for
1–2 minutes.
4. Agglutination occurs in the patients with RA factor in their serum.
5. Positive results are always confirmed with a semi-quantitative test.
6. Positive and negative controls are also tested.
1. The patient’s serum is serially diluted in 1 : 10, 1 : 20, 1 : 40 and 1 : 80 ratios.
2. One drop of each dilution is added to circles marked on the slide.
3. To this, one drop of the reagent is added and mixed well.
4. The slide is gently rotated for 1–2 minutes. The presence or absence of agglutination is
observed.
5. The highest dilution of the patient’s serum showing agglutination is the RA titre.
6. An approximate calculation of RA activity in serum is given by:
RA factor IU/ml = S × D
where,
S = 5 IU/ml (sensitivity of reagent)
D = Highest dilution showing agglutination
Immunology 281

Figure 9.8 RA factor slide test
COUNTERCURRENT IMMUNOELECTROPHORESIS
Aim
To identify the presence of antibodies in test serum by countercurrent immunoelectrophoresis.
Principle
The conventional way of detecting the presence of antigen or antibody by immunodiffusion is
generally slow and time-consuming. The problem can be complemented using countercurrent
immunoelectrophoresis, which was devised by Bussard in 1959. This technique is widely used in
pathology and disease investigation departments due to its speed, simplicity and sensitivity in diagnosis.
Countercurrent immunoelectrophoresis is absolutely a qualitative technique in which
antigen–antibody reaction occurs rapidly due to electro-endosmosis induced by electric current.
The migration of antibody towards the cathode and antigen towards the anode helps the molecule
to react more rapidly, which results in the formation of immune precipitate at an equivalence
point (where antigen–antibody are in optimal concentration) in less than one hour. Since antigen
and antibody cross each other to form precipitin line in a matter of few minutes, this technique
may also be called as crossover electrophoresis.
Materials required
Sample: Serum
Media : Agarose
Reagents : Antiserum, 5X electrophoresis buffer, standard antigen a, b, c, Test Ag 1and 2,
alcohol, distilled water
Equipment and other materials : Conical flask, measuring jar, pipette tips
Procedure
1. Glass slides are washed thoroughly and dried.
2. Agarose solution is prepared. 4 ml of buffer containing 40 mg of agarose is taken in a test
tube and kept in boiling water until the agarose powder dissolves completely.
3. The agarose gel is poured on the glass slide and allowed to solidify.
4. The template is placed and the wells are made with gel puncher. The gel pieces are removed
with the gel remover.
5. Then, the slide is placed on the bridges of the horizontal apparatus and paper wig connection
is established after pouring 25 ml of buffer to each reservoir.
6. Afterwards, the given antiserum (10µl) and antigen (10µl) are added in the appropriate wells.
7. The power supply is turned on and kept at 50V and run for 30 min.
8. After 30 minutes, electrophoresis is stopped, wires are disconnected and the slide is taken and
incubated in a Petri dish containing wet filter paper for 15 to 30 minutes at room temperature.
9. A precipitin line is observed, which shows the presence of antibody in the test serum.
+ –
Ag Ag
Ab
Ag
Ab
Figure 9.9 Countercurrent Immunoelectrophoresis
Observation
Note down the presence/absence of precipitin line between antigen and antisera wells.
Sample Precipitin line

Control
Test antisera 1
Test antisera 2
Test antisera 3
Interpretation
Precipitin line indicates the presence of antibody for the antigen in the test sera.
The absence of the precipitin line indicates the absence of any antibody for the antigen is
the test sera.
Immunology 283
IMMUNODIFFUSION
I. OUCHTERLONY’S DOUBLE DIFFUSION (ODD) TECHNIQUE
Aim
The objective of this experiment is to detect the immunological relationship between the two
antigens. It is used to find whether the given antigens are:
1. Identical to one another.
2. Not completely identical but share a common number of antigenic determinants
(partially identical).
3. Distinctly different or non-identical.
Principle
Ouchterlony’s double diffusion technique is a type of precipitation reaction, devised by
Ouchterlony. This method brings together the antigen and antibody through diffusion and when
the two meet at optimal concentration, they form visible form of precipitation lines. The reactants
move towards each other and are precipitated in the medium that originally contained neither.
Since both the components diffuse, the method is known as double immunodiffusion technique.
Both the antigens and antibodies are present as solutions in separate wells in an agar base.
They diffuse towards each other and based upon the pattern of precipitation, their relationship
is determined.
1. Identical antigens: Precipitation lines fuse.
2. Partially identical antigens: Spur formation.
3. Non-identical antigens: Precipitation lines cross each other.
Figure 9.10 Ouchterlony’s double diffusion technique

Materials required
Sample: Serum
Media : Agarose
Reagents : 10X assay buffer, antigen, test antiserum, distilled water
Equipment and other materials : Glass ware, conical flask, measuring jar, test tube, micropipette,
tips, moist chamber (box wet cotton)
Procedure
1. A clean microscopic slide precoated with 0.3% agar is coated with 1% agarose in phosphate
buffer solution (pH 7.2).
2. The agarose is allowed to solidify and the slide is placed on a template to punch wells.
3. Three wells of 4 mm diameter is made leaving an equal distance of 8 mm between each of
the wells (looks like an equilateral triangle).
4. The lower well is loaded with the antisera or antibody corresponding to the two antigens used.
5. The two upper wells are loaded with two different antigens, one in each well. Approximately
10µl of antigen and antibody are used.
6. The slides are then incubated overnight in a humid chamber (usually a Petri plate saturated
with the same buffer solution is used to prepare the agarose plates).
7. The slides are observed for the pattern of precipitation lines and the results are determined.
Staining of the slides Though the immunoprecipitation lines formed on agarose are quite
visible, visualization of weaker bands are achieved by staining with stains like Coomasie’s brilliant
blue. Staining makes photography easier. The staining method includes: a) removal of unrelated
proteins and excess of salts to avoid background staining, b) washing and thorough drying of
slides prior to staining.
The staining procedure involves the following steps:
i. The slides are placed in a Petri dish containing the PBS solution. The buffer is changed
thrice at regular intervals (2 hours).
ii. The slides are then placed in distilled water and rinsed repeatedly thrice to remove excess salt.
iii. The slides are now covered with a wet filter paper and allowed to dry in room temperature.
This is done to avoid cracking of the gel during drying.
iv. The slides are stained with Coomasie’s brilliant blue for 30 minutes, until bands become
visible.
v. Then, it is destained using the destaining solution (mixture of acetic acid, water and
methanol), for 3 to 4 times, until the background becomes clear.
vi. The slides are then dried at room temperature.
Immunology 285
Observation
Observe opaque precipitin line between the antigen and antiserum wells. Note down the height
dilution at which the precipitin line is formed.
Result
Presence of precipitin line was observed and titre value is 1:16
II. RADIAL IMMUNODIFFUSION (RID) TECHNIQUE

Aim
To detect and measure antigen in a mixture of antigens using radial immunodiffusion technique.
Principle
Radial immunodiffusion is a simple and rapid technique applied to detect and measure the
presence of small amount of antigen in a mixture of diverse antigens. Since only one of the
component, i.e., the antigen is allowed to diffuse into agar layer in which antibody is fixed, it is
known as the single diffusion technique. The diffusion of the antigen results in the formation of
precipitate at the optimal concentration of antigen–antibody reaction, which is visible in the form
of a halo around the antigen well. The halo shows the presence of antigen–antibody reaction.
The measurement of the diameter of the halo after it has reached maximum size is correlated
with concentration of the antigen.
Qualitative test For a qualitative test, the antiserum dilution is not critical, although the size
of the precipitin ring could be varied inversely with the antiserum concentration.
Quantitative test For a quantitative test to be performed, the concentration of the antiserum
becomes the more important part. Antiserum is diluted with barbital buffer to dilutions
1 : 10, 1 : 20, 1 : 30 and so on.
Materials required
Sample: Serum
Media : 1% agarose
Reagents : PBS, barbital buffer, sodium chloride, antiserum, alcohol, distilled water
Equipment and other materials : Glass ware, conical flask, measuring jar, micropipette, tips,
moist chamber, gel puncher
Procedure
Qualitative test
1. 1% agarose is prepared in PBS (pH 7.2) with 8% sodium chloride. The agarose is melted at
56°C in a water bath.
2. The antiserum to be used is brought to 56°C using water bath.
3. The antiserum is added to the molten agarose and carefully mixed without bubbles.
4. Using a pipette, required amount of (usually 3–3.5 ml per microscopic slide)
agarose–antiserum mixture is dispensed on the slide.
5. The agarose–antiserum mixture is allowed to solidify at room temperature in a chamber
(Petri plate) humidified with buffer.
6. A 2 mm well is made using a gel puncher.
7. To the well, about 2ml of antigen is added and incubated at room temperature in a humidified
chamber (Petri plate saturated with PBS).
8. The results (formation of precipitation rings) are observed after overnight incubation.
The results are best seen under fluorescent lamp.
Quantitative test
1. 1% agarose is prepared in 0.1 M of PBS(pH 7.2) or in 0.05 mM of barbital buffer (pH 8.6).
The agarose is melted and held at 56°C using a water bath.
2. Then, 3 ml of 1 : 10, 1 : 20, 1 : 30 and 1 : 40 dilutions of antiserum in barbital buffer is prepared.
3. About 1.5 ml of diluted antiserum is added to 1.5 ml of agarose. The solutions are mixed
gently by rotating between the two palms.
4. The slides are coated with agarose with antiserum at different concentrations.
5. The agarose is allowed to set and the wells are made using a gel puncher. Four wells are
made on each slide for different antigen dilutions.
6. Antigen dilutions like 1 : 10, 1 : 20, and 1 : 40 are made with barbital buffer.
7. About 2 µl of antigen is added to the wells on agarose. Each dilution is added to each slide
in the corresponding wells.
8. The slides are incubated in a moist chamber at room temperature until the reaction is
completed.
9. After incubation, the diameter of the halo formed around the antigen well is measured in
each slide.
10. A graph is plotted for the diameter of the halo in mm vs the concentration of the protein
or antigen added to the well.
Immunology 287
Figure 9.11 Radial immunodiffusion technique
Result
From the standard curve, determine and report concentration of antigen in the test sample.
ISOLATION AND CHARACTERIZATION OF ANTIGENS

Aim
To isolate and characterize Widal antigens.
Principle
Antigen is defined as any foreign substance, (non-self components, such as proteins, nucleoproteins,
polysaccharides and some glycolipids) which when introduced inside the body stimulates the
production of an antibody with which it reacts specifically in an observable manner. This
experiment deals with the isolation and characterization of Widal antigens by the use of whole
cell particulate antigens of Salmonella typhi, Salmonella paratyphi A and B.
Salmonella antigens are grouped into two:
 Flagellar (H) antigen
 Somatic (O) antigen
The H protein present in the flagella is called as flagellin protein. It is heat-labile and strongly
immunogenic inducing antibody formation. The somatic O antigen is a phospholipid– protein–
polysaccharide complex, which is less immunogenic than the H antigen.
Materials required
Culture : Salmonella culture
Media : Sterile nutrient agar, sterile MacConkey agar

Reagents : Saline bottles, 0.85% of NaCl, formalin
Equipment and other materials: Sterile saline bottles, incubator, sterile flask, water bath, Gram-
staining reagents, glass slides, Widal test reagents
Procedure
1. About 25 ml of sterile nutrient agar medium is coated as thin layer on the inner surface of
sterile saline bottles by rolling on ice beds.
2. Then, 2 ml of overnight Salmonella typhi culture is introduced into the bottle and rolled
thoroughly for uniform spreading of inoculum.
3. The bottles are incubated at 37°C for 48 hrs to obtain a pure growth of Salmonella sp.
4. The growth is harvested from each bottle using 40 ml of 0.85% saline.
5. This is transferred to a sterile flask and turbidity is adjusted to MacFarland’s opacity tube
no. 1 or optical density 1.
6. To obtain H antigens, the cells are mixed with formalin such that the final concentration of
formalin is 0.5% and is incubated for 48 hrs. Another batch of culture is boiled in a water
bath, to prepare O antigen.
7. Purity of the culture is checked by preparing Gram-stained smear.
8. The sterility of the antigen is checked by subculturing on MacConkey agar plates.
9. Final characterization is done by performing slide Widal test.
Result
In this experiment the antigens were prepared from Salmonella typhi. The characterization of
these antigens was done by slide agglutination. In this method, prepared antigen was mixed with
positive control serum and agglutination was confirmed. The H suspension shows large loose
fluffy clumps and O suspension shows or forms compact chalky granular clumps.
Note One of the important prerequisites in antigen preparation is the demonstration of the
purity and sterility of the antigen before immunization. The Gram-stained smear should show
only gram-negative rods and no growth should be obtained on MacConkey medium. The antigen
thus obtained can be used for several purposes, such as generation of polyclonal antibodies by
immunizing chicken.
Immunology 289
PURIFICATION OF IMMUNOGLOBULINS BY PRECIPITATION

AND DIALYSIS
Aim
To separate and purify the immunoglobulin fraction from the whole antiserum by salt precipitation
and dialysis.
Principle
Immunoglobulins are separated by precipitation of whole antiserum either by ammonium
sulphate or sodium sulphate. The changes on the protein in the solution can be neutralized by
the addition of these salts and thereby, it can be made to precipitate. Theoretically, any salt can
be used, generally ammonium sulphate is preferred mainly for two reasons:
 It has high solubility (840 g/l).
 Its distribution in water is exothermic (or) the solution gets cooled.
The precipitated immunoglobulins are dialysed in order to remove the salt content from the
protein and thereby, it is purified (the presence of proteins interfere in many ways). For dialysis,
special semi-permeable membrane, called dialysis tube is used. It has the property of allowing
the compounds with low-molecular weight to pass through it and retain those proteins with
high-molecular weight, such as immunoglobulins.
The protein sample to be desalted is taken inside the dialysis bag and the ends are secured
tightly to prevent any leakage. The bag should be filled to half the volume, leaving some space
so that during osmotic movement, the solution that goes inside can be accommodated. The bag
is now superloaded in a large vessel containing 100-fold of dilute buffer. The contents should be
continuously stirred. Salt molecules pass freely and gets diluted by the large volume of liquid or
fluid in the external medium. Repeated changes of the dialysis fluid helps in reducing the salt
concentration inside the bag to a negligible level.
Materials required
1. Antiserum against any antigen
2. Phosphate-buffered saline (pH 7.4)
3. Sodium phosphate (20%, 18%, 24%, 36%)
4. Dialysis bag
5. 2% sodium bicarbonate
6. 1M EDTA
7. Ammonium sulphate buffer
8. Nessler’s reagent (Appendix I)
Procedure I
1. To 1 ml of antiserum sample, 1 ml of PBS (pH 7.4) and 2 ml of 36% sodium sulphate is added.
2. It is mixed well and stirred gently for 30 minutes in room temperature.
3. Then, the mixture is centrifuged at 5000 rpm for 10 minutes. The precipitate is washed
twice with 18% sodium sulphate solution by centrifugation after which the supernatant is
discarded.
4. The precipitate is then dissolved in 0.8 ml of PBS and equal quantity of 24% sodium
sulphate solution is added and centrifuged at 5000 rpm for 10 minutes. It is then washed
and precipitated with 12% sodium sulphate.
5. The precipitate is then dissolved in 1 ml of PBS and transferred to a pretreated dialysis bag.
Procedure II
1. About 10 ml of antiserum is mixed with 6.62 g of ammonium sulphate to have 90% of
saturation (ammonium sulphate is dissolved into the sample slowly).
2. Then, this is kept at 4ºC overnight. The sediment is then centrifuged at 10,000 rpm for
15 minutes. The pellet obtained is used for dialysis against PBS (pH 7).
Preparation of dialysis bag required for dialysis The bag is cut and washed continuously in tap
water for 30 minutes. Then, it is boiled in a large volume of 2%(w/v) sodium bicarbonate and 1
mM EDTA(pH 8) at 80°C for 10 minutes and cooled down by acidification with 0.2% sulphuric
acid. It is then rinsed with hot water. Then, the treated bag is put in sterile water for use.
Dialysis One end of the dialysis bag is tied and after which it is half filled with serum sample.
The other end is also tied and the bag is immersed into a bag containing ammonium sulphate
buffer. The set is kept at 4ºC overnight. The buffer is changed 2–3 times to remove all the salts.
This is tested by adding Nessler’s reagent in the buffer.
Concentration of protein (immunoglobulins after dialysis) The dialysis bag with desalted
immunoglobulins are buried in a jar containing sucrose and this set-up is kept in refrigerator
(during this water will be removed out and absorbed by sucrose).
Result
The purified immunoglobulin was obtained as a small white pellet sticking on to the dialysis bag.
10
MEDICAL MICROBIOLOGY
ISOLATION AND CHARACTERIZATION OF PATHOGENS

FROM CLINICAL SAMPLES
I. SAMPLE COLLECTION
Sample Methodology of collection

1. Throat swab Good lighting is required when collecting the throat swab. Using the
handle of a disposable spatula, the tongue is depressed, the inside of
the mouth is examined and looked for inflammation and the presence
of any membrane exudate or pus. The affected area is swabbed using a
sterile cotton-wool swab. Care is taken not to contaminate the swab with
saliva. The swab is then kept in a sterile container and labelled. The label
must have the following details: first name, second name, date, location,
physician, time, date, age and sex.
2. Sputum sample Early morning is the best time to collect the sputum sample. Sputum is
a mixture of bronchial secretion and inflammatory exudate coughed up
into the mouth. Pooling of sputum is not recommended for culturing.
Sterile container should be provided to the patient. The container may
be silver-capped or white-capped sterile plastic container. The difference
between the sputum and spit should be explained to the patient. A deep
cough sample is taken. Patients with dentures should remove those and
all patients should rinse their mouth with water. The sample should be
collected directly into the container. The container must not be leaky
and the lid should be screwed properly. The container should be sealed
with sticky caps to prevent any leakage.
Table 10.1 Identification of microorganisms
Biochemical E. coli (gram- Pseudomonas Shigella sp. Proteus sp. Salmonella Vibrio cholerae Staphylococus Streptococcus Bacillus Klebsiella sp
tests negative (gram- (gram- (gram- sp. (gram- (gram- aureus (gram- pyogenes sp. (gram-
bacilli) negative negative negative negative negative positive cocci) (gram- (gram- negative
bacilli) bacilli) bacilli) bacilli) bacilli) positive positive bacilli)
cocci) bacilli)
Indole + – + – + – – –
MR + – + + + – + –
VP – – – – + + +
Citrate – – + – + +
Oxidase – + + – +
Coagulase +
H2S – – – + + – –
TSI butt/slant Acid/alkaline Acid/alkaline Acid/acid Acid/alkali –
–
ne
Nitrate +
– + + + + +
Gelatin Delayed
– + + + +
Catalase + + + + – + +
Urease – + + – – + +
Starch
hydro lysis – – +
Casein
– + – +
hydro lysis
Carbohydrate A/G, A/G, Acid Acid A/G Acid butt no Acid butt no Acid butt no Acid
fermentation A/G (oxidative gas gas gas butt no
(Glucose, metabolism, gas
sucrose, not
lactose) fermentative)
Table 10.1 (Continued)
Biochemical E.coli Pseudomonas Shigella sp. Proteus sp. Salmonella Vibrio Staphylococ Streptococcus Bacillus sp. Klebsiella sp
tests (gram- (gram-negative (gram- (gram- sp. (gram- cholerae us aureus pyogenes (gram- (gram-
negative bacilli) negative negative negative (gram- (gram- (gram- positive negative
bacilli) bacilli) bacilli) bacilli) negative positive positive bacilli) bacilli)
bacilli) cocci) cocci)
Selective Eosin Pseudomonas Deoxycholae SSA TCBS Mannitol Blood agar PLET medium MacConkey
media methylene isolation agar citrate agar (Thiosul- salt agar (Polymyxin agar
blue (EMB) (DCA), phate (MSA) lysozyme
agar Salmonella– citrate bile ethylene
Shigella (SSA) salt sucrose diamine
agar) tetraacetic
acid)
Motility Motile Motile Non-motile Motile Motile Motile Non-motile Non-motile Non-motile Non-motile
Medical Microbiology 293
3. Nasal secretions Nasopharyngeal aspirates, washings and swab specimens are collected
predominantly for the diagnosis of oral respiratory infections and also
for measles. Washings or swab specimens are collected for the detection
of Bordetella pertussis. An aspirate is collected with a plastic tube attached
to a 10 ml syringe or a suction catheter with a mucous trap.
4. Gastrointestinal The container, which is used for the sample collection, should be sterile
tract—stool and in nature. Screw-capped tubes are used. The collected
sample is rectal swab transported immediately to the laboratory. In case
of enteric fever, venous blood is collected for blood culture.
5. Urine sample Random and first morning specimens may be collected in clean, disposable
containers, large enough to hold at least 50 ml. The mid-stream urine
must be collected.
6. Blood sample The sample is collected intravenously, the arm of the patient is supported
on the edge of a table. The vein is located visually. The area is disinfected
with alcohol or spirit. A fresh sterile syringe and needle is introduced into
the skin with a firm and smooth motion. The vein is punctured a few
mm ahead of skin puncture and required amount of blood is drawn.
7. Skin scrapings The area of the skin from which the specimen is to be collected is first
cleaned with soap and water. Antiseptics or topical antibiotics should be
avoided as these may suppress the growth of pathogens, thereby defeating
the very purpose for which the specimen is being collected. Swabs are
firmly rubbed over the affected part of the skin and sent to the laboratory
for processing.
8. Pus sample Pus sample should be submitted in a small, screw-capped bottle or in a
sealed capillary tube. The pus is rubbed with a swab and kept in sterile
tube.
9. CSF The cerebrospinal fluid is usually collected by a lumbar puncture between
3rd and 4th lumbar vertebra using a flexible muscle about 10 cm long
and borer of 1.0–1.5 mm. Only 3–5 ml of fluid should be collected. The
procedure should be attempted only by well-trained physicians. Rigorous
aseptic precautions must be observed. The sample is collected in fresh,
sterile screw-capped containers.
10. Ear discharge The yellow-coloured pus from the wound is swabbed and taken as sample
and is kept in a sterile tube.
11 . Eye discharge The sample is collected from the inflamed portion of the conjunctiva with
a cotton tip swab and is kept in a sterile tube.
12. Vaginal swab The swab is held at its plastic lid end and the labia is parted and swab is
inserted into the vagina and swabbed 3 times inverted and dragged out
without touching the skin and the swab is inserted in the sterile plastic
container .
13. Endo cervical The endometrial lining is swabbed via the cervix using a swab.
swab
14. Urethral The tip of penis is cleaned and a special thin swab is inserted into
discharge the urethra and is twisted gently side to side and left for few
seconds before it is removed. This is done to allow the swab to collect
enough fluid to be cultured.
IDENTIFICATION OF MICROORGANISMS
II. SAMPLE PROCESSING
1. Pus
Pus is an inflammatory purulent exudate rich in leucocytes (mostly neutrophils), parenchymal
cell debris, deoxyribonucleoprotein and lysosomal enzymes derived from dead leucocytes. Hence,
pus-forming infections are termed as pyogenic infections and are caused by a wide variety of
bacteria and fungi. Pus samples are generally obtained from wounds, abscesses, burns and sinuses.
Possible pathogens
a. Bacteria
i. Staphylococcus aureus is the commonest pathogen in skin wounds.
ii. Clostridium perfringens exists in deep wounds where anaereobic conditions exist. The toxin
produced by the organism causes putrefactive decay of the tissue with gas production.
The death and decay of tissue by C. perfringens is called gas gangrene.
iii. Clostridium tetani is the commonest cause of neonatal death in the rural areas of developing
countries.
iv. Mycobacterium tuberculosis is associated with cold abscess.
v. Pseudomonas aeruginosa is associated with infected burns and hospital-acquired infections.
vi. Bacteroides are associated with abscess of the abdomen, respiratory infections, female genital
tract infections and Vincent’s angina.
vii. Proteus and Klebsiella species are also encountered in pus samples.
b. Fungi Candida albicans, Cryptococcus neoformans, Histoplasma sp.
c. Parasite Entamoeba histolytica
Laboratory diagnosis
a. Specimen collection Collection of pus can be done either by aspiration or with sterile swabs.
If the swabs are to be used, swabbing is done from the depth of the wound. Preferably two
swabs are used, where one can be used for the preparation of smear for microscopy and the other
for seeding of culture. Swabs are moistened with a little broth or saline to avoid drying of the
specimen and placed in a sterile screw-capped container.
Aspiration of pus from an abscess is done when the abscess is incised and drained or after it has
ruptured naturally. Special care is taken to avoid contamination of the specimen with commensal
organisms from the skin. Upto 5 ml of pus is aspirated and transferred to a leak-proof container.
b. Laboratory examination
Laboratory examination of pus includes:
i. Naked eye examination of the specimen
ii. Microscopic examination of a Gram film
iii. Culture onto different media
i. Naked eye examination The appearance of the specimen is described on the basis of colour
of the pus and the presence of granules:
 The pus of a staphylococcal lesion is creamy and thick in consistency.
 Straw-coloured and watery pus is an indication of Streptococcus pyogenes infection.
 A fishy smell in pus indicates Proteus infection.
 A sweet, musty odour and often blue pigmented pus is a direct indication of Pseudomonas
infection.
 Pus-containing anaerobic organisms often has an offensive putrid smell.
 Actinomycosis may contain small microcolonies that appear as “sulphur granules”.
 In fungal infections, like mycetoma, black or brown granules may be present.
ii. Microscopic examination
 A thick smear is made with the help of a swab or from a loopful of aspirated specimen
and Gram staining is done.
 A wet film of the specimen is done to reveal fungi, motile bacteria or acid crystals.
 A smear stained by Ziehl–Neelsen method is done to reveal tubercle bacilli.
 Dark-ground microscopy of a wet film is useful in the diagnosis of primary syphilis.
iii. Culture examination The pus sample is inoculated onto two blood agar plates, one
incubated at 37ºC aerobically and the other incubated with 5–10% CO2 anaerobically.
Inoculation of the specimen is done on the following plates:
 Chocolate agar
 MacConkey agar
 Sabouraud’s dextrose agar (SDA)
 Thioglycollate broth
 Mannitol salt agar (MSA)
 Cooked meat medium
 LJ medium
MacConkey, MSA and SDA agar plates are incubated aerobically at 37ºC for 24–48 hours
and thioglycollate broth is incubated anaerobically.
2. Sputum
Sputum is the material obtained from lower respiratory tract, which is a mixture of bronchial
secretion and inflammatory exudates coughed up into the mouth and expectorated. The first
expectorate coughed out in the morning is the most desirable specimen for laboratory investigation.
The mixing of saliva with sputum must be prevented for accurate results.
Possible pathogens in sputum
a. Bacteria
Gram-positive
i. Streptococcus pneumoniae is the common cause of lobar pneumonia.
ii. Staphylococcus aureus and Streptococcus pyogens are secondary invaders in patients with influenza
virus and pneumonia.
Gram-Negative
i. Haemophilus influenzae causes chest infection in post-surgical patients.
ii. Klebsiella pneumoniae causes pneumonia.
iii. Pseudomonas aeruginosa is found in patients with chronic lung cavities.
iv. Yersinia pestis is found in the sputum of patients with pneumonic plaque.
v. Mycobacterium tuberculosis is found in TB patients.
vi. Mycoplasma pneumoniae causes atypical pneumonia.
vii. Legionella pneumophila causes Legionnaire’s disease, a fatal form of pneumonia.
Sputum, when it is collected, passes through the pharynx and mouth. It therefore, becomes
contaminated with small numbers of commensal organisms from the upper respiratory tract and
mouth. These include: Staphylococcus aureus, S. epidermidis, Enterococcus, Micrococcus, Lactobacillus,
Diptheroids and Coliforms. Knowledge of these commensals is important for an accurate report.
b. Fungi and actinomycetes

Blastomyces dermatitides, Aspergillus fumigatus, Histoplasma capsulatum, Candida albicans, Cryptococcus
neoformans, Nocardia asteroides.
c. Parasites
Paragonium sp.
Specimen collection
The mouth is rinsed with water to avoid contamination. The early morning expectorate is
collected in a disposable, wide-mouthed, screw-capped, sterile plastic container of about 100 ml
capacity. The collected specimen is processed as soon as possible if suspected for S. pneumoniae or
H. influenzae and must not be refrigerated. The specimen can be kept at 4ºC for 2 hours, if the
suspected isolate is M. tuberculosis.
Laboratory examination
Laboratory examination of sputum includes:
i. Naked eye examination of the specimen
ii. Microscopic examination
 Gram staining
 Acid-fast staining for M. tuberculosis
 Saline preparation for Paragonium eggs
 Eosin preparation to observe the eosinophils in case of asthma or other allergic conditions
 KOH mount for fungal infection
 Giemsa staining to detect H. capsulatum and Y. pestis
iii. Culture onto different media
i. Naked eye examination: Appearance of the specimen Sputum from a bacterial infection is
purulent, containing yellow or green opaque material as well as clear mucoid secretion.
Note
Purulent Green looking sputum with pus
Mucopurulent Green looking with pus and mucus
Mucoid Mostly mucus
Mucosalivary Mucus with a small amount of saliva
ii. Microscopic examination A thick smear is prepared with the purulent part of the sputum
for Gram staining and also for Ziehl–Neelsen staining. If the clinical history suggests any other
infection, specific microscopic tests mentioned earlier, are done.
iii. Culture examination
 The sputum sample is inoculated onto the following medium: (i) Blood agar (ii) Chocolate
agar (iii) Lowenstein–Jensen medium (iv) MacConkey agar.
 The blood agar plate is incubated aerobically and chocolate agar, anaerobically, with
5–10% CO2 for 24 hours at 37ºC.
 The LJ slope is incubated at 37ºC for 6 weeks.
 The plates are examined after the incubation period and the results are recorded.
Processing of sample for Mycobacterium tuberculosis

Sputum Specimen should be preferably an early morning collection, received in a sterile wide-
mouthed, screw-capped jar. A good specimen is 5–10 ml of well-coughed-up material without
salivary contamination. Three to five consecutive samples are adequate for diagnosis.
Smear examination
i. Direct Use new slides. Pick up flecks of caseous, necrotic, cheesy, and rusty or blood-tinged
specimen to make uniform, thin smears. If the specimen is watery, centrifuge at high speed
at 3,500 rpm for 20 minutes and prepare smears from the sediment. Stain by Ziehl–Neelsen
technique/fluorescent dye and examine the smears for acid-fast pink staining bacilli or fluorescing
bacilli. Heat-fixing does not always kill Mycobacteria. Thus, slides should be considered potentially
infectious and handled appropriately.
ii. Concentration by Petroff’s method
1. Pick up similar portion of the sputum by means of a pipette and transfer to a sterile
screw-capped centrifuge tube or leak-proof tube or MacCartney bottle, until the volume is
about 5 ml.
2. Depending on the nature of the specimen, add one or two drops of equal volume of sterile
4% NaOH.
3. Mix well with a vortex mixer or in a shaker.
4. Place in a 37oC water bath for 20–25 minutes, depending on the nature of the specimen.
Shake well at 10-minute intervals to facilitate digestion of the mucopurulent material.
5. Add 1 drop of sterile 0.004% phenol red or 0.02% bromothymol blue indicator.
6. Add sterile 8% HCl slowly until a definite yellow colour is obtained.
7. Back titrate with sterile 40% NaOH until the first persistent pink or blue tinge appears.
8. Centrifuge at high speed for 15 minutes.
9. Pour or pipette off the supernatant into a sterile test tube.

