Textbook of Practical Microbiology

1 / Running Head 1
Textbook of
Practical
Microbiology
Subhash Chandra Parija
AHUJA
2 Textbook of Practical Microbiology
THE AUTHOR
Dr Subhash Chandra Parija MBBS, MD, PhD, FAMS, FICPath, FABMS, FICAI, FISCD and FIMSA is Director-Professor & Head,
Department of Microbiology, in the Jawaharlal Institute of Postgraduate Medical Education & Research, Pondicherry. Prof Parija completed his
MBBS at SCB Medical College, Cuttack, Utkal Unioversity, Orissa in 1977. He obtained his MD (1978-81) in Medical Microbiology from the
Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh. Prof Parija did his PhD from University of Madras in the year
1987 for his work on simple diagnostic methods in amoebiasis.
Prof Parija, after completion of the MD, began his carrier at the JIPMER, Pondicherry as senior resident in the year 1981. Subsequently He
became Professor of Microbiology in the year 1991, and Director-Professor of Microbiology in the year, 2004. During his tenure at JIPMER,
Pondicherry, Prof Parija was sent on deputation by Govt of India to set up and establish the departments of Microbiology & initiate an integrated
Clinical Laboratory Services at the B P Koirala Institute of Health Sciences, Dharan, Nepal, of which he was the founder head between 1995-98.
In recognition of his excellent contribution to the growth and development of the department of Microbiology, as well for the Institute, the
B P Koirala Institute of Health Sciences, Dharan, Nepal, conferred the most prestigious BP Koirala Internal Oration Award.
Prof Parija was awarded WHO fellowships for study of DNA probes, PCR and other molecular biological methods in the study of parasitic
diseases at the University of Aberdeen, UK. Prof. Parija is member of the ICMR task force on intestinal protozoal infections. He is also the
member of the Research Advisory Board of BP Koirala Institute of Health Sciences, Dharan, Nepal; Board of MD Examination in Parasitology,
Colombo University, Sri Lanka, and the visiting Professor of the College of Medicine & Health Sciences, Sultan Quaboos University, Muscat,
Oman.
Author of three books “Text Book of Medical Parasitology”, “Stool Microscopy ” and Sputum Microscopy: a Practical Manual”; editor of a book
“Review of Parasitic Zoonoses” and two monographs “Immunizing agents for tropics: success, failure and some practical issues” and “Kala-azar:
epidemiology, diagnosis and control in Nepal”; Prof Parija also has contributed several chapters for the books, compendium of lectures and
proceedings of scientific meetings. Prof Parija has published more than 137 papers both in the National and International journals of repute. Some
of his papers are quoted in text books and serial publications.
The development of simple, economical and rapid diagnostic tests in serodiagnosis of parasitic diseases such as amoebiasis and cystic
echinococcosis is the main field of his research. Prof Parija was the first to demonstrate excretion of hydatid antigen in urine and its detection for
diagnosis of cystic echinococcosis. He Developed for the first time the carbon-immunoassay (CIA) and staphylococci adherence test (SAT) as
two simple rapid diagnostic methods using a light microscope, and Co-agglutination (Co-A) and CIEP for the detection of antigen in the serum
in the patients with amoebic liver abscess and cystic echinococcosis, and for demonstration of antigen in the hydatid fluid and also in the urine for
the diagnosis of cystic echinococcosis. All these tests can be used in the field or in less equipped laboratories in the developing countries like India.
Prof Parija was first to report the use of LPCB and KOH in the wet mount preparation of stool for detection of intestinal parasites by light
microscopy.
Prof Parija is the recipient of the most prestigious Dr BC Roy National Award 2003 of the Medical Council of India in recognition of his
immense contribution to the development of Medical Microbiology. He was awarded the coveted Dr B P Pandey Memorial Oration Award of the
Indian Society for Parasitology, and BK Aikat Oration Award of the Indian Council of Medical Research for his research in diagnosis and
epidemiology of parasitic diseases. Other awards include Dr S.R.Memorial Award 2003 of the Bombay Veterinary College Alumni Association,
Dr SC Agarwal Oration Award 2001 of the Indian Association of Medical Microbiologists, Major General Saheb Singh Sokhey Award 1992 of the
Indian Council of Medical Research, Third Dr Datta Memorial Award 1999 of the Indian Association for the Advancement of Veterinary
Parasitology, IAPM (Orissa chapter) Oration Award 1993 of the Indian Association of Pathologists & Microbiologists (Orissa Chapter), Smt
Kuntidevi Malhotra Award 1990 of the Indian Association of Pathologists & Microbiologists, Dr S S Misra Memorial Award 1987 of the National
Academy of Medical Sciences, Young Scientist Award 1986 of the Indian Association of Medical Microbiologist and Best Scientific Paper Award 1987
of the JIPMER Scientific Society.
Professor Parija is the editor-in chief of the online journal Internet Journal of Parasitic Diseases, and member of the editorial editorial boards of
various journals both International (Parasitology International, BMC Infectious Diseases, BMC Clinical Pathology and Health Renaissance) and
National (Indian Journal of Medical Microbiology, Journal of Veterinary Parasitology, Journal of Parasitic Diseases and Indian Journal of Pathology &
Microbiology) .
He has been conferred with various fellowships such as FAMS, FICPath, FABMS, FICAI, FISCD and FIMSA by professional bodies. Prof
Parija has visited many countries, delivered invited talks in infectious diseases at universities, institutions conferences, seminars etc, and has
chaired scientific sessions in the international as well as national conferences. He has guided both MD and PhD students and has been examiner
for MBBS, MD and PhD of various universities of India and abroad.
Prof Parija was the former Secretary of the Indian Association of Medical Microbiologists and National Vice President of the Indian Association
of Pathologists and Microbiologists. He is currently the National Vice President of the Indian Association for Development of Veterinary Parasitolo-
gists . He is also life member and member of the executive council of many national and international scientific organizations.
1 / Running Head 1
Textbook of
Practical Microbiology
1 / Running Head 3
Textbook of
Practical Microbiology
Dr. Subhash Chandra Parija

MD, PhD, FAMS,FICAI, FABMS,
FISCD, FIMSA, FICPath
Director-Professor and Head
Department of Microbiology
Jawaharlal Institute of Postgraduate
Medical Education and Research
Pondicherry, India.
Ahuja Publishers
Bangalore, New Delhi
Textbook of Practical Microbiology
Copyright © 2006 Dr. Subhash Chandra Parija
All rights reserved. No part of the publication may be reproduced,

stored in retrieval system or transmitted by any means,
electronic, mechanical, photocopying or otherwise
without the prior writtenpermissionof the publisher.
First Edition : 2006
Printed in India
ISBN
Published by
Ahuja Publishers
1 / Running Head 5
To my mother
Late Smt. Nishamani Parija

1 / Running Head 7
Contents
Preface xi
Acknowledgments xii
UNIT I Microscope and Basic Microbiological Techniques 1
Introduction 2
1 Compound Microscope 3
2 Darkground Microscopy 7
3 Measurement of Microorganisms 9
4 Hanging drop Preparation 11
5 Isolation of Pure Cultures 14
UNIT II Bacterial Staining 19
6 Simple Staining 20
7 Gram’s Staining 23
8 Acid Fast Staining 27
9 Albert’s Staining 31
10 Capsule Staining 34
11 Spore Staining 37
12 Negative Staining 40
UNIT III Cultivation of Bacteria 43
13 Media for Routine Cultivation of Bacteria 44

14 Temperature Requirement for Growth of Bacteria 47
15 pH Requirement for Growth of Bacteria 49
16 Oxygen Requirement for Growth of Bacteria 51
17 Culture of Anaerobic Bacteria 53
18 Sterilization of Commonly Used Culture Media 56
19 Antiseptics and Disinfectants 59
UNIT IV Enzymatic and Biochemical Activities of Bacteria 61
20 Catalase Test 62
21 Oxidase Test 65
22 Coagulase Test 68
23 Urease Test 71
24 Indole Test 74
25 Methyl Red Test 76
26 Voges-Proskauer Test 78
27 Citrate Utilization Test 80
28 Triple Sugar Iron Agar (TSI) Test 82
29 Hydrogen Sulphide Test 85
30 Nitrate Reduction Test 88
UNIT V Antimicrobial Sensitivity Tests 91
31 Kirby-Bauer Method 92
32 Stoke’s Method 95
33 Agar Dilution Method 97
34 Broth Dilution Method 100
35 Epsilometer Test (E-test) 102
UNIT VI Immunology 105
Introduction 106
36 Bacterial Agglutination Test 108
37 Blood Grouping 110
38 Latex Agglutination Test 112
39 Co-agglutination Test 114
40 Widal Test 116
41 Weil Felix Test 119
42 Anti-Streptolysin O (ASLO) Test 121
43 VDRL Test 123
44 Radial Immunodiffusion Test 126
45 Immunoelectrophoresis Test 128
46 Counter-current Immunoelectrophoresis Test 130
47 Indirect Haemagglutination Test 132
48 Immunofluorescence Test 135
49 Enzyme-linked Immunosorbent Assay 138
UNIT VII Microbial Genetics and Molecular Techniques 143
Introduction 144
50 Isolation of Plasmids 145
51 Polyacrylamide Gel Electrophoresis 148
52 Isolation of Antibiotic Resistant Mutant 152
53 Bacterial Conjugation 155
Unit VIII Bacteriology 159
54 Normal Microbial Flora of the Mouth 160

55 Normal Microbial Flora of the Throat 162
56 Normal Microbial Flora of the Skin 164
57 Identification of Staphylococcus aureus 166
58 Identification of Streptococcus pneumoniae 169
59 Identification of b-haemolytic streptococci 172
60 Identification of Corynebacterium diphtheriae 175
61 Identification of Lactose Fermenting Enterobacteriacae 178
62 Identification of Vibrio cholerae 181
63 Identification of Pseudomonas aeruginosa 184
1 / Running Head 9
Unit IX Parasitology 187
Introduction 188
64 Saline Wet Mount of Stool 189
65 Iodine Wet Mount of Stool 192
66 LPCB Wet Mount of Stool 195
67 Acid-fast Staining of Stool Smears 198
68 Leishman’s staining of Peripheral Blood Smears 201
69 Concentration of Stool for Parasites 205
70 Culture of Stool for Entamoeba histolytica 208
Unit X Mycology 211
Introduction 212
71 Cultivation of Fungi 213
72 Gram’s Staining for Fungi 215
73 Lactophenol Cotton Blue (LPCB) Wet Mount of Fungi 217
74 Potassium Hydroxide Wet Mount of Fungi 219
75 Indian Ink Wet Mount Preparation 221
76 Slide Culture 223
77 Germ Tube Test 225
78 Urease Test 227
79 Carbohydrate Assimilation Test 229
80 Carbohydrate Fermentation Test 231
81 Identification of Common Fungi 233
Unit XI Virology 239
82 Cultivation of Viruses in the Cell lines 240

83 Cultivation of Viruses in Embryonated Egg 243
Unit XII Microbiology of Water, Milk and Air 247
84 Microbiology of Water 248

85 Microbiology of Milk 252
86 Microbiology of Air 254
Unit XIII Animal Experiments 257
87 Intravenous Inoculation into Mice Tail Vein 258

88 Collection of Blood from the Marginal Ear Vein of Rabbit 261
89 Animals and their uses in the Laboratory 263
Unit XIV Medical Entomology 267
90 Identification of Common Insects 268
Unit XV Common Viva Spots 273
91 Identification of Common Viva Spots 274
Index 295
1 / Running Head 11
Preface
Textbook of Practical Microbiology, is a performance–based text designed for use by students of medicine,
microbiology, medical laboratory technology, allied sciences; by laboratory workers and by others who are interested
in study of practical microbiology.
The intent of the book is to provide recent information and explain in detail the routine diagnostic methods performed
in a Microbiology laboratory. Every effort has been made to incorporate all aspects of practical microbiology. A
sincere effort is made to provide the essential underlying principles of practical microbiology, to help students to
perform various practicals, and to learn and apply the knowledge of practical microbiology in clinical medicine.
Textbook of Practical Microbiology consists of 15 learning units. Each unit contains many practical exercises.
Each exercise contains learning objectives, theoretical aspects of the practical, principle of the test, and experimental
procedure in detail. Important points of the practical experiment are highlighted, possible questions with answers are
provided and finally, useful additional informations are provided as box items. The book is profusely illustrated with
diagrams, and photomicrographs both black and white, and colour.
I owe a special debt of profound gratitude to my mother late Smt. Nishamani Parija and father Shri Mana Govinda
Parija without whose encouragement the book would not have been possible.
I welcome readers views and suggestions for further improvement of the book in future editions.

email: parijasc@vsnl.com
Acknowledgements
I gratefully acknowledge all my colleagues and friends for their valuable advice and assistance in preparation of the
manuscript. It is my pleasure to thank my niece Er(Ms) Kukumina Parija, son-in-law Er. Subhasis Ray, nephew Er.
Rajkumar Parija, daughter-in-law Mrs Smriti Parija, and daughters Dr. Madhuri Parija and Miss Mayuri Parija for
their untiring secretarial help towards the preparation of the manuscript.
I am very much thankful to Ahuja Publishers, New Delhi who have been very supportive of this venture.

Textbook of Practical Microbiology 1
UNIT
I
Microscope and Basic
Microbiological Techniques
Introduction
Lesson 1 Compound Microscope
Lesson 2 Darkground Microscopy
Lesson 3 Measurement of Microorganisms
Lesson 4 Hanging Drop Preparation
Lesson 5 Isolation of Pure Cultures
2
UNIT
Introduction
Microscope is the instrument, the most important characteristic of microbiology laboratories. The
magnification, it provides, enables us to see microorganisms and their structures otherwise invisible
to the naked eye. The magnitudes attainable by microscopes range 100X- 400000X. A microscope
may be defined as an optical instrument, consisting of a lens or a combination of lenses, for making
enlarged or magnified images of minute objects. (Micro: small; scope: to view).
Antony Von Leeuwenhoek is considered to be the first person who has seen a micro organism
through a simple microscope made by him with a magnification of 270-480 times. He described the
size, shape, movements of bacteria, protozoa and algae. These findings were later confirmed after
the development of compound microscope by Robert Hooke. The characteristic morphological
studies enabled by the discovery of powerful microscopes, helped the scientists to classify
microorganisms. Later improvements in the compound microscopes were made and Amici
discovered oil immersion lens, which enabled the scientists to study the characteristics more minutely.
Microscopes are continuously improved to enable us to have higher magnifications and better
resolutions.
Microscopes are of two categories: Light or optical microscopes and Electron microscopes
depending upon the principle on which the magnification is based. Light microscopy, in which the
magnification is obtained by a system of optical lenses uses light waves .The light microscopy
includes bright field microscopy, dark-field microscopy, fluorescence microscopy and phase contrast
microscopy. On the other hand the electron microscopy uses a beam of electrons in place of light
waves for visualization of objects .This includes transmission electron microscopy and scanning
electron microscopy.
LESSON
Compound Microscope
1
LEARNING OBJECTIVES Microscope stand
After completing this practical you will be able to: It is the main framework of the microscope. It consists of :
1 Become familiar with the principle, various parts of the a Main tube.
compound microscope and its usage in microbiology b Body tube.
laboratory. c An arm, which supports the main tube, body tube and the
2 Visualize the cellular morphology from stained slide stage.
preparation by using compound microscope. d A substage, and
e A foot or base upon which the whole instrument rests.
INTRODUCTION Main tube: The main tube primarily holds the objective and
eyepiece. The eyepiece, also known as ocular piece, is present
at the top of the main tube. The eyepiece is fitted loosely into
The commonly used microscope in microbiology laboratory is the upper end of the tube. This has a standard diameter so that
called compound microscope. In compound microscopy, the all the eyepieces are interchangeable. The lower end of the
microscopic field or area observed is brightly lighted and the tube contains objectives, which are screwed into what is known
objects being studied appear dark because they absorb some as a revolving nosepiece. A number of objectives of lenses of
of the light. Ordinarily microorganisms do not absorb much different magnifications are screwed into the nosepiece of the
light but staining them with a dye greatly increases their light microscope. These objectives can be revolved to increase or
absorbing ability resulting in greater contrast and color decrease the magnification of specimen being examined.
differentiation. Generally microscopes of this type produce a
useful magnification of about 1000x to 2000x. At magnifications Body and arm: The tube is attached to the microscope by the
greater than 2000x, the image becomes hazy. These component of the microscope called the body. The body of the
microscopes are provided by a coiled filament tungsten lamp. microscope and the tube attached to it are supported at the
The glowing filaments are prevented from causing glare by correct height by firm arm, which may also provide a lifting
focusing their light on the sub-stage condenser, rather than on handle for the microscope.
the object. This is known as Köhler illumination. Closing the
aperture of the condenser slightly may aid in detection of certain Substage: The substage lies immediately below the stage. This
organisms such as protozoa, fungi, etc. because the light will holds a condenser lens with an inbuilt diaphragm and a holder
be hitting the edges of the object at a sharper angle, increasing for light filter and stops.
contrast.
Foot: The microscope rests firmly on the laboratory bench
with the base called foot. This may be U-shaped or rectangular.
Parts of the compound microscope
Stage: A fixed platform with an opening in the center allows for
A microscope mainly consists of the passage of light from an illuminating source below to the
1 Microscope stand. lens system. It provides surface for the placement of a slide
2 Stage, and over the central opening. Stage can be of fixed or mechanical.
3 Microscope optics. Mechanical stage can be moved vertically or horizontally by
4 Compound Microscope
means of rack and pinion movement. Stage also contains clips The light source
on its surface to hold the slide.
A good source of light is needed to examine specimens
Microscope optics correctly. This may be daylight or electric light. Whatever the
source of light, it should fill field of view. It should also fill the
These include objectives, eye pieces, condenser and whole of the back lens of the objective regularly with light, if
illuminating source. not the image will not be clear. While using electric light, a blue
filter is placed between the source of illumination and the
Mechanical adjustment of a microscope substage condenser. In some other microscopes, one may find
that mirrors are also provided with artificial light. In such case,
It is being carried out to focus the specimen examined by the the flat side of the mirror should be used. When daylight is the
microscope. This adjustment includes coarse and fine focusing natural source of light, the concave mirror should be used
adjustments and condenser adjustments. without the substage condenser.
Coarse and fine focusing adjustments

PRINCIPLE
The coarse adjustment is required when focusing the specimens
with low power (10x) objectives. It is carried out by rapid and Magnification
relatively large movements of the stage, which contains the
specimen. The purpose of the microscope is to produce an enlarged, well-
The fine adjustment is carried out when finer focusing is defined image of objects too small to be observed with the
required by using high power (40x) objectives or oil immersion naked eye. The degree of enlargement is the magnification or
objectives. magnifying power and it is expressed as the number of times
the length, breadth or diameter but not the area of the object is
Condenser adjustment multiplied. The limit of useful magnification is set by the
resolving power. Magnification is effected in two stages: the
Condensers are classified depending on their uses such bright first by the objective lens and the second by the eye-piece lens.
field, dark field, phases contrast, etc. There are four principal
types of condensers with respect to correction of optical The three objectives most commonly used in microbiology
aberrations, as listed in the table 1-1. laboratory are:
The Abbe condenser has two optical lens elements that (i) A low power objective with focal length 16 mm and
produce an image of the illuminated field diaphragm that is not magnification 10X.
sharp and is surrounded by blue and red color at the edges. (ii) A high power objective with focal length 4 mm and
The next level of condenser correction is split between the magnification 40X.
aplanatic and achromatic condensers that are corrected (iii) An oil immersion objective with focal length 2 mm and
exclusively for either spherical (aplanatic) or chromatic magnification 100X.
(achromatic) optical aberrations.
The total magnification of the microscope can be calculated by
The condenser adjustment system consists of: multiplying the magnifying power of the objective by that of
a Focusing: It is done by moving the condenser up to down. the eye-piece. With the most powerful lenses, including the
b Adjustment of aperture: It is done by opening or closing iris 2mm oil immersion objective, the limit of resolution is about 0.2
diaphragm. µm and the greatest useful magnification 1000x or a little less.
Principle involved in the

magnification of the object
Table 1-1 Types of condensers
Condenser type Aberrations corrected

In biconcave lens, if the object is placed between focal length
Spherical Chromatic (f) and 2f, the image is enlarged. This image is real and can be
Abbe ------ ------- projected on to a screen. If the object is placed between the f
Aplanatic X ------ and the lens then an enlarged virtual image which cannot be
Achromatic ------ X projected on to a screen is produced. In the compound
Aplanatic achromatic X X microscope, the object is placed between the f and 2f of the
objective lens. The objective produces the primary image. The
primary image is real, inverted and magnified. The eyepiece
consists of two lenses, a field lens and an eye lens; and a
diaphragm between the two lenses. The field lens of the eyepiece
brings the real image to focus at the plane of the diaphragm. This examine for higher magnification. Focus the 100X objective
is within the focal length (f) of the eye lens. Then the eye lens using the fine adjustment. The condenser may be raised
produces the virtual magnified image that is seen by the eye. completely upward for obtaining better illumination.
12 Put off illumination and carefully clean all objective lenses
Importance of numerical aperture and eyepieces with lens paper.
13 Replace the microscope in its box.
The numerical aperture of a microscope objective is a measure
of its ability to gather light and resolve fine specimen detail at
a fixed object distance. All modern microscope objectives have OBSERVATIONS
the numerical aperture value inscribed on the lens barrel, which
allows determination of the smallest specimen detail resolvable Carefully observe the morphology and colour (after staining)
by the objective and an approximate indication of the depth of of bacteria present in the smear and also for uniformity in
field (Box 1-1). staining, size, shape, Gram’s reaction arrangement.
REQUIREMENTS RESULTS AND INTERPRETATION
I Equipment The organisms appear dark against a brightly lighted background.

Compound light microscope. The stained smear shows the presence of the bacteria. Sizes of
different organisms are summarised in the box 1-1.
II Reagents
Immersion oil and lens wiping paper.
BOX 1-1 TERMINOLOGY
III Specimen
Stained smear on a glass slide.
Numerical aperture
The numerical aperture may be defined as the ratio of the diameter

PROCEDURE of the lens to its focal length. The greatest possible numerical
aperture of a dry lens cannot exceed 1.0. Actually the highest
1 Place the microscope on a firm bench so that it does not practical numerical aperture of dry and oil immersion lenses is
vibrate. It should be preferably away from direct sunlight. 0.95 and 1.5 respectively.
2 Put on the illumination when using artificial light. Use the
flat side of the mirror to reflect the light up through the Resolution
condenser when using artificial light.
The limit of useful magnification of a microscope is set by its
3 Place the slide to be examined on the stage, making sure the
resolving power.
under side of the slide is completely dry. Resolving power is the ability to reveal two closely adjacent
4 Place the low power objective (10x) in position. Begin structural details as separate and distinct, expressed quantitatively
examination of the slide with 10x objective. Then to focus as microscope's limit of resolution i.e. the minimum distance
the objective, rack the objective carefully down and using between two visible bodies at which they are seen as separate and
the coarse focusing knob and looking it from the side until not in contact with one another. The greatest resolution in light
the lens is near the slide but not touching it. Then while microscopy is obtained with the shortest wavelength of visible
looking through the eyepiece, rack the objective slowly light and an object with maximum numerical aperture. Apochromatic
upward, still with the coarse adjustment, until the image objectives represent high degree of optical perfection used only
comes into view and is sharply focused. for critical research and photomicrography due to high expense.
5 Adjust the illumination in such a way that the illumination of
Illumination
the image is optimum.
6 Focus sharply on the specimen using the fine adjustment. Effective illumination is required for efficient magnification and
7 Then focus the condenser, for better visualization of the resolving power. Since the intensity of the daylight is an
specimen. uncontrolled variable, artificial light from a tungsten lamp is the
8 Examine the specimen, moving it by the mechanical stage. most commonly used light source in microscopy. The light is
9 Place the 40x objective in position and examine for higher passed through the condenser located beneath the stage. The
magnification. Focus the 40x objective using the fine condenser contains two lenses that are necessary to produce a
adjustment. The condenser may be raised upward for maximum numerical aperture. As the magnification of the lens
obtaining better illumination. increases, the distance between the objective lens and slide, called
10 Place a drop of immersion oil on the smear. working distance, decreases, whereas the numerical aperture of the
objective lens increases.
11 Place the oil immersion (100x) objective in position and
6 Compound Microscope
KEY FACTS
1 One should remember that proper use and care increases the life of a microscope many fold.
2 Microscope should be kept away from dust, moisture and direct sunlight.
3 After finishing work a cover should be put on the microscope.
4 Care must be taken while handling different parts of microscope.
5 Examination of the slide should always begin with the low power objective (10x).
6 Attempt should never be made to repair microscope by oneself.
BOX 1-2 SIZE OF DIFFERENT ORGANISMS
Bacteria Eg: Hymenolepis nana, Taenia saginata, Taenia soluim

Sizes are measured in µm Nematodes vary in size from 5 mm to even 1 meter in length
Cocci (spherical shaped bacteria): Size vary from 0.5 µm Eg: Trichinella spiralis, Dracunculus medinesis.
to 1 µm.
Eg. Staphylococcus Fungus
aureus
Bacilli (rod shaped bacteria): Size vary from 1 µm to 10 µm Sizes are measured in µm
in length and 0.3 µm to
1µm. In breadth. Yeast like - appearance
Eg. Bacillus anthracis. 1 Histoplasma capsulatum ___________ 2.3 µm x 3.4 µm
Viruses 2 Blastomyces dermatitides ___________ 8-15 µm
3 Paracoccidioides brasiliensis _______ 2-30 µm
Sizes are measured in nm. Show variable sizes . 4 Sporothrix schenckeii _____________ 2-10 µm
Smallest virus measures 20 nm in diameter Eg. Parvovirus 5 Candida albicans _________________ 3 µm x 5 µm
Largest virus measures 300 nm in diameter .Eg. Pox virus 6 Cryptococus neoformans ___________ 4-6 µm
7 Chromoblastomycosis _____________ 4-12 µm
Parasites
Mould appearance
For protozoa sizes are measured in µm 1 Aspergillus species ______ 2-5 µm wide hyphae
Most protozoa are around 50 µm in size 2 Zygomycetes ____________ 4-5 µm wide hyphae
Balantidium coli is an exception which measures 100 µm or
more in size . Spherule like appearance
For helminths sizes are variable.
Sizes are measured ranging from mm to meters 1 Coccidioides immitis ____ 5-60 µm thick walled spherule
Cestodes vary in size from 1mm to several meters in length 2 Rhinosporidium seeberi __ 200-300 µm sporangia
VIVA
1 Classify microscopes.
2 What is Köhler illumination?
Ans Compound microscopes are provided by a coiled filament tungsten lamp. The glowing filaments are prevented from
causing glare by focusing their light on the sub-stage condenser, rather than on the object. This is known as Köhler illumination.
3 List the parts of a compound microscope.
4 Define magnification, numerical aperture and resolution.
FURTHER READINGS
1 Brooks GF, Butel JS and Morse SA. Jawetz, Melnick and Adelberg. Medical Microbiology. 23rd ed. (McGrow Hill, USA.) 2004.
2 Duddington CL. The Microscope. (Museum Press, London) 1964.
3 Evans EGV, Killington RA, Heritage J. Introductory Microbiology. (Cambridge University Press, London.) 1996.
4 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC (Eds.). Color Atlas and Textbook of Diagnostic Microbiology.
5th ed. (Lippincott Williams and Wilkins, USA.) 1997.
LESSON
Darkground Microscopy
2
LEARNING OBJECTIVES PROCEDURE
After completing this practical you will be able to: 1 Take a clean lens wiping paper and carefully clean all the
lenses.
1 Observe the motility of microorganisms by dark ground 2 Place a drop of oil on the depression present on top lens of
microscopy. the condenser.
3 Keep the wet mount preparation on the stage.
4 Raise the condenser slowly so that oil touches the bottom
INTRODUCTION of the slide.
5 Place the low power objective in position in the light path
Visibility of an object by a microscope depends upon contrast and focus.
between the object and its background. This can be improved 6 Using the centering screws adjust the light to fall on the
by dark ground microscopy than a compound microscope. center of the microscopic field. The bright spot of the light
must be in the center of the field. If a bright spot is not
obtained, slightly raise or lower the condenser to get the
PRINCIPLE bright spot.
7 Place the dry high power objective in the light path, focus
In dark-ground microscopy, the object under examination is and examine the slide.
illuminated not directly but very obliquely (Box 2-1). This is 8 Remove the high power objective from the light path. Place
done by opening the aperture of the condenser completely a drop of oil carefully over the cover slip.
and inserting a funnel stop below the condenser, hence all 9 Now place the oil immersion objective in the light path,
rays from the condenser are made to pass outside the objective. focus and observe the slide.
The rays are reflected or diffracted by the object in the specimen. 10 Observe the entire field for the motile bacteria, and also
Hence field appears dark and object is illuminated. observe the type of motility of bacteria.
11 Record the observations in the note book.
REQUIREMENTS
OBSERVATIONS
I Equipments
Compound light microscope with dark ground condenser. Brightly illuminated motile bacteria are observed under dark
background.
II Reagents
Cedar wood oil, lens wiping paper.
RESULTS AND INTERPRETATION
III Specimen
Freshly prepared wet mount preparation of bacteria suspension. The wet mount preparation shows motile bacteria.
8 Darkground Microscopy
KEY FACTS
1 Most of the dark ground condensers have fixed focus and must be use with thin slides (1 mm thick) and cover slips (0.1 mm
thick).
2 Oil must be used below and above the slide.
3 The wet mount preparation must be thin and should not be dense.
BOX 2-1 PRINCIPLE OF DARK GROUND MICROSCOPY
Dark ground microscopy involves the alteration of microscopic technique rather than the use of dyes or stains to achieve the
contrast. By the dark field method, the condenser does not allow light to pass directly through the specimen but directs the
light to hit the specimen at an oblique angle.
Only light that hits objects, such as microorganisms in the specimens will be deflected upward into the objective lens for
visualization. All other light that passes through the specimen will miss the objective, thus making the background a dark field.
VIVA
1 Describe the principle of the dark ground microscopy.

2 List the uses of dark ground microscopy for demonstrating motility.
Ans Dark ground microscopy is used commonly for very thin slender bacteria such as spirochetes, which are not visible
under ordinary illumination. The motility of spirochetes such as treponemes is clearly seen in a dark ground microscope.
Dark ground microscope also is commonly used for demonstrating motility of the trophozoites of protozoa such as Trichomonas
vaginalis, Entamoeba histolytica, etc.
FURTHER READINGS
4 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC (Eds.). Color Atlas and Textbook of Diagnostic Microbiology. 5th
ed. (Lippincott Williams and Wilkins, USA.) 1997.
LESSON
Measurement of
3 Microorganisms
LEARNING OBJECTIVES counting the number of spaces occupied by the organism and
second by multiplying this number by the calculated calibration
After completing this practical you will factor for one ocular division.
1 Measure the size of bacteria using a microscope.

2 Become familiar with the calibration of an ocular REQUIREMENTS
micrometer.
I Equipments
Compound light microscope with ocular micrometer and stage
INTRODUCTION micrometer
Measurement of size of bacteria in a microscope can be done II Reagents

by micrometry. Cedar wood oil and lens wiping paper.
III Specimen
PRINCIPLE Heat fixed smear of Escherichia coli
First the diameter of the microscopic field must be established

by means of optic devices, namely, an ocular micrometer and a PROCEDURE
stage micrometer. The ocular micrometer is placed on a circular
shelf inside the eyepiece, which contains graduations on its 1 Carefully place the ocular micrometer into the eyepiece.
surface. The distance between these graduations will vary 2 Place the stage micrometer on the microscope stage and
depending on the objective being used, which determines the center it over the illumination source.
size of the field. This distance is determined by using a stage 3 With the stage micrometer in clear focus under the low-
micrometer, which also contains graduations that are 0.01 mm power objective, slowly rotate the eyepiece to superimpose
apart. The calibration procedure for the ocular micrometer the ocular micrometer graduations over those of the stage
requires that the graduations on both micrometers be micrometer.
superimposed on each other. This is accomplished by rotating 4 Add a drop of immersion oil to the stage micrometer; bring
the ocular lens. A determination is then made of the number of the oil immersion objective into position and focus.
ocular divisions per known distance on the stage micrometer. 5 Move the mechanical stage so that a line of the stage
Then the calibration factor for one ocular division is calculated micrometer coincides with the line of the ocular micrometer
by the formula: at one end. Fix another line on the ocular micrometer that
coincides with a line on the stage micrometer.
Known distance between two 6 Determine the distance on the stage micrometer (number of
lines on stage micrometer divisions X 0.01 mm) and the corresponding number of
One division on ocular micrometer = ——————————————
divisions on ocular micrometer.
Number of divisions on
ocular micrometer 7 Determine the value of the calibration factor for the oil
immersion objective.
Then the size of microorganisms can be determined, first by 8 Remove the stage micrometer.
10 Measurement of Microorganisms
9 Calculate the number of ocular divisions occupied by each

OBSERVATIONS
of three separate bacteria and determine the average.
10 Determine the size determined by multiplying the average
1 Observe the divisions on ocular micrometer.
by calibration factor.
2 Observe the calibration of ocular micrometer of oil immersion
objective.
3 Observe the calibration factor.
4 Determine the size by multiplying the average with calibration
QUALITY CONTROL factor.
A stained smear of standard bacterial strain (e.g., Klebsiella
pneumoniae) with known size is examined for comparison.
The size of E. coli is 2.5 µm in length and 0.6 µm in breadth.
KEY FACTS
1 Carefully clean the eyepiece and objective lenses before starting experiment.
2 Expert technician help must be taken for inserting ocular micrometer.
3 There must be proper coincidence between the lines of stage micrometer and ocular micrometer.
VIVA
1 Explain the method for the measurement of the size of a microorganism by microscopy.
2 Describe the principle involved in the measurement of the size of microorganisms by microscopy.
FURTHER READINGS
4 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC (Eds.). Color Atlas and Textbook of Diagnostic Microbiology. 5th
ed. (Lippincott Williams and Wilkins, USA.) 1997.
LESSON
Hanging Drop
4 Preparation
LEARNING OBJECTIVES Spirochaetes are the examples of bacteria which are motile
but without presence of any external flagella .
After completing this practical you will be able to: The list of motile and non-motile bacteria are summarized in
the table 4-1.
1 Observe motility, and also observe natural sizes and shapes
of the cells by hanging drop preparation.
REQUIREMENTS
INTRODUCTION
I Equipments
Hanging drop preparation is one of the easiest method to Compound light microscope.
observe motility in a clinical microbiological laboratory. This is
carried out by putting a loop full of bacterial suspension on II Reagents and glass wares
the cover slip and placing it over a cavity slide and observing Normal saline, inoculating loop wire, staining tray, cavity slides
it under a microscope. Advantage of this method is that by and cover slips.
this method live bacteria can be observed. Examination of
living organisms is useful to observe cell activities, viz. III Specimen
motility, binary fission, and also to observe natural sizes and Log phase broth culture of E. coli
shapes of the cells.
Motility of bacteria can also be demonstrated by i. Craigie ’s
tube method , ii. swarming of the bacteria on a non inhibitory PROCEDURE
medium ( e.g, blood agar ) and iii. by dark ground microscopy.
Capillary tube method is a useful method for demonstrating 1 Take a clean grease free cavity slide.
the motility of anaerobic bacteria (Box 4-1). 2 Take a clean coverslip, apply paraffin to four corners of
coverslip.
3 Place a drop of broth culture on the coverslip with the help
PRINCIPLE of inoculating loop.
4 Place the cavity slide (cavity down) over the coverslip so
Microorganisms such as bacteria, because of their small size that the drop is placed in center.
and a refractive index that closely approximates that of water, 5 Invert the slide, and observe under microscope.
do not lend themselves readily to microscopic examination in a 6 First observe under low power (10x), locate the edge of the
living, unstained state. drop, shift the focus to high power (40x) and observe.
Bacteria are motile due to the presence of flagella .Depending 7 Record the observation in a notebook.
on the location of the attachment of the flagella , bacteria can
be classified as i. monotrichous (single polar flagellum at one
end) e.g, Vibrio cholerae , ii. amphitrichous (single flagellum QUALITY CONTROL
at both the ends) e.g Pseudomonas aeruginosa,
iii. lophotrichous (tufts of flagella at one or both ends ) e.g, The test wet mount preparation is compared with known motile
Spirilla, and iv.peritrichous ( fagella arranged all around the culture of P. aeruginosa for appropriate details such as motility,
cell) e.g, Escherichia coli, Salmonella sp , etc . and natural sizes and shapes of the bacteria.
12 Hanging drop Preparation
their collision with water molecules. This movement is usually