10. Mix the sediment well, make smears and inoculate onto culture media.
Culture examination
1. Inoculate 2–3 slopes of Lowenstein–Jensen’s medium with the sediment after centrifuging.
2. Incubate at 35–37oC in a CO2 incubator with the caps loose.
3. Lay the inoculated tube horizontally for a few days, if space permits, otherwise for 24 hours
at least, to allow firm adherence of the inoculum to the surface of the medium.
4. Then keep the tubes vertically in racks for 8 weeks at 35–37oC in aerobic atmosphere.
Examine daily for 1 week for any rapid growers or for contamination, indicated by blowing,
digestion of media, etc., and thereafter, at weekly intervals for evidence of growth. Colonies
of M. tuberculosis are slow growing, dry, friable, warty and buff in colour. M. bovis colonies are
tiny, translucent and smooth. They appear usually in 3–4 weeks, some in 4–8 weeks. Colonies
of atypical Mycobacteria are quite variable—some are rough, but most are smooth, with colours
ranging from cream to orange to red. Incubation periods are also variable and colonies of some
may appear at any time after about 3–4 days of incubation. All positive cultures are examined
microscopically to be certain that acid-fast bacilli have grown and not non-acid-fast contaminants
that may form somewhat similar colonies.
Biochemical tests
The following biochemical tests may be useful in differentiating atypical Mycobacteria.
 Photochromogens: Nitrate reduction, Tween 80 hydrolysis, semi-quantitative catalase and
urease.
 Scotochromogens: Nitrate reduction, Tween 80 hydrolysis, urease and tolerance to
5% NaCl.
 Non-photochromogens: Niacin production, nitrate and tellurite reduction,
semi-quantitative and heat-stable catalase and Tween 80 hydrolysis.
 Rapid growers: Arylsulphatase, nitrate reduction, iron uptake, growth on LJ (Lowenstein–
Jenson) medium.
i. Niacin production All positive cultures are tested for niacin production after they are well-
matured. Strains of M. tuberculosis usually give positive niacin test results; Other Mycobacteria
usually give negative results.
Pipette 0.5 ml of the extract from the culture tube and place in another tube. Add 0.5 ml of
4% aniline in ethyl alcohol. This mixture should be colourless.
Add 0.5 ml of 10% cyanogen bromide. Look for immediate development of a yellow colour,
which is indicative of positive result. There is no colour change, if niacin has not been formed.
When the niacin test is negative, test for other properties must be carried out.
ii. Nitrate reduction M. tuberculosis, M. kansasii, M. szulgai and smooth colonies of M. fortuitum
are the clinically significant species, which have the property of reducing nitrates to nitrites,
though some saprophytic strains like M. flavescens also reduce nitrates.
 Place 3–4 drops of sterile distilled water into the tube and emulsify 1 loopful or spadeful of
colonies in the water.
 Add 2 ml of NaNO3 substrate. Shake by hand and place in 37oC water bath for 2 hours.
 Add 1 drop of 1 : 2 dilutions of concentrated HCl, shake by hand to mix.
 Add 2 drops of 0.2% aqueous sulphanilamide solution.
 Add 2 drops of 0.1% aqueous N-naphthyl ethylene diamine dihydrochloride solution.
Development of a red colour indicates a positive result. Grade the colour change from pink
to red as 1+ to 5+ reactions.
iii. Tween 80 hydrolysis (degradation) M. kansasii gives consistently positive results with this
test. Inoculate Tween 80 substrate with loopful of growth from solid medium of a slow growing
culture, or from a 7-day-old liquid culture.
Incubate at 35–37°C. Look for a colour change of the liquid—not the cells—to pink or red
as indicative of a positive result.
iv. Catalase activity, semi-quantitative test Nearly all Mycobacteria, with the exception of
certain isoniazid-resistant mutants of M. tuberculosis and M. gastri possess catalase enzymes.
Inoculate the surface of LJ medium, specially prepared as a butt with 0.1 ml or loopful of a
7-day-old liquid culture. Incubate with cap loose at 37oC for 2 weeks.
Add 1 ml of Tween–peroxide mixture made just before use. Keep the tube in upright position
at room temperature for 5 minutes.
Measure in mm the height of the column bubbles above the surface of the medium. Record
the result as follows:
Height of column < 45 mm Weak positive
Height of column > 45 mm Strong positive
No bubbling Negative
v. Arylsulphatase activity Test for this activity is usually positive in 3 days for potentially
pathogenic, rapidly growing Mycobacteria. Inoculate 1 tube each of the 0.001 ml and 0.003 M
liquid substrate medium with 0.1 ml of a 7-day liquid culture or a spadeful of organisms from
the surface of a freshly grown culture.
After 3 days incubation of the 0.001 M substrate culture, add 6 drops of 2N sodium carbonate
solutions.
Look for a colour change to pink or red as indicative of a positive result.

vi. Urease test Urea is hydolysed by all species of Mycobacterium except M. avium and
M. smegmatis
vii. Tellurite reduction All species reduce tellurite but M . phlei do not reduce tellurite.
vii. Iron uptake test All species of Mycobacteria utilize iron.
3. Blood
Blood is normally sterile, but bacteria occur transiently in the bloodstream, which is termed as
bacteraemia, during dental surgery, instrumentation of the genitourinary tract or bowel and also
in infections like typhoid fever, brucellosis and meningococcal infections. A more dangerous and
clinically alarming syndrome is septicaemia, a condition characterized by the rapid multiplication
of microorganisms with the elaboration of their toxins into the bloodstream. Blood culture is
requested mainly in two clinical situations:
 Where the possibility of septicaemia or bacteraemia is suggested by the presence of fever,
shock, suspected local infection, purpeural sepsis, pneumonia, meningitis, osteomyelitis or
endocarditis.
 In the investigation of fever which is difficult to diagnose, because of the absence of signs of
a specific infection or local infective lesion, i.e., pyrexia of unknown origin.
Possible pathogens
a. Bacteria
Gram-positive
Staphylococcus aureus and Streptococcus are associated with neonatal septicaemia.
Gram-negative
i. Salmonella typhi in typhoid fever is detected in the blood of 75–90% of patients during the
first 10 days of infection and in about 30% of patients during the 3rd week.
ii. Haemophilus influenzae is the cause of sub-acute infective endocarditis.
iii. Yersinia pestis is isolated from blood in septicaemic plaque.
iv. Organisms like Brucella, Pseudomonas, E. coli, Klebsiella, Proteus, Bacteroides, Neisseria meningitidis
can also cause bacteraemia.
v. Leptospira causes leptospirosis, Weil’s disease, and undifferentiated pyrexia.
vi. Borrelia is a commensal of mouth. Some species cause disease. For example, Lyme disease is
caused by B. burgdorferi.
Examples of parasites that may be seen in infected blood sample are Plasmodium, Ancylosoma,
Toxoplasma gondi.
a. Collection of blood
i. Using a sterile syringe of 21 gauge needle, 10–12 ml of blood is withdrawn from a suitable
vein, whose area has been cleansed with tincture of iodine followed by ethanol–ether.
ii. With care, the needle from the syringe is removed and replaced with another sterile needle
of the same size and is inserted into the rubber liner of the culture bottle cap.
iii. The specimen is used for microbiological analysis before it clots.
b. Microscopic examination The EDTA added anti-coagulated blood is centrifuged and a smear of
the buffy coat layer of the blood is done, dried and fixed with methanol for 2 minutes and stained by
Giemsa stain to observe the presence of malarial parasite, Borrelia, microfilariae and trypanosomes.
Using dark-field microscope, analysis of plasma can be done to observe motile Leptospira.
c. Culture examination Inoculate the blood sample into the following media:
i. Thioglycollate broth
ii. Blood agar
iii. Chocolate agar
iv. MacConkey agar
Observe the plates for growth of the microorganisms and record the observations.
4. Urine
The anatomical structure of the mammalian urinary system is such that the external genitalia
and the lower parts of the urethra are normally contaminated with a diverse population of
microorganisms. The other tissues and organs that consitute the urinary system, the bladder,
urethra and kidneys are sterile and therefore, urine that passes through these structures is also
sterile. When pathogens gain access to the system they can establish themselves and cause
infection.
Urinary tract infections may be limited to a single tissue or organ or they may spread upward
and involve the entire system. Infections, such as cystitis, involves the bladder but may spread
through the ureter to the kidneys and is called pyelitis. Glomerular nephritis is an inflammation
that results in the destruction of renal corpuscles; pyelonephritis results in the destruction of renal
tubes. Organisms may also act as etiological agents of urogenital infections.
Possible pathogens
a. Bacteria
Gram-positive
i. Heamolytic streptococci
ii. Staphylococcus aureus is associated with cystitis.

Gram-negative
E. coli, Klebsiella sp., Proteus mirabilis, Pseudomonas aeruginosa and Enterococcus faecalis—all these
are the commonest causative agents of cystitis.
b. Fungi Candida is present in immunocompromised patients.
C. Parasites Trichomonas vaginalis, Wuchereria bancrofti
a. Specimen collection There are many types of specimen collection procedures based on the type
of assay to be done. For a microbiological examination, midstream specimen of urine is required.
i. The genitalia is thoroughly cleaned and midstream urine (MSU) is collected in a sterile,
wide-mouthed container with a screw cap.
ii. The specimen must be immediately processed or if delay occurs, it can be stored at 4ºC for
1–2 hours. If it takes more than 5 hours, the specimen has to be discarded.
i. Physical examination of urine
Colour of urine Normal colour varies from pale yellow to deep yellow. If there is any colour
change it indicates a specific clinical condition.
Note
Reddish-brown high amount of urobilinogen
Bright red due to large amount of fresh blood
Smoky brown or brownish yellow or green presence of bile pigment
Milky white due to filariasis
Appearance of urine Normal urine is clear but at times cloudiness may be seen in urine because
of the presence of amorphous phosphate, fat, bacteria or fungi.
Odour of urine Fresh normal urine has a slight aromatic odour giving it an ammoniacal smell.
ii. Microscopic examination Microscopic examination of urine is done principally to detect the
presence of polymorphs, which is an indication of infection. A loopful of well-mixed urine sample
is placed on two clean glass slides and one is used for wet film and the other for Gram staining.
During observation if 1,00,000 bacteria/ml is found, then the specimen is considered to be the one
got from a proper bacteraemic condition and then it is processed; if not, the sample is discarded.
iii. Cultural examination A loopful of the urine sample is streaked onto the following media:
 Mannitol salt agar
 Blood agar
 MacConkey agar
 Nutrient agar
5. Throat Swab

Upper respiratory tract includes throat and nasopharynx. Many pathogens colonize and are
present in these sites causing symptomatic infections in deep, less accessible sites. Throat swabs
are collected to diagnose pharyngitis.
Possible pathogens
a. Bacteria
Gram-positive
i. Streptococcus pyogenes is the common cause of streptococcal pharyngitis (sore throat).
ii. Clostridium diptheriae causes diptheria.
Gram-negative
i. Vincent’s organism—Borrelia vincenti causes Vincent’s angina.
ii. Leptotricha buccalis, Bacteroides melaninogenicus.
b. Viruses Respiratory viruses, enteroviruses and Herpes simplex virus type I.
C. Fungi Candida albicans causes oral thrush in HIV patients.
a. Specimen collection
i. For bacteriological sampling, a plain, albumin-coated or charcoal-coated cotton wool swab
is used.
ii. The swab is rubbed with rotation over the tonsillar area and posterior pharyngeal wall and
other areas that is inflamed or bears exudates.
iii. The swab is then placed in its tube with care not to soil the rim and processed immediately;
otherwise, it can be placed at 4ºC for 1 hour.
i. Microscopic examination
 When a throat swab specimen is got, it is first moistened with sterile nutrient broth.
 Gram staining is done to examine the presence of pus cells, yeast-like cells, C.diptheriae and
Vincent’s organism.
ii. Culture examination The swab is used to inoculate the following plates :
 Blood agar plates—one plate is incubated anaerobically and on the other plate, an optochin
disc is placed.
 Tellurite blood agar—to identify C. diptheriae. The plate is incubated aerobically at 37ºC for
24 hours.
 SDA agar—to identify C. albicans.
6. Stool Sample

The feces are normally composed of food residue, materials secreted through the walls of the
intestine, and in bile, leucocytes, desquamated epithelial cells of bacteria. Average stool weighs
about 100–200 g in 24 hours varying with diet. In constipation, hard stool is evaporated like a
ball; soft stool is likely in diarrhoea and rice-watery stool in vegetarian diet. Stool is an important
specimen for diagnosis of diseases of gastrointestinal tract, diarrhoea, dysentery, parasitic infection,
gastrointestinal bleeding, peptic ulcer, carcinoma and malabsorption syndromes.
Possible pathogens
a. Bacteria
Gram-positive
i. Clostridium perfringens in food poisoning.
ii. Clostridium difficile in enterocolitis or pseudomembraneous colitis.
iii. Bacillus cereus in food poisoning.
Gram-Negative
i. Campylobacter, S. enteritidis, S. typhimurium, Shigella sonnei, Vibrio parahaemolyticus, Vibrio cholerae
in food poisoning.
ii. E. coli in enteric infection in children.
b. Fungi Candida albicans causes superinfection with various drug-resistant bacteria.
C. Parasites Hookworm, Ascaris sp.
d. Protozoa Giardia intestinalis, Paragonium sp., Entamoeba histolytica
a. Specimen collection Faeces specimen should be collected in the early stage of the disease
and preferably, before antibiotic treatment. For microbiological stool investigation, specimen is
preferred to a rectal swab.
The patient is given a clean, dry, disinfectant-free bed pan or a sterile container
(25 ml screw-capped, wide-mouthed glass or plastic bottle). The sample must be collected without
urine contamination.
The specimen is transported quickly to the laboratory and processed. If delay is unavoidable,
6 ml of buffered glycerol saline is used as the transport media.
i. Naked eye examination
 Colour of the stool (normally light-yellowish brown)
 Whether formed, semiformed, unformed or fluid.
 Presence of mucous, pus and blood indicates severe dysentery with helminthic infection.
ii. Microscopy Usually, a faeces sample is not examined microscopically unless the clinical
particular or failure to demonstrate alternative pathogens suggests that the patient’s illness may
be due to amoebiasis, giardiasis, balantidiosis, cryptosporidiosis or helminthiasis.
 A wet film is examined for protozoal cyst and helminthic ova.
 An eosin preparation to examine amoebic cysts.
iii. Culture for bacterial pathogens A faecal suspension is prepared for inoculating in different
media. Unless the sample is fluid, a portion of it is suspended to give a 1-in-10 dilution in
2–3 ml of phosphate buffered saline (pH 7.3) or 0.1 % peptone water.
The specimen is inoculated onto the following medium:
 XLD agar: Salmonella
 TCBS: Vibrio cholerae
 MacConkey or SS agar: Yersinia
7. Cerobro-spinal Fluid

CSF envelopes the brain and has several functions. CSF carries essential metabolites into the
neural tissue and cleans the tissues of waste as it circulates around the entire brain ventricles and
spinal cord.
Chemical and cellular changes in the CSF provides valuable information within the
subarachnoid space. The meninges is a collective term for three distinct layers surrounding the
brain and spinal cord—duramater (outer membrane layer), arachnoid and piamater (innermost
membrane layer). Infection within the subarachnoid space or through out the leptomeninges
(both piamater and arachnoid membrane) is called meningitis. Inflammation of the brain is called
encephalitis.
CSF is received in sterile test tube, preferably screw capped.
List of agents causing community-acquired pyogenic meningitis (in India).
Age group Etiological agent

Neonatal E. coli
Other Enterobacteriaceae
Group B streptococci
Enterococci
3 months–6 years Haemophilus influenzae
Streptococcus pneumoniae
Neisseria meningitidies
More than 6 years S. pneumoniae
N. meningitidis
Processing of sample
If turbid, make a wet preparation and smear for gram stain and methylene blue and inoculate media.
If not turbid centrifuge the specimen in a conical centrifuge tube at 1500 to 3000 rpm for
30 min. Pour supernatant into a sterile tube and store. The sediment is checked for free living
amoebae.
Culture media
The sample is inoculated on to blood agar, chocolate agar, MacConkey agar and later deep down
into thyoglycollate broth without creating air bubbles. The BA and CA are incubated in CO2
atmosphere at 37ºC for 48 hrs. The TB is incubated at 37ºC for 7 days. Transfer the stored
supernatant into the original CSF collection tube and incubate at 37ºC in CO2 atmosphere.
If B. anthracis is suspected, 5 ml nutrient broth and sodium are inoculated.
If Listeria monocytogens is suspected, triptycase soy agar plate and broth are inoculated.
Smear examination
Gram-positive cocci may be typical of S. pneumoniae and Staphylococcus species. Gram-negative
diplococci often but not always, within the polymorpho nuclear cell indicate Neisseria sps. Some
non-fermenting gram-negative bacilli can resemble Neisseria sps. which may also be seen as
intracellular organisms. Gram-negative bacilli may be typical of Haemophilus sps or any of
the Enterobacteriaceae. Large gram-positive bacilli in chains or single, showing bamboo stick
appearance with capsule ( in methylene blue) is suggestive of B. anthracis. Short gram-positive
diphteroid-like, coccoid, paired bacilli, intra or extra cellular organisms are suggestive of
L. monocytogens.
If many pus cells and no bacteria are seen a Ziehl–Neelsen staining has to be done, to look
for the presence of M. tuberculosis. If only one type is seen in the smear direct sensitivity is done
on appropriate media.
Culture and identification

Gram-positive cocci: Streptococcus pneumoniae
Colonies of Streptococcus pneumoniae on blood agar are hemolytic, circular, smooth and low
convex. On autolysis the center of the colony becomes depressed with edges being raised, this is
called pitting and some times take 48 hrs to develop. S. pneumoniae type 3 forms large, watery,
mucoid colonies in 24 hrs.
S. pneumoniae can be differentiaed from viridians streptococci by performing inulin fermentation,
bile solubility, and optochin susceptibility ( a zone of inhibition with diameter of 14 mm or more
is taken as susceptible).
Bile solubility test Inoculate 1 ml nutrient broth containing one drop of sterile 0x serum, with
3 or 4 colonies of S. pneumoniae and incubate overnight. Growth from BA suspended in 1 ml
of 85% saline can also be used. Add 1 drop 1% phenol red indicator and adjust pH to neutral
7–7.5 by adding two drops of 1N NaOH to prevent solidification on addition of sodiumdeoxy
cholate. Transfer 5 ml of this culture suspension to another test tube and add 2–3 drops of 10%
sodium deoxycholate to one of the tubes. The other tube will serve as negative control to which
an equal volume of sterile saline is added. Leave both tubes at room temperature. Clearing of
the solution indicates a positive solubility test while the negative control will remain turbid.
Pneumococci are generally dissolved by sodiumdeoxy cholate and broth cultures become clear
in 2–5 min.
Colonies of staphylococci are medium-sized to large, circular, entire, low convex, smooth,
moist, and opaque. They range in colour fron white (S. epidermidis) to golden yellow (S. aureus).
Usually colonies of S. aureus are surrounded by narrow zone of beta hemolysis. Test all staphylococci
for clumping factor and coagulase activity.
Slide test for clumping factor Prepare two suspensions of the test organism in sterile saline.
Add a drop of human plasma to one of the suspensions and mix by gently rocking the slide.
The other suspension serves as a negative control. A positive test is shown by immediate clumping
of organism while the control suspension remains uniformly turbid.
Tube coagulase test Measure about 0.5 ml of an over night broth culture of the test organism
and inoculate into a small sterile test tube. Add 0.5 ml plasma (diluted 1/4 or 1/5). Incubate at
37ºC for 30 min. to 2 hours and examine for coagulation of the suspension.
Gram-negative cocci Colonies of Neisseria meningitidis are medium-sized, circular, entire, low
convex and transparent on blood agar. They are oxidase positive. For further identification, sugar
fermentation should be done.
Gram-negative bacilli Haemophilus influenzae colonies are smooth and translucent on CA.
Gram-staining morphology is typical of H. influenenzae as it exhibits bipolar staining character.
Nutritionally Haemophilus influenzae prefers a complex medium and requires performed growth
factors that are present in blood, specifically X-factor (i.e., hemin) and V factor.
Testing for X and V factor requirement Subculture the test strain into one-half of the NA
plate. To obtain uniform growth, place cut pieces of X and V and XV factors at different points.
H.influenenzae grows around XV strip only, while H. parainfluenza grows around V and XV strip.
Other species can also be identified by their X and V factor requirement.
The following points can be considered for gram-positive bacilli suggestive of B.anthracis.
1. On nutrient agar, Bacillus anthracis produces large irregular dull opaque colonies with a
granular surface typically described as frosted glass appearance. On blood agar, they are
non-hemolytic.
2. Examine the nutrient broth for floccular deposit and surface pellicle. The deposit come
up as silky strands on shaking the broth gently.
3. Perform a hanging drop examination of the broth culture. B. anthracis is non-motile.
Amoeba resembling nagleriae or acanthamoeba These are seen in wet preparation. Examine
the incubated CSF for evidence of growth. A sub-culture is done on CA (chocolate agar). After
24 hours of incubation, examine sub-cultures for evidence of growth and proceed accordingly.
Antimicrobial susceptibility testing Once there is growth on culture, the organism is inoculated
on appropriate media for antimicrobial susceptibility testing.
IDENTIFICATION AND ENUMERATION OF LYMPHOCYTES

Introduction
A variety of blood tests are routinely performed as a part of physical examination because they
provide a significant amount of information about the health of a patient. Although these tests
have diagnostic value, the purpose of this test is not to diagnose but to understand the purpose
and nature of it.
Blood is the medium that transports substances to and from the cells. Its components include
plasma, which forms 55% of the blood volume, and the formed elements, which constitutes
the remaining 45%. The formed elements consist of erythrocytes (red blood cells), leucocytes
(white blood cells) and thrombocytes (platelets).
The appearance of these cells based on their features can be seen in stained blood smears. The
different types of leucocytes present in blood are given below. This is based on cellular morphology
and cytoplasmic staining characteristics.
1. Neutrophils It is also called polymorphonuclear leucocytes (PMNL). It has a multilobed
nucleus (3–4 lobed), which are very distinctive. They are of two types:
i. Band-type nucleus, which is sausage-shaped, non-segmented, also called stals.

ii. Segmented nucleus with definite lobes.
It also has granulated cytoplasm which stains with both acidic and basic dyes. These cells
differ from each other and two granulocytes in having smaller and polar granules in the cytoplasm.
2. Eosinophils Cytoplasm stains faint pink and contains large red and red-orange granules. They
have a bilobed nucleus. These are smaller cells when compared to neutrophils and monocytes,
and the granulated cytoplasm stains with the acidic dye, eosin (hence its name).
3. Basophils These cytoplasmic granules are large, dark and blue-black in colour, which fill
the cell. They have a single round nucleus and cells are comparatively smaller in size.
4. Lymphocytes Large-sized lymphocytes have clear blue cytoplasm at the margin of the nucleus.
In small lymphocytes, dark violet-coloured nucleus almost fills the entire cell and has a rim of
cytoplasm.
5. Monocytes The monocytes are larger than lymphocytes. They have a horse-shoe shaped nucleus.
HSC(G0) HSC Progenitor Progenitor
Promegakaryo
cyte
L–blast Mo–blast
Myeloblast
Pro–M Pro–E
Baso–E
Myelocyte
Meta–M Poly–E Megakaryocyte
(band) Ortho–E
(seg)
Lymphocyte Monocyte Neutrophil Eosinophil Basophil Erythrocyte Platelet
Diagram for the Blood cells
Aim
To enumerate the leucocytes in the given blood sample.
Principle
Enumeration of leucocytes Differential count is the per cent distribution of various
white cells in the peripheral blood. It is done with a stained smear under the microscope using
oil-immersion objective. Differential count is vital for the diagnosis of a number of blood disorders.
Its primary role is to identify the changes in distribution of white cells, which may be related
to specific types of disorders like infections and leukaemia.The normal values and functions of
various formed elements is shown in Table 10.2
Table 10.2 Formed elements of blood
Formed elements Concentration Function

Erythrocytes 4,000,000– Transport oxygen and carbon
6,000,000/mm3 dioxide
Leucocytes 5,000–10,000/mm 3 Destroy pathogens; neutralize
toxins
Granulocytes Neutrophils 50–70 % of leucocytes Phagocytosis
Eosinophils 1–3% of leucocytes Neutralize products of allergic
reaction; destroy parasitic worms
Basophils 0.5–1% of leucocytes Release heparin and histamine;
intensify inflammation and allergic
reactions
Agranulocytes Lymphocytes 20–30% of leucocytes B-lymphocytes form antibodies;
T-lymphocytes form chemicals
that destroy antigens and/or
stimulate phagocytosis
Monocytes 2–6% of leucocytes Phagocytosis
Thrombocytes 250,000–400,000/mm 3 Initiate clotting process
The major steps involved in differential count are the preparation of the smear, staining and
microscopic observation. The blood is taken directly from skin puncture. The staining is done
usually using Leishman’s stain or Giemsa stain (polychromatic stain). Following staining, the
basic components of the white cells (e.g., cytoplasm) are stained by acidic eosin dye and the acidic
components of the cell (e.g., nucleus and nucleic acids) take blue to purple shades of the basic
dye, methylene blue. The neutral components are probably stained by both the dyes.
Materials required
Sample: Blood sample
Reagents: Giemsa stain, alcohol
Equipment and other materials: Sterile needle, glass slide
Procedure
Preparation of blood smear and staining
The blood specimen is obtained from skin puncture, where the skin is punctured using a sterile needle
after disinfecting with 70% alcohol and drying it. The finger is squeezed and a drop of blood is placed
on one end of the slide. With the help of another slide, kept at 45o angle, a thin smear is made. The
smear is fixed with alcohol for 2–3 minutes. The smear is then washed with water, stained with
Giemsa stain for 5–10 minutes, washed, dried and observed under oil-immersion objective.
Differential counting of leucocytes The slide is examined and different types of leucocytes are
counted by serpentine counting pattern. The counting is continued until 100 cells are counted.
The various types of leucocytes and their corresponding number among the 100 cells give the
differential count pattern of the blood smear.
Figure 10.1 Serpentine counting pattern
Observation and interpretation

The slide was examined and different types of leucocytes were counted by serpentine counting
pattern. The counting was continued until 100 cells were counted. The various types of leucocytes
and their corresponding number among the 100 cells gives the differential count pattern of the
blood smear.
Result
Presence of neutrophil, eosinophil, basophil and monocyte were observed.
IDENTIFICATION OF FUNGAL PATHOGENS

A fungus is a eukaryotic organism that belongs to the Kingdom Fungi. The fungi are heterotrophic
organisms possessing a chitinous cell wall. The majority of species grow as multicellular filaments
called hyphae forming a mycelium; some fungal species also grow as single cells. Sexual and
asexual reproduction of fungi is commonly via spores, often produced on specialized structures
or in fruiting bodies. Some species lose their ability to form specialized reproductive structures
and propagate solely by vegetative growth. Yeasts, moulds, and mushrooms are examples of
fungi. The fungi are a monophyletic group that is phylogenetically clearly distinct from the
morphologically similar slime moulds (myxomycetes) and water moulds (oomycetes). The fungi
are more closely related to animals than plants, yet the discipline of biology devoted to the study
of fungi, known as mycology, often falls under the branch of botany.
Clinical Techniques in Mycology

A. Visualization of fungi in tissue preparations
1. Treatment with 10% potassium hydroxide
2. Positive stain with
i. Lactophenol cotton blue
ii. Grocott silver stain
iii. Haematoxylin
iv. Eosin
3. Negative stain with India ink
B. Fluorescence of fungi under ultraviolet light
C. Culture of fungi on
1. Sabouraud’s agar (favours fungal growth because of low pH)
2. Mycosal agar (selective for pathogenic fungi because of chloramphenicol and cyclohexidine
in medium)
D. Visualization of cultured fungi (25oC and 37oC)
1. Colonial morphology
2. Cellular morphology
a. Hyphal morphology
 Aseptate or coenocytic—non-septated hyphae, e.g., Mucor
 Septate—regular connection, clamp connection, hyphae with septa, e.g., Aspergillus
b. Spore morphology
Figure 10.2 Arthospore

Conidiospore: Asexual non-motile spore which are borne on stalks called conidiophores.
Sporangiospore: spore produced by a sporangium.
Arthospore: They are conidia produced simply by last cell hyphae breakage.
Chlamydospore: Thick-walled big resting spore
c. Yeast morphology
 Size
 Thickness of walls
 Capsule presence/absence
E. Identification of yeast by

1. Biochemical tests
2. Behaviour in broth and serum (germ-tube formation)
3. Behaviour on cornmeal agar (pseudohyphae formation)
When fungi do pass the resistance barriers of the human body and establish infections, the
infections are classified according to the tissue levels initially colonized. These are:
a. Cutaneous mycoses These are infections that extend deeper into the epidermis, as well as
invasive hair and nail diseases. The agents causing these diseases are termed as dermatophytes.
The diseases are referred to as ringworm or tinea. All of the dermatophytic diseases are caused
by members of the three genera, Microsporum, Trichophyton and Epidermophyton, which comprise of
41 species. Athletes foot is an example of a disease that belongs to cutaneous mycoses.
b. Subcutaneous mycoses These are infections involving the dermis, subcutaneous tissues, and
muscle. These infections initially involve the deeper layers of the dermis, subcutaneous tissue or
bone. They are initiated by trauma to the skin and are difficult to treat and surgical intervention
(excision or amputation) becomes necessary.
c. Systemic mycoses Infections that originate primarily in the lung and may spread to many
organ systems, e.g., aspergillosis
d. Opportunistic mycoses Infections in patients with immune deficiencies, e.g., candidiasis,
cryptococcosis.
Cutaneous Mycoses
These diseases are restricted to the keratinized layers of the skin, hair and nails.
Microsporum Microsporum species form both macro and microconidia on short conidiophores.
Macroconidia are hyaline, multiseptate, variable in form, fusiform, spindle-shaped to obovate,
ranging from 7–20 × 30–160 µm in size, with thin- or thick- echinulate to verrucose cell walls.
Their shape, size and cell wall features are important characteristics for species identification.
Microconidia are hyaline, single-celled, pyriform to clavate, smooth-walled, 2.5–3.5 × 4–7 µm

in size and are not diagnostic for any one species, e.g., Microsporum canis
Trichophyton The fungus of the genus Trichophyton is characterized by the development of both
smooth-walled macro and microconidia. Macroconidia are mostly borne laterally directly on the
hyphae or on short pedicels, and are thin- or thick-walled, clavate to fusiform, and range from
4–8 × 8–50 µm in size. Macroconidia are few or absent in many species. Microconidia are spherical,
pyriform to clavate or of irregular shape and range from 2–3 × 2–4 µm in size. Malabar itch,
a skin infection consisting of an eruption of a number of concentric rings of overlapping scales
forming papulosquamous patches, is caused by this fungus.
Figure 10.3 Trichophyton rubrum
Epidermophyton Septate, hyaline hyphae, macroconidia, and occasionally, chlamydoconidium-

like cells are visualized. Microconidia are typically absent. Macroconidia (10–40 × 6–12 µm)
are thin-walled, 3- to 5-celled, smooth and clavate-shaped. They are found singly or in clusters.
Chlamydoconidium-like cells, as well as arthroconidia, are common in older cultures.
Figure 10.4 Epidermophyton flocusum
Systemic Mycoses
It originates primarily in the lungs and may spread to many organ systems. Organisms that
cause systemic mycoses are inherently virulent. Generally, primary pathogens that cause systemic
mycoses are dimorphic. They are:
 Histoplasma capsulatum (causing histoplasmosis)

 Coccidioides immitis (causing coccidioidomycosis)
 Blastomyces dermatitidis (causing blastomycosis)
Histoplasma Parahyphae are septate and hyaline. Histoplasma capsulatum produces hyphae-like
conidiophores which arise at right angles to the parent hyphae. It has both macro and microconidia.
Macroconidia are tuberculate, thick-walled, round, unicellular, hyaline, large and often have finger-
like projections on the surface. These macroconidia are also referred to as tuberculochlamydospores
or macroaleurioconidia. Microconidia (microaleurioconidia) are unicellular, hyaline and round,
with a smooth or rough wall. At 37°C, narrow-based, ovoid, budding yeast cells are formed.
Yeasts of Histoplasma capsulatum are smaller.
Figure 10.5 Histoplasmosia capsulatum
Coccidiodes Microscopic appearance of the fungus depends on the temperature of isolation.