OBSERVATIONS seen around the axis of bacteria.
The bacteria showing motility are demonstrated in the hanging
drop preparation.
Note: It is important to differentiate active motility from
brownian movement. Brownian movement is not true motility,
The wet mount preparation shows motile bacteria.
instead it is exibited due to movement of organism as a result of
BOX 4-1 DEMONSTRATING MOTILITY OF ANAEROBIC BACTERIA
Capillary tube method is a useful method for demonstrating the motility of anaerobic organisms. A Robertson’s cooked meat
medium or thioglycollate broth culture, inoculated and incubated with anaerobic bacteria is taken. A broth culture of anaerobic
bacteria is prepared and is made to fill into the open capillary tube by capillary action. Once the tube is filled up, it is sealed at
both ends with clay to maintain anaerobiosis. The tube is then observed, under the high power objective of the light microscope
for the presence of motile anaerobic bacteria.
Table 4-1 Motile and non-motile bacteria
Motile bacteria Non motile bacteria
1 Other Bacillus species excluding Bacillus anthracis 1 Staphylococci

2 Clostridium tetani 2 Micrococci
3 Clostridium botulinum 3 Streptococci
4 Escherichia coli 4 Pneumococcus
5 Serratia 5 Meningococcus
6 Proteus 6 Corynebacterium diphtheriae
7 Salmonella except S.Gallinarum and S. Pullorum 7 Bacillus anthracis
8 Vibrio cholerae 8 Clostridium perfringens
9 Aeromonas 9 Lactobacillus
10 Pleisiomonas 10 Bifidobacterium
11 Campylobacter 11 Propionibacterium
12 Helicobacter 12 Bacteroides
13 Mobiluncus 13 Fusobacterium
14 Pseudomonas 14 Leptotrichia
15 Burkholderia pseudomallei 15 Klebsiella
16 Burkholderia cepacia 16 Shigella
17 Yersinia enterocolitica 17 Burkholderia mallei
18 Ligionella pneumophila 18 Yersinia spp other than Yersinia enterocolitica
19 Bordetella parapertusis 19 Pasteurella multocida
20 Bordetella bronchiseptica 20 Francisella tularensis
21 Spirochaetes 21 Haemophilus influenzae
22 Treponema 22 Moraxella
23 Borrelia 23 Gardenella
24 Leptospira 24 Bordetella pertusis
25 Listeria 25 Brucella
26 Spirillum minus 26 Mycobacterium
27 Chromobacterium violaceum 27 Mycoplasma
28 Eikenella corrodens 28 Actinomyces
29 Alcaligenes faecalis 29 Erysipelothrix
30 Streptobacillus
31 Flavobacterium
32 Calymnotobacterium
33 Cardiobacterium
34 Rickettsia
35 Chlamydiae
KEY FACTS
1 Always make a thin emulsion of bacterial suspension on the glass slide.

2 Drop should not touch the surface of cavity.
3 Observe the preparation immediately.
VIVA
1 List other methods to confirm motility of the bacteria.

2 Mention the bacterial appendage responsible for motility of the bacteria.
3 List motile and non-motile bacteria.
4 List a non-flagellated motile bacterium.
FURTHER READINGS
14
LESSON
Isolation of
5 Pure Cultures
LEARNING OBJECTIVES PRINCIPLE
Obtaining well discreet colonies and isolation of a pure culture

After completing this practical you will be able to:
initially require the number of organisms in the inoculum be
reduced. The resulting diminution of the population size
1 Know various procedures for the separation and isolation ensures that, following inoculation, individual cells will be
of the discrete colonies from a mixed culture or a clinical sufficiently far apart on the surface of the agar medium.
specimen. Streak plate method, a rapid qualitative isolation method,
2 Perform the streak plate inoculation procedure. involves spreading a loop full of culture over the surface of an
3 Perform the spread plate inoculation procedure. agar plate. Spread plate method depends on spread of the
4 Perform the pour plate inoculation procedure bacterial colonies over the surface of a solid agar medium with
a sterile, L- shaped bent gloss rod while the Petri dish is spun
on a turntable. Then the plates are incubated at 37°C for 48-72
hours. The well discrete colonies grown on the medium are
INTRODUCTION carefully transferred in to a fresh medium by any of pure culture
method. This confirms the purity of isolated colony.
The microbial population in our environment is large and Pour plate method is a rapid quantitative isolation method.
complex. Many different microbial species normally inhabit The method requires a serial dilution of the mixed culture by
various parts of our body such as oral cavity, intestinal tract, means of a pipette. This method involves pouring a specimen
skin , etc. These microorganisms may be present in extremely of molten agar (at 45°C) mixture in a Petri dish; and allowing it
large quantities. Environment, air, soil and water also consist to solidify and incubating at 37°C for 48-72 hours. Careful
of mixed populations of bacteria and other organisms. transfer of well discrete colonies in to a fresh medium by any of
Mixed culture is a culture that contains more than one kind pure culture method (generally streak plate method) confirms
of microorganisms, whereas pure culture contains only one the purity of isolated colony.
kind of microorganisms. Isolation of pure cultures is very
important in a clinical microbiology laboratory.
Most studies and tests of the physiological, immunological REQUIREMENTS
and other characters of bacteria are valid only when made on a
pure culture ( Box 5-1) . I Equipment and labwares
The preparation of pure culture involves not only the Bunsen flame, inoculating loop and marker pen (For all methods).
isolation of a given microorganism from a mixed natural Petri plates, turntable, L-shape glass rod and sterile beaker
population but also the maintenance of the isolated individual (Streak plate method).
and its progeny in an artificial environment to which the access Water bath, thermometer, test tube rack, sterile pipettes,
of other microorganisms is prevented. Obtaining well discreet mechanical pipetting device, sterile Petri dishes and
colonies and isolation of a pure culture initially require the disinfectant, (Pour plate method).
number of organisms in the inoculum be reduced. Pure culture
isolation methods include streak plate technique, spread plate II Reagents
technique and pour plate technique. Sterile trypticase soy agar plates (For all methods).
70% ethanol (Spread plate method).
Molten trypticase soy agar (Pour plate method).
III Specimen colony to a fresh agar plate with the help of sterile loop.
Mixed bacterial culture (24-48 hour nutrient broth cultures), Repeat the spread plate procedure.
exudates, stool and other clinical specimens. 11 After incubation confirm the purity of isolated colony.
For pour plate method

PROCEDURE
1 Liquify 2 agar deep tubes, each containing approximately
For streak plate method 20 ml of medium, in an autoclave or by boiling and cool the
molten agar tubes and maintain in a water bath at 45°C.
1 Take a sterile trypticase soy agar plate and label on the 2 Take 2 sterile test tubes and label as test and control.
back of the agar surface 1, 2, 3, 4 at four corners, 3 Then add mixed cultures to appropriate tube.
approximately 1 cm away from the edge of the plate. 4 Take two sterile dry Petri plates and label as test and control.
2 Take an inoculating loop and flame to deep red and cool. 5 Remove a tube from the water bath and check the
3 Charge the inoculating loop with the specimen to be temperature of molten agar medium (45°C).
cultured. 6 Wipe the outside surface dry with a tissue paper, and
4 Transfer the loop full of the specimen (control and test) on aseptically add 1 ml of test culture, mix the contents well by
to the surface of a well-dried agar plate (area 1), then spread rolling the tube between the two hands carefully and pour
over a small area at the periphery near the flame. the mixture in to test Petri plate. Allow the agar to solidify.
5 Re flame and cool the loop. 7 Take the second tube, wipe the outer surface aseptically,
6 Turn the Petri dish 90° and touch the loop to a corner of the add 1 ml of control culture, mix the contents well and pour
culture in the area 1 and drag it several times across the the mixture in to control Petri plate. Allow the agar to solidify.
agar in area 2. 8 After solidifying the agar, incubate the plates in inverted
7 Repeat the same in area 3 and area 4. position at 37°C for 48-72 hours.
Note: Flaming of loop can be decided based upon the 9 Select a well-discreet colony from each type and aseptically
turbidity of the sample. transfer to a fresh separate agar plate and repeat the streak
8 Incubate all plates in an inverted position for up to 48-72 plate method and incubate the plates at 37°C for 48-72
hours at 37°C. hours.
9 Carefully observe the colony morphology of different 10 After incubation confirm the purity of isolated organism.
colonies.
10 Transfer aseptically the single well-isolated colony on to
the surface of other agar plate. QUALITY CONTROL
11 Re flame and cool the loop and repeat the same for all colony
types. Known mixed culture containing standard strains.
12 Incubate all plates at 37°C for 48- 72 hours.
For spread plate method OBSERVATIONS
1 Take 2 trypticase soy agar plates and mark as test and control. For streak plate method
2 Take L shaped 2 glass rods, dip the lower bent portion into
70% ethanol. 1 Observe all the plates for discrete colonies after 24, 48 and
3 With a sterile pipette, transfer 0.1 ml of culture from tube 1 to 72 hours after first incubation.
the first agar plate that has been placed on the turntable. 2 Search the entire plate for colonies present outside the streak
Replace the cover. lines, if so discard the plate and repeat the experiment.
4 Remove the glass rod from ethanol and pass it through the
Bunsen burner flame, with the bent portion of the rod
pointing downward to prevent the burning alcohol from For spread plate method
running down to arm. Allow the alcohol to burn off the rod
completely. Cool the rod for 10-15 seconds. 1 Observe the control and test plates after 24, 48 and 72 hours
5 Remove the Petri dish cover and spin the turntable. after first incubation.
6 While the turntable is spinning, lightly touch the sterile bent 2 Note the results and colony morphology of different
rod to the surface of the agar and move it back and forth. colonies.
7 Remove the glass rod, dip in alcohol and re flame.
8 Stop the turntable. Replace the cover on agar plate.
For pour plate method
9 Remove the agar plate from turntable, and incubate in an
inverted position at 37°C for 48-72 hours.
1 Check the control plates for isolated similar looking colonies
10 Carefully observe the colonies and transfer a well-discreet
only.
16 Isolation of Pure Cultures
2 Note the colony characters of well- discrete colony of all For spread plate method
types of colonies (after both incubations).
3 Preserve the colonies for further characterization. 1 Check the control plates for isolated same colonies only.
2 Note the colony characters of well- discrete colony of all
types of colonies (after both incubations).
RESULTS AND INTERPRETATION 3 Preserve the colonies for further characterization.
For streak plate method For pour plate method

1 Check the control plates for isolated similar looking colonies only.
2 Note the colony characters of well- discrete colony of all 1 Check the control plates for isolated same colonies only.
types of colonies, which are present only on streak lines 2 Note the colony characters of well- discrete colony of all
(after both incubations). types of colonies (after both incubations) from the test plates.
3 Preserve the colonies for further characterization. 3 Preserve the colonies for further characterization.
VIVA
1 Define pure culture.
2 What are the uses of pure cultures in a microbiology laboratory?

Ans. In the microbiology laboratory pure culture are used to:
Isolate bacteria in pure culture.
Demonstrate their properties.
Obtain sufficient growth for preparation of antigens and for other tests.
Type the isolates.
Determine antibiotic sensitivity pattern.
Estimate viable counts.
Maintain stock culture.
3 List the selective methods used in the isolation of pure cultures.

Ans. Selective methods which help in the isolation of pure culture include:
Chemical methods
Use of a special carbon or nitrogen source
Use of dilute media
Use of inhibitory or toxic chemicals
Physical methods
Heat treatment
Incubation temperature
pH of the medium
Biological methods
Animal experiments
4 List the pure culture methods. Write their principles.

BOX 5-1 TERMINOLOGY
A culture that contains only one type of microorganisms is known as pure culture. Pure culture is also called axenic culture.
Separation of particular microorganism from a mixed population that exists in nature is called isolation.
The descendants of a single isolation in pure culture comprise a strain.
If a strain is developed from a single parent cell, it is termed as a clone.
KEY FACTS
1 Surfaces of agar plates must be dry.

2 Whole procedure must be done near the flame and inside a laminar flow inoculation chamber.
3 The loop should be properly sterilized and cooled.
4 Agar plates must be incubated at inverted position.
5 The exposure time of agar surface to the atmosphere should be minimized.
7 Loop should not touch the wall of the Petri dish any time.
8 Loop should not enter any other area except the specified in each step.
9 Contamination while transferring material from one tube to another should be avoided.
10 Occurrence of air bubbles while pouring the medium-culture mixture into plates should be avoided
FURTHER READINGS
18
UNIT
II
Bacterial Staining
Lesson 6 Simple Staining

Lesson 7 Gram’s Staining
Lesson 8 Acid Fast Staining
Lesson 9 Albert’s Staining
Lesson 10 Capsule Staining
Lesson 11 Spore Staining
Lesson 12 Negative Staining
20
LESSON
Simple Staining
6
LEARNING OBJECTIVES Preparation of polychrome methylene blue: This is prepared
by allowing Loeffler’s methylene blue to ‘ripen’ slowly and
After completing this practical you will be able to: shaking it at intervals to aerate the contents thoroughly. The
slow oxidation of the methylene blue forms a violet compound
1 Stain smears by simple staining method. that gives the stain its polychrome properties. The ripening
2 Compare various morphological forms and arrangement of takes 12 months or more to complete. The stain maybe ripened
bacterial cells. quickly by addition of 1% potassium carbonate to the stain.
This stain gives the acidic cell structures of the bacteria, a
red-purple hue.
INTRODUCTION
III Specimen
Simple staining employs staining of bacterial smears with a Pus, CSF, aspirations.
single staining reagent. The commonly used simple stains are
the basic stains such as methylene blue, crystal violet and
carbol fuchsin. They provide good colour contrast and impart PROCEDURE
the same colour to the stained bacteria (Box 6-1).
1 Make a thin exudate smear on a slide.
2 Heat fixes the smear by passing the slide 2–3 times gently
PRINCIPLE over the Bunsen flame with the smear side up.
3 Pour Loeffler’s methylene blue over the smear and allow it to
In simple staining, the bacterial smear is stained with a single stand for 3 minutes.
reagent. Basic stains with chromophore are used in simple 4 Wash the stained smear with water and air dry it.
staining methods.Bacterial nucleic acids and certain cell wall 5 Observe the smear first under low power (10x) objective, and
components carry a negative charge that strongly attracts and then under oil immersion (100x) objective.
binds to the cationic positively charged chromogen, and imparts 6 Record the observations in the note book.
same colour to all bacteria.
QUALITY CONTROL
REQUIREMENTS
The morphology of the cellular components and bacteria in the
I Equipments known methylene blue stained smear (positive control smears)
Compound light microscope. are compared with that of the blue stained structures in the test
II Reagents and glass wares smear.
Loeffler’s methylene blue, polychrome methylene blue, carbol
fuchsin, water, microscopic glass slides and Bunsen burner.
Preparation of Loeffler’s methylene blue stain: This is OBSERVATIONS
prepared by dissolving 30 ml of saturated solution of methylene
blue in alcohol, in 100 ml of distilled water containing 0.1 ml of 1 Blue coloured spherical cells, approximately 0.5–1 µm size
potassium hydroxide (0.01%). seen arranged in clusters (Fig. 6-1).
2 Blue coloured elongated rod shaped cells, approximately

3–5 µm length and 0.2–1.5 µm breadth seen.
3 Blue coloured round cells with darkly stained multilobed
nucleus in pus cells are also seen.
The stained smear contains mixture of methylene blue stained

cocci in clusters (Fig. 6-1), blue coloured bacilli and the blue
coloured multilobed pus cells.
FIGURE 6-1. Methylene blue stained smear of Staphylococcus

aureus, x 1000.
BOX 6-1 SIMPLE STAINS AND THEIR USES IN MICROBIOLOGY LABORATORY
Loeffler’s methylene blue: It demonstrates morphology of polymorphs, lymphocytes and other cells more clearly.
Polychrome methylene blue: It demonstrates the presence of bacteria in smear or wet mount preparation in addition to the
morphology of polymorphs and lymphocytes. It is also employed to demonstrate the acidic capsular material of the anthrax
bacilli as seen in the Mc Fadyean reaction.
KEY FACTS
1. Methylene blue staining is an example of simple staining.

2. Simple staining method uses a single reagent that imparts the same colour to all bacteria and cellular organelles.
3. Simple staining method shows the presence of bacteria and cellular contents in exudate smears and also in wet mount
preparations.
4. Loeffler’s methylene blue shows the characteristic morphology of polymorphs, lymphocytes and other cells more clearly
than stronger stains such as Gram’s stain.
5. Smears are required to be stained for 3 minutes and tissues for 5 minutes by simple Loeffler’s methylene blue stain.
VIVA
1 Describe differences between a simple stain and a differential stain.

Ans. Simple stains are those that contain only a single-coloured dye, and these when used impart the same colour to all the
bacteria in a stained smear. It shows the morphology of leucocytes and other cells more clearly. Methylene blue is an example
of a simple stain.
Differential stains are those stains that contain two different coloured dyes, hence they impart different colours to different
bacteria or bacterial structures, thus differentiating different groups of bacteria. But morphology of leucocytes and other cells
are stained less clearly by differential stains. Gram’s stain and acid-fast stain are two examples of differential stains.
2 Name other simple stains.
Ans. Loeffler’s methylene blue, polychrome methylene blue and dilute carbol fuchsin are the examples of other simple stains.
3 Mention the uses of methylene blue staining in a diagnostic microbiology laboratory.
Ans. Methylene blue staining is used to show
a. The presence of bacteria in a smear or wet mount preparation.
b. The nature of cellular contents in exudates, and
c. The characteristic morphology of polymorphs, lymphocytes and other cells more clearly than the Gram stain.
4 List the uses of polychrome methylene blue staining.
Ans. Polychrome methylene blue staining is employed for demonstration of the acidic capsular structure of anthrax bacillus,
as shown in the Mc Fadyean reaction.
22 Simple Staining
FURTHER READINGS
1 Evans EGV, Killington RA, Heritage J. Introductory Microbiology. (Cambridge University Press.) 1996.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5 th ed.
(Lippincott Williams and Wilkins.) 1997.
4 WHO. Guidelines on Standard Operating Procedures for Microbiology. Chapter 4: Staining Techniques.
5 WHO. Manual of Basic Techniques for a Health Laboratory, 1980.
LESSON
Gram’s Staining
7
LEARNING OBJECTIVES In the case of Gram-negative bacteria, the alcohol, being a lipid
solvent, dissolves the outer lipopolysaccharide membrane of
After completing this practical you will be able to: the cell wall and also damages the cytoplasmic membrane to
which the peptidoglycan is attached. As a result, the dye-iodine
1 Stain smears by Gram’s staining method. complex is not retained within the cell and permeates out of it
2 Differentiate between Gram positive and Gram negative during the process of decolourisation. Hence when a counter
bacteria. stain is added, they take up the colour of the stain and appear
pink.
Different modifications of Gram’s stainings are summarized
INTRODUCTION in the box7-1.
Gram’s stain was originally devised by histologist Christian

Gram (1884) as a method of staining bacteria in tissues. Gram REQUIREMENTS
positive bacteria stain purple, while Gram negative bacteria
stain pink when subjected to Gram staining. They stain I Equipments
differently because of the fundamental differences in the Compound light microscope.
structure of their cell walls. Approximately, 60%–90% of the
Gram positive bacterial cell wall is made up of peptidoglycan
and interwoven teichoic acid, while only 10%–20% of Gram II Reagents and glass wares
negative bacterial cell wall is composed of peptidoglycan, Bunsen flame and loop wire, clean grease free slides, marker
forming 2–3 layers. The Gram negative cell wall is also pen, methyl violet (basic dye), Gram’s iodine (mordant), 95%
surrounded by an outer membrane composed of phospholipids, ethanol (decolourising agent), and 1% safranine or dilute
lipopolysaccharide, lipoproteins and other proteins. carbol fuchsin (counter stain).
Preparation of methyl violet stain: This is prepared by

PRINCIPLE dissolving two solutions: solutions A and B. Solution A is
made by dissolving completely 2 grams of crystal violet in 120
The Gram reaction is dependent on permeability of the bacterial ml of ethyl alcohol. Solution B is made by dissolving 0.8 grams
cell wall and cytoplasmic membrane, to the dye-iodine complex. of ammonium oxalate in the above solution A. Then both
In Gram positive bacteria, the crystal violet dye-iodine solution A and B are mixed well.
complex combines to form a larger molecule which precipitates
within the cell. Also the alcohol/acetone mixture which act as Preparation of Gram’s iodine: This is made by first dissolving
decolourizing agent, cause dehydration of the multi-layered 20 grams of potassium iodide in 250 ml of distilled water and
peptidoglycan of the cell wall.This causes decreasing of the then 10 grams of iodine is further added to it with dissolution.
space between the molecules causing the cell wall to trap the Then 750 ml of distilled water is added and final volume is made
crystal violet iodine complex within the cell. Hence the Gram up to 1000 ml.
positive bacteria do not get decolourised and retain primary
dye appearing violet. Also, Gram positive bacteria have more Preparation of 1% safranine: This is prepared by dissolving 1
acidic protoplasm and hence bind to the basic dye more firmly. gram of safranine in 100 ml of distilled water.
24 Gram’s Staining
III Specimen Note: Decolourisation is a critical procedure. Hold the slide with
hand on one corner in a slant position and add drop by drop
Preparation of bacterial smear: (From liquid culture) of absolute alcohol till a faint colour comes out of the smear.
1 Take clean, and grease free glass slides for making the smears. 6 Rinse the smear again gently under tap water.
Note: Wipe the slide with a cotton swab dipped in alcohol 7 Cover the smear with dilute carbol fuchsin (counter stain)
and then holding its end with forceps roast it free from grease for 30 seconds to 1 minute.
by passing 6 – 12 times through a blue Bunsen flame. 8 Rinse the smear again gently under tap water and air dry it.
2 Take one or two loopfuls of the bacterial cell suspension 9 Observe the smear first under low power (10x) objective, and
and place on the clean slide with a bacteriological loop. then under oil immersion (100x) objective.
3 Then with circular movement of the loop, spread the cell 10 Record the observations in the note book.
suspension into a thin area. Note: List of Gram positive and Gram negative bacteria are
4 Allow the smear to air dry. summarized in the table 7-1.
5 Heat fix the smear while holding the slide at one end, and by
quickly passing the smear over the flame of Bunsen burner
two to three times. QUALITY CONTROL
Preparation of bacterial smear: (From solid media) On the same slide, smears of Staphylococcus aureus (Gram
1 Take clean and grease free glass slides for making the smears. positive bacteria) and Escherichia coli (Gram negative bacteria)
2 Take a loopful of 0.85% saline and place it on the centre of are made. The slide with control and test smears is stained by
the slide. Gram’s staining. The appearance of purple coloured Gram
3 With a straight wire touch the surface of a well isolated positive bacteria and pink coloured Gram negative bacteria in
colony from the solid media and emulsify in the saline drop the control smears indicate proper staining technique and
forming a thin film. stained test smear is compared with it.
4 Allow the smear to air dry.
5 Heat fix the smear while holding the slide at one end, and by OBSERVATION
two to three times. Presence of 0.5–1 µm sized purple coloured spherical bacteria
arranged in clusters ( Fig. 7-1) and 1–2 mm sized pink coloured
rod shaped bacteria seen ( Fig. 7-2).
PROCEDURE
1 Cover the smear with the methyl violet (basic dye). Allow it RESULTS AND INTERPRETATION
to stand for one minute.
2 Rinse the smear gently under tap water. The stained smear contains mixture of Gram positive cocci
3 Cover the smear with Gram’s iodine (mordant) and allow it to arranged in grape-like clusters (eg. Staphylococcus species)
stand for one minute. and Gram negative bacilli (eg. E. coli).
4 Rinse the smear again gently under tap water.
5 Decolourise the smear with 95% alcohol. Various uses of Gram’s staining are summarized in the box 7-2.
Table 7-1 List of Gram positive and Gram negative bacteria
Gram positive bacteria

Gram positive cocci Gram positive bacilli
In clusters: eg. Staphylococcus aureus In chains eg. Bacillus anthracis
In chains: eg., Streptococcus species with spores eg. Clostridium species
Lanceolate shaped in pairs: eg. Streptococcus pneumoniae
In pairs and short chains: eg., Enterococcus species
Gram negative bacteria

Gram negative cocci Gram negative bacilli
In pairs: eg.Neisseria gonorrhoea eg. Escherichia coli
Neisseria meningitidis Klebsiella pneumoniae
Comma shaped, curved Gram negative bacilli
eg. Vibrio cholerae
FIGURE 7-1 Gram stained smear of Staphylococcus aureus, x 1000. FIGURE 7-2 Gram stained smear of Gram negative bacilli, x 1000.
BOX: 7-1 MODIFICATIONS OF GRAM’S STAINING
1 Kopeloff and Beerman’s Gram method

This method uses acetone as a decolouriser.
2 Jensen’s Gram method for smears
This method uses alcohol as decolouriser and weak neutral red as counter stain. This modification is recommended for
examination of smears for gonococci and meningococci.
3 Preston and Morrell’s Gram method
This method uses iodine-acetone as decolouriser giving good results without the need for careful timing of the decolourisation
step.
BOX: 7-2 USES OF GRAM’S STAINING
1 Urine specimens
Identifies urine specimens that contain bacteria greater than 105 cfu/ml of urine.
Presence of at least 1 organism/ oil immersion field correlates with significant bacteriuria ( > 105 cfu/ml).
Presence of polymorphonuclear (PMN’s) in uncentrifuged urine correlates with number of PMN’s excreted per hour. In
patients with >400,000 PMN excreted into urine/hour are likely to be infected.
2 Stool specimens
Useful to detect
Candida.
Clostridium difficile.
3 Sputum specimens
Useful to detect
Streptococcus pneumoniae.
4 Pus specimen
Useful to detect
Staphylococcus aureus in pus from abscesses.
Streptococcus species in exudates from tonsillitis, pharyngitis and impetigo.
Gram negative rods in pus from cases of chronic otitis media and post operative wound infections.
26 Gram’s Staining
KEY FACTS
1 Gram staining is a differential stain, most commonly employed for diagnostic identification of bacteria in clinical specimens.
2 Gram staining differentiates bacteria into two categories: purple coloured Gram positive and pink coloured Gram negative
bacteria.
3 Tissue cells, leucocytes and the debris of inflammatory exudates all stain pink in Gram’s stained smears.
4 Gram positive bacteria have a thicker and denser peptidoglycan layer in the cell walls, which make them less permeable to the
stain, in comparison to those of the Gram negative bacteria.
5 The bacterial smear should not be overheated during heat fixation or over decolourised with alcohol, as it can cause in Gram
positive bacteria losing the dye-iodine complex and appearing Gram negative.
6 The age of culture can also influence the Gram stain reaction i.e. cultures more than 24 hours old can lose ability to retain
dye-iodine complex and appear pink coloured Gram-negative.
VIVA
1 Define differential stain. What are the other differential stains used for staining?
Ans. Differential stain is defined as those that contain two different coloured dyes, such that when the staining is completed,
different structures will take up different colours and can be differentiated.
The other differential stain that can be used for staining is acid fast stain and Albert’s stain.
2 State why Gram’s stain is said to be a differential stain?

Ans. Gram’s stain is said to be a differential stain because it differentiates bacteria as Gram positive bacteria (appears violet)
and Gram negative bacteria (appears pink).
3. Describe differences between a Gram positive and Gram negative cell wall.
Ans. The important differences between Gram positive and Gram negative cell wall are as follows:
i) Gram positives have a thick peptidoglycan layer in the cell wall when compared to Gram negatives which possess a
thinner peptidoglycan layer.
ii) Teichoic acid is present in Gram positive cell wall while it is absent in Gram negative cell wall.
iii) Lipopolysaccharide is absent in Gram positive cell wall and present in Gram negative cell wall.
4 Describe the conditions, which may result in a Gram-positive bacteria turning as Gram negative.
Ans. The conditions that can result in Gram positive bacteria appearing Gram negative are:
i) Excess decolourisation by ethanol/ acetone.
ii) Loss of cell wall due to action of lysozyme, penicillin, changes in the pH and aged cultures.
5 Give 2 examples each of Gram positive and Gram negative cocci and bacilli.
6 What are ‘Gram variable’ bacteria?

Ans. “Gram variable’ bacteria are those Gram positive bacteria that have lost their cell wall integrity because of antibiotic
treatment, old age, or action of autolytic enzymes. These changes allow crystal violet to come out of the cell wall during
process of decolourizing resulting in some cells staining pink and others staining purple.
7 Give other examples of decolourizing agents and counter stains.

Ans. Other examples of decolourizing agents are acetone, 95% ethanol, acetone- alcohol and iodine-acetone.
Other examples of counterstains are basic fuchsin and neutral red.
FURTHER READINGS
3 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th ed.
LESSON
Acid-Fast Staining
8
LEARNING OBJECTIVES stain (methylene blue) appearing blue, while the acid-fast cells
retain the red colour of primary stain (carbol fuchsin).
After completing this practical you will be able to :
Different modifications of acid-fast staining is summarized in
1 Stain smears by acid-fast staining method. the box 8-1.
2 Become familiar with the chemical basis of the acid-fast stain
REQUIREMENTS
INTRODUCTION
I Equipments
The acid fast staining method is a modification of Ehrlich’s Compound light microscope.
(1882) method. Ehrlich suggested the method for the differential
staining of tubercle bacilli and other acid-fast bacilli with aniline- II Reagents and glass wares
gentian violet followed by strong nitric acid. The ordinary Bunsen flame/ torch soaked in methylated spirit, loop wire,
aniline dye solutions do not readily penetrate the substance of glass slides, slide rack, strong carbol fuchsin, acid-alcohol (3
the tubercle bacillus and therefore is unsuitable for staining. ml HCl + 97 ml ethanol) (decolourising agent), and Loeffler’s
The Ziehl-Neelsen acid-fast staining method has proved to be methylene blue (counter stain).
most useful for staining acid fast bacilli belonging to the genus
Mycobacterium especially Mycobacterium tuberculosis and Preparation of strong carbol fuchsin: This solution is prepared
Mycobacterium leprae, and also for Nocardia. These acid fast by dissolving 5 grams basic fuchsin powder in 25 grams
bacilli have a high concentration of the lipid-mycolic acid in crystalline phenol by placing them in a 1 litre flask. The flask
their cell walls. When stained they appear pink against a blue containing solution is kept over a boiling water-bath for about
background. 5 minutes, shaking the contents from time to time. When the
solution is complete, 50 ml of 95% alcohol or 100% ethanol is
added to the solution and mixed thoroughly. Then 500ml of
PRINCIPLE distilled water is added to it and the mixture is filtered before
use.
Acid fastness of acid-fast bacilli is attributed to the presence
of large quantities of unsaponifiable wax fraction called mycolic Preparation of 20% sulphuric acid : 800ml of water is collected
acid in their cell wall and also the intactness of the cell wall. in a large flask. The 200ml concentrated sulphuric acid (about
The degree of acid fastness varies in different bacteria. 98% or 1.835g / ml)) is poured slowly down the side of the flask
In this staining method, application of heat helps the dye into the water, about 50 ml at a time. The mixture becomes hot.
(a powerful staining solution containing carbol fuchsin and Remainder of acid is added in same manner.
phenol) to penetrate the tubercle bacillus. Once stained, the Note: The acid must be added to the water. It is dangerous to
stain cannot be easily removed. The tubercle bacilli resist the add the water to the acid. Great care must be taken to avoid
decolourizing action of acid-alcohol which confers acid fastness spilling the acid on skin, clothing or elsewhere.
to the bacteria. The other microorganisms, which are easily
decolourised by acid-alcohol, are considered non-acid fast. Preparation of 95% alcohol : This is prepared by adding 95
The non-acid fast bacilli readily absorb the colour of the counter ml of ethanol and adding water to it to make 100ml.
28 Acid Fast Staining
Preparation of acid-alcohol decolouriser: This solution 8 Rinse the smears again under tap water and air dry it.
contains 75 ml concentrated hydrochloric acid (HCl) and 25 ml 9 Observe the smear first under low power (10x) objective,
of industrial methylated spirit. Methylated spirit is poured into and then under oil immersion (100x) objective.
a large flask. The flask is placed in 5–8 cmm of cold water in the Note: The smear should be examined following a zig-zag
sink. Then hydrochloric acid is added slowly and the top of the pattern for at least 10 minutes or 300 fields, before declaring
flask is covered to stop the fumes from escaping. It is left for 10 the smear negative.
minutes. It is then decanted into a labeled bottle for use. The 10 Record the observations in the note book. Findings are
final concentration of HCl is 3%. recorded, together with grading of the positive smear.
III Specimen
Sputum smear positive for tubercle bacilli / culture smear of QUALITY CONTROL
Mycobacterium species.
Note: Frequently examined specimens for the detection of A positive control sputum smear from a known case of
Mycobacterium tuberculosis are summarized in the box 8-2. tuberculosis patient, stained with Z-N stain is compared with
the stained test smear for appropriate morphology and staining
appearance.
PROCEDURE With appropriate staining, the acid-fast bacillus appears
pink against blue background of pus cells.
1 Heat fixes the smears by passing the slide 2–3 times gently
over the flame with the smear side up. Allow the smear to
be air dried. OBSERVATION
2 Put the smears on a slide rack and cover the smears with
strong carbol fuchsin. Allow it to stain for 5 minutes. Presence of 3 x 0.3 µm sized pink coloured slender rod shaped
3 During this period, heat the slides from below intermittently structures are seen with curved ends, and are scattered amidst
by Bunsen flame or torch soaked in methylated spirit blue coloured round cells with darkly stained multilobed
without boiling the solution, until the steam rises. Do not nucleus.
allow the stain to dry on the slide, and if necessary add
more carbol fuchsin to cover the smear.
4 Rinse the smears gently under tap water. RESULTS AND INTERPRETATION
5 Cover the smear with 20% sulphuric acid for at least 10
minutes for decolourisation. The stained smear contains pink coloured acid fast bacilli seen
6 Wash the slides thoroughly with water to remove all traces among the blue coloured multilobed pus cells.
of acid. The smear is positive for acid fast bacilli.
Note: Decolourisation with 95% alcohol for 2 minutes is Probably, the smear contains Mycobacterium tuberculosis
only optional and may be omitted. (Fig. 8-1).
7 Cover the smear with Loeffler’s methylene blue for 15–20 Note: List of other acid–fast structures are provided in the
seconds. table 8-1.
FIGURE 8-1 Acid fast stained smear of Mycobacterium FIGURE 8-2 Acid fast stained smear of Mycobacterium leprae, x 1000
tuberculosis, x 1000.
Table 8-1 List of acid –fast structures
Bacteria Parasites Fungi Other structures

Mycobacterium tuberculosis ( Fig. 8-1) Cryptosporidium parvum Fungal spores Spermatozoa head
Mycobacterium leprae ( Fig. 8-2) Cyclospora cayetanensis
Mycobacterium smegmatis Isospora belli
Mycobacterium fortuitum
Nocardia asteroids
Nocardia brasiliensis
Nocardia caviae
BOX 8-1 DIFFERENT MODIFICATIONS OF ACID FAST STAIN AND THEIR USES
1 5% sulphuric acid is used as a decolourizing agent for staining Mycobacterium leprae.