At 25°C, hyphae and arthroconidia are produced. Hyphae are hyaline, septate and thin. Racquet
hyphae may occasionally be observed on slides prepared from young cultures. Arthroconidia are
thick-walled, barrel-shaped, and 2–4 × 3–6 µm in size. Typically, these arthroconidia alternate
with empty disjunctor cells. On the released arthroconidia, annular frills that are the remnants
of the disjunctor cells are observed. At 37ºC, large, round, thick-walled spherules (10–80 µm
in diameter) filled with endospores (2–5 µm in diameter) are observed. Production of spherules
in vitro requires inoculation into a special synthetic medium, such as converse liquid medium, at
incubation temperature of 37–40°C in the presence of CO2 at a concentration as high as 20%.
Blastomyces At 25°C, the growth rate is slow to moderately rapid. The colony diameter is
0.5–3 cm following incubation for 7 days on potato glucose agar. The texture is membranous
and downy to woolly. The surface colour is white to beige and reverse is pale to brownish.
At 37°C, conversion to a yeast form at 37°C usually requires inoculation onto an enriched medium.
The growth rate is slow to moderately rapid. The colony diameter is 0.5–3 cm following incubation
for 7 days. The texture is typically creamy and yeast-like. It appears granular to verrucose on the
surface. The colour of the colony is white to beige.
A common laboratory diagnosis procedure for all of the organism is given as follows:
1. Microscopic examination of the material
2. Growth on appropriate media
3. Biochemical or special test for confirmation
4. Pathogenicity test for the disease-causing agent
OPPORTUNISTIC MYCOSES
Candida It causes systemic mycoses due to opportunistic pathogens (infections of patients with
immune deficiencies who would otherwise not be infected). Examples of immunocompromised
conditions include AIDS, alteration of normal flora by antibiotics, immunosuppressive therapy
and metastatic cancer. Examples of opportunistic mycoses include candidiasis, cryptococcosis
and aspergillosis. On cornmeal, following 72 hours of incubation at 25°C, abundant branched
pseudohyphae and true hyphae with blastoconidia are present. The blastoconidia are formed in
grape-like clusters along the length of the hyphae. Terminal chlamydoconidia may be formed
with extended incubation.
YEASTS AND YEAST-LIKE FUNGI

Introduction
Yeasts are considered to be normal flora of oral cavity and gastrointestinal tract of man.
These may be isolated from clinical specimens, such as throat cultures, respiratory secretions,
gastric washings, vaginal secretions and stool. They are also recovered from urine, skin and nail
scrapings. They are not normally present in sterile body fluids but when they are present, they
assume clinical importance. Routinely Candida and Cryptococcus neoformans are identified from
clinical specimens. However, in immunocompromised or immunosuppressed and AIDS patients,
different yeasts and yeast-like organisms are recovered and they assume clinical importance and
their identity need to be established.
Cryptococcus
The genus Cryptococcus contains many species. Among them, C. neoformans is considered to be the
only organism causing infections in man. In recent years, C. albicans and few others have been
isolated from few severely debilitated patients.
Table 10.3 Characteristics of Yeast-like fungi
Species Growth Glu- Mal- Suc- Lac- Galac- Melib- Cello- Inosi- Xy- Raff- Treh- Dulc Ure Nitrate Phenol Asco-
at cose tose rose tose tose iose biose tol lose inose alose -itol -ase utilization oxidase spore
37°C
Cr. neoformans + + + + – + – + + + + + + + – + –
C. albicans – + + + + + + + + + + + + + + – –
Cr. laurentii + + + + _ + + + + + + + + + – – –
C. luteolus – + + + – + + + + + + + + + – – –
C. terreus – + + – + + – + + + – + + – + – –
C. ungittulatus – + + + – – – – + + + – – – – –
Morphology of Cryptococci
1. It is a round to oval yeast-like fungi.
2. The size varies from 3.5–8 µm or more in diameter.
3. It has a single bud and a narrow neck between mother and daughter cells (occasionally several
buds may be seen).
4. Rarely pseudohyphae are seen.
5. Cell wall is fragile and collapsed cells or crescent-shaped cells are seen in tissues.
6. Characteristic mucopolysaccharide capsules are seen around the cells, which may be thick or thin.
Colony Morphology
1. Colonies are mucoid due to the presence of capsular material.
2. They exhibit a wide range of colours (cream, tan, pink, yellow).
Biochemical Characteristics All members produce urease, utilize various carbohydrates
and are non-fermentative.
Candida
Morphology of Candida
It is 2–7× 3–8.5 micro meter in size.
Colony morphology
1. Small, round, moist and colouress colonies.
2. Profound growth on SDA plate.
Figure 10.6 Candida albicans
I. GERM TUBE TEST

Aim
To demonstrate the production of germ tube by Candida species.
Principle
Candida albicans produces characteristic germ tube when incubated with serum within 3 hours
of incubation at 37ºC. This property can be used to identify Candida albicans.
Materials Required
Sample: Candida albicans, C. parapsilosis, test Candida strain
Equipment and other materials: Test tubes 12×75 mm, other standard labware.
Reagents: Human or rabbit serum
Procedure
1. Take three 12×75mm test tubes.
2. Add 0.5 ml of serum into all of them (human or rabbit serum).
3. With a sterile loop, pick up half the portion of a single colony from 24-hour old plate and
suspend in the following order:
 Into tube 1, inoculate control C. albicans.
 Into tube 2, inoculate Candida parapsilosis.
 Into tube 3, inoculate the test organism.
4. Incubate the tubes at 37ºC for 2–3 hours (not more than 3 hours).
5. Place one drop of suspension from tube 1 onto slide 1 and cover it with a coverslip.
6. In the same way, transfer one drop from tubes 2 and 3 onto slides 2 and 3, respectively.
7. Examine them under 100 X or 450 X magnification.
Result
Tube 1 will show germ tube. Tube 2 will not show germ tube. Read the test comparing it with
the above controls.
II. GROWTH ON CORNMEAL AGAR FOR CHLAMYDOSPORE

OR TRUE HYPAE PRODUCTION
Aim
To demonstrate the production of chlamydospores or true hyphae by yeasts and yeast-like fungi
(Candida).
Principle
Different Candida species show various kinds of sporulation when grown on cornmeal agar.
C. albicans produces characteristic chlamydospores on cornmeal agar.
Materials required
Culture: Candida albicans
Media: Corn meal agar
Equipment and other materials: Inoculation loop, Tween 80 , Petri plates
Procedure
1. Suspend 1.9g of HiMedia cornmeal agar in 100 ml of distilled water and heat to boil to
dissolve the agar. Add 1ml of Tween 80, mix and autoclave at 121°C for 15 minutes, pour
plates and allow to set.
2. Pick up a small portion of a single colony of the yeast and inoculate as for isolation. Make
few cuts into the agar with the loop in a slanting manner near the secondary and tertiary
streak area and place a coverslip over the cut area.
3. Incubate the plate at 30°C for 24–72 hours.
4. Examine the plates under the microscope (100X, 45X) for the characteristic blastoconidia
(yeast cells) formation along the streak line. Look for pseudohyphae, true hyphae and
chlamydospores.
Result
The hyphae formed is observed on the culture plate as shown in the figure.
Figure 10.7 Hyphae of Candida

III. CARBOHYDRATE ASSIMILATION TEST

Aim
To find out the pattern of assimilation of carbohydrate by yeast and yeast-like organisms.
Principle
Yeast and yeast-like organisms are identified by the pattern of carbohydrate assimilation. They
are inoculated on carbohydrate-free yeast nitrogen base agar on which different filter paper discs
containing various carbohydrates are placed. After incubation for appropriate time, growth around
the discs is observed and carbohydrate utilization pattern is assessed.
Materials required
Sample: Pure growth of test organism on SDA
Media: Yeast nitrogen base agar (Appendix III)
Reagents: MacFarland’s standard, Sterile saline
Equipment and other materials: Filter paper discs containing different carbohydrates, general
laboratory equipment and glasswares
Procedure
1. Pipette 2 ml of sterile saline into a test tube.
2. With a sterile loop, pick up a few isolated colonies of the organisms from SDA plate and
emulsify to a turbidity equal to MacFarland’s 4 units.
3. Cover the surface of liquid nitrogen base agar with the suspension of yeast cells.
4. Remove the excess fluid and allow the surface of the agar to dry.
5. With sterile forceps, place the carbohydrate disc onto the agar surface in such a way that at
least 30 mm space is present between each disc.
6. Incubate the plate at 30°C/37°C for 24–48 hours.
7. At the end of the incubation period, observe the plate for growth around the disc.
III. CARBOHYDRATE FERMENTATION TEST
Aim
To determine the ability of different yeasts and yeast-like fungi to ferment various carbohydrates.
Principle
Fermentation tests are used as supplement tests when there is a difficulty in making a definitive
identification using carbohydrate assimilation test.
Materials required
Sample: Preparation of inoculum: From the pure growth of yeast, make suspension in sterile
saline equivalent to MacFarland’s 4 opacity standard.
Media: Basal medium—Indicator broth medium (IBM) [Refer Appendix III]
Reagents: Different carbohydrates
Equipment and other materials: Standard laboratory glassware
Procedure
Inoculate each sugar tube with 0.2 ml (5 drops) of culture and incubate at 30–37°C for
2–19 days. Do not screw cap the tubes tightly.
Result
Presence of bubbles, or drop in the fluid level in Durham’s tube indicates fermentation.
Development of yellow colour is not a reliable indicator of fermentation and is ignored.
IV. UREASE TEST

Introduction
Certain fungi possess the enzyme urease that hydrolyses urea releasing ammonia into the medium.
This produces a change in the pH of the medium that can be detected by the colour change in
the indicator dye. This test can be used to differentiate different groups of fungi.
Aim
To demonstrate the production of urease by the given fungus.
Principle
Urea is a diamide of carbonic acid. Urease, the enzyme possessed by fungi, hydrolyses urea and
release ammonia and carbon dioxide. Ammonia reacts in solution to form ammonium carbonate,
which is alkaline, leading to the increase in pH. Phenol red that is incorporated in the medium
changes its colour from yellow to red in alkaline pH, thus indicating the presence of urease activity.
Materials required
Christensen’s urea agar (Appendix III), test tubes, yeast culture, inoculating loop, Bunsen burner,
marker, etc.
Procedure
Inoculate the slant with one colony of yeast or a small bit of inoculum from the mould colony.
Result
When positive, the colour of the medium changes to pink. An uninoculated medium is incubated
along with the test to compare the colour change.
EXAMINATION OF BLOOD SMEAR FOR MALARIAL PARASITE

Aim
 To prepare a thick smear and stain with eosin.
 To prepare a thin smear and stain with Leishman’s stain.
Materials required
Sample: Blood
Reagents: Leishman stain
Equipment and other materials: Clean glass slide, sterile needle
Principle
Malaria is an acute parasitic disease caused primarily by four different species of the parasite,
Plasmodium, namely Plasmodium vivax, P. falciparum, P. malariae and P. ovale. The most common
malarial parasite encountered in South India is P. vivax. Demonstration of the parasite in blood
can make a definite diagnosis of malaria. For this purpose two types of blood smears—thick and
thin smear—are prepared.
A thick blood smear is a drop of blood on a glass slide. Thick blood smears are most useful
for detecting the presence of parasites because they examine a larger sample of blood. (Often
there are few parasites in the blood at the time the test is done.) A thin blood smear is a drop of
blood that is spread across a large area of the slide. Thin blood smears helps discover what species
of malaria is causing the infection.
Procedure
1. Finger tip or ear lobe is wiped with alcohol and pricked with a sterile needle or lancet.
2. One or two drops of blood are placed at one end of two separate slides, one for a thick smear
and the other for a thin smear.
3. For thick smear, the sample is spread evenly throughout the slide.
4. This slide is allowed to air-dry, and is not heat fixed. The smear is then flooded with warm
water to dehaemoglobinize, and the eosin stain is flooded over the smear and kept for 30
seconds. The slide is then washed with water and then stained with methylene blue for one
minute, again washed with water, air-dried and then observed under microscope.
5. For thin smear, a second clean slide having a smooth edge is held by its edge at one end and
the other end touching the drop. The blood is allowed to spread along the edge. The slide is
held at 45° angle while the edge still touches the blood. Now, the slide is drawn in a quick
motion in such a way that blood is spread evenly on the flat slide. Then it is air-dried.
6. The thin smear is flooded with 2 drops of Leishman’s stain, kept for 1 minute, and
air-dried.
7. Then, the slide is washed with 1 ml of buffered saline and kept for 10–15 minutes and
air-dried.
8. The slide is then observed under oil-immersion objective.
EXAMINATION OF PARASITES FROM FAECES

Aim
To examine the faecal specimen for parasites, their trophozoites, cysts, ova, larva and adult forms.
Principle
The examination of faeces for parasites can be done by three methods:
1. Direct smear method
2. Iodine preparation
3. Concentration flotation
Examples of parasites in faeces are: Entamoeba coli, E. histolytica, Endolimax nana, Blastocystis
hominis. The first three organisms cause amoebiasis.
Materials required
Sample: Faeces
Reagents: Iodine, saline.
Equipment and other materials: Clean glass slide, cover slip, microscope
Procedure
I. Direct smear method
1. A drop of normal saline is placed on a clear glass slide. With the help of an applicator stick,
a small portion of the stool specimen is picked and emulsified in the drop of saline.
2. A coverslip is placed over it and the emulsion is examined under microscope.
3. Three emulsions for 3 consecutive days are done to examine the presence of cysts, ova and
trophozoites.
II. Iodine preparation method
1. A thick smear of the stool sample is prepared and a drop of iodine is placed. It is mixed well
and a coverslip is placed over it.
2. This preparation is then examined microscopically. Cysts and ova, if present, are not stained
by iodine and will be seen as clear halos.
III. Concentration flotation method

(a) Saturated salt solution technique
This technique is done as the eggs of most of the intestinal parasites have low specific gravity in
saline. As the gravity of saline is more than that of the ova, they begin to float.
1. A sterile 15–20 ml capacity test tube is taken and filled with saturated salt solution.
2. About 2 g of stool sample is added and mixed; care is taken to prevent spilling.
3. A grease-free coverslip is placed over the test tube in such a way that the top of the fluid
surface touches the surface of the coverslip and is left undisturbed for 15 minutes.
4. After 15 minutes, the coverslip is removed with the help of forceps and placed on a clean
glass slide containing a drop of Lugol’s iodine or saline and then examined.
Formal ether sedimentation technique

1. Transfer half teaspoonful of faeces in 10 ml of water in a glass container and mix thoroughly.
2. Place 2 layers of gauze in a funnel and strain the contents into a 15 ml centrifuge tube.
3. Centrifuge for 2 minutes at about 500 g.
4. Discard the supernatant and resuspend the sediment in 10 ml of physiological saline.
Centrifuge at 500 g and discard the supernatant.
5. Resuspend the sediment in 7 ml of 10% formaldehyde (1 part of 40% formalin in 3 parts
of saline).
6. Add 3 ml of ether (or ethyl acetate).
7. Close the tube with a stopper and shake vigorously to mix. Remove the stopper and centrifuge
at 500g for 2 minutes.
8. Rest the tube in a stand. Four layers now become visible, the top layer consists of ether,
second is a plug of debris, third is a clear layer of formalin and the fourth is the sediment
as show in the figure.
9. Detach the plug of debris from the side of the tube with the aid of a glass rod and pour off
the liquid leaving a small amount of formalin for suspension of the sediment.
10. With a pipette, remove the sediment and mix it with a drop of iodine. Examine under the
microscope.
Ether
Stool Particles
Formol saline
Deposit of parasite
Deposit of Parasites
Result
Cyst, motile, non-motile Strongyloides were observed
ANTI MICROBIAL SUSCEPTIBILITY TESTING (KIRBY–BAUER

DISC DIFFUSION METHOD)
Introduction
Antibiotics are a group of metabolic products, which are produced by one group of
microorganisms and are effective against other group of organisms at minimal concentration,
e.g., Penicillin produced by the genus Penicillium. Chemotherapeutic agents are chemically
synthesized antimicrobial agents, used for treatment against a variety of microorganisms, e.g.,
Sulphonamides. Most of the antibiotics and chemotherapeutic agents have either bacteriostatic
(inhibits essential metabolic processes of targeted microorganisms) or bactericidal (kills the
susceptible microorganisms) activities.
Generally most of the antibiotics have the following modes of action:
1. Inhibit cell wall synthesis, e.g., Penicillin, Cephalosporin
2. Inhibit protein synthesis, e.g., Streptomycin
3. Inhibit nucleic acids and DNA replication, e.g., Quinolones
4. Inhibit important enzymes required for metabolic process, e.g., sulphonamide and
trimethoprim
In vitro testing of sensitivity of bacterial isolates to antimicrobial agents can be carried out
using different techniques and principles. The classical method is the Kirby–Bauer disc diffusion
method.
Aim
To perform antimicrobial susceptibility test using Kirby–Bauer method.
Principle
Kirby–Bauer technique of testing antibiotic is based on disc diffusion method. The disc is
impregnated with known concentration of antibiotics and is placed on agar plates that have been
inoculated with a bacterium to be tested. The antibiotic diffuses through the media and produces
a zone of inhibition around the disc, if the organism is susceptible to that particular antibiotic. If
the organism is resistant to that antibiotic, then it grows up to the edge of the disc.
Materials required
Sample: E.coli
Media: Muller Hinton agar plates
Equipment and other materials: Forceps, antibiotic discs, Standard chart
MacFarland’s Standard
The turbidity of the standard is equivalent to overnight broth culture.
1. Prepare 1% sulphuric acid solution.
2. Prepare 1.175% solution of barium chloride by dissolving 2.35g of dehydrated barium
chloride (BaCl2.2H2O)in 200ml of distilled water.
3. To make the turbidity standard, add 0.5ml of barium chloride solution to 99.5ml of
sulphuric acid solution and mix.
The standard can be stored in dark at room temperature for upto 6 months.
Procedure
1. About 100 ml of Muller–Hinton agar is prepared, sterilized and poured into sterile Petri
plates.
2. The plates are dried properly so that there are no deposits of moisture on the agar surface.
3. The density of the culture to be inoculated is standardized by diluting with sterile saline or
broth to a density visually equivalent to the 0.5 MacFarland’s unit.
4. The medium is inoculated by even streaking of the swab over the entire surface of the plate.
5. After the inoculum had dried, selected antibiotic discs are applied with forceps aseptically on
agar plates and pressed gently to ensure even contact with the medium.
6. Plates are incubated for 24 hours at 35–37ºC.
7. After incubation, the diameter of zone of inhibition is measured.
8. Each zone size is interpreted according to the organisms by using the reference standard chart
DETERMINATION OF MINIMUM INHIBITORY

CONCENTRATION
Aim
To find the antibiotic susceptibility pattern of the given organisms.
I. ANTIBIOTIC SUSCEPTIBILITY TESTING

Principle
The antimicrobial activity of an antibiotic may be tested by several methods, designed to determine
the smallest amount of the agent needed to inhibit the growth of microorganisms. The resulting
value is known as the minimum inhibitory concentration (MIC). It can be determined by
several methods. In the agar diffusion procedure, a Petri plate containing a suitable medium
is heavily inoculated with the microorganism, whose antibiotic sensitivity is to be determined.
Commercially available filter paper discs, each containing defined concentrations of specific
antibiotics, are released from an automatic disc dispenser or removed from individual containers
and placed on the agar surface.The preparation is incubated for a definite period of time, during
which the antibiotics diffuse from the discs into the agar. At a particular distance from each
disc, the MIC for the antibiotic is reached. The MICs are recognized by the presence of growth
inhibition (clear) zones surrounding the various antibiotic discs used. The diameter of such zones
can be measured with a ruler. The results constitute an antibiogram.
Materials required
Culture: 24-hour nutrient broth culture of test organisms (Staphylococcus aureus, Proteus vulgaris),
Equipment and other materials: Individual antibiotic discs, one ruler with a millimetre scale,
one pair of forceps, one beaker containing 70% ethanol, four sterile 1 ml pipettes, two nutrient
agar deeps and 2 sterile Petri plates.
Procedure
1. Prepare one pour plate with Staphylococcus aureus and another with Proteus vulgaris.
2. Place the given antibiotic discs using dispenser.
3. Repeat the procedure with each plate in which pour-plate technique is followed.
4. Invert and incubate all plates at 37°C for 24 hours.
5. Examine each plate and look for the presence of growth inhibition zones. Using the millimetre
scale, measure the diameter of such zones.

We follow HiMedia standard chart and a copy is attached along with interpretation.
II. E-TEST
Principle
A novel approach to test antimicrobial activity is provided by the E-test. This in vitro susceptibility
method is designed to determine the minimum inhibitory concentrations (MIC) of antimicrobial
agents. The E-test involves the use of a thin, plastic strip, one side of which contains a continuous
concentration gradient of a stabilized and dried antibiotic. The drug diffuses from the strip
producing both qualitative and quantitative results showing the antibiotic susceptibility of the
organism being tested. The effective concentrations of an antibiotic can be readily determined
from the growth inhibitory zone produced after incubation. The E-test has been used for several
purposes including studies of antibiotic resistance mechanisms and quality control of media used
for susceptibility testing.
Materials required
Culture: 48 hours culture of ampicillin-resistant Staphylococcus aureus, Escherichia coli and Haemophilus
influenzae
Media: Blood agar plates
Equipment and other materials: Sterile cotton swabs, E-test strips of doxycycline and penicillin-V,
container with disinfectant.
Procedure
1. Label two agar plates for each of the bacterial cultures provided.
2. Obtain one sterile cotton swab, and dip it into one of the cultures provided.
3. Remove any excess fluid by gently pressing the swab against the side of the tube.
4. Then, swab the agar plate completely by covering the surface with the culture material on
the swab.
5. Allow the agar surface to dry.
6. Place an E-test strip at the centre of the swabbed agar surface.
7. Repeat steps 2 to 6 with the same culture and the second E-test strip provided.
8. Repeat steps 2 to 7 with the remaining bacterial cultures.
9. Incubate all plates at 37°C for 24–48 hours.
10. Read the minimal inhibitory concentration (MIC) values on each plate. The MIC value is
read where the zone of growth inhibition intersects the E-test strip.

After the incubation period, when an even lawn of growth is distinctly visible, read the MIC value
where the edge of the inhibition ellipse intersects the side of the strip as shown in the figure.
III. BROTH DILUTION METHOD

Aim
To identify the MIC by performing antibiotic dilution tube technique.
Principle
The minimum inhibitory concentration of the particular antibiotic is checked by inoculating
the test organism in the broth with antibiotic tubes and comparing with the control. In this
method, the dilutions of antibiotic concentrations are made and the best organism is inoculated.
The lower concentration of the agent that inhibits the growth of the organism is detected by
the lack of visual turbidity, which is designated as the minimum inhibitory concentration (MIC).
Organisms with MIC at or below the break point are considered “susceptible”. (An intermediate
result indicates that a moderately susceptible category also exist for certain organisms’ in antibiotic
combinations.)
Materials required
Media: Sterile tubes with broth
Equipment and other materials: Antibiotics, test organism, agar plates and sterile Petri plates
Procedure
1. About 1ml of test organism suspension (1×106 CFU/ml) is added to tubes containing
1 ml of broth and concentration of antibiotic, such as 4µg, 8µg, 16µg, 32µg, 64µg,
per ml.
2. Control tube is maintained with 1 ml of broth and 2 ml of test organisms without antibiotics
and 0.5 ml of broth, and immediately, 0.1 ml from tube is subcultured onto agar plates.
3. Now, all the tubes and plates are incubated at 37ºC.
4. Visual turbidity is noted in broth tubes and 0.1 ml from tube is subcultured on agar plates.
5. The plates are incubated overnight at 37ºC.
6. Colony forming units on subcultured plate is determined by colony count.
7. From the incubated plates, the particular concentration of antibiotics at which the growth of
organisms is completely absent, is considered as MBC/MLC (minimum lethal concentration/
minimum concentration of drug required to kill the organism).
Antibiotic susceptibility tests
Minimum inhibitory concentration test Disk diffussion test
A
Susceptible
organism
0 2 4 8 16
B
Resistant
organism
0 2 4 8 16
µg/ml antibiotic 10 µg antibiotic in discs

The colonies formed by plating the visible growth dilution tubes are counted and the plate in
which there was no growth was noted and compared to the dilution and that dilution is recorded
as MIC.
11
BIOCHEMICAL METHODOLOGY
CENTRIFUGATION
INTRODUCTION
Centrifugation is a process in which a particle (can be a macromolecule or a cell organelle) is
subjected to centrifugal force, that is, rotated at a high speed. This is given by,
F = mω2r
where,
F = Intensity of centrifugal force
m = Effective mass of the sedimenting particle
ω = Angular velocity of rotation
r = Distance of the sedimenting particles from the central axis of rotation.
F can also be represented in terms of earth’s gravitation force, g, as relative centrifugal force
(RCF). Centrifuged particles settle depending on their mass, shape, density of the particle and
density of the medium.
When particles are centrifuged, they settle down as pellet at the bottom of the container
and the upper liquid portion, called the supernatant, can be decanted (separated).
COMPONENTS
Any basic centrifuge contains two important components, namely, (i) an electric motor with a
drive shaft and (ii) a rotor that holds the tubes and other containers. The motor with shaft spins
the samples present in the container placed in the rotor.
TYPES
Centrifuges are of different types, ranging from those that are used for mere separation of particles
to those that are used for making analytical measurements during centrifugation process itself.
The three common types are:
1. Low-speed centrifuge
2. High-speed centrifuge
3. Ultracentrifuge
Low-speed Centrifuge
1. Used for separation of heavy particles, like cell debris.
2. Used at a speed of 4000 to 5000 rpm.
3. Works at room temperature without any separate control of temperature.
4. Samples of 12–50 ml can be used.
5. Uses either fixed angle or swinging bucket rotors.
Figure 11.1 Low-speed centrifuge
High-speed Centrifuge
1. Used for the separation of biological samples, like cellular organelles, protein precipitates
and microorganisms.
2. They are spun at a speed of 12,000 to 15,000 rpm.
3. These instruments always have speed and temperature control systems.
4. Amount of sample centrifuged depends on the type of rotor used.
5. Three different types of rotors are used:
Biochemical Methodology 337
 Fixed angle Rotors that are fixed at particular angles.

 Swinging bucket Rotors that move perpendicular to the axis of rotation during
centrifugation.
 Vertical rotors Sample tubes remain in upright position.
6. Rotor chamber of most of these are maintained at 4ºC.
Figure 11.2 High-speed centrifuge
Ultracentrifuge
1. These centrifuges reach a maximum speed of 1,00,000 rpm.
2. These are sophisticated instruments that generate intense heat, so the spin chamber should
be placed under refrigeration and high vacuum to reduce friction.
3. Metal rotors break into small pieces due to the high stress applied on them, so the rotor
chamber is covered with steel armour plates.
4. The drive shaft is made of flexible material to withstand any “wobble” of the rotor due to
any imbalance of the samples.
Figure 11.3 Ultracentrifuge
Operation of centrifuge
Switch on the centrifuge. Samples of equal volumes are placed in rotor. Slowly raise the speed of
the machine from zero to required speed by slowly turning the speed-controlling knob. When
the required speed is reached it is left to spin for the required time for the precipitation of the
sample. Increasing the effective gravitational force will more rapidly and completely cause the
precipitate to gather on the bottom of the tube as a ‘pellet’. The remaining solution is called
the ‘supernate’ or ‘supernatant’. The supernatant liquid is then quickly decanted from the tube
without disturbing the pellet.
Factors affecting rate of settling The time it takes for the precipitate to settle depends on:
 The size, shape and density of the precipitate particles
 The density and viscosity of the solution.
APPLICATIONS
1. Preparative centrifugation This involves placing the sample container in the
rotor, and spinning at a fixed speed, after which the
sample is separated into two phases, the pellet and the
supernatant. Low-speed and high-speed centrifuges
are used only for preparative purposes.
2. Analytical centrifugation Analytical centrifugation helps in determining the
molecular weight, density and purity of biological
samples. All analytical measurements are made only
with the ultracentrifuge. This is classified into two
types, namely, (a) differential centrifugation and (b)
density-gradient centrifugation. Differential
centrifugation involves the sedimentation of sample in
the medium of homogenous density. Density-gradient
centrifugation involves the centrifugation of the
sample in a fluid medium that gradually increases in
density from top to bottom. When sample is
centrifuged in a preformed gradient, it is called zonal
centrifugation. Isopycnic centrifugation is the one
in which a self-generating gradient forms during
centrifugation.
SEPARATION OF AMINO ACIDS

I. PAPER CHROMATOGRAPHY
Introduction
Chromatography is a biochemical technique introduced by Micheal Tswett in 1906. It is the most
powerful technique to separate chemically closely related substances into individual components
on the basis of their physicochemical properties. The compounds are separated on the basis of their
partition coefficients between two immiscible phases. The static phase may be a solid or liquid
while the mobile phase may be a solid, liquid or gas. The separation is called as chromatography
and the preparation is called chromatogram.
Aim
To separate and identify amino acids using paper chromatography.
Principle
The separation of the solutes (amino acids) is based on the liquid–liquid partitioning of amino
acids in paper chromatography. The partitioning takes place between the water molecules (static
phase), adsorbed to the cellulosic matter of the paper and the organic (mobile) phase.
Paper chromatography is an example of partition chromatography. In this, the cellulose support
is extensively hydrated, so distribution of solutes occur between the immobilized water (stationary
phase) and the mobile developing solvent. As the developing solvent molecules move through
the stationary phase, polar solvent molecules bind to the immobilized support and become the
stationary phase. Paper chromatography is used for the identification of unknown samples and
isolation of compounds in a mixture.
Paper chromatography requires only a minute sample size, the analysis is fast and inexpensive
and the detection is straight forward. The separation of different molecular components of the
sample is measured in terms of a unit called Rf (relative front or relative motility):
Distance run by the substance
Rf =
Distance run by the solvent
Under a given set of physical conditions, such as, temperature, pH, etc., the Rf of a compound
in a particular solvent system is constant, and this can be used to identify the unknown compounds.
If two compounds, A and B give the same Rf values in a solvent system, they are probably the
same compounds, but they can separate in a different solvent system.
The mechanism of carrying out a paper chromatographic analysis varies but the concept is
the same. The various methods are:
1. Ascending chromatography, where the solvent is allowed to rise by capillary action.

2. Descending chromatography, where the solvent is fed to the paper from top.
3. Horizontal or circular chromatography, where the plane of the paper is kept horizontal.
4. Two-dimensional chromatography, an improved method in which the mixture of
compounds is subjected to chromatography in two solvent systems, in the same sheet of
paper. First, the sample is chromatographed in one solvent in one direction. Then it is
chromatographed in the second solvent in a direction 90º to the first one.
Materials required
Sample: Preparation of sample Dissolve different individual amino acids in distilled water at a
concentration of 1 mg/ml. Use very dilute (0.05N) HCl to dissolve the free amino acids, tyrosine
and phenylalanine. Dissolve tryptophan in very dilute 0.05N NaOH.
Reagents:
1. Preparation of mobile phase—solvent system: Mix n-butanol, glacial acetic acid and
water in the ratio 4 : 1 : 5 in a separating funnel and stand to equilibrate for 30 minutes.
Drain off the lower aqueous phase into a beaker and place it inside to saturate the
chromatography chamber. Save the upper organic phase and use it for developing the
chromatogram.
2. Ninhydrin reagent: Dissolve 100 mg of ninhydrin in 100 ml acetone.
3. Elution mixture: Prepare 1% copper sulphate solution, and mix ethanol and copper
sulphate solution in the ratio 80 : 20 (v/v).
Equipments and other materials: Whatman no.1 filter paper, chromatography chamber,
hair-dryer or spot-lamp, atomizer, microsyringe or micropipette
Procedure
1. Extraction of sample Grind a known quantity of the sample material, [bacterial or fungal
cells, plant materials (dry/wet)] in a pestle and mortar with 10-fold volume of 70% ethanol.
Shake the contents at 55ºC for 30 minutes. Centrifuge the contents at 10,000 rpm for
10 minutes and collect the supernatant. Repeat the extraction of the pellet at 55ºC at least
twice. Pool the supernatants of leaf extracts, treat with equal volume of petroleum ether
(40–60ºC) and shake vigorously. Discard the petroleum ether layer containing chlorophyll.
Evaporate the alcohol fraction to dryness under vacuum using either water pump or
rotary evaporator at 40–45ºC. Dissolve the residue in a known volume of absolute ethanol
or water for analysis.
2. Cut the chromatography sheet carefully to a convenient size (40×24 cm). Draw a line
with pencil across the sheet about 1 cm away from one end. Make a number of points at an
interval of 3 cm.
3. Apply a small volume (say, 25 ml) of each amino acid as a separate small spot using a
microsyringe. A stream of hot air from a hair-dryer facilitates fast drying of spot. The spot
should be as small as possible for better resolution.
4. Similarly, spot different known aliquots of sample extract.
5. After spotting, place the sheet in a stainless steel trough in the chromatography chamber,
firmly hold it by placing a long steel rod over the sheet. The spot-end of the sheet should
be in the trough (descending chromatography). Otherwise, the sheet may be rolled as a
cylinder, tied together with fine thread and placed upright with the spots as the bottom in
a large Petri dish for upward movement of solvent (ascending chromatography).
6. Add the organic (phase) solvent to the trough/Petri dish and close the chamber air-tight.
Develop the chromatogram, preferably overnight or longer, until the solvent moves almost
to the other end.
7. Note the solvent front and dry the chromatogram free of solvent in a fume chamber.
8. Spray the chromatogram with the ninhydrin reagent using an atomizer. Dry the paper
for about 5 minutes at room temperature followed by drying at 100ºC in an oven for
2–3 minutes. Amino acids appear as purple spots; hydroxyl proline and proline give yellow-
coloured spots. Mark all the spots and calculate their Rf values by the formula:
Distance (cm)moved by the solute from the origin

Rf =
Distance (cm) moved by the solvent from the origin
9. The amino acids present in the sample are then identified by comparing the Rf values with
that of the authentic amino acids chromatographed.
10. For quantitative estimation, cut each spot into several small bits and transfer to the bottom
of the test tube. Add 3 ml of elution mixture. Shake the tube vigorously for 15 minutes.
Decant the liquid and elute the pieces with another 2 ml of elution mixture. Repeat the
elution with small aliquots until the bits are colourless. Combine and clear the eluate by
centrifuging at 10,000 rpm for 10 minutes. Read the intensity of purple colour at 570 nm
in a colorimeter. Use the spot of leucine (50 mg), run as standard, for comparison.
Paper strip in jar
Pencil
Barely
Touching
Figure 11.4 Paper Chromatography
Result
The given amino acids can be identified using the standard chart.
Amino acid R f value

Histidine 0.07
Serine 0.10
Lysine 0.10
Arginine 0.11
Aspartic acid 0.13
Glutamic acid 0.16
Glycine 0.17
Hydroxy proline 0.20
Alanine 0.22
Threonine 0.22
Proline 0.30
Tyrosine 0.32
Methionine 0.40
Valine 0.42
Tryptophan 0.47
Isoleucine 0.55
Phenylalanine 0.58
Leucine 0.60
Cysteine 0.80
II. THIN-LAYER CHROMATOGRAPHY

Introduction
Izmailor and Schraiber described the separation of plant extract on a thin layer of alumina in 1938.
Later, after twenty years, the equipment for the preparation of thin layer became available. Thin-
layer chromatography (TLC) is a widely employed laboratory technique and is similar to paper
chromatography. However, instead of using a stationary phase of paper, it involves a stationary
phase of a thin layer of adsorbent like silica gel, alumina or cellulose on a flat, inert substrate.
Fluorescent dyes can be incorporated into the medium to assist the identification of spots.
The separation of solute particles may be due to absorption, ion exchange, and partition
chromatography or gel permeation, depending upon the nature of the medium employed.
The method is very rapid and many separations can be completed in an hour. Very low
concentrations of compounds can be detected. Separated compounds can be detected by corrosive
sprays at elevated temperatures with some thin layer materials, which is not possible in paper
chromatography.
Aim
To separate and identify amino acids using thin-layer chromatography.
Principle
The general principle involved is that the solute competes with the solvent for the surface sites
of the adsorbent. Depending on the distribution coefficients, the compounds are distributed on
the surface of the adsorbent. Of course, in TLC, the partition effect in the separation is also not
ruled out. The adsorbent normally used contains a binding agent, such as calcium sulphate, which
facilitates the holding of the adsorbent to the glass plate.
Materials required
Sample: Sample should be extracted following the procedures indicated for each group of
compounds, e.g., extraction with 80% alcohol for amino acids and sugars.
Reagents: Standards, spraying agent—ninhydrin, developing solvents—n-butanol, acetic acid
and water in the ratio 4:1:1, adsorbent silica gel.
Equipments and other materials: Glass plate (20 × 20 cm or 20 × 10 cm), glass tank with lid,
spreader.
Procedure
1. Commercially available thin sheets are used.
2. The sheets are cut according to the size of the chromatographic jar.
3. The samples are loaded on the plate above 1 inch from one end of the sheet.
4. The chromatographic chamber is saturated with n-butanol, acetic acid and water in the ratio
4 : 1 : 1 and the solvent system is loaded in the jar.
5. The thin layer with the sample loaded is kept inside the chromatographic jar and closed with
glass plate.
6. After the solvent reaches the top, the plate is dried.
7. A solution of 0.3% ninhydrin is then sprayed over the plate. It is then activated at 60ºC for
30 minutes.
8. Coloured spots developed are compared with the values in the standard chart and the given
amino acids are identified.
Solvent
tank
Separated
sample
Sample
Spotted
Solvent Solvent
a) Time zero
b) After ten minutes
Figure 11.5 Thin-layer chromatography

As the chemicals being separated may be colourless, several methods exist to visualize the spots:
1. Often a small amount of a fluorescent compound, usually manganese-activated zinc silicate,
is added to the adsorbent that allows the visualization of spots under a blacklight (UV254).
The adsorbent layer will thus fluoresce light green by itself, but spots of analyte quench this
fluorescence.
2. Iodine vapours are generally unspecific colour reagents.
3. Specific colour reagents exist into which the TLC plate is dipped or which are sprayed onto the plate
 Potassium permanganate is a strong oxidant.
 Iodine
4. In the case of lipids, the chromatogram may be transferred to a PVDF (polyvinylidane

fluoride) membrane and then subjected to further analysis, for example mass spectrometry,
a technique known as Far-Eastern blotting.
Once visible, the Rf value, or retention factor, of each spot can be determined by dividing the
distance travelled by the product by the total distance travelled by the solvent (the solvent
front).
The Rf value of the given sample is _______. Thus the given sample can be separated and
identified.
COLUMN CHROMATOGRAPHY
Introduction
In this technique, a column of tiny particles of inert substances is prepared that contain small
pores. These inert substances have an open network formed by cross-linking of the polymeric
chains. These are hydrophilic and thus are capable of absorbing water. The absorption of water
induces swelling and causes opening of the structure. When a solution containing molecules of
various dimensions is passed through such a column, the molecules larger than the pores of the gel
move only in the space between the particles and thus are not retarded by the column material.
On the other hand, the molecules which are smaller than the pores of the gel diffuse in and out of
the particles. As a result, the rate of downward movement of the particles is considerably slowed
down. In case the particles of the inert substances do not absorb the molecules, the determining
factor for the rate of movement of the molecules through the column is the probability of their
penetration inside the gel. Accordingly, the molecules are eluted from the column in order of their
decreasing molecular weight, provided the shape is relatively constant. Gels with high degree of
swelling are used to fractionate high-molecular weight compounds, whereas the lower swelling
gels are used for separation of low-molecular weight compounds.
The following types of gels are used in column chromatography: dextran, agarose, and
polyacrylamide.
Dextran It is commercially available under the trade name, Sephadex and is generally used
for the separation of proteins. Sephadex is available in the form of dry beads that swell when
water is added. Swelling is the process by which the pores become filled with the liquid to be
used as eluent.
Agarose The word ‘agar’ comes from the Malay word agar-agar (meaning jelly). Agar consists
of a mixture of agarose and agaropectin. Agarose is a linear polymer, made up of the repeating
monomeric unit of agarobiose. Agarobiose is a disaccharide made up of d-galactose and
3, 6-anhydro-l-galactopyranose. Agar exhibits hysteresis, melting at 85°C and solidification at
a temperature of 32–40°C
Polyacrylamide Polyacrylamide [IUPAC poly (2-propenamide) or poly(1-carbamoylethylene)]

is a polymer (-CH2CHCONH2-) formed from acrylamide subunits. It can be synthesized as a
simple linear-chain structure or cross-linked, typically using N, N’-methylenebisacrylamide.
Polyacrylamide is not toxic. However, unpolymerized acrylamide, which is a neurotoxin, can be
present in very small amounts in the polymerized acrylamide, therefore it is recommended to
handle it with caution. In the cross-linked form, the possibility of the monomer being present
is reduced even further. It is highly water-absorbent, forming a soft gel when hydrated, used in
such applications as polyacrylamide gel electrophoresis and in manufacturing soft contact lenses.
In the straight-chain form, it is also used as a thickener and suspending agent.
Packing of the column The prepared slurry (using either one of the gels) is poured down
through the column with the help of a rod. If the slurry is prepared in the right manner with
the calculated amount of the gel, the entire amount can be added in one step. Liquid should be
added carefully otherwise the gel surface will be disturbed.
Application of the sample The sample is applied from the top of the gel bed. The flow is
started and the sample solution is allowed to pass through the gel. A small volume of the eluent
is added and the last traces of it are washed into the bed.
Aim
It is a method used to purify individual chemical compounds from mixtures of compounds on
scales from micrograms up to kilograms.
Materials required
Sample: Wet and dry samples are used.
Reagents: Column buffer (pH 7.0), NaCl (0.1M), Tris–HCl (12 mM), EDTA (2.5 mM), Sephadex
G50 (sephadex G50, 0.4 g; column buffer, 6 ml).
Equipments and other material: 50 ml sterile beaker, 10 ml syringe, glass wool.
Procedure
1. 400 mg of Sephadex G50 is dissolved in 6 ml of column buffer in 50 ml sterile beaker and
kept for swelling process.
2. A sterile silicon glass wool is placed at the neck of the closed 10 ml syringe so as to serve as
a support for the gel matrix.
3. The 3/4th of the syringe is filled with column buffer and then swollen sephadex is gradually
added to the column to 3/4th height.
4. The column outlet is opened to allow the drainage of the buffer and the Sephadex G50
column is equilibrated several times with column buffer and then the sample is added at the
surface of the column.
5. Now, the column outlet is again opened to allow the sample to enter into the gel particles.
The column buffer is added on the surface continuously and the sample is collected.
Buffer Sample
Column
Separated Sample
Figure 11.6 Column chromatography
COLORIMETER AND SPECTROPHOTOMETER

INTRODUCTION
A colorimeter or spectrophotometer can be used to measure any test substance that is itself coloured
or can be reacted to produce colour. Colorimetry refers to the measurement of colour and colorimetric
method is a technique used to evaluate an unknown colour with reference to known colours.
In colorimetric chemical test, the intensity of colour from the reaction must be proportional to
the concentration of the substance being tested.
In the most basic colorimetric method, the reacted test sample is visually compared to known
colour standard. A colorimeter or spectrophotometer can be used to photoelectrically measure
the amount of coloured light absorbed by a coloured sample with reference to blank. Intensity
of transmitted light is directly proportional to concentration.
A spectrophotometer is a photometer (a device for measuring light intensity) that can
measure intensity as a function of colour or more specifically the wavelength of light. There are
many kinds of spectrophotometers. They are classified on the basis of: (a) the wavelengths at
which they work, (b) the measurement technique they use, (c) how they acquire a spectrum and
(d) the sources of intensity variation they are designed to measure. Other important features
of spectrophotometers include the spectral bandwidth and linear range. The most common
application of spectrophotometers is measurement of light absorption.
Colorimeters rely on the principle that the absorbent substance is proportional to
its concentration, i.e., a more concentrated solution gives a high absorbance reading.
Spectrophotometer uses a quartz halogen lamp as a source of white light. The white light passes
through an entrance slit and is focused on a ruled grating consisting of 12 lines/mm. The grating
causes the light to be dispersed into its various component wavelengths. The monochromator
design allows the user to select a specific wavelength of interest when passed through an exit slit
and into the sample. The use of mirrors and additional filter papers prevents light of undesired
wavelengths overtones, stray light from making its way into the sample. A photodetector measures
the amount of light, which passes into the sample.
LAWS OF ABSORPTION
There are fundamental laws related to the absorption of monochromatic radiant energy by
homogenous transparent systems:
1. Lambert’s Law
When monochromatic light passes through a transparent medium, layers of equal thickness
of that homogenous absorbing medium absorb equal proportions of incident radiation.
2. Beer’s Law
The fraction of the monochromatic radiant energy absorbed, on passing through a solution,
is directly proportional to the concentration of absorbent.
From Law 1,
dp = –k1 P0 db
where,
b = Thickness; P0 = Radiant power of beam
From Law 2,
dp = k 2 P1 dc
log P0/P = abc =A (absorbent)
where,
a= Molar extinction coefficient or
Molar absorptivity (or absorption coefficient)
b= Path Length
c= Concentration
I. VISIBLE SPECTROPHOTOMETER
Most spectroscopic methods are differentiated as either atomic or molecular, based on whether or
not they apply to atoms or molecules. Along with that distinction, they can be classified based on
the nature of their interaction. Absorption spectroscopy uses a range of electromagnetic spectra in
which a substance absorbs. This includes atomic absorption spectroscopy and various molecular
techniques, such as infrared spectroscopy in that region and nuclear magnetic resonance (NMR)
in the radio region. Emission spectroscopy uses the range of electromagnetic spectra in which a
substance emits. The substance must absorb energy. This energy can be from a variety of sources,
which determines the name of the subsequent emission, like luminescence. Molecular luminescence
techniques include spectrofluorometry. Scattering spectroscopy measures the amount of light
that a substance scatters at certain wavelengths, incident angles and polarization angles. The
scattering process is much faster than the absorption/emission process. One of the most useful
applications of light scattering spectroscopy is Raman spectroscopy.
Types of Visible Spectrophotometers

They are divided to three groups:
1. Those with glass optics sensitive from 300 – 800 m
2. Those with quartz optics sensitive from 200 – 100 m
3. Those converting the range from 100 m
They can be:
i. Manual or non-recording
ii. Automatic or recording
Non-recording types include Cenco-sheard Backmann, Baush & Lamb, etc.
Recording types include Perkin Elmer, Backmann, etc.
They employ single beam or double beam optical systems.
The simplest and least expensive spectrophotometers are direct reading spectrophotometers.
Applications of visible spectrophotometers
i. They are used for analysis of various elements such as Al , NH3, As , Co, Cu, F, Fe, Mg, Mn,
Ni, Ti, V, salicylic acid, urea and glycine.
ii. They are used for identifying compounds.
iii. They help to decide the constituents of compounds.
iv. They measure the concentration of solutions.
v. They help to study H+ ion concentration.
vi. They are used to study the structure of inorganic complexes.
Instrumentation
Spectrophotometers are preferred than filter photometer. As filters have certain disadvantages,
they are replaced by monochromator.
Light source Incandescent tungsten lamp in the visible region 110–320 nm is used as source of light.
Monochromator Monochromator, such as prism or diffraction grating may be used. In few
monochromator lenses, mirror or other optical components are required.
Detectors A detector is a transducer that converts EMR into an electron flow and subsequently
into a current flow or voltage in the read-out circuit. They include photoiodides, photoemissive
tubes and photomultiplier tubes.
Working Principle
C
Grating device
(G)
Cells
Detector
Reflection G Mirror
Slit (T)
Lens (L)
Light source
Figure 11.7 Principle of a visible spectrophotometer
‘S’ is a source of radiation, which is a tungsten lamp. Light from the radiation source ‘S’ is
allowed to pass, by means of a lens ‘L’, through a narrow slit ‘T’ and then by means of a mirror to
an optical grating ‘G’ which divides light into narrow spectral regions corresponding to different
wavelengths. The light of a desired wavelength emerging from the grating is allowed to pass
through the cuvette B containing the solution under examination. The light further passes to
photoelectric cell D, which is in contact with galvanometer. The intensity of light can be measured
with the help of cell D. The cuvette B is now replaced by another cuvette C containing pure
solvent and the same light is allowed to pass through it and then to cell D.
If I0 = Intensity of light which passes through the solution
It = Intensity of light which passes through the solvent
Then, I0 / It = absorbance
The absorbance obtained is also called as optical density. The value increases with the increase
in the concentration of the solution or the sample.
II. UV SPECTROPHOTOMETER
All atoms absorb light in the UV region because these photons are energetic enough to excite
outer electrons. If the frequency is high enough, photoionization takes place. UV spectroscopy
is also used in quantifying proteins and DNA concentration as well as ratio of protein to DNA
concentration in a solution. The utilization of near ultraviolet absorption spectra as an analytical
tool has increased in recent years, due to their efficiency.
Applications of Ultraviolet Spectroscopy

i. It is used for identification of vitamins, sterols, hydrocarbons, enzymes and pharmaceuticals.
ii. Vitamin A can be assayed by measuring its absorbance at 324 nm.
iii. It is used to determine inorganic substances, e.g., lead measurement in bone ash.
iv. It is used for identifying compounds.
v. It is used to decide the constituents of compounds.
vi. It is used to measure the concentration of solutions.
vii. It helps in studying the H+ concentration.
viii. It helps to study the structure of inorganic complexes.
Principle
The instrument used in ultraviolet visible spectroscopy is called as UV/Visible spectrophotometer.
It measures the intensity of light passing through a sample [I] and compares it to the intensity
of light before it passes through the sample [I0]. The ratio I/I0 is called transmittance, and is
usually expressed as a percentage [%T]. The absorbance, A is based on the transmittance.
A = – log [%T]
Instrumentation
Light source Both high and low voltage hydrogen lamps give rise to continuous spectrum in
the region between 180–375 nm. A deuterium lamp produces very high radiation intensity than
H2 lamp. The basic parts of a spectrophotometer are a light source, often an incandescent bulb
for the visible wavelengths, or a deuterium arc lamp in the ultraviolet, a holder for the sample, a
diffraction grating or monochromator to separate the different wavelengths of light and a detector.
Monochromator They must not have glass optics, so prisms or quartz or fused silica must be
used as dispersive device. The performance of spectrophotometer related to the design of the
monochromator is evaluated by taking the amount of stray radiant energy and resolution into
condition.
Cuvette Quartz or fused silica may be used for UV regions. Two cells may be identical but their
absorption characteristics may be different in the UV regions. Hence, it is necessary to use one cell
as a reference cell and the other as a sample cell always. Samples for UV/Vis Spectrophotometer
are most often liquids, although the absorbance of glasses or even of solids can also be measured.
Samples are typically placed in a transparent cell, known as cuvette. Cuvette is typically
rectangular in shape, commonly with an internal width of 1 cm. Test tubes can also be used as
cuvette in some instruments. The best cuvettes are made of high quality quartz, although glass
or plastic cuvettes are common (Glass and most plastics absorb in the UV, which limits their
usefulness to visible wavelengths).
Detectors UV spectrophotometers use photomultiplier cells or vacuum photoemissive phototubes as
detectors. The detector is typically a photodiode or a CCD. Photodiodes are used with monochromator,
which filter the light so that only light of a single wavelength reaches the detector. Diffraction gratings
are used with CCDs, which collect light of different wavelengths on different pixels.
Special Methodolgy in UV Spectrometric Analysis

A spectrophotometer can be either single beam or double beam. In a single beam instrument
such as the Spectronic 20, all of the light passes through the sample cell. I0 must be measured by
removing the sample. This was the earliest design, but is still in common use in both teaching
and industrial labs. In a double-beam instrument, the light is split into two beams before it
reaches the sample. Some double-beam instruments have two detectors (photodiodes) and the
sample and reference beam are measured at the same time. In other instruments, the two beams
pass through a beam chopper, which blocks one beam at a time. The detector alternates between
measuring the sample beam and the reference beam.
III. FLUORESCENCE SPECTROSCOPY

Fluorescence spectroscopy or spectrofluorometry, is a type of electromagnetic spectroscopy which
analyses fluorescence from a sample. It involves using a beam of light, usually ultraviolet light
that excites the electrons in molecules of certain compounds and causes them to emit light of a
lower energy, typically, but not necessarily, visible light. A complementary technique is absorption
spectroscopy. Devices that measure fluorescence are called fluorometers or fluorimeters.
Molecules have various states, referred to as energy levels. Fluorescence spectroscopy is
primarily concerned with electronic and vibrational states. Generally, the species being examined
will have a ground electronic state (a low energy state) of interest, and an excited electronic state
of higher energy. Within each of these electronic states are various vibrational states.
In fluorescence spectroscopy, the species is first excited, by absorbing a photon, from its ground
electronic state to one of the various vibrational states in the excited electronic state. Collisions
with other molecules cause the excited molecule to lose vibrational energy until it reaches the
lowest vibrational state of the excited electronic state.
The molecule then drops down to one of the various vibrational levels of the ground electronic
state again, emitting a photon in the process. As molecules may drop down into any of the several
vibrational levels in the ground state, the emitted photons will have different energies, and thus
frequencies. Therefore, by analysing the different frequencies of light emitted in fluorescent
spectroscopy, along with their relative intensities, the structure of the different vibrational levels
can be determined.
In a typical experiment, the different frequencies of fluorescent light emitted by a sample
are measured, holding the excitation light at a constant wavelength. This is called an emission
spectrum. An excitation spectrum is measured by recording a number of emission spectra
using different wavelengths of excitation light.
Sample cell
Exitation
monochromator
Xa lamp
Emission
monochromator
Photodetector
Figure 11.8 Schematic of a fluorometer with 90º geometry utilizing a Xe light source
Two general types of instruments exist:

 Filter fluorometers use filters to isolate the incident light and fluorescent light.
 Spectrofluorometers use diffraction grating monochromators to isolate the incident light
and fluorescent light.
Both types utilize the following scheme: The light from an excitation source passes through
a filter or monochromator, and strikes the sample. A proportion of the incident light is absorbed
by the sample, and some of the molecules in the sample fluoresce. The fluorescent light is emitted
in all directions. Some of this fluorescent light passes through a second filter or monochromator
and reaches a detector, which is usually placed at 90° to the incident light beam to minimize the
risk of transmitted or reflected incident light reaching the detector.
Various light sources may be used as excitation sources, including lasers, photodiodes, and
lamps; xenon arcs and mercury-vapour lamps, in particular. A laser only emits light of high
irradiance at a very narrow wavelength interval, typically under 0.01 nm, which makes an
excitation monochromator or filter unnecessary. The disadvantage of this method is that the
wavelength of a laser cannot be changed much. A mercury-vapour lamp is a line lamp, meaning
it emits light near peak wavelengths. By contrast, a xenon arc has a continuous emission spectrum
with nearly constant intensity in the range from 300–800 nm and a sufficient irradiance for
measurements down to just above 200 nm.
Filters and/or monochromators may be used in fluorimeters. A monochromator transmits
light of an adjustable wavelength with an adjustable tolerance. The most common type of
monochromator utilizes a diffraction grating, i.e., collimated light illuminates a grating and exits
with a different angle depending on the wavelength. The monochromator can then be adjusted
to select which wavelengths to transmit. For allowing anisotropy measurements, the addition
of two polarization filters is necessary: one after the excitation monochromator or filter, and one
before the emission monochromator or filter.
As mentioned before, the fluorescence is most often measured at a 90° angle relative to
the excitation light. This geometry is used instead of placing the sensor at the line of the
excitation light at a 180° angle in order to avoid interference of the transmitted excitation light.
No monochromator is perfect; it will transmit some stray light, i.e., light with other wavelengths
other than the targeted. An ideal monochromator would only transmit light in the specified
range and have a high wavelength-independent transmission. When measuring at a 90º angle,
only the light scattered by the sample causes stray light. This results in a better signal-to-noise
ratio, and lowers the detection limit by approximately a factor 10000, when compared to the
180° geometry. Furthermore, the fluorescence can also be measured from the front, which is
often done for turbid or opaque samples. The detector can either be single-channelled or multi-
channelled. The single-channelled detector can only detect the intensity of one wavelength at a
time, while the multi-channelled detects the intensity at all wavelengths simultaneously, making
the emission monochromator or filter unnecessary. The different types of detectors have both
advantages and disadvantages.
The most versatile fluorimeters with dual monochromators and a continuous excitation light
source can record both an excitation spectrum and a fluorescence spectrum. When measuring
fluorescence spectra, the wavelength of the excitation light is kept constant, preferably at a
wavelength of high absorption, and the emission monochromator scans the spectrum. For measuring
excitation spectra, the wavelength passing through the emission filter or monochromator is kept
constant while the excitation monochromator is scanning. The excitation spectrum generally is
identical to the absorption spectrum as the fluorescence intensity is proportional to the absorption.
Analysis of Data
At low concentrations, the fluorescence intensity will generally be proportional to the concentration
of the fluorophore.Unlike in UV/visible spectroscopy, ‘standard’ device independent spectra are
not easily attained. Several factors influence and distort the spectra, and corrections are necessary
to attain ‘true’, i.e., machine-independent, spectra. The different types of distortions will here
be classified as being either instrument- or sample-related. First, the distortion arising from the
instrument is discussed. The light source intensity and wavelength characteristics varies over time
during each experiment and between each experiment. Furthermore, no lamp has a constant
intensity at all wavelengths. To correct this, a beam splitter can be applied after the excitation
monochromator or filter to direct a portion of the light to a reference detector.
Additionally, the transmission efficiency of monochromators and filters must be taken into
account. These may also change over time. The transmission efficiency of the monochromator also
varies depending on wavelength. This is the reason that an optional reference detector should be
placed after the excitation monochromator or filter. The percentage of the fluorescence picked up
by the detector is also dependent upon the system. Furthermore, the detector quantum efficiency,
i.e., the percentage of photons detected, varies between different detectors, with wavelength and
with time, as the detector inevitably deteriorates.
Correction of all these instrumental factors for getting a ‘standard’ spectrum is a tedious process,
which is only applied in practice when it is strictly necessary. This is the case when measuring the
quantum yield or when finding the wavelength with the highest emission intensity, for instance.
As mentioned earlier, distortions arise from the sample as well. Therefore, some aspects of the
sample must be taken into account too. First, photodecomposition may decrease the intensity of
fluorescence over time. Scattering of light must also be taken into account. The most significant
types of scattering in this context are Rayleigh and Raman scattering. Light scattered by Rayleigh
scattering has the same wavelength as the incident light, whereas in Raman scattering, the
scattered light changes wavelength usually to longer wavelengths. Raman scattering is the result of
a virtual electronic state induced by the excitation light. From this virtual state, the molecules may
relax back to a vibrational level other than the vibrational ground state. In fluorescence spectra,
it is always seen at a constant wavenumber difference relative to the excitation wavenumber, e.g.,
the peak appears at a wavenumber 3600 cm–1 lower than the excitation light in water.
Other aspects that have to be considered are the inner filter effects. These include
reabsorption. Reabsorption happens because another molecule or part of a macromolecule absorbs
at the wavelengths at which the fluorophore emits radiation. If this is the case, some or all of the
photons emitted by the fluorophore may be absorbed again. Another inner filter effect occurs
because of high concentrations of absorbing molecules, including the fluorophore. The result
is that the intensity of the excitation light is not constant throughout the solution. As a result,
only a small percentage of the excitation light reaches the fluorophores that are visible for the
detection system. The inner filter effects change the spectrum and intensity of the emitted light
and they must therefore be considered when analysing the emission spectrum of fluorescent light.
Tryptophan Fluorescence
Tryptophan is an important intrinsic fluorescent probe (amino acid), which can be used to estimate
the nature of microenvironment of tryptophan. When performing experiments with denaturants,
surfactants or other amphiphilic molecules, the microenvironment of tryptophan might change.
For example, if a protein containing a single tryptophan in its hydrophobic core is denatured with
increasing temperature, a red-shift emission spectrum will appear. This is due to the exposure
of the tryptophan to an aqueous environment as opposed to a hydrophobic protein interior.
In contrast, the addition of a surfactant to a protein, which contains a tryptophan, which when
exposed to the aqueous solvent, will cause a blue-shift emission spectrum, if the tryptophan is
embedded in the surfactant vesicle or micelle. Proteins that lack tryptophan may be coupled to a
fluorophore. At 295 nm, the tryptophan emission spectrum is dominant over the weaker tyrosine
and phenylalanine fluorescence.
Applications
Fluorescence spectrocopy is used in biochemical, medical, and chemical research fields for analysing
organic compounds. There has also been a report of its use in differentiating malignant, bashful
skin tumours from benign.
ATOMIC ABSORPTION SPECTROPHOTOMETER (AAS)

The technique was first introduced for analytical purpose by Walsh and Alleemade, Mihaz
(1956) under the designation Atomic absorption spectroscopy. It is found to be superior to
other techniques as it can be used to determine 50–60 elements from trace to large quantities.
They may include metals and non-metals.
Principle
The sample is first converted to atomic vapour and then the absorption of atomic vapour is
measured at selected wavelength characteristic of atoms of each element. The amount of light
absorbed is determined because the absorption is proportional to the concentration of the element.
Instrumentation
The apparatus consists of:
1. Radiation source
2. Atomizer
3. Monochromator
4. Lenses and slits
5. Detectors
Radiation source Hollow cathode lamp is widely used. It is a thick-walled tube with a transparent
window at one end. Tungsten wires are sealed into other end of the tube, which acts as anode.
Other wire is attached to a hollow metal cylinder which acts as cathode. The tube is filled with
helium or argon at 1–2 mm pressure.
Atomizers It is the one which introduces a spray into the flame. Atomization refers to the
dispersion of liquid into particles by rapidly moving gas, liquid stream or by mechanical means.
Flame atomizer The burner and nebulizer help in the atomization of the element. The flame is
produced in the burner where the combustion occurs and an atomic vapour of the element to be
analysed is produced. The selection of flame temperature is important for ionization. When it is
low, atomization will be incomplete. When it is high, the atoms may be ionized.
Graphite furnace atomizer A graphite furnace atomizer is used in graphite furnace atomic
absorption spectroscopy. The atomizer may be elongated enough in its axis to increase the distance
between the optical path and the sample deposition point. The elongation of atomizer increases
the analytical sensitivity.
Oxidants and fuels Fuels used are H2, propane, butane, acetylene and natural gas. The most
widely used is acetylene. Oxidants used are enriched with O2 and nitrous oxide.
Flame Temperature Elements
( ºC )
Air–coal gas 1800 Zn, Cu, Cd, Pb
Air–propane 1900 Volatile and noble elements
Air–acetylene 2300 Sn, Ba, Cr, etc.
N2O–acetylene 2955 Al,V, Bl, Tp, Se, etc.
Monochromator It is important that this instrument is capable of providing a narrow band

width to separate the line chosen for determination from other undesirable lines. Usually used
devices are gratings or prisms.
Lenses and slits They are used for isolation of required spectral line from the total spectrum.
Detectors Photomultipliers are used as detectors. In some instruments, two filters and two
detectors are used to compensate the fluctuations in the output of source. The output of
photomultiplier is taken to amplifier, which helps in source modification.
Hollow cathode Flame nebulizer Detector and

Monochromator read out
lamp unit
device
Figure 11.9 Read-out device
Working of the Instrument

A blank solution is sprayed into the flame and the meter is adjusted for zero absorbance or 100%
transmittance. Now, the solution under investigation is sprayed. The atoms in the excited state
absorbs certain part of light resulting in decrease in transmitted light or increase in absorbed light
falling on photomultiplier. This gives a deflection in the meter needle. With the help of standard
graph, the concentration of a particular element in the sample can be found out.
Interferences Though atomic absorption spectrophotometers are free from spectral interferences,
they are prone to chemical interferences as given below:
Chemical interferences Normally in AAS, chemical interferences could occur, due to the nature
of the ions involved. For example, the phosphate ions interfere with the determination of calcium
and magnesium. The reason is the formation of phosphates of calcium and magnesium, which
prevents the easy breakage of Ca and Mg ions into the flame. This interference could be reduced
by the addition of salt of Lanthanum or Thorium. These salts will form a bond with phosphate
ions. Therefore, the calcium and magnesium ions can be determined easily.
Ionization interferences Such interference can also occur in AAS along with chemical
interference. Such interference occurs when free metal atoms undergoes ionization as follows:
M M+ +e–
This occurs with alkali metals as they need very low energy for their ionization. If a deionizer
or a radiation buffer is added, then this type of interference may be overcome.
Applications
 It can be used to determine about 60 metals.
 It can be used to determine Al, Mg, Zn, Cu, Pb, Ni, etc.
 Na and K in biological fluids can be determined.
 Willis had determined Ca and Zn in urine samples of industrial workers.
 Fe in blood samples can also be found out.
 Council of British Archeology (1964) has used ASS to analyse polluted samples.
 In agriculture, soil extracts, plant materials and fertilizers have been analysed for determination
of Ca, Cu, Fe, K, Fe, Mn, Mg, Mo, Sr, Zn.
 Oils, such as crude oil, lubricating oil and feedstocks can be analysed in the determination
of metals.
 Impurities in petrol and refined oils can be determined.
 Electroplating effluents can be analysed for Cd, Fe, N and Cu.
TURBIDOMETRY
Turbidometry is based upon the scattering of light by non-transparent suspended particles in
the solution.
Principle
When a light passes through a suspension, part of the incident radiant energy is dissipated by
absorption, refraction and reflection, while remainder is transmitted. The measurement of the
intensity of transmitted light as a function of the concentration of dispersed phase is the basis of
turbidimetric analysis. Turbidimetry is more satisfactory for the determination of relatively high
concentration of suspended particles.
Light source Lens Sample Detector and

read-out device
Figure 11.10 Schematic diagram of turbidometer
Intensity can be determined by

log I0 / Ic = K'Ic
where,
I0 = Intensity of the incident light
c = Concentration of the absorbing particles in solution
I = Thickness of the absorbing medium
K = Turbidity coefficient
Instrumentation
The instruments used in turbidimetry are similar to that used in spectrophotometry.
They include:
1. Source Generally, a mercury arc with special filter combination is highly preferred.
2. Cell A cell with a rectangular cross-section is usually selected for the study.
3. Detectors Phototubes are used in turbidimeters. They are fixed on the circular disc, which
allows measurement from 35º to 135º. A simple turbidimeter, consists of a cylinder to
contain the turbid solution, a lamp filament of fixed intensity at the base and an adjustable
plunger through which visual observations can be made. The depth of turbid solution needed
to extinguish the image of lamp is noted. A standard graph is drawn using the standard
suspension. The graph is drawn by plotting depth versus concentration.
Light is passed through blank solution placed in the turbidometer. Part of the incident
radiant energy is dissipated by absorption, refraction and reflection. The remaining energy is
transmitted. The transmitted light reaches the photovoltaic cell. The light energy is converted
into electrical energy which reaches the read-out device. The intensity of transmitted light is
measured as a function of concentration dispersed. The light scattered will increase depending on
the turbidity of the solution. Thus high concentration of suspended particles can be determined
using turbidometer.
Procedure
Turbidimetry determination of bacterial numbers.
1. Put the original tube of E. coli and four tubes of the sterile NB in a test-tube rack. Each tube
of NB contains 5 ml of sterile broth. Use four of these tubes (tubes 2 to 5) of broth to make
four serial dilutions of the culture.
2. Transfer 5ml of E. coli to the first tube of NB, thoroughly mixing the tube afterwards. Transfer
5ml from that tube to the next tube, and so on until the last of the 4 tubes has 5ml added
to it. These tubes will be 1/2, 1/4, 1/8, and 1/16 dilutions.
3. The directions for spectrophotometer use are as follows.
a. The wavelength is preset somewhere between 550– 600 nm. Do Not change it.
b. Standardize the spectrophotometer as directed.
c. Obtain the 6 micro cuvettes. The cuvettes will look like either of the 2 shown to the
right. The lined or etched sides of the cuvettes face you, with the clear sides facing the
light source. The micro cuvette must contain 1ml for the spectrophotometer to read the
fluid, but you can and estimate the amount by eyesight.
d. The blank used to standardize the machine is sterile nutrient broth: it is called the blank
because it has a sample concentration equal to zero. Pipette 1ml of the sterile NB into
one of the micro cuvettes. Place into the blank cuvette holder (red line towards you),
close the cover and read. Save blank to re-standardize the machine to infinity absorbance
and zero absorbance before each reading because the settings tend to drift.
e. Pipette 1ml of the original bacterial specimen into a second micro cuvette. Place in cuvette
holder and read. When read, discard micro cuvette into bleach container on your table.
Next pipette the 1/4 dilution into the third cuvette and read it. Repeat this with the 1/4
1/8, and 1/16 dilutions.
4. Record your values, along with the dilutions that they came from. Using the plate count
data, calculate the colony-forming units per milliliter for each dilution.
Observation and result
Dilutions Absorbance Approximate number

of bacteria (Y)
Original of E.coli
1/2
1/4
1/8
1/16
1. Fill in your absorbance values for the 5 tubes read in the spectrophotometer.
2. Calculate the number of bacteria in the original tube of E. coli, and place that value in the
top right cell of the table.
3. Calculate the approximate numbers of bacteria in the 1/2, 1/4, 1/8, and 1/16 by having the
number in the cell above.
Applications
 They are of higher importance in water treatment plants, power and steam generating
plants, in beverage plants bottling industry, paper pulp industry, petroleum refineries and in
pharmaceutical industries.
 In water analysis, they are used to determine clarity and to understand the efficiency of
treatment process.
 They are used to determine the CO2 concentration.
 Turbidimetry is used to analyse turbidity in sugar products and citrus juices.
 It helps to determine the contents of carbonate as BaCO3, Cl– and cyanide as AgCN.
 These methods are more precise than calorimetric methods. For example, ‘P’ can be detected
at a concentration of 1 ppm too.
 The turbidity caused by BaSO4 can be analysed using the instrument. The turbidity could be
set at 100 NTU using standard solution (hydrazine sodium + hexamethylene tetraamine).
Then, BaSO4 solution can be fed into the instrument and the turbidity could be read directly.
BOMB CALORIMETER
Aim
To determine the enthalpy of combustion of a substance using bomb calorimeter.
Principle
A bomb calorimeter is used to measure heat flow for gases and high temperature reactions.
In a bomb calorimeter, the reaction takes place in a sealed metal container, which is placed in the water
in an insulated container. Heat flow from the reaction crosses the walls of the sealed container to the
water. The temperature difference of the water is measured. Analysis of the heat flow is a bit more
complex because the heat flow into the metal parts of the calorimeter must be taken into account.
Motorized stirrer
Electrical leads for
igniting sample
Thermometer
Insulated container
Oinlet
2
Bomb
(reaction chamber)
Fine wire in contact

with sample
Cup holding sample
Water
Figure 11.11 Bomb Calorimeter

The bomb has a fixed mass and specific heat. The mass multiplied by its specific heat is
sometimes termed the calorimeter constant, denoted by the symbol ‘C’ with units of joules
per degree Celsius. The calorimeter constant is determined experimentally and will vary from
one calorimeter to the other.
Once the calorimeter constant is known, the heat flow can be calculated. The pressure
within a bomb calorimeter often changes during a reaction, so the heat flow may not be equal
in magnitude to the enthalpy change.
Procedure
1. A little less than 1g of the sample is formed into a pellet by means of a pellet press.
2. The pellet is weighed and placed in the sample pan.
3. The fuse wire of measured length, about 10 cm, and known heat of combustion per unit
length, is attached to the two terminals and adjusted to give a firm contact with the pellet.
4. The cover is carefully assembled with the bomb and tightened.
5. The bomb is then connected to the oxygen tank, and oxygen is admitted slowly until the
pressure is 25 atm. The valves are then closed, the pressure in the line is relieved, and the
bomb is removed.
6. About 2000 ml of water, the temperature of which has been adjusted so as to be at least
several degrees below the upper limit of the thermometer range and preferably close to room
temperature, is placed in the calorimeter can; the latter is then placed within the adiabatic
jacket. The ignition leads are connected, and the bomb is immersed in the water.
7. The water in the can must cover the bomb.
8. The cover of the adiabatic jacket is set in place and the thermometer lowered into position
and read for a few minutes to be sure that equilibrium has been attained. This temperature
is recorded as the initial temperature T1.
9. The ignition switch is then closed until fusion of the wire is indicated by extinction of the lamp.
10. If combustion has occurred, the temperature of the water in the can will be seen to rise
within a few seconds. After a successful ignition, the temperature of the calorimeter rises
quickly. After several minutes, the rate of change of the temperature becomes small.
The final steady temperature of the can is then determined by extrapolation and recorded as T2.
11. The difference in the temperature dT(T2 – T1) gives the enthalpy of combustion.
Note It is important to avoid getting kinks in the fuse wire since fusion may occur at such points
before the portion of wire in contact with the pellet becomes hot enough to initiate combustion.
The surfaces at which closure of the bomb is to be effected must be kept scrupulously clean and
every precaution must be taken to avoid marring them. The parts of the dismantled bomb should
be placed on a clean, folded towel.
Result and Interpretation

In addition to measuring the energy release of one particular reaction, bomb calorimetry is an
important tool for determining the enthalpy of formation for the compound under study. This
information can then be applied to any reaction involving the compound.
The enthalpy of combustion for the reaction can be written as:
comb H (C x H y Oz ) =n (C x H y Oz ) f H °(C x H y Oz )+n (O2 ) f H °(O2 )+
n (CO2 ) f H °(CO2 )+n (H2O) f H °(H2O)
where v(i) is the stoichiometric coefficient of i. Since ∆ f H °(C x Hy Oz ) and ∆ f H °(H2O) are known
[and ∆ f H°(O2 ) equals zero], measurement of ∆ comb H (C x Hy Oz ) allows calcualtion of f H °(C x Hy Oz )
The enthalpy of formation for the given compound is ________.
TOTAL PROTEIN ESTIMATION

I. LOWRY’S METHOD
Aim
To determine the concentration of protein by Lowry’s method.
Principle
The blue colour developed by the reduction of the phosphomolybdic phosphotungstic components
in the Folin–Ciocalteau reagent, by the amino acids, tyrosine and tryptophan, present in the
protein, and the colour developed by the biuret reaction of the protein with the alkaline cupric
tartarate, are measured in the Lowry’s method.
Most protein estimation techniques use bovine serum albumin (BSA) universally as a standard
protein, because of its low cost, high purity and ready availablility. The method is sensitive down to
about 10mg/ml and is probably the most widely used protein assay method despite of its being only
a relative method, subject to interface from Tris buffer, EDTA, non-ionic and cationic detergents,
carbohydrates, lipids and some salts. The incubation time is very critical for a reproducible assay.
The reaction is also dependent on pH and a working range of 9–10.5 pH is essential.
Materials required
Sample: Protein solution (Stock standard) Weigh accurately 50 mg of bovine serum albumin
(Fraction V) and dissolve in distilled water and make up to 50 ml in standard flask.
Reagents:
1. Alkaline copper solution: 2% sodium carbonate in 0.1N sodium hydroxide (Reagent A),
0.5% copper sulphate (CuSO4.5H2O) in 1% potassium sodium tartarate (Reagent B).
Mix 50 ml of Reagent A and 1 ml of Reagent B prior to use (Reagent C).
2. Folin–Ciocalteau reagent (Reagent D): Reflux gently for 10 hours, a mixture consisting of
100 g of sodium tungstate (Na2W2.2H2O), 25 g of sodium molybdate (Na2MoO4.2H2O),
700 ml of water, 50 ml of 85% phosphoric acid, and 100 ml of concentrated hydrochloric
acid in a 1.5 L flask. Add 150 g of lithium sulphate, 50 ml of water and a few drops of
bromine water. Boil the mixture for 15 minutes without condenser to remove excess
bromine. Cool, dilute to 1 L and filter. The reagent should have no greenish tint.
(Determine the acid concentration of the reagent by titration with 1N NaOH to a
phenolphthalein end point).
3. Working standard: Dilute 10 ml of the stock solution to 50 ml with distilled water in
a standard flask. One ml of this solution contains 200µg protein.
Procedure
1. Pipette out 0.2, 0.4, 0.6, 0.8 and 1ml of the working standard into a series of test tubes.
2. Pipette out 0.1 ml and 0.2 ml of the sample extract in two other test tubes.
3. Make up the volume to 1ml in all the test tubes. A tube with 1ml of water serves as the blank.
4. Add 5 ml of Reagent C to each tube, including the blank. Mix well and allow to stand for
10 minutes.
5. Then add 0.5 ml of Reagent D, mix well and incubate at room temperature in the dark for
30 minutes. Blue colour is developed.
6. Take the readings at 660 nm.
7. Draw a standard graph and calculate the amount of protein in the sample.
8. Prepare different dilutions of BSA solutions by mixing stock BSA solution (1mg/ml) and
water in the test tube as given in Table 11.1. The final volume in each of the test tubes is
5 ml. The BSA range is 0.05 to 1mg/ml.
9. From these different dilutions, pipette out 0.2 ml of protein solution to different test tubes
and add 2 ml of alkaline copper sulphate reagent (analytical reagent). Mix the solutions well.
1 0. Incubate this solution at room temperature for 10 minutes.
11. Then add 0.2 ml of Folin–Ciocalteau solution (reagent solutions) to each tube and incubate
for 30 minutes. Zero the colorimeter with blank and take the optical density (measure the
absorbance) at 660 nm.
12. Plot the absorbance against protein concentration to get a standard calibration curve.
13. Check the absorbance of unknown sample and determine the concentration of the unknown
sample using the standard curve plotted above.
Table 11.1 Preparation of BSA solutions
BSA Water Sample conc. Sample Alk.CuSO4 Lowry O.D. at

(ml) (ml) (mg/ml) vol.(ml) (ml) reagent 600 nm
0.25 4.75 0.05 0.2 2 0.2
0.5 4.5 0.1 0.2 2 0.2
1 4 0.2 0.2 2 0.2
2 3 0.4 0.2 2 0.2
3 2 0.6 0.2 2 0.2
4 1 0.8 0.2 2 0.2
5 0 1.0 0.2 2 0.2
60
BSA standard (g)
50
40
30
20
10
0
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
A660
Data from Lowry Assay for Protein

A sample Lowry protein assay standard curve is produced using BSA at triplicate points of 0,
10, 20, 30, 40, and 50mg. The data are fit with a linear regression by the line y=153.06×+0.179
with and R2 value pf 0.992. The data table is used to generate the figure and depiction of a typical
Lowry assay.
Result
The concentration of the protein in the sample can be identified by extrapolating the graph.
II. BRADFORD’S METHOD
Aim
To determine the concentration of protein by Bradford’s method.
Principle
The assay is based on the ability of proteins to bind Coomasie brilliant blue G250 and form a
complex whose extinction coefficient is much greater than that of the free dye.
Materials required
Reagents:
1. Dye concentrate: 100 mg of Coomasie brilliant blue G250 is dissolved in 50 ml of 95%
ethanol. 100 ml of concentrated (ortho) phosphoric acid is added. Then distilled water
is added to make the final volume to 200 ml. This is stored in amber bottles under
refrigeration. The solution remains stable for at least six months.
2. Diluted dye: One volume of concentrated dye solution is mixed with four volumes of
distilled water for use. This is then filtered with Whatman no. 1 paper, if any precipitate
occurs.
3. Phosphate buffered saline (PBS).
Procedure
1. A series of protein samples are prepared in test tubes in different concentrations. This is
preferably prepared in PBS.
2. The experimental sample (a few dilution) is prepared in 100 µl.
3. 5ml of the diluted dye binding solution is added to each tube.
4. This is well-mixed and the colour is allowed to develop for at least 5 minutes but not longer
than 30 minutes. Blue colour is developed, when the dye is bound to the protein.
5. The absorbance at 595 nm is read.
6. A standard curve is plotted using the standard protein absorbance vs concentration.
The protein in the experimental sample is calculated using the standard curve.
Result
The concentration of protein in the sample is µg/ml
CREATININE ESTIMATION
Aim
To estimate creatinine by alkaline picrate method.
Principle
Creatinine reacts with alkaline picrate to give a yellowish red colour, which is measured
colorimetrically using green filter.
Materials required
Sample: Serum
Reagents: 10% sodium tungstate, 2/3N sulphuric acid, Saturated picric acid, 10% sodium
hydroxide, Creatinine stock standard (100 mg)
1. Preparation of creatinine working standard: Take 0.1ml of creatinine stock standard, add
9.9ml of distilled water and mix well.
2. Preparation of alkaline picrate solution: Take 0.1ml of sodium hydroxide, add 5ml of
saturated picric acid and mix well.
Procedure
Preparation of protein-free filtrate
Table 11.2 (a)
Contents Test (ml) Standard (ml)
Distilled water 7 7
Serum 1 –
Creatinine working standard – 1
10% Sodium tungstate 1 1
2/3N Sulphuric acid 1 1
Centrifuge for 5 minutes and take the supernatant fluid.

Table 11.2 (b)
Contents Test (ml) Standard (ml)
Supernatant fluid 6 6
Alkaline picrate solution 3 3
Mix well, wait for 10 minutes at room temperature and take readings on a colorimeter using
green filter (640 nm).
Calculations
Test O.D. = 0.04
Standard O.D. = 0.03
Standard concentration = 1 mg%
Serum creatinine in mg % = Test O.D./Standard O.D. × Concentration
= 0.04/0.03 × 1
= 1.3 mg%
Result
The level of serum creatinine present in the given blood sample is 1.3 mgs %
Normal value: 0.4–1.4 mg%
Clinical significance The serum creatinine level increases in renal failure, uraemia, pregnancy
and fever.
FRACTIONATION AND SIZE DETERMINATION OF PROTEINS

USING SDS–PAGE
Introduction
Polyacrylamide is the first choice for synthetic gel in electrophoresis. It can be prepared in
laboratory with defined texture and porosity. Hence, it has been generally used in the separation of
wide range of protein molecules even in the extreme experimental conditions like pH, temperature
and electrical conditions. Discontinuous (Disc) electrophoresis by Davis (1964) is a novel method.
It involves
i. preconcentration stacking of samples by several-fold leading to ultrathin starting zone

prior to electrophoretic separation,
ii. Separation of molecules in small pore gel according to molecular size Combination of
these two phenomenon results in higher resolution. This Disc–PAGE has become very
popular and is widely used.
Disc electrophoresis can be carried out using vertical gel apparatus in which electrophoresing and
comparing the profiles of several samples can be done simultaneously under the uniform conditions.
There is a choice of varying the gel thickness and the number of wells according to investigation.
Vertical slab gel apparatus, in general, consists of two reservoirs, a lower reservoir (anode) and
an upper reservoir (cathode). Platinum electrodes housed in the reservoir are connected to the two
terminals. The supporting sheet of the upper reservoir and one of the glass plates are noted to
have buffer connection between the reservoir and the gel. The gel plate is mounted accordingly
and buffer is prevented from any leakage. Recently, a number of modifications have been done
for slab gel apparatus, so as to circulate buffer or cool water to maintain desirable temperature
and pH during electrophoresis.
Glass plate sandwich is used to make gel slabs. Two rectangular glass plates, one of which
is notched, are separated by thin spacers of 0.5–1.5 mm thickness for the glass sandwich.
This arrangement is sealed on three slides by means of vacuum, grease, agar gel sealing or by using
gasket. It is then kept vertically for preparing gel mixture. First, separating gel is polymerized
and over which stacking gel is made. Before polymerizing the stacking gel, suitable well-forming
combs are inserted.
Discontinuous buffer system based on the method proposed by Laemeli (1970) is widely
employed for separating different polypeptides. Prior to electrophoresis, protein samples are
heated with 5% mercaptoethanol and 10% of sucrose or glycerol. SDS denatures proteins by
binding with individual polypeptides and mercaptoethanol disrupts sulphide bonds to keep the
polypeptides apart. In this buffer system, polypeptides stack before they start separating in the
small pore gel. They migrate according to their size. By comparing the migration of known
peptides, the sizes of unknown peptides can be determined. Estimation of molecular weight is
also determined by employing standard protein.
Polyacrylamide Gel
O O O
CH2 = CH C NH2 + CH3 CH2 C NH C CH2 CH3
(Acrylamide) N, N!-methylene bisacrylamide
Photochemical polymerization Polymerization chemical

APS+TEMED (Riboflavin+TEMED+Long wavelength)
CONH2 CONH2 CONH2 CONH2
CH3 CH CH2 CH CH2 CH2 CH CH CH2 CH2 CH CH2 CH CH2 CH2
CONH CONH CONH
CH2 CH2 CH2
CONH CONH CONH
CH3 CH CH2 CH CH2 CH2 CH2 CH CH2 CH CH2 CH2 CH CH2 CH3
CONH CONH
CH2 CH2
CONH CONH CONH2 CONH2
CH3 CH CH2 CH2 CH2 CH2 CH2 CH CH2 CH CH2 CH2
Materials required
Sample: Protein samples
Reagents: Acrylamide monomer, 4X separating gel buffer (pH 8.8), 4X stacking gel buffer (pH
8.8), 10% SDS, ammonium persulphate (APS), 10% gel overlaying solution (pH 8.8), tank
buffer (pH 8.3), water saturated in butanol.
Equipments and other materials: Rectangular glass plates, notched glass plates, spacers, metal
clips, electrophoresis unit, power supply, etc.
Procedure
1. Glass plates are assembled for preparing a slab gel.
2. The thoroughly cleaned rectangular plates are plated.
3. The notched glass plates are placed over the gasket and two spacers are inserted along the gaskets.
4. The rectangular and notched glass plates assembly are clamped together using metal clips.
5. The whole assembly is kept in a vertical position and after placing them in a gel casting stand,
it is tightened gently.
6. Then, 15 ml of 10% separating gel mixture is prepared by adding acrylamide stock solution,
4X separating gel buffer,10% SDS solution, distilled water, 10% APS,TEMED. The gel
mixture is poured in the side arm flask and the solution is degassed in vacuum for 5–10
minutes and then added.
7. The gel mixture is poured immediately using a 10 ml pipette into the glass sandwich
up to the mark and no air bubbles are allowed.
8. Immediately after pouring the separating gel mixture, about 0.5–1 ml of n-butanol saturated
with water is taken and injected slowly, pointing the needle tip at about 45ºC towards the
entire top surface of the separating gel mixture.
9. The gel is left undisturbed for polymerization for about 15–20 minutes. A clean
liquid-gel interface is visible on polymerization.
10. After polymerization, the overlay solution is discarded. The surface is washed with separating
gel solution.
11. About 5ml of 5% of stacking gel mixture is poured by adding acrylamide stock solution,
4X stacking gel buffer,10% SDS solution, distilled water,10% APS,TEMED.
1 2. Stacking gel mixture is poured above the separating gel and the comb is inserted into the gel.
13. The polymerization is allowed to complete for about 15–30 minutes. Samples are prepared
in the mean time.
14. Equal volume of protein samples are mixed with 2X sample buffer and kept in a boiling
water bath for 90 seconds. The samples are placed in ice after boiling.
15. Now, the bottom spacer is removed and the gel is clamped strengthened with the
electrophoresis unit.
16. Afterwards, the top and the lower chambers are filled up with tank buffer very gently using
a pipette and followed by this, the comb is removed carefully.
17. Using a micropipette, 40 µl of the sample is applied to each well. Equal volume of 1X sample
buffer is applied to any well that is free.
18. The tank is connected to a power pack and the power supply is turned on. The power supply
is adjusted to a constant current mode of 10–15mA. However, the stacking and separating
steps are carried out under optimum conditions.
19. The run is continued till the marker dye reaches the bottom of the gel.
20. The power is decreased and the power supply is turned off. The power cord is disconnected.
21. Upper reservoir is drained and the gel is taken out carefully. The top notched plate is removed.
22. The separating gel is placed in dye (coomassie brilliant blue) solution. After cutting the
stacking gel, enough dye solution is poured to cover the gel and stained for 3–4 hours.
23. The staining solution is then removed and destaining solution is added. It is soaked intermittently
and destaining solution is changed several times until a clear background is obtained.
24. The bands are examined in white light transilluminator and are shown in the photograph.
Unique protein profiles are found in the gel for the fractioned portion samples and results
are photographed.
25. Using a ruler, the distance travelled can be identified by measuring the marker dye
from the origin and each band position (standard as well as unknown) from the origin.
Relative mobility (Rf value) can be calculated using the formula.
26. The values are plotted in a semi-log graph by entering molecular weight of standard protein
in X-axis and Rf values on Y-axis.
27. By calculating the Rf values of unknown proteins, molecular weight can be found out from
the standard graph.
SDS–PAGE-12%T (Tris–Glycine buffer)
RNA polymerase
β - Galactosidase
4
10×10
4 Phosphorylase b
9×10
4
8×10
7×104 Bovine serum albumin
Molecular we ight
4
6×10
4
5×10 Ovalbumin
4 Lactate dehydrogenase
4×10
4 Carbonic anhydrase
3×10
Trypsin inhibitor
4
2×10
Lysozyme
4
1×10
0.0 0.2 0.4 0.6 0.8 1.0
R f value
Laemmli Plot
Result and Discussion