2 1% sulphuric acid is used as a decolourizing agent for staining Nocardia species, Cryptosporidium and Isospora oocysts
(Kinyoun’s modification of acid-fast stain).
3 0.25% sulphuric acid is used as a decolourizing agent for staining spores.
BOX 8-2 FREQUENTLY EXAMINED SPECIMENS FOR THE DETECTION OF

MYCOBACTERIUM TUBERCULOSIS
Pulmonary tuberculosis
Sputum.
Bronchial or laryngeal washings.
Gastric lavage (when sputum is swallowed as in children).
Miliary tuberculosis
Bone marrow.
Liver biopsy.
Tuberculous meningitis
Cerebrospinal fluid.
Renal tuberculosis
Urine.
KEY FACTS
1 Ziehl-Neelsen stain is intended for the differential staining of tubercle bacilli from other acid fast bacilli.
2 Acid fast bacilli appear pink coloured in stained smears.
3 At the end of the process of decolourisation by sulphuric acid, the smear will appear faintly pink.
4 Acid fastness of the bacteria is attributed to presence of mycolic acid in high concentration in the cell walls of tubercle bacilli
and also to the intactness of the cell wall.
5 Positive sputum smear is graded only under oil-immersion as follows:
3–9 AFB in entire smear = 1+
10 or > AFB in entire smear = 2+
10 or > AFB in each oil immersion field = 3+
30 Acid Fast Staining
VIVA
1. List the acid fast organisms.
2. Why are some bacteria acid fast?

Ans. Some bacteria are acid-fast because their thick waxy cell wall is made up of long chain fatty (mycolic) acids. These mycolic
acids render the cells resistant to decolourisation, even with acid-alcohol decolourisers. Thus when acid is added as a decolouriser,
these acid fast bacteria retain the dye because the dye is more soluble in the cytoplasm compared to sulphuric acid.
3 How do you grade a positive sputum smear?
4. List another method that can be used for detecting acid fast bacteria in a smear?
Ans. Fluorescent staining methods using fluorochrome dyes such as auramine O and rhodamine is another method that can
be used for detecting acid fast bacteria in a smear. By this method acid fast bacteria fluoresces bright yellow or orange
against a greenish background.
5. What are the various specimens obtained in the laboratory for the diagnosis of tuberculosis?
6. List different modifications of acid fast staining and their uses.
FURTHER READINGS
LESSON
Albert’s Staining
9
LEARNING OBJECTIVES II Reagents and glass wares
Bunsen flame, loop wire, glass slides, Albert’s stain I and II.
Preparation of Albert’s stain I: This stain is composed of 1.5
1 Stain smears by Albert’s staining to demonstrate the presence grams toluidine blue, 2 grams malachite green, 10 ml glacial
of metachromatic granules in the diphtheria bacillus. acetic acid, 10 ml alcohol (95% ethanol) and 1 litre distilled
water. Toluidine blue and malachite green are dissolved in the
alcohol and then added to the water and acetic acid. The stain
INTRODUCTION is then allowed to stand for one day and then filtered.
The diphtheria bacillus, Corynebacterium diphtheriae has well Note: Toluidine blue stains the granules bluish black due to
developed granules within their bacterial cytoplasm. These metachromatic effect and malachite green stains the bacilli
granules are known as Volutin granules, Babes Ernst granules, green.
polar bodies or metachromatic granules. These granules are
made up of polymetaphosphate and are seen in unstained wet Preparation of Albert’s stain II: Albert’s II (also known as
preparations as round, refractile bodies within the bacterial Albert’s iodine) is composed of 6 gram iodine, 9 gram potassium
cytoplasm. With basic dyes, granules tend to stain more iodide and 900 ml distilled water. The solution is made by first
strongly than the rest of the bacterium. With toluidine blue or dissolving 2 gram potassium iodide in 10 ml distilled water and
methylene blue, they stain metachromatically, and appear then 1 gram of iodine is further added to it with dissolution.
reddish purple in colour. These granules are clearly Then 290 ml of distilled water is added and final volume is made
demonstrated best by special stains such as Albert’s, Neisser’s up to 300 ml.
or Puch’s stain. With Albert’s staining, the bacilli appear green
with bluish-black metachromatic granules. III Specimen
Exudate smear collected directly from pseudomembrane
obtained using a throat swab / culture smear of C. diphtheriae.
PRINCIPLE
The granules present in the diphtheria bacilli exhibit PROCEDURE

metachromasia property and hence appear bluish-black
coloured when stained with the toluidine blue present in Albert 1 Heat fix the smears by passing the slide 2–3 times gently over
I reagent, while the diphtheria bacillus appears green due to the flame with the smear side up. Allow the smears to be air dried.
malachite green present in Albert I reagent. 2 Put the smears on a slide rack and cover the smears with
Albert’s stain I. Allow it to stain for 3-5 minutes.
3 Rinse the smears gently under tap water and blot those dry.
REQUIREMENTS 4 Then cover the smear with Albert’s stain II. Allow it to act
for 1 minute.
I Equipments 5 Rinse the smears again under tap water and blot those dry.
Compound light microscope. 6 Observe the smear first under low power (10x) objective, and
then under oil immersion (100x) objective.
32 Albert’s Staining
7 Record the observations in the note book. Findings are The smear is positive for bacilli showing bluish-black
recorded, together with grading of the positive smear. metachromatic granules.
Probably, the smear contains Corynebacterium
diphtheriae.
QUALITY CONTROL
A known positive control smear of the diphtheria bacillus stained

with Albert’s stain and the stained test smear are compared for
appropriate morphology and staining appearance.
With appropriate staining, the bacillus should appear green
with bluish black metachromatic granules.
OBSERVATION
1–2 µm sized green coloured bacilli showing Chinese letter

arrangement at angles to each other, containing bluish-black
metachromatic granules, are seen (Fig. 9-1).
RESULTS AND INTERPRETATION FIGURE 9-1 Albert’s stained smear of Corynebacterium diphtheriae
showing volutin granules, x 1000.
The stained smear contains malachite green stained bacilli
showing bluish-black metachromatic granules.
BOX 9-1 RAPID STAINING BY DIRECT FLUORESCENT ANTIBODY METHOD
Direct fluorescent antibody testing for Corynebacterium diphtheriae is a rapid diagnostic method like that of Albert’s
staining. This method involves the use of specific antibodies raised against C. diphtheriae conjugated with a fluorescent dye
to detect directly the C. diphtheriae antigen in the throat swab or smear taken from pseudomembrane. The smear is viewed by
immunofluorescent microscopy. The method is highly specific as well as sensitive.
VIVA
1. What is metachromasia?
Ans. When a substance is stained with a particular coloured dye, and if a change in original colour is observed, this phenomenon
is called metachromasia. This phenomenon is observed with C. diphtheriae. The granules present in the bacteria are called
metachromatic granules because the blue colour of the stain is changed to bluish-black by those granules.
2 Mention the other names for metachromatic granules.
3 What is the composition of metachromatic granules?
Ans. Metachromatic granules are composed of polymetaphosphate, ribonucleic acid and protein.
4 What is the significance of metachromatic granules?
Ans. The significance of metachromatic granules is that they represent storage depots of materials needed to form high-
energy phosphate bonds. They are not found during active growth period and are depleted under starvation conditions.
Their presence in thin slender bacilli helps to distinguish C. diphtheriae from short, thick, plumpy, non- pathogenic
diphtheroids which lack them.
5 What is the typical arrangement of C. diphtheriae? What is the reason for such an arrangement?
Ans. The typical arrangement of C. diphtheriae is Chinese letter pattern or Cuneiform arrangement, (bacilli are seen
arranged at various angles to each other resembling the letters V or L. This is due to incomplete separation of the daughter
cells after longitudinal binary fission.
6 Mention the other stains used for the detection of C. diphtheriae.
KEY FACTS
1 Volutin granules of C. diphtheriae are made up of polymetaphosphate.

2 Special stains like Albert’s stain, Neisser’s stain and Puch’s stain are used to demonstrate metachromatic granules.
3 Granules exhibit metachromasia and are seen in unstained wet preparations as round refractile bodies within bacterial
cytoplasm.
4 The diphtheria bacillus gives its characteristic volutin staining reactions best in a young culture (18–24 hours) on Loeffler’s
serum slope or serum medium.
FURTHER READINGS
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company,
St. Louis) 2002.
3 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic
Microbiology. 5th ed. (Lippincott Williams and Wilkins.) 1997.
34
LESSON
Capsule Staining
10
LEARNING OBJECTIVES the capsular materials are water soluble and may be dislodged
and removed with vigorous washing. Bacterial smears should
After completing this practical you will be able to : not be heated, because the resultant cell shrinkage may create
a clear zone around the organism, an artifact that can be mistaken
1 Demonstrate capsule of the bacteria present in animal tissues, for capsule.
blood, serous fluids and pus and artificial cultures , by The capsule is non-ionic, so that the dyes commonly used
positive staining and negative staining methods. will not bind to it. Two dyes, one acidic and one basic, are used
2 Differentiate between capsulated and non-capsulated to stain the background and the cell wall, respectively.
bacteria.
REQUIREMENTS
INTRODUCTION
I Equipments
Some bacteria secrete chemical substances that accumulate on Compound light microscope.
the outer surfaces of the cell wall and form capsules. When the
capsule has a diameter of 20 nanometer or more and seen under II Reagents and glass wares
light microscope, it is referred to as macrocapsule. When the Bunsen flame, inoculating loop, staining tray, glass slides, 1%
diameter is less than 20 nm and seen under electron microscope, crystal violet and 20% copper sulphate (CuSO4 5H2O) for
it is called microcapsule. Capsules may be seen in stained or positive staining; and India ink or Nigrosin stains for negative
unstained preparations as a clear zone around the bacteria. staining. Nigrosin staining is prepared by adding 0.03 grams of
Two types of staining procedures can be employed to nigrosin in 100 ml of distilled water.
demonstrate the capsule. They are the positive and negative
staining procedures. In the positive staining technique, the III Specimen
capsule is stained and coloured whereas in the negative staining Culture of Streptococcus pneumoniae (A capsulated
procedure, the background is stained and the capsule is seen bacterium).
as unstained hallow around the organism.
The best method for staining capsules on bacteria in either PROCEDURE
liquid or solid media is the wet-film India ink method. Dry-film
negative staining methods using India ink, nigrosin or eosin For positive staining of smears
are somewhat less reliable, since occasionally shrinkage spaces 1 Make a smear from colony of S. pneumoniae on a clean
give the appearance of capsules around bacteria that are non- grease free glass slide, and allow it to air dry.
capsulated. Note: The smear is not heat fixed.
2 Put the smear on a slide rack and flood smear with crystal
violet. Allow it to stain for 5-7 minutes.
PRINCIPLE 3 Wash the smear with 20% copper sulphate solution and blot
it dry.
Chemically, the capsular material is a polysaccharide, a 4 Observe the smear first under low power (10x) objective, and
glycoprotein or a polypeptide. Capsule staining is more difficult then under oil immersion (100x) objective.
than other types of differential staining procedures because 5 Record the observations in the note book.
For negative staining of smears

1 Take a clean grease free glass slide.
2 Put a large loopful of undiluted India ink on the slide.
3 Then add a small loopful of liquid bacterial culture to the
India ink and emulsify.
4 Take a clean, grease free cover slip and place on the ink drop
and press it down, so that the film becomes very thin and
thus pale in colour.
5 Observe the wet film under high power (40x) objective.
QUALITY CONTROL
The test smear subjected to capsular staining (e.g. both positive

and negative staining technique) is compared with known FIGURE 10-1 India ink staining of Streptococcus pneumoniae showing
stained control smear of S. pneumoniae (capsulated bacteria) capsule, x 1000.
for appropriate structural details and staining appearance.
OBSERVATION
Observation of positive staining method

In the culture smear, the capsule is seen as a light blue hue in
contrast to the deep purple colour of the cell.
Observation of negative staining method

The capsule in negative staining method is seen as clear
refractile, halo around the organism against a black background.

FIGURE 10-2 Gram’s staining showing clear unstained capsulated
Positive staining method: The smear shows capsulated bacteria. area around Streptococcus pneumoniae, x 1000.
e.g. S. pneumoniae.
Negative staining method: The wet India-ink film contains
capsulated bacteria. e.g. S. pneumoniae (Fig. 10-1).
KEY FACTS
1 Capsule of the bacteria, Bacillus anthracis in the stained blood smears is demonstrated by McFadyean reaction which uses
polychrome methylene blue.
2 Capsules of the bacteria can be demonstrated by two methods: positive staining and negative staining procedures.
3 In the positive staining technique, the capsule is stained and coloured. In negative staining, the background is stained, and
the capsule is seen as an unstained halo around the organism.
4 The wet India ink preparation can also be employed for demonstration of slime which are irregular masses of amorphous
material seen lying between the bacteria and outside the capsules of capsulate ones.
5 Modified India ink preparation using 2% mercurochrome helps clearly demonstrate the internal structure of capsulated
budding yeast.
6 India ink film should have appropriate thickness if the film is too thick, the capsule will be obscured by overlying ink and if
too thin, the capsules get crushed or flattered and the India ink background becomes too pale to give a good contrast.
36 Capsule Staining
VIVA
1 Give examples of bacteria having polysaccharide or polypeptide capsules.

Ans : a) Polysacchride capsule
Staphylococcus aureus
Group B streptococci
Group D streptococci
Streptococcus pneumoniae
Neisseria meningitidis
Haemophilus influenzae
Anaerobic Gram negative bacilli
b) Polypeptide capsule
Bacillus anthracis
2 List various capsulated bacteria

Staphylococcus aureus (microscopically visible capsules)
Streptococcus pyogenes (some stains)
Group C Streptococci ( capsules made of hyaluronic acid).
Group D streptococci (polysacchaide capsules)
Streptococcus pneumoniae.
Neisseria memigitidis (fresh isolates capsulated)
Bacillus anthracis (polypeptide capsule)
Clostridium perfringens
Bacteriodes fragilis
Escherichia coli (some strains)
Klebsiella pneumoniae
Vibrio parahaemolyticus
Yersinia pestis
Francisella tularensis
Bordetella pertusis
3 Describe the functions of the capsule.

Ans: i) Capsules contribute to the virulence of pathogenic bacteria by inhibiting phagocytosis, ii) They protect the bacteria
from antibody, and iii) Capsular material is antigenic in nature and may be demonstrated by serological methods such as
Quellung reaction widely employed for typing of pneumococci.
4 Describe the capsular antigens

Ans: i) Type specific capsular polysaccharide antigen of pneumococcus is also known as specific soluble substance.
ii) Capsular polysaccharide antigens of meningococci have been used to classify them into 13 serogroups, and iii) K antigen
(acidic capsular polysaccharide antigen) of Escherichia coli protect it from bactericidal effect of complement and phagocytes.
FURTHER READINGS
LESSON
Spore Staining
11
LEARNING OBJECTIVES stain (malachite green) and counter stain (0.5% safranine or
0.05% basic fuchsin).Ordinary tap water acts as decolourising
After completing this practical you will be able to: agent.
Unlike most of the vegetative cells that are stained by
1 Stain smears for bacterial endospores by malachite green stain. common procedures, the spore, because of its impervious coats,
2 Differentiate between bacterial spore and vegetative cell are not stained by the primary stain easily. The application of
forms in the bacterial smear. heat facilitates penetration of the primary stain, malachite green.
After the primary stain is applied and the smear is heated, both
the vegetative cell and spore appear green.
INTRODUCTION Once the spore is stained with the malachite green, it cannot
be decolourised by tap water, which removes only the excess
Spore production is a very important characteristic of some primary stain. The spore remains green. On the other hand,
bacteria such as members of anaerobic genera Clostridium water removes the stain from the vegetable cells, because the
and aerobic genus Bacillus. They are highly resistant and stain does not demonstrate a strong affinity for the vegetative
metabolically inactive forms. They occur when environmental cell components and these vegetable cells therefore become
conditions become unfavorable for continuing vegetative colourless.
cellular activities, particularly with the exhaustion of nutritional Red coloured-safranine as counterstain is used as the
carbon source. Because of the chemical composition of spore second reagent to colour the decolourised vegetative cells,
layers, the spore is resistant to deleterious effects of excessive which will absorb the counterstain and appear red. The spores
heat, freezing, radiation, desiccation, and chemical agents, as retain the green of the primary stain.
well as to the commonly employed microbiological stains.
The morphology of the bacterial endospores is best
observed in unstained wet films under the phase-contrast REQUIREMENTS
microscope, where they appear freely or within the bacterial
cells as large, refractile, oval or spherical bodies. I Equipments
Different staining techniques are available for staining of Compound light microscope.
spores. These are malachite green stain (Schaeffer and Fulton
method), modified Ziehl-Neelsen stain using 0.25% sulphuric II Reagents and lab wares
acid as decolouriser, toluidine blue stain and also Gram’s stain. Bunsen burner, beaker of boiling water, staining tray, glass
With the Gram’s stain, the body of the bacillus appears slides, inoculating loop, malachite green and safranine.
deeply stained, whereas the spore is unstained and appears as
a clear area in the organism. The spores on staining with Preparation of malachite green stain: This stain is prepared
modified Ziehl-Neelsen stain appear red and bacilli blue. by dissolving 5 gram of malachite green in 100 ml of distilled
water.
PRINCIPLE Preparation of safranine stain: This stain is prepared by

dissolving 0.5 gram of safranine in 100 ml distilled water.
Malachite green stain, also known as Schaeffer-Fulton Method
for bacterial endospores uses two different reagents: primary
38 Spore Staining
III Specimen
Smear collected from 48 hours to 72 hours nutrient agar slant
OBSERVATION
culture of Bacillus cereus/ thioglycollate culture of Clostridium
butyricum. On a clean glass slide, a smear from the culture is Bacterial endospores stain green, and vegetative bacilli stain
made in saline, then air dried and fixed with heat. red. (Fig. 11-1)
PROCEDURE RESULTS AND INTERPRETATION
1 Heat fix the smears by passing the slide 2–3 times gently over A 2 – 3 µm red coloured rod-shaped structure seen along with
the flame with the smear side up. Allow the smear to be air dried. an intracellular 0.5 µm sized spherical green coloured structure.
2 Put the slide with the smear over a beaker of boiling water, It represents red coloured vegetative bacilli with green coloured
resting it on the run with the bacterial film upper most. spores by the malachite green staining method. May be spore-
3 When, within several seconds, large droplets have bearing bacilli (eg. Bacillus species or Clostridium species)
condensed on the underside of the slide, flood the smear
with 5% acqueous solution of malachite green and allow to
act for 1 minute, while the water continues to boil.
3 Wash the smears with cold water.
4 Then cover the smear with 0.5% safranine or 0.05% basic
fuchsin. Allow it to act for 30 seconds.
5 Rinse the smears again under tap water and blot those dry.
6 Observe the smear first under low power (10x) objective ,
and then under oil immersion (100x) objective.
7. Record the observations in the note book.
QUALITY CONTROL
A check smear of a known positive control of bacteria with

spores is stained with the malachite green staining method,
FIGURE 11-1 Spore staining, x 1000.
and its appropriately stained morphology is observed under
the microscope and compared with the stained test smear.
VIVA
1 Name spore bearing bacilli causing anthrax and gas gangrene.

Ans : Spore bearing bacilli causing anthrax is Bacillus anthracis.
Spore bearing bacilli causing gas gangrene are Clostridium perfringens , Clostridium novyi , Clostridium septicum , and
Clostridium histolyticum.
2 Spores of which .bacilli are used to test efficacy of sterilization using i) dry heat and ii) moist heat?
Ans: The spores of non toxigenic strain of Clostridium tetani are used for testing efficacy of sterilization by dry heat. The
spores of Bacillus stearothermophilus are used for testing efficacy of sterilization by moist heat.
3 Which sterilization method is employed for destruction of spores?
Ans: Autoclaving at 120°C for 15 minutes is the sterilization method employed for destruction of spores in clinical specimens.
KEY FACTS
1 Spores are highly resistant, metabolically inactive forms occurring when environmental conditions are unfavorable.
2 The morphology of spores is best observed in unstained wet films under the phase-contrast microscope.
3 The staining methods available for demonstrating spores are Gram’s staining, modified acid fast staining using 0.25%
sulphuric acid as decolouriser, toluidine blue staining and malachite green staining (Schaeffer-Fulton Method).
4 With malachite green staining vegetative bacilli appear red and spores green.
5 With toluidine blue staining , vegetative bacilli appear light blue and spores dark blue.
FURTHER READINGS
40
LESSON
Negative Staining
12
LEARNING OBJECTIVES REQUIREMENTS
After completing this practical you will be able to: I Equipment

Compound light microscope.
1 Perform a negative staining procedure for demonstration of
bacteria II Reagents and glass wares
These include Bunsen flame, staining tray, glass slides and
coverslips nigrosin stains. Nigrosin staining solution is prepared
INTRODUCTION by adding 0.03 gram of nigrosin in 100 ml of distilled water.
Negative staining procedure is so called because the III Specimen

background gets stained and the organism remains 24 hour broth culture of Klebsiella pneumoniae (A capsulated
colourless. It is also known as ‘Indirect staining. The bacterium).
procedure requires the use of acidic stains such as India ink
or Nigrosin. Negative staining finds its utility for the
demonstration of capsule and bacteria difficult to stain such PROCEDURE
as Treponema palladium. Wet film India-ink method is the
best method for staining capsules of bacteria from cultures 1 Take a clean grease free glass slide.
in either liquid or solid media. 2 Put a small drop of nigrosin close to one end of a clean slide.
A modification of India ink using 2% mercurochrome helps 3 Using a sterile loop, a loopful of broth culture of the
to clearly demonstrate the capsulated budding yeast, capsulated organism is mixed with the nigrosin drop.
Cryptococcus neoformans. Indian ink method for 4 With the edge of a second slide, held at 30° angle and held in
demonstration of capsule is described earlier in the chapter 10. front of the bacterial suspension mixture, spread the drop
In this chapter dry film negative staining by using nigrosin to along the edge of the applied slide. The slide is then pushed
detect bacteria in dry smears will be described. away from the previously spread drop of suspended organism,
forming a thin smear.
5 Air dry the preparation without any heat fixation.
PRINCIPLE 6 Observe the stained smear under oil immersion (100x) objective.
The acidic dyes such as India ink, nigrosin or eosin have
negatively charged chromogen, and will not readily combine
with the negatively charged bacterial cytoplasm. Instead it forms QUALITY CONTROL
a deposit around the organism, leaving the organism itself
colourless. Therefore, the unstained cells are easily discernible The nigrosin-stained preparation of test organism is compared
against the coloured background. with the preparation of capsulated control organism such as
Streptococcus pneumoniae, for appropriate staining and
capsule demonstration.
OBSERVATION
The capsule or the bacterial organism is seen as a clear halo

against a black or dark background in the wet film or dry film
preparation (Fig. 12-1).
The nigrosin-stained smear contains capsulated bacteria. e.g. FIGURE 12-1 Nigrosin-stained smear showing capsulated
K. pneumoniae. K. pneumoniae, x 400.
BOX 12-1 LIST OF MOST COMMON CAPSULATED ORGANISMS THAT CAN BE

DEMONSTRATED BY NEGATIVE STAINING
Bacteria Fungus
Streptococcus pneumoniae Cryptococcus neoformans
Klebsiella pneumoniae
Group D streptococci
KEY FACTS
1 Negative staining procedure is so called because the background gets stained and the organism remains colourless.
2 In negative staining method only background is stained, the organism and capsule remained unstained and refractile.
VIVA
1 List the different stains that can be employed for negative staining.
Ans: The different stains employed for negative staining are: India ink, modified India ink (using 2% mercurochrome) and
10% nigrosin.
2 List the capsulated organisms and bacteria that can be demonstrated by negative staining.
3 What is negative staining? Which bacteria can be demonstrated by India Ink.

Ans: In negative staining, the bacteria are mixed with dyes such as India ink or nigrosin that provide a uniformly coloured
background against which the unstained bacteria stand out in contrast. This is particularly useful in the demonstration of
bacterial capsules.
FURTHER READINGS
42
UNIT
III
Cultivation of Bacteria
Lesson 13 Media for Routine Cultivation of Bacteria

Lesson 14 Temperature Requirement for Growth of
Bacteria
Lesson 15 pH Requirement for Growth of Bacteria
Lesson 16 Oxygen Requirement for Growth of Bacteria
Lesson 17 Culture of Anaerobic Bacteria
Lesson 18 Sterilization of Commonly Used Culture Media
Lesson 19 Antiseptics and Disinfectants
44
LESSON
Media for Routine
13 Cultivation of Bacteria
LEARNING OBJECTIVES 2 Enriched medium: These are solid selective media. These
media, in addition to basal nutrients also contain nutritional
After completing this practical you will be able to: supplements like blood, serum, etc., which favour the growth
of fastidious bacteria. e.g. blood agar (Fig. 13-2), chocolate
1 Know the following commonly used culture media as well as agar (Fig. 13-3) , Löwenstein- Jensen medium (Fig. 13-4), etc.
their uses in a clinical microbiology laboratory: 3 Enrichment media: These are liquid selective media. They
favor the growth of some bacteria by extending the lag phase
a. Basal medium of others eg. Selenite F broth.
b. Differential medium 4 Selective media: These media contain ingredients that
c. Selective medium selectively enable the growth of some species, while
d. Enriched, and inhibiting others eg. Deoxycholate citrate agar (DCA) medium.
e. Enrichment media This medium is a selective medium for growth of Salmonella
spp. present in stool which contains a mixed bacteria flora.
This medium inhibits Escherichia coli and other Gram-
INTRODUCTION negative bacteria.
5 Differential media: These media differentiates between
Cultivation of bacteria is often the first step in the diagnosis of species of bacteria depending on a specific property.
infectious disease. Since bacteria have varied growth Example: MacConkey (Fig. 13-5) agar is a differential medium.
requirements, a wide range of media is available. The choice of This medium is used to demonstrate lactose fermenting
most appropriate media depends on many factors including properties, and differentiate between lactose and non-lactose
nutritional and growth requirements of the bacteria, biochemical fermenting bacteria.
properties and many other properties of the bacteria.
PRINCIPLE
A number of media have been formulated for growing bacteria

(Table 13-1). Media generally contain a carbon source, nitrogen
source and some essential minerals and salts. Some media may
contain additional nutritional supplements. In addition solid
media contain agar as a solidifying agent. Meat extract and
peptone are the commonest sources of carbohydrates and
amino acids.
Media are of different types. These are:
1 Basal media: These contain nutrients that support the

growth of non-fastidious bacteria. They do not confer any
selective advantage, e.g. nutrient agar (Fig. 13-1). FIGURE 13-1 Nutrient agar.
QUALITY CONTROL
1 One un inoculated set of media as sterility control

2 Nutrient agar: Colonies of non-fastidious bacteria such as
S. aureus.
3 Blood agar: Haemolytic strain of S. aureus streaked on the
plate surrounded by a zone of hemolysis.
4 MacConkey agar: Pink, lactose fermenting colonies of
E. coli and colorless colonies of Proteus spp.
5 Selenite F broth: Growth positive Salmonella spp, and growth
negative Proteus spp.
FIGURE 13-2 Blood agar.

OBSERVATIONS
All the inoculated bacteria (e.g. S. aureus, E. coli, P. mirabilis

and Salmonella spp) produce colonies on the nutrient agar
(basal medium) and blood agar (enriched medium). In addition
S. aureus may or may not produce haemolysis on the blood
agar.
FIGURE 13-3 Chocolate agar.
REQUIREMENTS
I Equipments
Bacteriological incubator. FIGURE 13-4 Lowenstein-Jenson’s medium.
II Reagents and media

Different kinds of media such as nutrient agar, blood agar,
MacConkey agar and Selenite F broth.
III Specimen
24 hour broth cultures of Staphylococcus aureus, E. coli,
Proteus mirabilis and Salmonella spp.
PROCEDURE
1 Inoculate a loopful of the test organism, using a sterile

inoculating loop, into appropriately labeled plates and tubes.
2 Incubate the plates and tubes for 18 hours at 37°C.
3 Examine the plate and tubes for growth and record
observations. FIGURE 13-5 Mc Conkey agar.
46 Media for Routine Cultivation of Bacteria
On MacConkey agar E. coli produces pink, lactose

fermenting colonies where as Proteus spp. produces colorless
non-lactose fermenting colonies.
In SF broth most bacteria are inhibited. Different colonies of various bacteria on different media show
Selenite F broth inhibits all except S. Typhi different growth requirement of these bacteria.
Table 13-1 List of different type of media commonly used for isolation of bacteria
Type of medium Example Bacteria grown/isolated
Basal media Nutrient agar S.aureus, E.coli, Klebsiella, Proteus, Pseudomonas

Peptone water S.aureus, E.coli, Klebsiella, Proteus, Pseudomonas.
Enriched media Blood agar S. pyogenes.
Brain heart infusion agar H. influenzae.
Enrichment media Alkaline peptone water V. cholerae.
Selenite F Broth Salmonella spp.
Selective media Pikes medium Streptococcus spp.
Cetrimide agar Pseudomonas spp.
Differential media MacConkey agar, E.coli, Proteus spp., S.aureus, Klebsiella.
CLED medium Proteus, Pseudomonas, Enterobacter, Citrobacter.
KEY FACTS
1 Bacteria have different nutritional requirements. Most of the bacteria grow on enriched media.
2 S. aureus can grow on MacConkey medium.
3 Media serve many purposes such as culture and isolation of the bacteria from clinical specimens, demonstration of growth
and biochemical characteristics of bacteria, and for storage of bacterial isolates, etc.
VIVA
1 Give examples of different kinds of media used for culture of bacteria.
2 What are the constituents of MacConkey agar?

Ans. These are the peptone (2%), sodium taurocholate (0.5%), lactose (1%), neutral red (0.7%), and agar (2%).
3 Name the enrichment medium used for Vibrio cholerae.
4 Mention an important difference between enriched medium and enrichment medium.

Ans Enriched medium (e.g., blood agar) is a solid medium where as enrichment medium (e.g., alkaline peptone water) is a
liquid medium.
FURTHER READINGS
1 Bhattacharya S, Vijayalakshmi N, Parija SC. Uncultivable bacteria: Implications and recent trends towards identification. Indian J Med
Microbiol. 2002: 20; 174-177.
2 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995.
3 Isenberg HD. (Ed) Clinical Microbiology Procedures Handbook. American Society for Microbiology, Washington DC, 1992.
4 PHLS Standard Operating Procedures. Inoculation of Culture Media. No B.SOP 54 Version: 1, 1998.
5 WHO. Guidelines on standard operating procedures for Microbiology. Blood safety and clinical technology. Chapter 6: Cultivation of
bacteria on laboratory media. (World Health Organisation, Geneva) 1997.
LESSON
Temperature
14 Requirement
for Growth of Bacteria
LEARNING OBJECTIVES bacteria prefer to grow in the bodies of warm blooded hosts
and include most bacterial pathogens that cause diseases in
After completing this practical you will be able to understand: humans.
3 Thermophilic bacteria: These bacteria can grow above 35°C.
1 Effect of temperature on growth of the bacteria in culture They are either facultative with an optimum temperature of
media. 45°C -60°C but capable of growth at 37°C or obligate, growing
only at temperature above 50°C.
INTRODUCTION Examples of bacteria showing different temperatures for their

growth is summarized in the table 14-1.
A number of factors influence the growth and culture of bacteria
on culture media. Apart from nutritional requirements, which
vary greatly, environmental factors also play an important role. REQUIREMENTS
These factors include availability of proper temperature, pH
and oxygen in the culture environment. I Equipments
Bacteriological incubator.
PRINCIPLE II Reagents and media

Nutrient agar plates.
Cellular enzymes of the bacteria are optimally active at a certain
temperature. Above this point, they get denatured. On the other III Specimen
hand, at low temperatures, they tend to get inactivated. Either A 24 hour broth culture of Serratia marcescens, Escherichia
event causes biochemical reaction in bacterial cells to cease, coli, Enterococcus faecalis and Flavobacterium spp
thereby interfering with the survival of the organism. In general,
bacteria can survive across a wide range of temperatures from
5 0° C to 80°C. However, each bacterial species has an optimum PROCEDURE
growth temperature at which reproduction is most rapid and
also a maximum/minimum growth temperature beyond which 1 Mark the nutrient agar plates into 4 quadrants each.
growth does not occur. The ideal temperature for growth may 2 Using a sterile loop, aseptically inoculate a drop of each
not coincide with that for specific enzyme activities. culture, taking care to label each quadrant correctly.
Based on their temperature requirements, bacteria can be 3 Incubate the plates at 4°C, 20°C, 37°C and 60°C for 24-48 hr.
classified into the following groups: 4 Observe the plates for growth of bacteria and pigment
production if any.
1 Psychrophilic bacteria: These bacteria can grow within a
temperature range of 5°C to 20°C. Optimum temperature for
growth of these bacteria varies between 10 °C to 15°C. QUALITY CONTROL
2 Mesophilic bacteria: These bacteria can grow within a
temperature range of 20°C to 45°C. Optimum temperature for 1 Nutrient agar plate inoculated with Proteus mirabilis as
growth of these bacteria is 37°C (human body temp). These quality control for mesophilic bacteria.
48 Temperature Requirement for Growth of Bacteria
2 Nutrient agar plate inoculated with Pseudomonas aeruginosa

as quality control for psychrophilic bacteria.
3 Nutrient agar plate inoculated with Bacillus
stearothermophilus as quality control for thermophilic bacteria. 1 Growth of all species tested will be absent at 4°C except for
Yersinia enterocolitica and Flavobacterium spp.
2 At 20°C growth will be minimal/inhibited for E. coli, S.
OBSERVATIONS marcescens and Flavobacterium spp. Flavobacterium spp
is capable of growth at low temperature as well.
1 Reproduction of bacteria is maximal at optimal temperature 3 At 37°C all the bacterial species tested will produce good
and will be seen as a luxuriant growth of colonies. growth. Pigment production will be seen with the cultures of
2 Pigment production is often optimum at a temperature both Flavobacterium and S. marcescens. Pigment production
different from that for growth. S. marcescens produces a is influenced by temperature with a greater intensity on color
red/magenta pigment. at 20°C for both the species tested. There is a different
temperature requirement for optimal growth and pigment
production.
4 At 60°C, only E. faecalis will show growth.
Table 14-1 Examples of bacteria showing different temperatures for their growth
Type of bacteria Example

Psychrophilic Psychrobacter immobilis, Methanosarcina spp.
Mesophilic Escherichia coli.
Thermophilic Thermus aquaticus, Bacillus stearothermophilus.
KEY FACTS
1 Temperature requirement for optimal growth and enzyme activity are often different.
2 Different bacteria require different optimal temperature for their growth.
3 Most of the medically important bacteria grow at 35°C -37°C.
4 Campylobacter grows at 42°C.
VIVA
1 How does temperature affect bacterial growth?