Plot the relative mobility of each protein against the log of its molecular weight. Relative mobility
is the term used for the ratio of the distance the protein has moved from its point of origin (the
beginning of the separating gel) relative to the distance the tracking dye has moved (the gel front).
The ratio is abbreviated as Rf . Molecular weight is expressed in daltons. The above graph presents
a plot of the relative mobility of protein standards against the log of their molecular weight.
The Rf value of the protein present in the given sample is _________. With the Rf value,
the protein present in the given sample can be determined.
BUFFER SOLUTIONS
Aim
To prepare buffers of varying pH.
Principle
Microorganisms produce acids or alkalies due to their metabolic activities. They alter the pH
of the environment bringing about various changes. For example, when bacteria are grown in a
medium containing sugar, they may produce acids as intermediate or end-products. If these acidic
products are allowed to accumulate in an unbuffered medium, the organisms will be killed by
the low pH produced. It is therefore suggested to include buffers in culture media. These buffers
will resist the change in hydrogen ion concentration.
A buffer solution is defined as a solution, one which resists changes in pH when small quantities
of an acid or an alkali are added to it.
Acidic buffer solutions An acidic buffer solution is a solution that has a pH less than 7. Acidic
buffer solutions are commonly made from a weak acid and one of its salts, often a sodium salt,
e.g., acidic acid (HC2H3O2), sodium acetate (NaC2 H3O2).
Alkaline buffer solutions An alkaline buffer solution has a pH greater than 7. Alkaline buffer
solutions are commonly made from a weak base and one of its salts. These buffers will resist the
change in H+ ion concentration. Both the acid and alkaline buffer solutions resist large change in
pH by partially absorbing the H+ and OH– ions in a solution. Buffered solutions do not change
in pH upon the addition of H+ and OH– ions. However, the change is much less than that which
could occur, if buffer was present, e.g., sodium bicarbonate (NaHCO3), potassium bicarbonate
(KHCO3 ).
Henderson–Haselbach equation
 A – 
pH = pka + log
[HA ]
This equation is useful in the preparation of buffer solution and in determining the pH of
the buffer solution. The pH of buffer is determined by the value pKa, which is a constant for a
particular acid, and by the logarithm of ratio of salt to the acid solution. When the concentration
of salt is equal to that of acid, then pH will be equal to the pKa value. The effective range of
buffering system is 1.5 pH units below and above pKa value. The other weak acid with suitable
pKa values is selected so that by their means it is possible to prepare buffer solutions of any pH.
Procedure
1. Preparation of acetate buffer
Solution A 0.2 M solution of acetic acid (11.5 ml in 1000 ml)
Solution B 0.2 M solution of sodium acetate (16.4 g of C2H2Na or C2H3O2Na. 3H2O in
1000 ml)
X ml of Solution A, Y ml of Solution B, diluted to a total of 100 ml.
Table 11.3 Acetate buffer
X (ml) Y (ml) pH
46.3 3.7 3.6
44.0 6.0 3.8
41.0 9.0 4.0
36.8 13.2 4.2
30.5 19.5 4.4
25.5 24.5 4.6
20.0 30.0 4.8
14.8 35.2 5.0
10.5 39.5 5.2
8.8 41.2 5.4
4.8 45.2 5.6
2. Preparation of boric acid–borax buffer

Solution A 0.2 M solution of boric acid (12.4 g in 1000 ml)
Solution B 0.05M solution of borax (19.05g in 1000 ml; 0.2M in terms of
sodium borate)
50 ml of solution A, X ml of solution B, diluted to a total of 200 ml.
Table 11.4 Boric acid–borax buffer
X (ml) pH
2.0 7.6
3.1 7.8
4.9 8.0
7.3 8.2
11.5 8.4
17.5 8.6
22.5 8.7
30.0 8.8
42.5 8.9
59.0 9.0
83.0 9.1
115.0 9.2
3. Preparation of carbonate–bicarbonate buffer

Solution A 0.2 M solution of anhydrous sodium carbonate (21.2 g in 1000 ml)
Solution B 0.2 M solution of sodium bicarbonate (16.8 g in 1000 ml)
X ml of solution A, Y ml of solution B, diluted to a total of 200 ml.
Table 11.5 Carbonate–bicarbonate buffer
X (ml) Y (ml) pH
4.0 46.0 9.2
7.5 42.5 9.3
9.5 40.0 9.4
13.0 37.0 9.5
146.0 34.0 9.6
19.5 30.5 9.7
22.0 28.0 9.8
25.0 25.0 9.9
27.5 22.5 10.0
30.0 20.0 10.1
33.0 17.0 10.2
35.5 14.5 10.3
38.5 11.5 10.4
40.5 9.5 10.5
42.5 7.5 10.6
45.0 5.0 10.7
4. Preparation of citrate buffer

Solution A 0.1 M solution of citric acid (21.01 g in 1000 ml)
Solution B 0.1 M solution of sodium citrate (29.41 g of C6H5O7Na3. 2H2O in 1000 ml)
Table 11.6 Citrate buffer
X (ml) Y (ml) pH X (ml) Y (ml) pH X (ml) Y (ml) pH

46.5 3.5 3.0 33.0 17.0 4.0 18.0 32.0 5.2
43.7 6.3 3.2 31.5 18.5 4.2 16.0 34.0 5.4
40.0 10.0 3.4 28.0 22.0 4.4 13.7 36.3 5.6
37.0 13.0 3.6 25.5 24.5 4.6 11.8 38.2 5.8
35.0 15.0 3.8 23.0 27.0 4.8 9.5 40.5 6.0
20.5 29.5 5.0 7.2 42.8 6.2
5. Preparation of glycine–HCl buffer

Solution A 0.2 M glycine (15.01 g in 1000 ml)
Solution B 0.2N HCl
Table 11.7 Glycine–HCl buffer
X (ml) pH
22.0 2.2
16.2 2.4
12.1 2.6
8.4 2.8
5.7 3.0
4.1 3.2
3.2 3.4
2.5 3.6
6. Preparation of phosphate buffer

Solution A 0.2 M solution of monobasic sodium phosphate (27.8 g in 1000 ml)
Solution B 0.2 M solution of dibasic sodium phosphate (53.65 g of Na2HOP4.7H2O or 71.7g
of Na2HPO4.12H2O in 1000 ml)
Table 11.8 Phosphate buffer
X (ml) Y (ml) pH X (ml) Y (ml) pH

93.5 6.5 5.7 45.0 55.0 6.9
92.0 8.0 5.8 39.0 61.0 7.0
90.0 10.0 5.9 33.0 67.0 7.1
87.7 12.3 6.0 28.0 72.0 7.2
85.0 15.0 6.1 23.0 77.0 7.3
81.5 18.5 6.2 19.0 81.0 7.4
77.5 22.5 6.3 16.0 84.0 7.5
73.5 26.5 6.4 13.0 87.0 7.6
68.5 31.5 6.5 10.5 89.5 7.7
62.5 37.5 6.6 8.5 91.5 7.8
56.5 43.5 6.7 7.0 93.0 7.9
51.0 49.0 6.8 5.3 94.7 8.0
7. Preparation of tris (hydroxymethyl) amino methane–HCl (Tris–HCl) buffer

Solution A 0.2 mol/litre solution of Tris(hydroxymethyl)amino methane (24.2 g in
1000 ml)
Solution B 0.2 mol/litre HCl
Table 11.9 Tris–HCl buffer
X (ml) pH
5.0 9.0
8.1 8.8
12.2 8.6
16.5 8.4
21.9 8.2
26.8 8.0
32.5 7.8
38.4 7.6
41.4 7.4
44.2 7.2
8. Preparation of citrate–phosphate buffer

Solution A 0.1 mol/litre solution of citric acid (19.21 g in 1000 ml)
Solution B 0.2 mol/litre solution of diabasic sodium phosphate (28.39 g of Na2HPO4 or
71.7 g of NaHPO4.12H2O in 1000 ml)

Table 11.10 Citrate–phosphate buffer
X (ml) Y (ml) pH
44.6 5.4 2.6
42.2 7.8 2.8
39.8 10.2 3.0
37.7 12.3 3.2
35.9 14.1 3.4
33.9 16.1 3.6
32.3 17.7 3.8
30.7 19.3 4.0
29.4 20.6 4.2
27.8 22.2 4.4
26.7 23.3 4.6
25.2 24.8 4.8
24.3 25.7 5.0
23.3 26.7 5.2
22.2 27.8 5.4
21.0 29.0 5.6
19.7 30.3 5.8
17.9 32.1 6.0
16.9 33.1 6.2
15.4 34.6 6.4
13.6 36.4 6.6
9.1 40.9 6.8
6.4 43.6 7.0
CONCENTRATION UNITS
( MOLARITY, NORMALITY, MOLALITY )
INTRODUCTION
The concentration of a chemical solution refers to the amount of solute that is dissolved in a
solvent. We normally think of a solute as a solid that is added to a solvent (e.g., adding table
salt to water), but the solute could just as easily exist in another phase. For example, if we add
a small amount of ethanol to water, then the ethanol is the solute and the water is the solvent.
If we add a smaller amount of water to a larger amount of ethanol, then the water could be the
solute. Concentration may be expressed in several different ways, using per cent composition by
molarity, molality, or normality, etc.
MOLARITY
Molarity of a solution is defined as the number of gram-moles of solute dissolved in 1 litre of a
solution.
Number of moles of solute
Molarity =
Volume of solution in litres
If ‘X’ grams of the solute is present in ‘V’ cm3 of a given solution, then,
X 1000
Molarity = ×
Mol.mass V
Molarity is represented by the symbol ‘M’. Molarity can also be calculated from the strength
as follows:
Strength in grams per litre
Molarity =
Molecular mass of the solute
Example A 0.1 M solution of sugar, C12H22O11 (Mol. mass = 342), means that 34.2 g of sugar
is present in one litre (1000 cm3) of the solution.
Problem 4.5 g of urea (Molar mass = 60 g mol–1) is dissolved in water and the solution is made
to 100 ml in a volumetric flask. Calculate the molarity of the solution.
Solution
Mass of urea = 4.5 g
Mass 4.5g
Moles of urea = =
Molar mass 60 gmol –1
= 0.075 mol
100 L
Volume of solution =
1000
0.1L
Mass of solute in grams

Molarity of solution =
0.075
= mol 0.75M
0.10
NORMALITY
Normality of a solution is defined as the number of gram equivalents of the solute dissolved per
litre of the given solution.
Number of gram - equivalents of solute

Normality =
If ‘X’ grams of the solute is present in ‘V’ cm3 of a given solution, then,
X 1000 ml
Normality = ×
Eq.mass V
Normality is represented by the symbol ‘N’. Normality can also be calculated from strength
as follows:
Strength in grams per litre
Normality =
Eq.mass of the solute
Example A 0.1N (or deci-normal) solution of H2SO4 (Eq. mass = 49) means that 4.9 g of H2SO4
is present in one litre (1000 cm3) of the solution.
Problem Calculate the normality of the solution containing 3.15 g of hydrated oxalic acid
(H2C2O4.2H2O) in 250 ml of the solution (Mol. Mass = 126).
Solution
Mass of oxalic acid = 3.15 g
Mol. mass
Equivalent mass of oxalic acid =
Basicity
126
= 63g equiv –1
2
Mass of solute
Equivalents of oxalic acid =
Eq. mass
3.15
= 0.05equiv –1
63
250
Volume of solution =250 ml = L 0.25L
1000
Equivalent of solute
Normality =
0.05
= 0.25N
0.25
MOLALITY
Molality of a solution is defined as the number of gram-moles of solute dissolved in 1000 g
(or 1kg) of a solvent. Mathematically,
Number of moles of solute

Molality =
Mass of solvent in kilograms
If ‘X’ grams of the solute is dissolved in ‘b’ grams of the solvent, then,
X 1000
Molality = ×
Mol. mass b
Molality is represented by the symbol ‘m’.
Example A 0.1 m solution of glucose, C6H12O6 (Mol.mass=180), means that 18 g of glucose
is present in 1000 g (or one kilogram) of water.
Problem Calculate the molality of an aqueous solution containing 3.0 g of urea (Mol.mass =
60) in 250 g of water.
Solution
Mass of solute = 3.0 g
Mass of solute
Moles of solute =
Molar mass
3.0
= = 0.05 mol
60
Mass of solvent = 250 g
250
= 0.25kg
1000
Moles of solute
Molality of solution =
Mass of solvent in kg
0.05
= 0.2m
0.25
12
VIROLOGY
ISOLATION OF COLIPHAGES
Aim
To isolate coliphages from sewage sample.
Principle
The presence of coliphages in the natural environment is relatively low and therefore, it is essential
to use desired host bacteria along with the nutrients as an enrichment technique. After incubation,
the bacteriophages can be separated by centrifugation and further by membrane filtration, which
uses 0.4 µm pore size filters to physically remove the bacteria from the liquid. The final step is
to produce plaques by seeding a layer of bacteria with phages in the filtrate.
Materials required
Nutrient broth, nutrient soft agar, nutrient agar plate, sewage sample, test tubes, pipettes,
membrane filter, filtration apparatus.
Procedure
1. About 45 ml of sewage is added to 5 ml of 10X nutrient broth in a sterile flask.
2. About 5 ml of E. coli or other bacteria is added (any other desirable bacteria for which phage
isolation is needed). It is mixed gently and incubated for 24 hours at 37º C.
3. In order to remove bacterial cells, 10 ml of this enrichment culture is centrifuged at
5000 rpm for 10 minutes.
4. The supernatant is filtered through membrane filter.
5. Four tubes of soft nutrient agar are liquefied and cooled to 50ºC. The tubes are kept in a
water bath at the same temperature to prevent solidification.
6. About 0.3 ml of the E. coli culture and 0.1 ml of supernatant (or other desirable host) is
transferred to each of the four tubes of soft agar and mixed by rolling the tube between our
hands.
7. The contents of the tube are poured over four nutrient agar plates and labelled.
8. Once the agar is cooled, the plate is kept inverted and incubated at 37ºC for 24 hours.
9. The plaque formation and the size of the plaques are observed and counted.
Plate showing plaques
Result
Plaques are clear zones formed in a lawn of cells due to lysis by phage. Plaque formation indicates
presence of coliphages in the given sample. Thus coliphages are isolated.
PHAGE TITRATION
Introduction
In 1915 and 1917, respectively, F. Twort and F.D’Herelle independently discovered that bacteria
are susceptible to infection with viruses and these viruses were called bacteriophages (φ).
These eaters of bacteria, also referred to as phages, are widely distributed in nature and have
been isolated from faeces, sewage, human gastrointestinal tracts, nasopharyngeal areas and
sputum specimens.
Aim
To perform phage titration technique.
Virology 387
Principle
Bacteriophage assays or titrations can be performed in the following manner: serial dilutions
10–1, 10–2, 10–3 of phage-containing specimens are made. Specific aliquots of such dilutions and
a standard suspension of a suitable bacterial host are added to a standard quantity of melted soft
agar and is then poured over the surface of an appropriate medium and incubated. During the
incubation period, the lysis of bacterial cells by bacteriophages occurs.
The lytic activity of bacteriophages may be observed by the clearing of a cloudy broth
suspension of infected bacterial cells or by the formation of clear zones known as plaques against
the opaque bacterial lawn background.
Materials required
1. Test tubes, pipettes, Petri plates
2. T2 bacteriophage suspension (10–3 diluted)
3. Tryptose phosphate broth culture of Escherichia coli strain
4. Tryptose phosphate broth, tryptose agar
Procedure
1. Label the test tubes consecutively from 10–4 through 10–7, and the last tube as control. Also
label the tryptose agar plates corresponding to each of the labelled tubes.
2. Aseptically add 4.5 ml of the tryptose phosphate broth into each tube.
3. Prepare a serial 10-fold dilution of the 10–3 bacteriophage preparation. Aseptically transfer
0.5 ml of bacteriophage suspension to the 10–4 labelled tube.
4. Shake the tube well and transfer 0.5 ml from the contents of the 10–4 tube to the labelled
10–5 tube.
5. Repeat this procedure till the tube labelled 10–7 is reached. Discard 0.5 ml from the last tube.
6. Place tubes of soft agar in the boiling water bath. After the contents have been melted, cool
the tubes and keep them in the 45°C water bath.
7. Remove the soft agar and add 0.5 ml of the Escherichia coli strain B suspension to the tubes
of soft agar.
8. To each of the tubes of soft agar, transfer 0.1 ml of the respective phage dilution and control.
9. Mix the contents of each of the soft agar tubes and pour onto the surface of the appropriate
labelled plate.
10. Rotate the plates and allow the soft agar to harden.
11. Incubate at 37°C for 24 hours.
12. After incubation, using the colony counter, examine the plates and count the number of
plaques or the clear halos in the lawn of bacteria.
13. Average the number of plaques for each dilution and calculate the number of plaque-forming
units for 1 ml of the original bacteriophage suspension.
1:100
1:10 1:10 1:10 1:10 1:10

Virus
Serial dilution
–2 –3
10 10 10
–4
10
–5
10–6 10–7
Plate 1 ml
Plaques
(100,000) (10,000) (1000) 100 10 1
Titre = 1×107 Pfu/ml
Plaque assay method
Calculations
The number of plaques on each plate are counted. Plates with 25 to 250 plaques are used to
determine the number of coliphages in 1ml of original enriched sample.
The number of plaque-forming units present in the original sample used is calculated by
using the following formula:
Number of plaques formed by the original specimen
Plaque forming units (PFU) =
Dilution of the original specimen used×Volume used
Sample calculation
An average of 50 plaques formed in the 1:10,000 dilution wells. Volume of diluted virus added
is 0.2 ml
number of plaques
∴Plaque count =
0.2×Dilution
50
= = 2.5×106 pfu ml
0.0001×0.2
The number of plaque-forming units present in the original sample used is 2.5×106 pfu/ml.
Virology 389
EGG INOCULATION
Introduction
Viruses can only replicate in living cells. Before cell culture was developed, fertile hens’ eggs
were used to cultivate viruses in the laboratory. The use of eggs for virus propagation was first
demonstrated by Woodruff, Goodpasture, and Burnet in 1930, and much early progress in the
field of virology was due to the use of this system. Chicken embryos continue to have certain uses
in virology. Under natural conditions, many viruses are relatively host-specific. Moreover, they
may show a marked predilection for certain tissues of the host such as nervous tissue, epithelial
tissue, etc. While a number of viruses display host-specificity and tissue affinity or ‘tropism,’ the
majority can be adapted to foreign hosts by passage.
The cells and extraembryonic membranes of the chicken embryo provide varied substrates
that allow the growth of many viruses. Because of the ability to alter their tropism and to adapt
to a new host species, many viruses become capable of growing in chicken embryo tissues and
may even attain a higher concentration than in the tissues of the natural host. Before describing
the various methods by which fertile eggs can be inoculated, it is essential to summarize the
structure, development, and physiology of the chicken embryo to understand which tissues are
most prominent at each stage of development. By analysing the target tissues growth in the
developing embryo, the inoculation period can be decided. The chicken embryo develops from
a single cell to a hatchling chick in 21 days of incubation in a humid 38°C environment.
The extraembryonic membranes of the chick embryo arise from three germinal layers: the
endoderm, mesoderm, and ectoderm. The chorion and amnion develop from the fused ectoderm
and mesoderm; the allantoic and yolk sac membranes develop from the mesoderm and endoderm.
The yolk sac is very large early in embryonic development. As the embryo grows and uses the
enclosed nutrients, the yolk sac becomes less prominent. The amniotic membranes grow rapidly
and fuse to form the amniotic sac by the fifth day. The allantois grows out as a bud from the
hindgut of the embryo and enlarges rapidly. By the 10th day the allantois becomes attached to
the outer layer of the amniotic sac and the inner layer of the chorion to form the chorioallantoic
sac, which separates the chorion from the amnion. The fused chorionic and allantoic membranes
are referred to as the chorioallantoic membrane. Because the chorioallantoic sac represents a
diverticulum of the gut, it serves as the excretory receptacle for the embryo. It contains about
5 to 10 ml of fluid with dissolved solids, the solution being clear in early stages but becoming
turbid after the 12th day due to the presence of urates. The chorioallantoic membrane is the
respiratory organ of the embryo and thus is richly supplied with blood vessels. The embryo is
surrounded by the amniotic sac and lies bathed in about 1 ml of amniotic fluid. The amniotic
fluid serves as a source of protein that is ingested during swallowing movements the embryo is
seen to make from the 9th day onward.
The egg itself has a blunt end where there is an air space or air sac. Underlining the shell is
the fibrous eggshell membrane. Unlike the other tissues in the egg, the shell membrane does not
contain live cells; consequently, it will not support virus replication. In the beginning stages of
development, the chicken embryo can be recognized with difficulty as a small dark area attached
to the very large yolk sac. After 4 to 5 days, the embryo can be readily detected by candling. The
embryo is of moderate size by the 10th day of development, after which the embryo rapidly increases
in size and feathers appear. The respiratory tract develops between the 12th and 15th day.
Routes of inoculation Routes of inoculation include the chorioallantoic sac, chorioallantoic
membrane, yolk sac, amniotic sac, intracerebral, and intravascular. Although many viruses are
now cultivated in cell culture, for some viruses no suitable cell culture system exists and egg
inoculation is the method of choice. Influenza virus vaccines are still cultivated in eggs, and
hence people with egg allergies cannot tolerate the influenza vaccines.
1. Intraamniotic inoculation The embryo lies within the amniotic sac. Virus inoculated into
this uses the embryo’s respiratory endothelium for its replication. The embryo age is usually
10–11 days. This route of inoculation has been used for the following viruses: influenza A
and B, parainfluenza 1,2,3,4 and mumps. The amount of inoculum is strictly limited by the
relatively small quantity of amniotic fluid and the yield is therefore, small in amount. This
route is hazardous to the chick embryo because it is very easy either to tear the amniotic
membrane (or) to damage the embryo. All viruses mentioned above cause haemagglutination
and therefore, the amniotic liquid is collected from inoculated eggs after 48 hours of addition
of the inoculum. The eggs are examined for plaque formation after 48–72 hours by candling.
2. Intraallantoic inoculation This is the simplest method of inoculation with a relatively
large yield. Its use is confined to Influenza A and B and the Paramyxovirus. After primary
isolation in the amniotic sac, the viruses can readily be adapted to grow intraallantoically.
HA tests are used to detect the presence and to titrate the amount of virus present. The
method, which utilizes embryos at 11–12 days of incubation, is now used mainly by those
who require large amounts of a particular Influenza virus rapidly. The eggs are examined for
plaque formation after 42–72 hours by candling.
The most convenient method of propagating Newcastle disease virus in the laboratory is by the
inoculation in the allantoic cavity of embryonated eggs. All strains of Newcastle disease virus
will grow in the cells lining the allantoic cavity. The virus enters these cells and multiplies.
As the cells are disrupted, viruses are shed into the allantoic fluid.
Virulant strains of the virus invade cells beyond the lining of the allantoic cavity and kill
the embryo. The time taken for this to occur is the basis of the “Mean Death Time Assays”,
which indicates the level of virulence.
The avirulent 1-2 strains of Newcastle disease virus will not kill embryos inoculated by the
allantoic cavity. Inoculation of the allantoic cavity of embryonated eggs is a technique used
for the following purposes:
Virology 391
1. Newcastle disease vaccine production

2. Establishing the infectivity titre of a suspension of Newcastle disease virus.
3. Isolation of Newcastle disease virus field specimens for laboratory diagnosis.
3. Yolk sac inoculation The lining membrane of the yolk sac contains the primitive
haemopoietic cells of the chick embryo. 7–9-day old embryonoted eggs are used, at which
time the haemopoietic tissue is well established. The inoculation route is through the
blunt end, under vision after removal of the shell and the shell membrane over the air sac.
This method of inoculation is used for Chlamydia sp. The eggs are examined for plaque
formation after 48–72 hours by candling.
4. Chorioallantoic membrane inoculation CAM is inoculated mainly for growing poxvirus.
Herpes simplex virus is also grown. Virus replication produces visible lesions, grey white
area in transparent CAM. Each pock is derived from a single virion. Pocks produced by
different viruses have different morphologies. Under optimal conditions each infectious
virus particle can form one pock. Pock counting, therefore can be used for the assay of
pock-forming virus such as vaccinia.
Air sac
Chorioallantoic
membrane
inoculation
Amniotic cavity
Shell Chorioallantoic
membrane
Amniotic cavity
inoculation
Allantoic cavity Allantoic cavity
inoculation
Albumin
Yolk sac
Figure 12.1 Routes of egg inoculation
Aim
To cultivate virus using egg inoculation method.
Materials required
1. 9-day old or 10-day old embryonated eggs. Candle the eggs. Mark the inoculation sites.
Eggs should be placed in egg rack with the inoculation site uppermost.
2. Egg shell punch
3. Cotton wool
4. 70% alcohol solution in water
5. Syringe 1 ml
6. Needles, preferably
7. Stationary tape, also called cello or sticky tape or melted wax to seal the inoculation site
8. Inoculum (this must be free of microbial contamination)
9. Discard tray
10. Disposable surgical gloves
Routes of inoculation of chicken embryos
Allantoic Cavity in chicken embroyos is inoculated by given in the following steps.
1. Candle the egg and select an area of the chorioallantoic membrane distant from the
embryo and amnionic cavity and free of large blood vessels about 3 mm below the base
of the air cell. In this area make a pencil mark at the point of inoculation.
2. Make a similar mark at the upper extremity of the shell over the air cell.
3. Drill a small hole through the shell at each mark but do not pierce the shell membrane.
4. Apply tincture of metaphen or another suitable disinfectant to the holes and allow to dry.
Allantoic Cavity inoculation employs embryos of 9- to 12-days incubation. The inoculum is
generally 0.1-0.2 cc. Some of the viruses which grow well in the allantoic entoderm are those of
fowl plague, Newcastle disease, infectious bronchitis, influenza, mumps, and Eastern, Western, and
Venezuelan encephalitis. This route has the advantage of simplicity of inoculation and collection
of specimens when large quantities of virus-infected fluid are to be obtained for use in chemical
analysis, vaccine production, and preparation of antigen for serologic tests.
For Amnionic Cavity inoculation, embryos from 7- to 15- days incubation,inoculum
01.-0.2 cc, may be used. The age chosen is largely determined by the virus used or the study to
be undertaken. Slow-growing viruses are benefited by the longer incubation period. The inner
epithelial lining of the amnion and the epidermal epithelium of the embryo are exposed to infection.
Swallowing and respiratory movements of older embryos further serve to bring the infectious
agent into contact with the mucous membranes of the upper respiratory and gastrointestinal
tracts. This route is particularly effective for primary isolation of influenza and mumps viruses
from throat washings.
Virology 393
Replication of a virus in embryos may be determined by several methods such as