Ans: Bacterial growth is dependent on enzyme activity. Enzyme activity in turn is influenced by temperature. At high
temperatures, they are denatured, while at low temperatures, they tend to get inactivated. Each species has an optimum
range of temperature within which its reproductive rate and hence growth is optimum.
2 Give 2 examples each of psychrophilic, mesophilic and thermophilic bacteria.
3 Name some other enzymatic activities of the bacteria that are affected by temperature.
Ans: Pigment production, production of flagella, and expression of stress proteins.
FURTHER READINGS
Microbiol. 2002: 20; 174-177.
3 Isenberg HD. (Ed) Clinical Microbiology Procedures Handbook. American Society for Microbiology, Washington, DC, 1992.
LESSON
pH Requirement for
15
15 Growth of Bacteria
After completing this practical you will be able to understand: 1 Using sterile pipettes, inoculate 0.1 ml of each organism into
1 The pH requirement of bacteria. nutrient broth tubes of pH, 3, 5, 7 and 9.
2 Incubate the tubes for 24-48 hr.
3 Check for growth of bacteria in these tubes.
INTRODUCTION
Like temperature, pH also plays an important role in the growth QUALITY CONTROL
and reproduction of bacteria. Each species of bacteria can grow
within a particular pH range, and maximum growth occurs in an 1 Nutrient broth tube inoculated with Candida spp as quality
optimum pH range. This is a reflection of this natural environment control for growth at acidic pH.
eg. enteric bacteria such as Escherichia coli have a broad pH 2 Nutrient broth tube inoculated with E. coli as quality control
range, similar to that of the gut. Generally, bacteria grow best at for growth at pH 7.2
a pH between 5.5-8 optimum pH being 6.5-7.5 Fungi thrive in an 3 Nutrient broth tube inoculated with A. faecalis as quality
acidic environment of about pH 4-6. Most laboratory media control for growth at alkaline pH.
have a neutral pH which suits nearly all organisms.
OBSERVATIONS
PRINCIPLE
1 Reproduction of bacteria is maximal at optimal pH.
Since pH changes can occur during growth of the bacteria 2 Ability to tolerate and grow at a particular pH will be seen as
due to the accumulation of metabolic byproducts, buffers are turbidity in culture tubes.
incorporated into the medium to prevent pH change. These
may include natural buffers like proteins, peptone and amino
acids which retard the shift because of their amphoteric nature. RESULTS AND INTERPRETATION
Salts of weak acids and weak bases may also be added.
1 All 3 microorganisms (E. coli, A. faecalis and Candida spp)
show growth and turbidity at pH 7.
REQUIREMENTS 2 No microorganism shows any growth at pH 3.
3 E. coli and A. faecalis do not show any growth at pH 3 or pH 5.
I Equipments 4 Only A. faecalis shows growth and turbidity at pH 9.
Bacteriological incubator. 5 Only Candida spp shows growth and turbidity at pH 5.
II Reagents
The ability to grow at different pH varies among bacteria.
Nutrient broth tubes, 3 each of pH, 3, 5, 7 and 9.
Most pathogens show an optimum pH close to that of their
III Specimen preferred habitat.
A 24 hour broth culture of Escherichia coli, Alcaligenes
faecalis and Candida spp.
50 pH Requirement of Growth of Bacteria
KEY FACTS
1 pH is a critical factor affecting growth and metabolism of bacteria.

2 Most laboratory media has a pH of 7 and incorporate buffers for maintaining the same.
3 Extremes of pH can be used to selectively grow some microorganisms.
VIVA
1 Name some buffers used in preparing culture media.

Ans: Citrate buffer, bicarbonate buffer and phosphate buffer.
2 Name bacteria that can be selectively grown in alkaline medium.

Ans: Vibrio cholerae and Enterococcus faecalis.
3 Why does optimum growth of most pathogenic bacteria occur at pH 7.2?

Ans: Most body fluids have a pH of 7.2. Pathogenic bacteria have adapted to their hosts, and hence are best capable of
survival at a pH identical to that of the host.
FURTHER READINGS
Microbiol. 2002: 20; 174-177.
LESSON
Oxygen Requirement
16 for Growth of Bacteria
LEARNING OBJECTIVES pathway is followed in absence of oxygen substrate when

substances like nitrate, sulphates, etc. can act as terminal
After completing this practical you will be able to understand: electron acceptor.
1 The oxygen requirement of bacteria.
When inoculated into a tube of semisolid or liquid medium,
aerobes grow only on the surface while obligate anaerobes
INTRODUCTION grow only in the depths of the tube. Facultative anaerobes are
evenly dispersed throughout the medium while microaerophiles
Oxygen is one of the most important growth limiting factor for grow slightly below the surface (subsurface growth).
microorganism. It plays a vital role in many biological processes Some bacteria require the presence of 5 to 10% C02 for better
of the microorganisms. Oxygen requirement vary widely among growth of the bacteria by using candle jar (Fig. 16-1).
bacteria and this is reflective of the different bio-oxidative
enzyme systems present in bacterial cells.
REQUIREMENTS
PRINCIPLE I Equipments
Bacteriological incubator and water bath.
On the basis of oxygen requirement, bacteria can be classified
into 5 groups as follows: II Reagents and media
Nutrient broth tubes and brain heart infusion (BHI) agar tubes.
1 Aerobes: These bacteria can grow only in the presence of
free oxygen. Enzyme systems present in these bacteria require III Specimen
oxygen to be the final electron (hydrogen) acceptor A 24 hour nutrient broth culture of Escherichia coli,
especially for the oxidative breakdown of high energy Pseudomonas aeruginosa, and 48 hour thioglycollate broth
molecule like glucose. culture of Clostridium sporogenes.
2 Microaerophiles: These bacteria require small amount of
oxygen for their growth and survival. An excess of oxygen
is inhibitory to their growth.
3 Obligate anaerobes: These bacteria cannot survive in the
presence of free oxygen, hence other molecule act as the
final electron acceptor.
4 Aerotolerant anaerobes: They do not use oxygen as a final
electron acceptor but possess enzymes like superoxide
dismutase and catalase, which can prevent the accumulation
of toxic metabolites produced in the presence of oxygen.
Therefore, oxygen is not lethal to them.
5 Facultative anaerobes: These bacteria can grow in the
presence or absence of free oxygen. They generally follow
an aerobic respiratory pathway, an anaerobic respiratory
FIGURE 16-1 Candle jar used for incubation and culture of bacteria..
52 Oxygen Requirement for Growth of Bacteria
and facultative anaerobes throughout the medium in the tube

PROCEDURE (Table 16-1).
2 Growth will be seen as turbidity in the appropriate section of
1 Warm the BHI agar tube in a water bath so that the agar melts. the tube.
2 Cool the medium to 45°C.
3 With a sterile inoculating loop, introduce a loopful of the
culture into appropriately labeled tube.
4 Rotate the tubes between palms to evenly dispense the organisms.
5 Incubate the tubes for 24 hours at 37°C.
Ps. aeruginosa will grow only on the surface of the medium. E.
6 Innoculate thioglycollate broth with Cl. sporogenes and
coli will grow throughout the tube and Cl. sporogenes only in
incubate at 37°C for 48 hours.
the depths at the bottom of the tube.
Ps. aeruginosa is an aerobe, Cl. sporogenes is an obligate
QUALITY CONTROL anaerobe and E. coli is a facultative anaerobe.
1 BHI agar tube inoculated with P. aeruginosa, the bacteria

that does not grow anaerobically Table 16-1 Examples of bacteria grouped depending on their
2 Thioglycollate broth culture inoculated with Cl. sporogenes, requirement of oxygen
the bacteria that does not grow aerobically
Type Example
OBSERVATIONS Obligate aerobes Vibrio cholerae

Microaerophiles Clostridium perfringens
1 Bacteria will grow at different depths in the tube depending Obligate anaerobes Clostridium tetani
on their oxygen requirement. Aerobes will grow on the Aerotolerant anaerobes Clostridium histolyticum
surface of the medium, anaerobes in the depth of the medium, Facultative anaerobes Escherichia coli
KEY FACTS
1 Oxygen is both beneficial and toxic to living organisms. The benefits are because of their ability to act as a final electron
acceptor in the respiratory pathway with the liberation of much energy. However it also results in the accumulation of toxic
byproducts.
2 Bacteria may possess enzyme system to remove these toxic substances and thus help in their survival in the presence of
oxygen.
3 Anaerobic bacteria can be cultured by the exclusion of air using anaerobic jars, prereduced media, etc.
VIVA
1 Give example of obligate aerobes, microaerophiles, obligate anaerobes, aerotolerant anaerobes and facultative anaerobes.
2 What are the media commonly used to culture anaerobes?

Ans: Robertson’s cooked meat (RCM) medium, thioglycollate broth, blood agar incorporating antibiotics like neomycin or
kanamycin.
FURTHER READINGS
Microbiol. 2002: 20; 174-177.
LESSON
Culture of
17
17 Anaerobic Bacteria
LEARNING OBJECTIVES bearing a screened catalyst chamber. The catalyst, consisting

of pellets of sodium borohydride, cobalt chloride, citric acid
After completing this practical you will be able to understand: and sodium bicarbonate is contained in sachets. Water is added
to the sachet and it is immediately placed in the jar, which is
1 The methods of culture of anaerobic bacteria. then sealed tightly. The resulting reaction liberates hydrogen
and carbon dioxide. An indicator is also added to demonstrate
anaerobiosis.
INTRODUCTION List of anaerobic bacilli and cocci are summarized in the
box 17-1.
Obligate anaerobes can grow in media only in the absence of
oxygen, ie. from which air is excluded. These organisms die
rapidly on exposure to air, hence for maintaining anaerobiosis REQUIREMENTS
various methods have been devised for anaerobic culture.
I Equipments
Anaerobic culture systems: McIntosh and Fildes jar and Gaspak
PRINCIPLE system.
There are different ways of creating anaerobic conditions II Reagents and media
suitable for the growth of obligate anaerobes. Deep nutrient Blood agar plates and thioglycollate broth culture.
agar tubes are the simplest method. The tubes are inoculated
while still molten, cooled rapidly and incubated. Anaerobes III Specimen
grow in the depths of the medium, and the number of colonies A 48 hour thioglycollate broth culture of anaerobic bacteria
becomes fewer towards the surface. Strict anaerobes will not (Clostridium sporogenes, Bacteroides spp), and 24 hour
grow within a centimeter of the surface. cultures of facultative anaerobes (Escherichia coli), and
Alternatively, reducing agents like 0.5-1% glucose, 0.1% obligate aerobes (Pseudomonas aeruginosa).
ascorbic acid, 0.1% cysteine, 0.1% thioglycollate can be added.
Cooked meat particles also act as a good reducing agent
Example. Robertson Cooked meat medium (Fig. 17-1). PROCEDURE
For culture of anaerobes, oxygen must be excluded either
by combustion or by replacing it with an inert gas. 1 Divide each plate into 4 quadrants.
In many laboratories, combustion involves the combining 2 Inoculate a loopful of each organism into a quadrant.
of oxygen with hydrogen to form water in the presence of a 3 Stack the plates into the anaerobic jars, introduce the catalyst
catalyst like palladium or palladinized asbestos. Anaerobic jars and quickly seal the lid.
are a constant feature of anaerobic culture. They include the Note: Anaerobic condition should be checked by alkaline
McIntosh and Fildes jar (Fig. 17-2) , which has inlets to admit methylene blue indicator.
hydrogen and carbon dioxide, a vacuum pump for evacuating 4 Incubate the plates at 37ºC for 48 hours.
oxygen, and a catalyst fitted into the lid. 5 Incubate one plate aerobically.
A simpler but more expensive technique is the Gaspak 6 Remove the plates from the jars and examine for growth.
system. This utilizes a transparent polycarbonate jar with a lid
54 Culture of Anaerobic Bacteria
FIGURE 17-1 Robertson’s cooked meat medium. FIGURE 17-2 Macintosh Filde’s jar.
QUALITY CONTROL Cl. sporogenes and Bacteroides are strict anaerobes that cannot
grow if oxygen is present.
1 Blood agar inoculated with P. aeruginosa, the bacteria that
does not grow anaerobically E. coli will grow on all the plates, either incubated aerobically
2 Thioglycollate broth culture inoculated with Cl. sporogenes, or anaerobically. E. coli is a facultative anaerobe and can grow
the bacteria that does not grow aerobically either in the presence or absence of oxygen.
OBSERVATIONS BOX 17-1 LIST OF ANAEROBIC

BACILLI AND COCCI
If anaerobiosis is complete, obligate anaerobes like Cl.
sporogenes will grow, while obligate aerobes like P. aeruginosa Anaerobic bacilli
will not grow. Gram positive bacilli Gram negative Bacilli
Bifidobacterium Bacteroides
Propionobacterium Fusobacterium
RESULTS AND INTERPRETATION Eubacterium
Lactobacillus
P. aeruginosa will not show growth on the plates incubated Actonomyces
anaerobically, while Cl. sporogenes and Bacteroides spp will
grow on the blood gar plates. P. aeruginosa is a strict aerobe Anaerobic cocci
that cannot grow in the absence of oxygen. Gram positive cocci Gram Negative cocci
Peptosteptococcus Veillonella
P. aeruginosa will show growth on the aerobically incubated Coprococcus Acidaminococcus
plate while Cl. sporogenes or Bacteroides spp will not grow. Ruminococcus Megasphaera
VIVA
1 List some methods of anaerobic culture not utilizing anaerobic jars.
2. What is the Hungate procedure of anaerobiosis?

Ans. The Hungate procedure is a method for the anaerobic culture of bacteria. In the presence of an oxygen free gas, usually
nitrogen, a thin layer of agar is coated on the inside of the culture tube. The organisms are then inoculated, also in the
presence of the same gas. The tubes are then sealed. This ensures anaerobic conditions inside the tube.
KEY FACTS
1 If anaerobiosis is inadequate, obligate anaerobes may fail to grow.

2 The addition of indicators to an anaerobic system is a useful way of ascertaining absence or presence of oxygen.
FURTHER READINGS
Microbiol. 2002: 20; 174-177.
56
LESSON
Sterilization of
18 Commonly Used
Culture Media
LEARNING OBJECTIVES Chamber land),b) asbestos filters (e.g., Seitz filter (Fig. 18-1), c)
sintered glass filter (Fig. 18-2), and d) cellulose membrane filters.
After completing this practical you will be able to become Of these cellulose membrane filter is most extensively used now
familiar with: a days.
Different methods of sterilization of various substances are
1 The commonly used techniques for the sterilization of culture summarized in the table 18-1.
media.
REQUIREMENTS
INTRODUCTION
I Equipments
Sterilization is the process by which an article is freed of all Autoclave (Fig. 18-3), hot air oven (Fig. 18-4) and Seitz filter.
living organisms, including spores. This is vital for isolation
and maintenance of microbes. The common methods used for II Reagents
sterilization of media is heat and filtration. Sterile nutrient broth, Browne’s tube no. 3, and filter paper strips
impregnated with spore of Clostridium tetani and filter paper
strips impregnated with spores of Bacillus stearothermophilus.
PRINCIPLE
III Specimen
Sterilization by heat Spore suspensions of Bacillus spp and suspension of
Escherichia coli
This can be performed by two methods as mentioned below:
Dry heat: Sterilisation by dry heat kills the bacteria by oxidizing
PROCEDURE
essential cell components of the cell. The hot air oven is commonly
used in a microbiology laboratory for sterilization of laboratory
1 Soak strips of filter paper in the spore suspensions of
glassware, oils, powders etc. In a hot air oven, temperatures
Bacillus spp and dry them in a Petri dish in the incubator.
maintained at 160°C, for an hour is effective for sterilization.
2 Take 7 test tubes and place a dried strip in each of them.
Moist heat: Moist heat kills the microorganisms by coagulat-
3 Place the first three tubes in a hot air oven at 160°C for 20, 30
ing their proteins and denaturing their enzymes. Moist heat has
and 60 minutes respectively.
greater penetrating power than the dry heat and so relatively lower
4 Autoclave the 4th, 5th and 6th tubes for 10, 20, and 30 minutes
temperature is required for sterilization by this method. Pasteuri-
respectively.
sation is an example of sterilization by moist heat (Box 18-1).
5 After cooling, aseptically add 5 ml of nutrient broth to each
tube and incubate for 24 hours at 37°C.
6 The 7th tube acts as control.
Sterilization by filtration 7 Inoculate a drop of the E.coli suspension onto a nutrient
agar plate.
Heat labile fluids such as serum, antibiotics, etc. are sterilized by 8 Filter the suspension through a Seitz filter.
passing them through special filters. There are different types 9 Aseptically inoculate the filtrate onto a nutrient agar plate
of filters. These include a) earthenware candles (e.g., Berkefield, and incubate both plates at 37°C for 24 hours.
FIGURE 18-1 Seitz filter.
FIGURE 18-3 Laboratory autoclave.