1. Sampling of the virus in the extraembryonic fluids and membranes or in the embryo proper
for quantitative assay of infectivity,
2. Pathologic alterations,
3. Serologic tests,
4. Hemagglutination,
5. Antigenicity, and
6. Immunogenicity.
Chicken Embryo inoculation routes
Chorioallantoic SAC (CAS) Route
1. Embryos of 9 to 11 days of age.
2. Candle the embryos for viability. Mark an area on the side of the egg about 1/8 inch below
the air cell in the chorioallantoic membrane that is unoccupied by blood vessels.
3. Disinfect using Bioguard, punch a hole directly in the top of the air cell (optional).
4. With egg puncher, make a hole where you marked. (Using sterile technique.)
5. Use a 25-gauge needle, 7/8 in. length. Insert the needle at a 45 degree angle into the allantoic
cavity about 1/8 in. in depth and inoculate.
6. Use Elmer’s glue to close holes. This route of inoculation is used mainly to isolate Newcastle
disease, infectious bronchitis and adenovirus.
YOLK SAC (YS) ROUTE
2. Rotate the egg until blood vessels can be seen close to the margin of the air cell. These vessels
may appear as nothing more than an array of faint lines, orange in color, extending from a
clear halo. The embryo is within the area of the halo.
3. With an egg punch, make a hole in the top of the shell.
4. Use a 25-27 gauge, 1 1/2-in. length needle. Insert the needle straight down into the yolk
sac until its point is one-third to one-half the depth of the egg. This route is mainly used to
isolate avian encephalomyelitis.
Artificial air SAC Route
1. Embryos of 9-11 days of age.
2. Candle embryos for viability.
3. Mark an area about 1/4 inch below and parallel to the base of the air cell. Disinfect with
Bioguard.
4. Drill or punch a hole at this mark being very careful not to tear the shell membrane. Punch
a hole directly at the top of the air cell.
5. Place the embryo horizontally in the tray, with the hole facing up.
6. Holding the embryo in the same position and using a rubber bulb, draw air out of the air cell
by placing the bulb over the hole at the top of the embryo. This negative pressure creates
the artificial air cell by pulling the CAM down.
7. Using a 25-27 gauge needle, insert it into the artificial air sac about 1/8 inch and release the
inoculum. Make sure the embryo is laying horizontally for 24 hours then return to upright
position.
This route is used mainly for fowl pox and IBDV.
Chorioallantoic Membrane (CAM) Top Route
2. Candle the embryos for viability. Disinfect with Bioguard and punch a hole directly in the
top of the air cell.
3. Use a 26 or 28-gauge, 1/2 in. needle. Insert the needle straight down the top of the egg the
full length of the needle. Pull the needle back out about 1/4 in. and release the inoculum.
This procedure as well as the artificial air cell route (dropped CAM) are used mainly for
isolation of pox and laryngo tracheitis virus. Usually, the titer will not be as high as if the
dropped CAM or artificial air sac method is used.
Intravenous inoculation does not have wide practical application for study of experimental
infections of the avian embryo. The procedure is generally employed for hematologic studies.
Embryos of 10- to 15-days incubation are most suitable for this route. The amount of inoculum
may vary from 0.02 to 0.05 cc.
Intracerebral inoculation can be performed with 8 to 14-day-old embryos and inoculum of 0.01-
0.02 cc. This route may be employed in studies of pathologic alterations of the brain following
infection. The viruses of herpes simplex and rabies may be cultivated by this route.
Interpretation
Embryos are incubated after inoculation for a period appropriate for the virus employed and they
are examined at least once daily. Death of the embryo within the first 24 hours after inoculation is
generally considered to be due to nonspecific causes such as trauma. Some viruses kill all embryos
and mortality is the criterion of infection. Newcastle disease virus is an example in which embryos
are killed in two to four days depending upon the strain of the virus. With some viruses such
as influenza virus the mortality rate varies on initial passage but may increase with subsequent
passage. The criterion of infection with herpes and pox viruses is the formation of pock lesions on
the chorioallantoic membrane. Other gross pathologic manifestations of infection of the embryo
may be curling and dwarfing of the embryo, fibrosis of the amnionic membrane, edema of the
Virology 395
chorioallantoic membrane, and urates in the kidney and meso nephros such as produced by avian
corona viruses on initial and low passage in the embryo. Various types of cytologic changes,
including inclusion bodies with certain viruses, may be detected by microscopy. The embryo
should be examined soon after death so that postmortem changes do not obscure any specific
pathologic alterations. Chilling of the embryos for several hours or for overnight before collection
of extra embryonic fluids is recommended to reduce hemorrhage into the fluids.
CULTIVATION OF ANIMAL AND PLANT VIRUSES

Introduction
The viruses do not reproduce independent of living host cells, they cannot be cultured in the
same way as bacteria and eukaryotic microorganisms. However, the cultivation of viruses can be
discussed under the following headings (i) cultivation of animal viruses, (ii) cultivation of plant
viruses, and (iii) cultivation of bacteriophages.
I. CULTIVATION OF ANIMAL VIRUSES

1. Animal Inoculation
Suitable living mammals (such as sheep, calves, mice or rabbits) are selected for cultivation of viruses.
The selected animals should be healthy and free from any communicable diseases. The specific virus
is introduced into the healthy animals. The site of administration (intracerebral, subcutaneous,
intiraperitoneal or intranasal) varies according to the type of virus. At the end of the incubation
period, the animals are slaughtered and washed thoroughly and viruses are obtained from them.
Advantages of animal inoculation
1. To isolate those viruses which do not grow in cell lines/eggs.
2. To understand pathogenesis/immune response.
3. To test efficacy of vaccine/drugs.
4. Animals still in use are suckling mice, rabbits, hamsters, monkeys.
Disadvantages of animal inoculation
1. Expensive.
2. Difficult to handle.
3. Maintenance- is difficult.
4. Show biological diversity.
5. Presence of latent viruses in the animals to be inoculated.
6. Pressure from animal friends groups and human volunteers.
2. Chick Embryo

The animal viruses can be successfully cultivated using chick embryo technique. In this method,
fertile hen eggs are selected. Eggs must not be more than 12 days old. To prepare the egg for virus
cultivation, the shell surface is first disinfected with iodine and penetrated with a small sterile
drill. After inoculation, the drill hole is sealed with gelatin and the egg is then incubated. The
myxoma virus grows well on the chorioallantoic membrane, whereas the mumps virus prefers the
allantoic cavity. The infection may produce a local tissue lesion known as pock, whose appearance
often is characteristic of the virus.
Advantages of chick embryo
1. The eggs are much simpler to handle than animals.
2. The eggs are very economical and easily available.
3. They are clean and bacteriologically sterile.
4. They do not need feeding and caging.
5. They do not have an immune mechanism like animals to counteract virus infection.
6. Chick embryo offers several sites for virus cultivation.
Disadvantages of chick embryo
1. Some virus do not show growth on primary inoculation into the eggs.
2. Slight amount of bacterial contamination in the inoculum may kill the embryo.
3. Eggs may be contaminated with mycoplasma and latent fowl viruses which may interfere
with the growth of other viruses.
3. In vitro Culture (Tissue Culture Technique)
More recently developed in vitro cultivation of animal viruses has eliminated the need to kill animals.
This technique has become possible by the development of growth media for animal cells and
by the availability of antibiotics which prevent bacterial and fungal contaminations in cultures.
Cultivating animal viruses using tissue culture technique involves the following three main steps:
i. Monolayer preparation Live tissues of vital organs (e.g., heart or kidney) are taken and the cells
are separated from the tissue by digesting the intracellular cement substance with dispersing
agents such as trypsin or collagenase or ethylene diamine tetraacetic acid (EDTA). The cell
suspension is passed through screen filters so that the coarse particles are removed from the
separated cells. The cells are washed free of dispersing agents. The cells are centrifuged,
if required and resuspended in nutrient medium contained in glass or plastic vessels. The
composition of medium and other conditions of incubation depends on the type of cells
used. Upon incubation, the cells quickly settle and attach firmly to the bottom of the flask.
If undisturbed, these cells grow and spread to form monolayers.
Virology 397
ii. Clonal cell line preparation The monolayer cells are first removed and washed with saline
solution devoid of calcium and magnesium ions and then added to the dilute solution of EDTA
(1 : 3000) to chelate intracellular magnesium or calcium ions. After sometime, the loosened
cells are shaken and resuspended in growth medium in fresh culture vessels and incubated.
The cells are cultivated under 5% CO2 condition. The cultures of cells so obtained are called
diploid cell strains. It is extremely difficult to distinguish primary cell and the diploid cell
strain. On repeated subculturing, each cell starts multiplying to form separate colonies. If
each colony is removed and cultivated separately, it forms pure culture. These bunch of cells
from a single cell is called clonal cell lines.
iii. Infection with virus The clonal cell lines suspended in suitable media are infected with
any desired virus which replicates inside the multiplying cells. If the virus is virulent, they
cause lysis of cells and virus particles are released in the surrounding medium. These newly
produced virus particles (virions) infect the adjacent cells. As a result, localized areas of cellular
destruction and lysis (called plaques) are often formed. Plaques may be detected if stained
with dyes, such as neutral red or trypan blue, which can distinguish living from dead cells.
Viral growth does not always result in the lysis of cells to form a plaque. Animal viruses,
in particular, can cause microscopic or macroscopic degenerative changes or abnormalities
in host cells and in tissues called cytopathic effects, cytopathic effects may be lethal, but
plaque formation from cell lysis does not always occur.
Advantages and disadvantages of tissue culture
Tissue culture offers a number of advantages for a variety of studies and applications, but there
are also limitations, so that tissue culture should not be used indiscriminately. These advantages
and disadvantages are listed in the table.
Advantages and limitations of the use of animal tissue culture.
I. Advantages
1. Controlled physiochemical environment (pH, temperature, osmotic pressure, O2, CO2, etc.).
2. Controlled and defined physiological conditions (constitution of medium, etc.).
3. Homogeneity of cell types (achieved through serial passages).
4. Economical, since smaller quantities of reagents are needed than in vivo.
5. Legal, moral, and ethical questions of animal experimentation are avoided.
II. Disadvantages
1. Expertise is needed, so that behaviour of cells in culture can be interpreted and regulated.
2. Ten times more expensive for same quantity of animal tissue; therefore reasons for its use
should be convincing.
3. Unstable aneuploid chromosome constitution (Abnormal number of chromosome).
Types of tissue cultures

1. Organ culture Small bits of organs can be maintained in vitro for days and weeks, preserving their
original architecture and function. Formalin is used for the preservation. Organ culture is useful
for the isolation of some viruses which appear to be highly specialized parasites of certain organs.
Example: Tracheal ring organ culture is employed for the isolation of coronavirus, a respiratory
pathogen.
2. Explant culture Fragments of minced tissues can be grown as ‘explants’ embedded in plasma
clots. They may also be cultivated in suspension. This was originally called as tissue culture. This
method is now seldom employed in virology.
Example: Adenoid tissue explant culture was used for the isolation of adenovirus.
3. Cell culture The cell culture is the method routinely employed nowadays for identification
and cultivation of viruses. Cells of various types of tissues of animals may be cultivated. But more
commonly, fibroblast and muscle epithelial cells are used for the propagation of virus.
The tissue is first removed from the organism concerned. This tissue is then broken down
into its constituent cells by utilizing suitable physical means. Homogenization in a homogenizer
is common method utilized. The complete tissue is then converted into many small pieces.
The tissue fragments are washed with salt solutions (to avoid contamination). Sterile physiological
saline or other types of solution like saline or other types of solution like Hank’s solution or
Eagle’s solution are used. The pieces are converted into their constituent cells by a process called
dispersion of the cells from tissue. It is done by breaking down the proteinaceous cementing
material (i.e., Hyaluronic acid), joining the cells with the help of proteolytic enzymes like trypsin
and mechanical shaking. This step is called as trypsinization.
The washed tissue fragments are then placed in a flask with sterile trypsin solution at 4°C for
about 18 h. During this period, the tissue fragments are gradually dispersed into their cellular
components. Presence of chemicals like EDTA helps in dispersion of cells. The cells are then
centrifuged and resuspended in washing medium. It is done repeatedly. The washed suspended
cells are then cultivated in a suitable growth medium. The essential constituents of growth
medium are physiological amounts of essential amino acids, and vitamins, salts and glucose and
5% carbondioxide. This is supplemented with 5% calf or foetal calf serum. Antibiotics are added
to prevent bacterial contamination and phenol red is added as indicator. Such media will allow
most cell types to multiply with a division time of 24–48 h.
Cultivation is done after adjustment of the number of cells per unit volume. The required
number of cells is suspended in the growth medium taken in a tube or flask. The entire culture
is then incubated at 36°C for 72h. The cells in culture multiply and cover the bottom of the
glass container with a thin but continuous layer, which is often one cell thick. Such cell layers are
called as monolayer. This technique was improved by Dr. Dulbecco and his associates in the USA.
Virology 399
They found that the monolayer can be developed on agar containing necessary nutrients. Virus
particles grown on such monolayer are extremely uniform in growth. Sometimes the dispersed
cells are not allowed to settle down at the bottom of the container. Rather they are kept floating
by shaking the flasks continuously on the mechanical shaker. This type of culture is called as
suspension culture. A vigorously growing monolayer or suspension culture is then inoculated with
the types of viruses to which it is susceptible. The inoculation is done by mixing or spreading
the viral suspension with the cultivated host cells. The virus particle infects the host cells in due
course. They multiply in number within the host cell and eventually come out by destroying the
host cell. They are thus liberated into the surrounding medium and infect the neighboring cells.
The cell culture looks disintegrated. The initially formed virus particles soon lead to the production
of many more viruses. These areas appear to be completely disintegrated and take shapes of white
patches called as plaques.
Types of cell cultures On the basis of origin, chromosomal characters, and the number of generations
through which they can be maintained, cell cultures are classified into three types.
1. Primary cell culture These are normal cells obtained from fresh organs of animals and cultured.
Once the cells get attached to the vessel surface, they undergo mitosis until a confluent monolayer
of cells covers the surface. These layers are capable of limited growth in culture and cannot be
maintained in serial culture. They are commonly employed for primary isolation of viruses and
in preparation of vaccine. Primary cell cultures are generally best for viral isolation and Rhesus
monkey kidney cells cultures are widely used, which are sensitive to a wide range of viruses.
Examples are Rhesus monkey kidney cell culture, human amnion cell culture.
2. Diploid cell culture It is also called as semi-continuous cell lines. These are subsequent cultures
derived from primary cell cultures. These are cells of single type that retain the original diploid
chromosome number and karyotype during serial sub-cultivation for a limited period of time.
There is rapid growth rate and after 50 serial subcultures, they undergo senescence and the cell
strain is lost. The diploid cell strains are susceptible to a wide range of human viruses. They are
also used for isolation of some fastidious viruses and production of virus vaccines. Examples are
human embryonic lung strain (WI-38) and Rhesus embryo cell strain (HL-8)
3. Continuous cell culture These are cells of a single type, usually derived from the cancer cells
that are capable of continuous serial cultivations indefinitely. These cells grow faster and their
chromosomes are haploid. (The isolation and characterization of a near-haploid cell line which has
only a disomy of chromosome 8 from the heterogeneous human leukemia cell line KBM-7. This
cell line remains karyotypically stable for many weeks in culture and near-haploid subclones can
be repeatedly isolated from this population of cells, allowing for the continuous maintenance of
near-haploid cells in culture. These properties should make this cell line useful for somatic cell
genetics). They are also called as permanent cell lines. Permanent cell lines derived from a single
separated cell are called as clones. One common example of such clone is HeLa strain derived
from cervical cancer of lady HeLa, by name. Continuous cell lines are maintained either by serial
subculture or by storing in deep freezer at –70°C so that these can be used when necessary. Some
cell lines are now permitted to be used for vaccine manufacture.
Methods to detect virus growth in cell culture

Haemagglutination assay Many viruses attach to molecules present on the surface of RBCs.
A consequence of this is that at certain concentrations, a viral suspension may bind together
(agglutinate) the RBCs, thus preventing them from settling out of suspension. Since agglutination
is rarely linked to infectivity, attenuated viruses can therefore be used in assays.
By serially diluting a virus suspension into an assay tray (a series of wells of uniform volume)
and adding a standard amount of blood cells, an estimation of the number of virus particles
can be made. While less accurate than a plaque assay, it is cheaper and quicker (taking just
30 minutes).
This assay may be modified to include the addition of an antiserum. By using a standard amount
of virus, a standard amount of blood cells, and serially diluting the antiserum, one can identify
the concentration of the antiserum (the greatest dilution which inhibits haemagglutination).
Detecting viruses by the plaque assay One of the most important procedures in virology
is measuring the virus titre — the concentration of viruses in a sample. A widely used approach for
determining the quantity of infectious virus is the plaque assay. This technique was first developed
to calculate the titres of bacteriophage stocks. Renato Dulbecco modified this procedure in 1952
for use in animal virology, and it has since been used for reliable determination of the titres of
many different viruses.
To perform a plaque assay, 10-fold dilutions of a virus stock are prepared, and 0.1 ml aliquots
are inoculated onto susceptible cell monolayers. After an incubation period, to allow virus to
attach to cells, the monolayers are covered with a nutrient medium containing a substance, usually
agar, that causes the formation of a gel. When the plates are incubated, the original infected
cells release viral progeny. The spread of the new viruses is restricted to neighbouring cells by
the gel. Consequently, each infectious particle produces a circular zone of infected cells called a
plaque. Eventually the plaque becomes large enough to be visible to the naked eye. Dyes that
stain living cells are often used to enhance the contrast between the living cells and the plaques.
Only viruses that cause visible damage of cells can be assayed in this way. An example of plaques
formed by poliovirus on a monolayer of HeLa cells is shown in the figure. In this image, the cells
have been stained with crystal violet, and the plaques are readily visible where the cells have
been destroyed by viral infection.
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Pvwt PV-3A-Flag-Y
PV-3A-HA PV-3A-myc
The titre of a virus stock can be calculated in plaque-forming units (PFU) per millilitre.
To determine the virus titre, the plaques are counted. To minimize error, only plates containing
between 10 and 100 plaques are counted, depending on the size of the cell culture plate that is
used. Statistical principles dictate that when 100 plaques are counted, the sample titre will vary
by plus or minus 10%. Each dilution is plated in duplicate to enhance accuracy. In the example
shown below, there are 17 plaques on the plate made from the 10–6 dilution. The titre of the
virus stock is therefore 1.7 x 108 PFU/ml.
0.1 ml 0.1 ml
0.9 ml
Virus stock
10
–1
10
–2
10–3 10–4 10
–5
10–6 10–7
0.1 ml 0.1 ml 0.1 ml
The plaque assay
II. CULTIVATION OF PLANT VIRUSES

There are several methods of cultivation of viruses, such as plant tissue cultures, cultures of
separated cells, cultures of protoplasts, etc.Viruses also can be grown in whole plants. Leaves
are mechanically inoculated by rubbing with a mixture of viruses and an abrasive, such as
carborundum. When the cell wall is broken by the abrasive, the viruses directly come in contact
with the plasma membrane and infect the exposed host cells. A localized necrotic lesion often
develops due to the rapid death of cells in the infected area. Even when lesions do not arise, the
infected plant may show symptoms, such as change in pigmentation or leaf shape. Some plant
viruses can be transmitted only if a diseased part is grafted onto a healthy plant.
Purification (or Isolation) of Plant Viruses

Purification or, as it is usually called, isolation of viruses, is necessary to know about their structure
and other properties. By employing the method of purification, a virus is finally obtained in its
pure form as a colourless pellet in a test tube and may be used for various purposes. Following
are the steps involved in virus purification (isolation):
1. Infected leaves are thoroughly homogenized in water or preferably in phosphate, borate or
citrate buffer in an electric grinder or in a mortar with pestle.
2. Tissue homogenate is strained through a piece of muslin cloth (or cheese cloth). Crude sap,
which comes out and contains virus, is collected and then poured into a centrifuge tube.
The tube is spun at low-speed (3000–17000 g). As a result, the crude sap differentiates into
supernatant and pellet. The pellet is discarded and the supernatant with virus is collected.
3. The supernatant with virus is poured into a centrifuge tube. The tube is placed in fixed-
angle rotor of ultracentrifuge and spun at high speed (40000–150000 g). In the tube, the
virus sediments and forms a tiny pellet at the bottom of the tube and a supernatant over it.
Supernatant is discarded and the pellet of virus is mixed with a buffer and stirred with rod
so that it resuspends in buffer.
4. Low- and high-speed centrifugation steps are repeated 2–3 times and the virus is purified
by density-gradient centrifugation, the most frequently used technique. A tier of layer of
sucrose solutions of different concentrations (e.g., 10–40%), and hence, densities is formed
in the centrifuge tube: the layer at the bottom being the most dense and the one at the top,
the least dense, with layers of intermediate concentrations. Virus suspension is placed at the
top of the top-most layer of the centrifuge tube, and centrifuged in swimming-bucket rotors
at high-speed ultracentrifuge.
5. When settled, virus particles move together as a band in the gradient solution of sucrose.
The virus band is collected as separate fraction through puncture at the bottom of the
centrifuge tube. The virus fraction is placed in cellulose dialysis tubing and sucrose is removed
by dialysis in buffer solution or water. Thus, the virus is obtained in pure form.
Virology 403
Tissue homogenate
Leaf debris
Electric grinder Muslin cloth
Crude sap with virus
(a)
Pellet discarded
Pellet Supernatent
with virus
Supernatant with
Crude sap poured Low-speed centrifuge virus is collected
into centrifuge tube
(b)
Supernatant discarded Stirring rod
Supernatant poured in High-speed centrifuge Pellet virus Virus in pellet is

centrifuge tube resuspended in buffer
(c)
Virus suspension at the

top of sucrose gradient
Gradient 10%
sucrose
solution in 20%
centrifuge 30%
tube 40%
Ultracentrifuge
(d)
Buffer solution or water
Virus band
Pure virus
Dialysis to remove
Sucrose solution remaining sucrose
Removal of sucrose solution

to obtain the band with virus
(e)
Figure 12.2 Plant virus purification
The concept of purity of viruses is an optional one because the virus preparation
obtained after purification is, however, rarely absolutely pure as it usually contains some
impurities. For practical purposes, a virus preparation is considered to be pure if its properties
(e.g., amino acid composition, nucleotide composition, percentage of protein, sedimentation
profile, etc.) do not change upon further purification. However, the purification of a virus is always
done with some particular experimental work in mind so that the degree of purity is tested with
reference to that work.
Virology 405
Result
Sucrose gradient centrifugation allows concentration of particles from a sample. The sucrose
cushion method causes no mechanical stress and allows the collection of morphologically intact
particles. The dialysis process removes remaining sucrose, thus pure virus is obtained.
APPENDIX I
PREPARATION OF REAGENTS
A. BIOCHEMICAL TEST REAGENTS

1. Kovac’s reagent (for detection of indole)
p-dimethylaminobenzaldehyde 5.0 g
Amyl alcohol 75.0 ml
Hydrochloric acid (concentrated) 25.0 ml
Distilled water 1000 ml
Note Dissolve the p-dimethylaminobenzaldehyde in the amyl alcohol. Add the hydrochloric
acid.
2. Methyl red solution (for detection of acid)
Methyl red 0.1 g
Ethyl alcohol 300.0 ml
Distilled water 200.0 ml
Note Dissolve the methyl red in 95% ethyl alcohol. Dilute to 500 ml with distilled water.
3. Barritt’s reagent (for detection of acetylmethyl-carbinol)
Solution A
Alpha-naphthol 5.0 g
Absolute ethanol 95.0 ml
Note Dissolve the alpha-naphthol in the ethanol with constant stirring.
Solution B
Potassium hydroxide 40.0 g
Creatinine 0.3 g
Note Dissolve the potassium hydroxide in 75 ml of distilled water. The solution will become
warm. Allow to cool to room temperature. Add the creatinine and stir to dissolve. Add the
remaining water. Store in a refrigerator.
408 Appendix
4. 3% Hydrogen peroxide (for detection of catalase activity)

Preparation of H2O2 3ml of H2O in 97 ml of sterlie distilled water.
Note Refrigerate when not in use.
5. Gram’s iodine (for detection of starch)
As in Gram’s stain.
6. p-aminodimethylaniline oxalate (for detection of oxidase activity)
p-aminodimethylaniline oxalate 0.5 g
Note To dissolve fully, gently warm the solution.
7. Nessler’s reagent (for detection of ammonia)
Potassium iodide 50.0 g
Distilled water (ammonia-free) 35.0 ml
Note Add saturated aqueous solution of mercuric chloride until a slight precipitate persists.
8. Potassium hydroxide (50% aqueous) 400.0 ml
Note Dilute to 1000 ml with ammonia-free distilled water. Let it stand for 1 week, decant
supernatant liquid and store in a tightly capped amber bottle.
9. Trommsdorf’s reagent (for the detection of nitrite)
Slowly add 100 ml of 20% aqueous zinc chloride solution to a mixture of 4 g of starch in
water. Heat until the starch is dissolved as much as possible and the solution is almost clear.
Dilute with water and add 2 g of potassium iodide. Dilute to 1000 ml, filter, and store in an
amber bottle.
10. Nitrate test solutions (for nitrate reduction test)
Solution A
Sulphanilic acid
Dissolve 8.0 g of sulphanilic acid in 1000 ml of 5N acetic acid.
Solution B
Alpha-naphthylamine
Dissolve 5.0 g of alpha-naphthylamine in 1000 ml of 5N acetic acid.
11. Anthrone reagent
Dissolve 200 mg of anthrone in 100 ml of ice-cold 95% sulphuric acid. Prepare fresh before
use.
Appendix 409
12. Schiff’s reagent

Dissolve 0.05g of pure fuchsin (4 – rosaline hydrochloride) in 50ml of distilled water.
Add 2 ml of saturated sodium bisulfide solution. After allowing the solution to sit for
1 h, add 1 ml of concentrated HCl. Allow to stand overnight.
B. STAINING REAGENTS
I. GRAM STAINING

1. Crystal violet (Hucker’s)
Solution A
Crystal violet (90% dye content) 2.0 g
Ethyl alcohol (95%) 20.0 ml
Solution B
Ammonium oxalate 0.8 g

Note Mix solutions A and B.
2. Gram’s iodine
Iodine 1.0 g
Potassium iodide 2.0 g
3. Ethyl alcohol (95%)
4. Safranin
Safranin O 0.25 ml
II. NEGATIVE STAINING
1. Nigrosin
Nigrosin, water-soluble 10.0 g
410 Appendix
Note Immerse in boiling water bath for 30 minutes.

Formalin 0.5 ml
Note Filter twice through double filter paper.
III. ACID-FAST STAINING

1. Carbol fuchsin (Ziehl’s)
Solution A
Basic fuchsin (90% dye content) 0.3 g
Solution B
Phenol 5.0 g
Note Mix solutions A and B. Add 2 drops of Triton-X per 100 ml of stain for use in heatless
method.
2. Acid-Alcohol
Hydrochloric acid 3.0 ml
3. Methylene blue
Methylene blue 0.3 g
IV. CAPSULE STAINING
1. Crystal violet (1%)
Crystal violet (85% dye content) 1.0 g
2. Copper sulphate solution (20%)
Copper sulphate (CuSO4.5H2O) 20.0 g
V. GIEMSA STAIN
Giemsa powder 0.5 g
Methyl alcohol, absolute, acetate-free 33.0 ml
Note Mix thoroughly, allow to sediment, and store at room temperature.
Appendix 411
VI. SPORE STAINING

1. Malachite green
Malachite green 5.0 g
2. Safranin
As in Gram’s stain.
VII. FLAGELLA STAINING
Leifson’s Flagellar stain
Solution A
Dissolve 0.9 g of p-rosaniline acetate and 0.3 g of p-rosaniline hydrochloride in 100 ml of
95% ethyl alcohol. Let it stand overnight at room temperature for complete dissolution.
Solution B
Dissolve 3 g of tannic acid in 100 ml of distilled water.
Solution C
Dissolve 15 g of sodium chloride in 100 ml of distilled water.
Mix equal volumes of solutions A, B and C and allow it to stand for 2 hours. Store in a
stoppered bottle in the refrigerator. Discard the precipitate that forms at the bottom
of the bottle. Do not filter. The stain can be stored indefinitely. If frozen, frozen stain
solution should be thoroughly mixed after thawing, because the water separates from
the alcohol. After mixing, the precipitate should be allowed to settle to the bottom.
VIII. FUNGAL STAINS
1. Lactophenol cotton blue solution
Lactic acid 20.0 ml
Phenol 20.0 g
Glycerol 40.0 ml
Aniline blue 0.05 g
Note Heat gently in hot water to dissolve, and then add aniline blue dye.
2. KOH mount
10% potassium hydroxide is used.
3. Eosin stain
Eosin Y is typically used in concentrations of 1 to 5% weight by volume, dissolved in water
or ethanol.
412 Appendix
IX. LEISHMAN’S STAIN

It is a readymade double stain, used for staining blood films. It gives blue colour to the nucleus
and pink colour to the cytoplasm.
Composition:
Leishman stain powder 15.0 g
Ethyl alcohol (solvent) 100 ml
Note For good results, this stain is kept in dark-coloured bottle.
X. GRANULE STAINING

Ponder’s stain
Toluidine Blue 0.02 g
Glacial acetic acid 1.0 ml
Absolute alcohol 2.0 ml
Grind toluidine blue in alcohol, mix with water and then add glacial acetic acid. Filter through
a filter paper.
Albert’s stain
Toluidine blue 0.15 g
Alcohol (95%) 2.0 ml
Grind and dissolve the dyes in alcohol; add water and then finally acetic acid. Let stand for
24 h and filter through a filter paper.
MACFARLAND’S STANDARD
The turbidity of the standard is equivalent to overnight broth culture.
1. Prepare 1% sulphuric acid solution.
2. Prepare 1.175% solution of barium chloride by dissolving 2.35 g of dehydrated barium
chloride (BaCl2.2H2O) in 200 ml of distilled water.
3. To make the turbidity standard, add 0.5 ml of barium chloride solution to 99.5 ml of
sulphuric acid solution and mix.
The standard can be stored in dark at room temperature for up to 6 months.
Appendix 413
APPENDIX II
REAGENTS USED IN MOLECULAR BIOLOGY
A. ISOLATION OF CHROMOSOMAL DNA

1. STE I
0.3 M sodium chloride 17.5 g
1M Tris 121.14 g
0.5M EDTA 146.1 g
pH 8.0
2. STE II
1mM EDTA 0.1 g
100 mM Sodium chloride 10 g
10 mM Tris 1g
pH 8.0
3. Phosphate Buffer Solution
Na2HPO4 (anhydrous) 10.9 g
NaH2PO4 (anhydrous) 3.2 g
NaCl 90 g
pH 7–11
4. STET buffer
Sucrose 8%
Triton-X 100 5%
Tris–HCl 50 mM
EDTA 50 mM
pH 8.0
Filter sterilize and store it at 4°C
5. TE buffer
10 mM Tris 1.57 g
1 mM EDTA 2.95 g
414 Appendix

pH 7.5
6. TAE buffer
Tris–base 24.2 g
0.5 M EDTA 100 ml
pH 1000 ml
Distilled water 8.0
7. Supaquick buffer
1 M Tris
5 M Sodium chloride
0.5 M EDTA (pH 8)
10% SDS
H2O 1000 ml
8. TBE buffer
53 g of Tris base
27.5 g of boric acid
20 ml of 0.5 M EDTA (pH 8.0)
TBE can be diluted to 0.5 X prior to use in electrophoresis, 1 X is acceptable as well.
9. Ethidium bromide solution
Stock 10 mg/ml
Note Dissolve 0.2 g in 20 ml of distilled water, mix well and store at 4ºC.
10. Sample buffer/ loading buffer (10X )
Sucrose 40.0%
Bromophenol blue 0.25%
B. ISOLATION OF PLASMID DNA

1. Solution I
50 mM glucose
25 mM Tris (pH 8.0)
10 mM EDTA (pH 8.0)
2. Solution II
SDS
Appendix 415
0.2N NaOH
3. Solution III
5 M sodium acetate
Glacial acetic acid
4. Loading dye
Xylene xylanol 0.25%
Glycerol in water 3.0%
C. REAGENTS FOR SDS–PAGE

1. Stock acrylamide solution
Acrylamide 30.0% (pH 6.8)
Bisacrylamide 0.8%
(Stored at 4ºC in dark)
Note Make up to 100 ml with double-distilled water and filter (it is light-sensitive, store it in
an amber-coloured bottle)
Tris–HCl 1.5 M
Note Adjust the pH with 2N HCl (Take 4.16 ml and make to 25 ml).
2. Separating gel buffer 4X (pH 8.8)
Tris–HCl 0.5 M
Note Dissolve 3.02 g, adjust pH with HCl and make it up to 50 ml and store at 4ºC.
3. Stacking gel buffer 4X (pH 6.8)
4. Polymerizing agent
Ammonium persulphate 10%
TEMED (it is added for activation of polymerization)
SDS 10%
Note Dissolve 5 g in 50 ml and keep it at 40ºC for half a day. Store at room temperature.
Tris–HCl 0.25 M
Glycine 1.92 M
SDS 10%
Water 500 ml
5. Electrode buffer 5X (pH 8.2–8.4)
416 Appendix
6. Sample buffer (4X)

Tris–HCl (pH 6.8) 0.5 M
SDS 10%
Glycerol 40%
2-mercaptoethanol 20%
Note Store frozen in small aliquots and dilute to 1X concentration just before use.
7. Destaining solution
Methanol (40%) 40 ml
Acetic acid (10%) 10 ml
8. Staining solution
Coomassie brilliant blue R 250 (1%): 100 mg is dissolved in destaining solution (first the
dye is dissolved in methanol. Fresh preparation is used every time).
Note Acrylamide, bisacrylamide, buffer and water can be prepared in large batches and either stored
at 4ºC for one month or frozen in aliquots and used indefinitely. Remove the required amounts,
warm to room temperature, and add ammonium per sulphate and TEMED before use only.
9. Separating gel preparation (for 30 ml)
12.5 % gel
Acrylamide stock 12.5 ml
Separating gel buffer 6.0 ml
SDS (10%) 0.30 ml
APS (5%) 0.15 ml
TEMED 0.03 ml
10% gel
Separating gel buffer 6.0 ml
SDS (10%) 0.30 ml
APS (5%) 0.15 ml
TEMED 0.03 ml
The stacking gel is cast over the separating gel using 4% stacking gel mixture prepared as
follows for 10 ml:
Appendix 417
10. Stacking gel preparation

Stacking gel buffer 1.38 ml
SDS(10%) 100 µl
APS(5%) 100 µl
TEMED 10 µl
APPENDIX III
MEDIA COMPOSITION
1. Alkaline Peptone Water
Peptone 10.0 g
pH 8.6
Note Tryptone water (tryptone 10.0 g, sodium chloride 5 g, distilled water 1000 ml) can also
be used.
2. Ammonium Sulphate Broth
Ammonium sulphate 2.0 g
Magnesium sulphate.7H2O 0.5 g
Ferric sulphate.7H2O
Magnesium carbonate 10.0 g
Dipotassium
hydrogen phosphate 1.0 g
pH 7.3
3. Bacteriophage Broth (10X)
Peptone 100.0 g
Beef extract 30.0 g
Yeast extract 50.0 g
Potassium
418 Appendix
dihydrogen phosphate 80.0 g

pH 7.6
4. Bile esculin agar
Ingredients per litre of deionized water
Dipeptone 10.0 g
Pancreatic digest of casein 6.0 g
Beef–heart infusion 2.0 g
Yeast extract 2.0 g
Esculin 1.0 g
Ferric citrate 0.5 g
Vitamin K 10.0 mg
Hemin 5.0 mg
Agar 15.0 g
Final pH 7.0 ± 0.2 at 25oC.
5. Blood Agar (for optochin sensitivity test)
Nutrient agar 1000 ml
Note After sterilization, add 50 ml of defibrinated sheep blood.
6. Blood Agar Base
Beef-heart muscle infusion 375.0 g
Tryptose or peptic
digest of animal tissue 10.0 g
Agar 15.0 g
Note Sterilize, cool to 50ºC and add 5% defibrinated sheep blood.
7. Brain–Heart Infusion Broth (BHIB)
Peptone 10.0 g
Dextrose 10.0 g
Sodium phosphate 5.0 g
Sodium citrate 1.0 g
Brain infusion broth 250 ml
Heart infusion broth 750 ml
Appendix 419
Sodium
polyanethol sulphonate 0.25 g
pH 7.4
Note Obtain ox brain and heart; remove all fat from the heart. Cut into small pieces and grind.
Add distilled water three times (v/w). Keep at 4°C overnight. From the brain, remove meninges
fully and then weigh. Add distilled water (3 times, v/w) and mash by using hand. Keep in
the cooler overnight. Next morning, boil the brain and heart separately for 30 minutes. Then
filter through cotton layer. Measure each broth separately. Mix both infusions and remaining
ingredients. Dissolve well and adjust pH of the entire solution to 7.4–7.6. Autoclave at 121°C for
15 minutes. Filter through filter paper and distribute in bottles in 50–100 ml amounts. Autoclave
once more at 115°C for 10 minutes. BHIB is used for direct inoculation of whole blood, bone
marrow and body fluids for culture.
8. Bismuth Sulphite Agar (Wilson and Blair Medium)
Polypeptone or Peptone 10.0 g
Beef extract 5.0 g
Dextrose 5.0 g
Disodium phosphate 4.0 g
Ferrous sulphate 0.3 g
Bismuth sulphite indicator 8.0 g
Brilliant green 0.025 g
Agar 20.0 g
pH 7.5 ± 0.2
9. Buffered Peptone Water
Peptone 10.0 g
Dibasic sodium potassium
phosphate 3.5 g
Monobasic potassium
phosphate 1.5 g
pH 7.2
10. Carbohydrate Fermentation Broth
Trypticase 10.0 g
420 Appendix

Phenol red 0.018 g
Desired sugar
(dextrose, lactose, sucrose, glucose) 5.0 g
pH 7.4
11. Carboxy methyl cellulose agar composition
MgSO4. 7H2O 1 g
Yeast extract 1 g
Carboxymethyl cellulose 26 g
Agar 3 g
pH 7.0
12. Chocolate Agar
Proteose peptone 20.0 g
Dextrose 0.5 g
Disodium phosphate 5.0 g
Agar 15.0 g
pH 7.2
Note Aseptically add 5% defibrinated sheep blood to the sterile molten agar. Heat at 80°C until
a chocolate colour develops.
13. Christensen’s Urea Agar
Peptone 0.1 g
Glucose 0.1 g
Mono potassium phosphate 0.2 g
Phenol red 1 ml
Agar 2 g
pH 6.8
Note Prepare the base, sterilize by autoclaving at 121°C for 15 minutes. Cool at 50°C in a water
bath and then add 5 ml of filter sterilized 40% urea solution. Mix, distribute in 2–4 ml amounts
in 12 × 100 mm test tubes. Allow the medium to solidify in a slanting position in such a way
to get half-inch butt and one-inch slant.
Appendix 421
14. Citric Acid Fermentation Medium

Sucrose medium (g/l)
Sucrose 310.0 g
NH4NO3 25.0 g
MgSO4.7H2O 2.5 g
KH2PO4 1.0 g
CuSO4 0.04 g
Methanol 4% v/w
pH 4.0
15. Corn meal agar
Formula/litre
Corn Meal, Infusion from solids 50 g
Agar 15 g
Final pH:6.0+ 0.2 at 25º C
Formula may be adjusted and/or supplemented as required to meet performance specifications.
16. Deoxycholate Citrate Agar (DCA)
Meat extract 5.0 g
Peptone 5.0 g
Lactose 10.0 g
Sodium thiosulphate 5.4 g
Sodium deoxycholate 5.0 g
Neutral red 0.02 g
Agar 15.0 g
pH 7.5
17. Egg Yolk Agar (Ingredients per litre of deionized water)
Peptone 20.0 g
Yeast Extract 5.0 g
Disodium Phosphate 5.0 g
Sodium Chloride 2.5 g
Glucose 2.0 g
422 Appendix
Reducing Agents/Peroxide Inhibitors 1.5 g

Pyruvate 0.5 g
L-Cystine 0.4 g
L-Tryptophan 0.2 g
Hemin 5.0 ml
Tween 80 1.0 ml
Vitamin K 1.0 ml
Magnesium Sulphate solution 0.2 g
Agar 20.0 g
Final pH 7.0 ± 0.2 at 25o C.
18. Emmon’s Medium
Neopeptone 10 g
Glucose 20 g
Agar 20 g
19. Endo Agar
Peptone 10.0 g
Lactose 10.0 g
Dipotassium phosphate 3.5 g
Sodium sulphite 2.5 g
Basic fuchsin 0.4 g
Agar 15.0 g
pH 7.5
20. Eosin Methylene Blue Agar (Levine)
Peptone 10.0 g
Lactose 5.0 g
Eosin Y 0.4 g
Methylene blue 0.065 g
Agar 15.0 g
pH 7.2
21. Glycerol Yeast Extract Agar supplemented with aureomycin
Glycerol 5.0 ml
Appendix 423
Yeast extract 2.0 g

Agar 15.0 g
pH 7.0
Note Aseptically add aureomycin, 10 µg per ml, to the sterile, molten and cooled agar. For plain
glycerol yeast extract agar medium, aureomycin is not added.
22. Inoculum Media for Glutamic Acid Production
Glucose 200 g
Meat extract 3.0 g
Peptone 3.0 g
pH 7.35–7.45
23. Glutamic Acid Production Media
Glucose 500 g
Urea 8.0 g
Dipotassium hydrogen phosphate 1.0 g
Magnesium sulphate 0.25 g
Manganous sulphate 0.01 g
Ferrous sulphate 0.01 g
Protein (casein peptone,
tryptic digest) 0.6 g
Calcium carbonate 13.0 g
pH 7.2
24. K–F Broth
Peptone 10.0 g
Yeast extract 10.0 g
Sodium glycerophosphate 10.0 g
Sodium carbonate 0.636 g
Maltose 20.0 g
424 Appendix
Lactose 1.0 g
Sodium azide 0.4 g
Phenol red 0.018 g
pH 4.0
25. Lactose Fermentation Broth
Beef extract 3.0 g
Peptone 5.0 g
Lactose 5.0 g
pH 6.9
26. LJ Medium (Lowenstein Jensen’s Medium)
Potato starch 30.0 g
Asparagine 3.6 g
Potassium dihydrogen phosphate 2.4 g
Magnesium citrate 0.6 g
MgSO4.7H2O 0.24 g
Homogenized whole egg 1.0l
Glycerol 12 ml
pH 6.9
27. LB medium ( Luria–Bertani medium)
Bacto tryptone 10.0 g
Yeast extract 5.0 g
pH 7.0
Add the ingredients to 950 ml of deionized water, stir for complete solubility. Adjust pH to
7.0 with 5N NaOH (~0.2 ml). Adjust the volume to 1000 ml with deionized water. Sterilize
by autoclaving for 20 minutes at 15 lb/sq.in.
28. MacConkey Agar
Bacto peptone 17.0 g
Proteose or polypeptone 3.0 g
Appendix 425
Lactose 10.0 g
Bile salts 1.5 g
Agar 15.0 g
Neutral red 0.030 g
Crystal violet 0.001 g
pH 7.1
29. Malonate broth
Approximate Formula Per litre
Ammonium Sulphate 2.0 g
Monopotassium phosphate 0.4 g
Sodium malonate 3.0 g
Bromothymol blue 25.0 mg
30. Mannitol Salt Agar
Beef extract 1.0 g
Peptone or polypeptone 10.0 g
Mannitol 10.0 g
Agar 15.0 g
Phenol red 0.025 g
pH 7.4
31. M-Endo broth
Yeast extract 1.2
Casein hydrolysate 3.7
Peptone from meat 3.7
Dryptose 7.5
Lactose 9.4
Di-potassium hydrogen phosphate 3.3
Potassium hydrogen phosphate 1.0
426 Appendix
Sodium chloride 3.7

Sodium deoxycholate 0.1
Sodium lauryl sulphate 0.05
Sodium sulshite 1.6
Pararosanilin (fuchsin) 0.8
32. M-FC broth
Typtose or biosate. 10.0 g
Proteose peptone No. 3 or polypeptone. 5.0 g
Yeast extract 3.0 g
Sodium chloride, NaCl 5.0 g
Lactose 12.5 g
Bile salts No. 3 or bile salts mixture 1.5 g
Aniline blue 0.1 g
Agar (optional) 15.0 g
Reagent-grade water 1 L
33. Milk Agar
Skim milk powder 100.0 g
Peptone 5.0 g
Agar 15.0 g
pH 7.2
34. Minimal Agar
Solution A
Disodium hydrogen phosphate 6.0 g
Ammonium chloride 2.0 g
pH 7.0
Solution B
Glucose 8.0 g
MgSO4. 7H2O 0.1 g
Agar 15.0 g
Appendix 427

pH 7.0 g
Note Autoclave solutions A and B separately and then mix.
35. Moeller Decarboxylase broth
(1) Moeller Decarboxylase Broth (Control):
Peptic Digest of Animal Tissue 5.00 g
Beef Extract 5.00
Bromocresol Purple 0.01
Dextrose 0.50
Cresol Red 5.00 mg
Pyridoxal 5.00
Final pH 6.0 ± 0.2 at 25°C
(2) Moeller Arginine Dihydrolase Broth
Same as (1) with the addition of 10.0 g of l-Arginine HCl.
(3) Moeller Lysine Decarboxylase Broth
Same as (1) with the addition of 10.0 g of l-Lysine HCl.
(4) Moeller Ornithine Decarboxylase Broth
Same as (1) with the addition of 10.0 g of l-Ornithine HCl.
36. MR–VP Broth (Methyl red–Voges Proskauer)
Peptone 7.0 g
Dextrose 5.0 g
Potassium phosphate 5.0 g
pH 6.9
37. Muller–Hinton Agar
Beef infusion 300.0 ml
Casein hydrolysate 17.5 g
Starch 1.5 g
Agar 15.0 g
pH 7.4 ± 0.2
38. Nitrate Broth
Peptone 5.0 g
428 Appendix
Beef extract 3.0 g

Potassium nitrate 5.0 g
pH 7.2
39. Nitrogen-free Mannitol Agar
Mannitol 15.0 g
Calcium sulphate 0.1 g
Agar 15.0 g
pH 7.3
Nitrogen-free mannitol medium without agar is called nitrogen-free manitol broth.
40. Nutrient Agar
Peptone 5.0 g
Beef extract 3.0 g
Agar 15.0 g
pH 6.8
41. Nutrient agar with 0.5% NaCl
Beef extract 0.3g
Yeast extract 0.3g
Nacl 0.5g
Peptone 0.5g
D.water 100 ml.
42. Nutrient agar with 5% NaCl
Beef extract extract 0.3g
Yeast extract 0.3g
Nacl 5g
Peptone 0.5g
D.water 100 ml.
Appendix 429
43. Nutrient agar with 10% NaCl

Beef extract extract 0.3g
Yeast extract 0.3g
Nacl 10g
Peptone 0.5g
D.water 100 ml.
44. Nutrient agar with 0.5% sucrose
Sucrose 0.5g
Yeast extract 0.3g
Nacl 0.5g
Peptone 0.5g
D.water 100 ml.
45. Nutrient agar with 15% sucrose
Sucrose 15g
Yeast extract 0.3g
Nacl 0.5g
Peptone 0.5g
D.water 100 ml.
46. Nutrient agar with 60% sucrose
Sucrose 60g
Yeast extract 0.3g
Nacl 0.5g
Peptone 0.5g
D.water 100 ml.
47. Nitrogen-free mannitol broth
Mannitol 15.0 g
Magnesium sulphate (MgSO4) 0.2 g
Calcium sulphate 0.1 g
pH 7.3
430 Appendix
48. Nutrient Broth

Peptone 5.0 g
Beef extract 3.0 g
pH 7.0
49. Nutrient Gelatin
Peptone 5.0 g
Beef extract 3.0 g
Gelatin 120.0 g
pH 6.8
50. OF Basal Medium
Approximate formula per litre
Pancreatic digest of casein 2.0 g
Bromothymol blue 0.08 g
Agar 2.0 g
51. Peptone Broth
Peptone 4.0 g
pH 7.2
52. Peptone Iron agar
Peptone 15.0 g
Sodium glycerophosphate 1.0 g
Ferric ammonium citrate 0.5 g
Sodium thiosulfate 0.08 g
pH 6.7 ± 0.2 at 25°C
53. Pikovskaya’s Medium
Glucose 10.0 g
Tricalcium phosphate 5.0 g
Appendix 431
Potassium chloride 0.2 g

Yeast extract 0.5 g
Manganese sulphate Trace
Ferrous sulphate Trace
Agar 15.0 g
54. PLET Agar (Polymyxin B–Lysozyme–EDTA – Thallous Acetate Agar)
PLET agar medium is the best selective medium for isolation and cultivation of Bacillus
anthracis from environmental specimens, animal products or clinical specimens; it inhibits
the growth of Bacillus cereus.
Composition:
Beef heart infusion agar 40.0 g
EDTA 0.3 g
Thallous acetate 0.04 g
Polymyxin 30,000 units
Lysozyme 0.04 g
Final pH 7.3 ± 0.2 at 25°C
Note Dissolve the first three ingredients in the water at 100°C. Autoclave at 121°C for
15 minutes. Cool to about 60°C. Then add polymyxin and lysozyme in small volumes of filter-
sterilized solutions. Pour plates and dry their surfaces. Do not re-melt the complete medium as
the heating would destroy the lysozyme.
55. Potato Dextrose Agar
Potato dextrose agar is recommended for the isolation and enumeration of yeasts and moulds
from dairy and other food products.
Composition
Ingredients gms/litre
Potato, infusion from 200
Dextrose 20
Agar 15
Final pH (at 25ºC) 5.6 ± 0.2
432 Appendix
Directions
Suspend 39 in 1000 ml distilled water. Heat to boiling to dissolve the medium completely.
Sterlize by autoclaving at 15 lbs pressure (121ºC) for 15 minutes. Mix well before dispensing.
In specific work, when pH 3.5 is required, acidify the medium with strelie 10% tartaric acid.
The amount of acid required for 100 ml of sterlie, cooled medium is approximately 1 ml. Do
not heat the medium after addition of the acid.
56. Preparation of BOD reagent
1. Manganese sulphate solution: Dissolve 3.46 g of manganese sulphate in distilled water. Filter
and dilute it to 1 litre.
2. Alkaline iodide azide solution: Dissolve separately 700 g of potassium hydroxide and 15 g of
potassium iodide in distilled water. Mix them and make the volume up to one litre. Dissolve
separately by adding 10 g of sodium azide in 40 ml of distilled water. Add the solution to
960 ml of alkaline iodide reagent.
3. Sodium thiosulphate titrant (0.025 N): Dissolve 6.250 g of sodium thiosulphate to freshly
boiled and cooled distilled water and dilute to 100 ml, add one pellet of NaOH as preservative.
4. Starch indicator: Filtered extract of boiled potato is used.
(or)
Dissolve 1 g of starch in 200 ml of hot distilled water and add few drops of toluene as
preservative.
57. Protease Production Media
Glucose 5.0 g
Peptone 7.5 g
Salt solution 50 ml
pH 9.0
Salt solution
MgSO4.7H2O 5.0 g
KH2 PO4 5.0 g
FeSO4.7H2O 0.1 g
58. Protease Inoculum Media
Glucose 2.0 g
Casein 0.5 g
Peptone 0.5 g
Yeast extract 0.5 g
Appendix 433
Salt solution 50 ml
pH 7.0
Salt solution
KH2 PO4 5.0 g
MgSO4.7H2O 5.0 g
FeSO4.7H2O 0.1 g
59. Robertson’s Cooked Meat Medium
Beef heart solid 98.0 g
Dextrose 2.0 g
pH 7.2
60. Sabouraud’s Dextrose Agar
Peptone 10.0 g
Dextrose 40.0 g
Agar 15.0 g
pH 5.6
If Sabouraud’s medium is added without agar, it is called Sabourand’s broth.
61. Salmonella–Shigella Agar (SSA)
Beef extract 50.0 g
Peptone 50.0 g
Bile salt mixture 5.0 g
Lactose 10.0 g
Neutral red 0.25 g
Brilliant green 0.03 g
Agar 20.0 g
pH 7.6
434 Appendix
62. SIM Agar/Peptone Iron Agar

Peptone 30.0 g
Beef extract 3.0 g
Ferrous ammonium sulphate 0.2 g
Agar 8.0 g
pH 7.3
63. Simmons Citrate Agar
Ammonium dihydrogen phosphate 1.0 g
Agar 20.0 g
Bromothymol blue 0.08 g
pH 6.9
64. Soft agar
Peptone 5 g
Beef extract 3 g
Agar 7.5 g
pH 7
65. Starch agar
Peptone 5.0 g
Beef extract 3.0 g
Starch (soluble) 2.0 g
Agar 15.0 g
pH 7.0
66. Starch fermentation medium.
Agar 15g
Soluble starch 10g
Appendix 435
Potassium phosphate dibasic 2g

Potassium nitrate 2g
Sodium chloride 2g
MgSO4. 7H2O 0.05g
CaCO3 0.02g
FeSO4. 7H20 0.01g
67. TCBS Agar (Thiosulfate citrate bile salt sucrose medium)
Yeast extract 5.0 g
Agar 15.0 g
pH 8.6
68. Tellurite Blood Agar
Nutrient agar 1000 ml
Potassium tellurite (1% solution) 2.0 ml
Defibrinated sheep blood 70.0ml
pH 7.8
69. Thioglycollate Broth
Casein 15.0 g
Glucose 5.0 g
Yeast extract 5.0 g
l-cystine 0.5 g
Sodium thioglycollate 0.5 g
pH 7.1 ± 0.2
70. Tributyrin Agar
Special peptone 5.0
Yeast extract 3.0
Agar 12.0
436 Appendix
Final pH 7.5 ± 0.2 at 37º C

Store prepared medial below 8ºC, protected from direct light. Store dehydrated powder, in
a dry place, in tightly-sealed containers at 2–25ºC.
Directions:
Dissolve 20 g in 1 litre distilled water. Sterilize by autoclaving at 121ºC for 15 minutes. Let it
cool to 80ºC and add 10 g neutral tributyrin (Fluka No. 91010). Mix throughly to emulsify
the tributyrin completely. Pour plates in order to maintain uniform turbidity. If the emulsion
separates, the effectiveness of the culture medium is affected.
71. TSI Agar (Triple sugar Iron agar)
Peptone 20.0 g
Lactose 10.0 g
Sucrose 10.0 g
Glucose 1.0 g
Beef extract 3.0 g
Yeast extract 3.0 g
Phenol red 0.025 g
Agar 20.0 g
pH 7.4 ± 0.2
Note Dissolve the ingredients, check the pH, dissolve the agar by boiling and check the pH again.
Distribute in 3–4 ml quantities in 12×100 mm test tubes. Autoclave at 121°C for 15 minutes
and allow it to set in such a way that about 1 inch butt and a slope is obtained.
72. Tryptose Phosphate Broth
Tryptose 20.0 g
Disodium hydrogen phosphate 2.5 g
Glucose 2.0 g
pH 7.3 ± 0.2
73. Urea Broth
Urea broth concentrate
Appendix 437
(filter-sterilized solution) 10.0 ml

Sterile distilled water 90.0 ml
pH 6.8
Note Aseptically add the urea broth concentrate to the sterilized and cooled distilled water.
Under aseptic conditions, dispense 3-ml amounts into sterile tubes.
74. Yeast extract glucose agar composition (g/litre)
Yeast extract 5
d (+) glucose 20
Chloramphenicol 0.1
Agar 14.9
pH 7
Preparation: Suspend 40 g/ litre and autoclave.
75. XLD Agar (Xylose Lysine decarboxylase agar)
Yeast agar 3.0 g
Lactose 7.5 g
l-lysine 5.0 g
Sucrose 7.5 g
Xylose 3.5 g
Sodium deoxycholate 2.5 g
Ferric ammonium citrate 0.8 g
Phenol red 0.08 g
Agar 15.0 g
pH 7.4
76. Yeast Broth
Glucose 1.0 g
Yeast extract 0.5 g
438 Appendix

pH 6.3–6.8
77. Yeast Extract Mannitol Agar (YEMA)
Mannitol 10.0 g
Yeast extract 0.4 g
Agar 15.0 g
pH 6.8
78. Yeast Nitrogen Base
Nitrogen source
Amino acids
l-histidine monohydrochloride 10.0 mg
l, d-methionine 20.0 mg
l, d-tryptophan 20.0 mg
Biotin 2.0 µg
Calcium pantothenate 400.0 µg
Folic acid 2.0 µg
Inositol 2,000.0 µg
Niacin 400.0 µg
p-aminobenzoic acid 200.0 µg
Pyridoxine hydrochloride 400.0 µg
Riboflavin 200.0 µg
Thiamine hydrochloride 400.0 µg
pH 5.4
Vitamins
Appendix 439
Compounds supplying trace elements

Boric acid 500.0 µg
Copper sulphate 40.0 µg
Potassium iodide 100.0 µg
Ferric chloride 200.0 µg
Manganese sulphate 400.0 µg
Sodium molybdate 200.0 µg
Zinc sulphate 400.0 µg
Salts
Monopotassium phosphate 1.0 g
Calcium chloride 0.1 g
REFERENCES
Ananthanarayanan. R and Jayaram Panikar.C.K, Textbook of Microbiology, 2002, 6th edition, Orient
Longman Ltd., Chennai.
Aneja, K.R., Experiments in Microbiology, Plant Pathology and Biotechnology, 2003, 4th edition, New
Age International.
Atlas,R.M. and Bartha,R., Microbial Ecology: Fundamentals and Applications, 1981, Addison-Wesley
publishing Company.
Atlas.R.M. Microbiology: Fundamentals and Applications, 1986, MacMillan Publishing company.
New York.
Brown,T.A., Gene Cloning-An Introduction, 1995, 3rd edition. Chapman and Hall, Madras.
Diagnostic Manual. CMC Hospital. Vellore.
Gerard J Tortora, Berdell R Funke, Christine L Case, Microbiology: An Introduction, 2003, Pearson
Education, Benjamin Cummings Publishers.
Gunasekaran, P., Laboratory Manual in Microbiology, 2008, New Age International Publishers,
New Delhi.
Jacquelyn G Black, Laura J Black., Microbiology- Principles and explorations., 2008, 7th edition,
John Wiley & Sons.
James Cappuccino and Natalie Sherman, Microbiology – A laboratory Manual, 2004. 6th edition.
Pearson Education, Singapore pvt.Ltd.
Jawetz E, J.L. Melnic and E.A. Adelberg, Medical Microbiology, 2001, Twenty second Edition,
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Kannan ,N., Laboratory Manual in General Microbiology, 2002, Panima publishing corporation,
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442 References
Keith Wilson and John Walker, Practical Biochemistry Principles and Techniques, 2004, 5th edition.
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Publishers, Boston.
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Sambrook,J., Fritsch,E.F. and Maniatis,T., Molecular Cloning: A Laboratory Manual, 2001, Third
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The Benjamin/Cummings Publishing Company, Inc.

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Contents i

Department of Microbiology and Biotechnology

ISBN 978-81-8094-107-8 MJP PUBLISHERS

4. Microbial Physiology 117

Mushroom Cultivation 183

Isolation of RNA 239

Identification of Fungal Pathogens 313

Fractionation and Size (Determination of Proteins Using SDS–PAGE 369

Outer membrane Storage granule

Figure 1.1 Structure of a bacterial cell

STAGES OF BACTERIAL GROWTH

Figure 1.2 Growth Curve

FACTORS AFFECTING BACTERIAL GROWTH

3. Osmotic Pressure

4. Carbon, Nitrogen and other Growth Factors

5. Gaseous Requirements

 Streptococcus intermedius has also been described as microaerophilic.

Figure 1.3 Nostoc

Figure 1.4 Oscillatoria

GENERAL PRINCIPLES OF MICROSCOPY

Total magnification = Magnification of objective lens × Magnification of ocular lens

Figure 2.1 Simple microscope

Objective lenses Arm

Stage with clips

Illuminator Stage Controls

Figure 2.2 Compound microscope

2. Dark-field Microscope

Figure 2.3 Dark-field microscope

3. Phase Microscopes

Elevated (or excavated) ring on phase plate is coated to

Objective Phase plate

Phase annulus creates a ring of oblique

Dim, indirect light deviated by passage through specimen

Now the direct light has been dimmed

Figure 2.4 Phase-contrast microscope

ii. Differential interference contrast microscope In the mid-1950s, a French optics

Figure 2.5 Differential interference contrast microscope

4. Fluorescence Microscope

Figure 2.6 Fluorescent microscope

1. Transmission Electron Microscope

2. Scanning Electron Microscope

(a) Transmission electron microscope (b) Scanning electron microscope

Figure 2.7 Electron microscope

1. Scanning Tunnelling Microscope

Control voltages for piezotube

Tunneling Distance control

Figure 2.8 Schematic diagram of scanning tunneling microscope

2. Atomic Force Microscope

USE AND CARE OF MICROSCOPES

 Bring the high-power objective into place.

 Remove the slide.

10× 4 (scanning) = 40×

10× 10 (low) = 100×

10× 40 (high) = 400×

Microscope type Distinguishing features Principal use

background; inexpensive and very easy to use.

Microscope type Distinguishing features Principal use

Steam exhaust pipe

Steam from jacket into chamber

Inner chamber Door

To drain Steam supply

Figure 2.10 Autoclave

Checking the Efficiency of Autoclave