QUALITY CONTROL
1 Autoclave: Filter paper strips impregnated with spores of

B.stearothermophilus are autoclaved along with the normal
load and then transferred to nutrient broth and incubated. If RESULTS AND INTERPRETATION
the correct sterilization conditions have been achieved no
growth should occur. 1 There will be growth in tubes, kept in the hot air oven for 20
2 Hot air oven: This can be tested by using a) Browne’s tube minutes and 30 minutes but not for 60 minutes.
no. 3: Color change from red to green is a satisfactory result; 2 Only the tube autoclaved for 10 minutes will show growth.
b) Filter paper strips impregnated with spores of non The other two tubes will remain free of growth.
toxigenic strain of C. tetani heated along with the load and 3 There will be no growth in the culture filtrates.
incubated in appropriate media. No growth should occur. Both dry and moist heats are effective sterilization methods.
3 Filters: Efficient filters should be able to retain Serratia However moist heat is more effective requiring less time and
marcescens. temperature. Filtration is also a suitable method of
sterilization.
OBSERVATIONS
If the spores have been killed by the heat process there will be
no growth as demonstrated by a lack of turbidity. Similarly, if
the bacteria have been held back by the filter there should be
no growth in the sample of filtrate.
FIGURE 18-4 Hot air oven.

FIGURE 18-2 Sintered glass filter.
58 Sterilization of Commonly Used Culture Media
BOX 18-1 PASTEURISATION
Pasteurization is a method of sterilisation by moist heat. It is sufficient to kill heat labile bacteria, but not spores. Two methods
are available: holder method (60°C for 30 minutes) and flash method (72° C for 15 seconds). Some pathogens such as Coxiella
burnetii is not destroyed, so this is not a very efficient method of sterilization.
Table18-1 Different methods of sterilization of various substances
Methods of sterilization Substances sterilized
Dry heat
Flaming Inoculating wires, loops.
Hot air oven Glassware, oils, powders, paraffin .
Incineration Biohazardous material- used gloves, needles, etc.
Moist heat
Pasteurization Serum, milk.
Inspissation Löwenstein Jensen media, Loeffler’s serum slope.
Boiling Needles, glass syringes.
Tyndallization Sugar solution.
Autoclaving Heat stable media such as nutrient agar.
Filtration Heat labile media, serum, antibiotics, etc.
KEY FACTS
1 Various factors influence sterilization by heat. These include time, temperature, number of microorganisms and spores and
their type, nature of material, etc.
2 It is important not to overfill the media container.
3 The load should be properly plugged or wrapped to ensure sterility.
4 The load should be cooled before being removed from the autoclave/hot air oven.
5 Filters should be assembled and autoclaved prior to use.
VIVA
1 Why should media not be sterilized in large quantities?

Ans: Large quantities of media are difficult to sterilize by heat. It will not be possible to ensure that all parts of the media have
attained sterilization temperature, and thus sterilization cannot be guaranteed. If methods like filtration are used, large
volumes cannot be handled by the filtration apparatus.
2 What are the different methods of sterilizing by dry heat?

Ans: These are sterilization by red heat, flaming, hot air oven and incineration.
3 How would you sterilize a) sera, b) Löwenstein Jensen medium, c) nutrient agar and d) paraffin?
FURTHER READINGS
Microbiol. 2002: 20; 174-177.
LESSON
Antiseptics and
17
19 Disinfectants
LEARNING OBJECTIVES II Reagents and media

Sterile nutrient broth, nutrient agar plates, used as well as fresh
After completing this practical you will be able to become disinfectant (e.g. lysol).
familiar with:
III Specimen
1 The commonly used antiseptics and disinfectants and A 24 hour broth culture of Escherichia coli.
methods of testing them.
PROCEDURE
INTRODUCTION
1 With a sterile pipette, transfer 1 ml of the used disinfectant
Disinfection is defined as the destruction of microorganisms into 9 ml sterile nutrient broth.
not including bacterial spores. The process reduces 2 Also mix 1 ml of fresh disinfectant with 9 ml of E coli culture.
microorganism to a level acceptable for a defined purpose. A 3 Inoculate this mixture onto 10 different areas of two well
chemical agent that disinfects a substance is called a dried nutrient agar plates each.
disinfectant. A disinfectant that can be safely applied to living 4 Incubate one plate for 3 days at 37°C and the other for 7
tissue is called an antiseptic. days at room temperature.
Table 19-1 summarises the list of commonly used
disinfectants and antiseptics.
QUALITY CONTROL
PRINCIPLE 1 Fresh disinfectant should be a) sterile, and b) should kill a

test inoculum of E coli within 10 minutes.
Disinfectants are used for medical devices where sterility is
not required. They have markedly different activity against
different microorganisms and are generally most effective OBSERVATION
against Gram positive bacteria. The concentration at which
they are used should be accurate for optimal activity. The article If the disinfectant has lost activity it will fail to kill microorganism
to be disinfected should be cleaned thoroughly (to reduce on objects immersed in it. This will show a growth on the nutrient
organic matter which may inactivate the agent) or immersed in agar plate.
the disinfection for the required amount of time.
Commonly used disinfectants and their mechanism of actions
are summarized in the box 19-1. RESULTS AND INTERPRETATION
If growth occurs on 5 or more spots on either plate, the used

REQUIREMENTS disinfectant is said to have failed the test.
With time and use, the activity of a disinfectant decreases. The
I Equipments fresh disinfectant will be able to bring about disinfection more
Sterile pipette. effectively than the used one.
60 Antiseptics and Disinfectants
Table 19-1 List of commonly used disinfectants and antiseptics
Disinfectants Antiseptics
Lysol 70% ethyl alcohol
Zephiran Chlorhexidine
Glutaraldehyde Iodine
Sodium hypochlorite Dettol
BOX 19-1 COMMONLY USED DISINFECTANTS AND

THEIR MECHANISM OF ACTIONS
1 Phenols (e.g. lysol, chloroxylenol): They act by a number of mechanisms such as disruption of cells, precipitation of
protein inactivation of enzyme, etc. Phenol is the standard disinfectant against which other disinfectants are compared.
2 Alcohols (e.g. ethyl alcohol, usually at a concentration of 70%): They can also be used as antiseptics and are active
against viruses as well. They act by denaturing proteins damaging lipid complexes and dehydration.
3 Halogens (eg. iodine, chlorine and sodium hypochlorite): Iodine is commonly used as an antiseptic. It acts by oxidation.
Chlorine is a widely used disinfectant. The compounds act by the liberation of nascent oxygen and are effective against
most bacteria and viruses.
4 Heavy metals and their compounds (e.g. copper sulphate): They act by inactivating cellular protein.
5 Synthetic detergents (e.g. sodium lauryl sulfate): They help in mechanical removal of microorganism.
6 Quaternary ammonium compound (e.g. zephiran): They act on the cell membrane of bacteria.
KEY FACTS
1 Disinfectant does not guarantee sterility. They merely ensure that an object is relatively free of microbial contamination.
2 Antiseptics can be used for decontamination of living tissue.
3 Disinfectants are used for decontamination of non-living tissue.
4 It is essential to test disinfectant from time to time to detect loss of activity.
VIVA
1 What are the commonly used disinfectants and antiseptics?

2 What is the difference between sterilization and disinfection?
Ans: Sterilization frees an article of all infectious materials including spores, whereas disinfection merely frees an article of
infectious, vegetative cells. It doesn’t guarantee the removal or inactivation of spores. In general, the temperatures used in
sterilization are higher than those used for disinfection, and the concentration of chemical agents is higher when they are
used for sterilization than disinfection.
3 What are different methods used for testing disinfectant?
Ans: These are : a) Rideal Walker test, b) Chick Martin test and c) In-use test.
FURTHER READINGS
Microbiol. 2002: 20; 174-177.
UNIT
IV
ENZYMATIC AND BIOCHEMICAL
ACTIVITIES OF BACTERIA
Lesson 20 Catalase Test

Lesson 21 Oxidase Test
Lesson 22 Coagulase Test
Lesson 23 Urease Test
Lesson 24 Indole Test
Lesson 25 Methyl Red Test
Lesson 26 Voges-Proskauer Test
Lesson 27 Citrate Utilization Test
Lesson 28 Triple Sugar Iron (TSI) Agar Test
Lesson 29 Hydrogen Sulphide Test
Lesson 30 Nitrate Reduction Test
62
LESSON
Catalase Test
20
After completing this practical you will be able to: I Reagents and glass wares
3% hydrogen peroxide, glass slides, test tubes, glass rod /
1 Demonstrate the presence of catalase, an intracellular enzyme platinum loop / plastic loop and other standard lab wares.
in the bacteria.
2 Distinguish the bacteria based on the catalase activity. II Specimen
Pure growth of bacteria from solid media preferably from non-

INTRODUCTION blood agar plates (Examples: nutrient agar, Muller-Hinton agar)
is tested.
Catalase is an enzyme produced by many bacteria. The enzyme
splits hydrogen peroxide into water and oxygen. Hydrogen
peroxide is a by product of aerobic respiration and is lethal if it PROCEDURE
accumulates in the bacterial cell. Catalase degrades the
hydrogen peroxide in the bacterial cell before it can do any Test can be done by 2 methods as follows:
damage to the bacterial cell.
1 Slide method
2 Tube method
PRINCIPLE
Slide method
Chemically, catalase is a haemoprotein, similar in structure to
haemoglobin, except that the four iron atoms in the molecule 1 Transfer pure growth of the organism from the agar to a
are in the oxidized (Fe3+) rather than the reduced (Fe2+) state. clean slide with a loop or glass rod.
The enzyme converts hydrogen peroxide into water and oxygen. 2 Immediately add a drop of 3% hydrogen peroxide to the growth.
3 Observe for bubble formation.
2H202 —— 2H2 0+02 (gas bubbles)
Tube method
Production of the enzyme catalase can be demonstrated by
adding hydrogen peroxide to colonies of the bacteria. If catalase 1 Take 1 ml of 3% hydrogen peroxide in 12 x 100 mm test tube.
is present it is indicated by the presence of free gas bubbles. If 2 Introduce small quantity of bacterial growth into the fluid
catalase is absent, no bubbles will be seen. with the help of a glass rod or plastic loop and touch the side
The catalase test is most commonly used to differentiate of the tube.
members of the family Micrococcaceae from members of the 3 Observe the release of bubbles.
family Streptococcaceae (Box 20-1).
Catalase test is also carried out for Mycobacteria to dif- Note: Hydrogen peroxide must be stored in amber coloured
ferentiate tubercle bacilli from atypical mycobacteria (Box 20-2). bottles. If stored in colourless penicillin vials, the hydrogen
List of catalase positive and negative bacteria are peroxide on exposure to light will be broken down into oxygen
summarized in the table 20-1. and water. Use of this solution will give false negative results.
Table 20-1 Catalase positive and negative bacteria

QUALITY CONTROL
Catalase positive bacteria Catalase negative bacteria
Positive control: Staphylococcus aureus (catalase positive bacteria).
Staphylococci Streptococcus pyogenes
Negative control: Streptococcus species (catalase negative bacteria). Micrococci Gardnerella vaginalis
Corynebacterium diphtheriae Fusobacterium species
Enterobacteriaceae Eikenella corrodens
OBSERVATIONS Kingella kinge
Shigella dysenteriae type 1
Slide method Fatumella ptysees
Gas bubbles are formed immediately when 3% H2 02 is added to
the colony.
Tube method
Gas bubbles are released when colonies are introduced into BOX 20-1 USES OF CATALASE TEST
the hydrogen peroxide in the test tube.
The catalase test is useful to differentiate:
RESULTS AND INTERPRETATION 1 Staphylococci (catalase +ve) from streptococci and

enterococci (catalase - ve).
1 The rapid and sustained appearance of bubbles or 2 Listeria monocytogenes and Corynebacteria (catalase
effervescence constitutes a positive test. It means bacteria +ve) from other Gram positive, non-spore forming bacilli
possesses the enzyme catalase, hence is catalase positive. (catalase - ve).
2 Some bacteria possess enzymes other than catalase that 3 Members of family Enterobacteriacae.
can decompose hydrogen peroxide. Hence, forming a few 4 Aerobic bacteria (catalase +ve) from anaerobic bacteria
tiny bubbles after 20-30 seconds is not considered a (catalase - ve).
positive test.
BOX 20-2 CATALASE TEST FOR MYCOBACTERIA
It helps to differentiate tubercle bacilli from atypical mycobacteria. Most atypical mycobacteria are strongly catalase positive,
while tubercle bacilli are weakly positive.
Most strains of mycobacteria, except certain strains of Mycobacterium tuberculosis complex (some isoniazid resistant
strains) and M. gastri, produce the intracellular enzyme catalase.
The test is performed by mixing equal volumes of 30% hydrogen peroxide and 0.2% catechol in distilled water and then
adding it to 5ml of a mycobacterial test culture. It is allowed to stand for a few minutes. Effervescence indicates catalase
production.
Catalase production is assessed by
a) Relative activity of the enzyme catalase determined by the height of the column of oxygen bubbles formed by the action of
untreated enzyme produced by the organism (Semi quantitative catalase test). On the basis of the semi quantitative
catalase test, mycobacteria are classified in to 2 groups: i. those producing <45mm column height of bubbles, and ii.those
producing >45mm column height of bubbles.
b) By the ability of the enzyme catalase to remain active after heating, a measure of the heat stability of the enzyme (Heat
stable catalase test). When heated to 68°C for 20 minutes, the catalase of M. tuberculosis. M. bovis, M. gastri and M.
haemophilum becomes inactivated.
64 Catalase Test
KEY FACTS
1 Catalase enzyme splits hydrogen peroxide into oxygen and water.

2 In aerobic organisms, 02 serves as H2 acceptor and hydrogen peroxide (H2 02) is formed in the cell.
3 High concentration of H2 02 is toxic to the cell. Therefore, aerobic bacteria possess catalase to split toxic hydrogen peroxide.
4 Most of the aerobes are catalase positive.
5 Most of the anaerobes do not possess the enzyme, catalase.
6 Care must be taken while performing catalase test in growth from blood agar plate. Blood (RBC) contains catalase, which
gives false positive reactions.
VIVA
1 What is catalase test?

2 What are the positive and negative controls used in the catalase test?
3 What is the importance of catalase test?
4 Can you take colonies from blood agar plate for testing catalase reaction? Give explanations.
5 Name some catalase positive and negative organisms.
FURTHER READINGS
2 Forbes BA, Sahm DF and Weissfeld AS (Eds). Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis)
2002.
3 Introduction to Clinical Microbiology. University of Texas- Houston Medical School, DPALM Medic. 1995.
4 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC (Eds). Color Atlas and Textbook of Diagnostic Microbiology. 5th
ed. (Lippincott Williams and Wilkins, USA). 1997.
LESSON
Oxidase Test
17
21
LEARNING OBJECTIVES dihydrochloride (1%), and dimethyl – p – phenylene diamine
dihydrochloride (1%).
After completing this practical you will be able to: Wood stick/platinum loop/glass rod, and filter paper.
1 Demonstrate the presence of oxidase, an intracellular enzyme
in the oxidase-positive bacteria. II Specimen
2 Distinguish the bacteria based on the cytochrome oxidase Young culture of bacteria to be tested, preferably less than 24
activity. hours old, growing on an agar plate or agar slant.
INTRODUCTION PROCEDURE
The enzyme oxidase plays a vital role in the operation of the The test is performed by following two methods:
electron transport system during aerobic respiration.
Cytochrome oxidase catalyzes the oxidation of a reduced 1 Direct plate technique, and
cytochrome by molecular oxygen, resulting in the formation of 2 Indirect paper strip procedure.
water or hydrogen peroxide.Aerobic bacteria, as well as some
facultative anaerobes and microaerophiles exhibit oxidase Direct plate technique
activity.
1 Take a nutrient agar plate with colonies of bacteria to be tested.
2 Add 2 to 3 drops of reagent (tetramethyl p-phenylene diamine
PRINCIPLE hydrochloride or dimethyl-p- phenylene diamine
dihydrochloride) directly to the bacterial colonies growing
The cytochromes are iron containing haemoproteins that act on medium in the plate.
as the last link in the chains of aerobic respiration by 3 Note the change of colour of the colonies.
transferring electrons (hydrogen) to oxygen, with the formation
of water. The cytochrome oxidase test uses certain reagent Indirect filter paper strip procedure
dyes such as p-phenylene diamine dihydrochloride which acts
as a substitute for oxygen as artificial electron acceptors. This 1 Take a filter paper strip.
enzyme oxidises the reagent N-N tetramethyl para-phenylene 2 Moisten the filter paper strip with freshly prepared 1% oxidase
diamine hydrochloride (a colour less reagent in reduced form) reagent.
to indophenol blue, a purplish blue coloured product. Note: Oxidase reagent is freshly prepared in distilled water
List of oxidase positive and negative bacteria is presented every day.
in the table 21-1. 3 Pick up the colonies to be tested with the help of a glass rod
or plastic loop or platinum wire.
4 Smear the colonies into the reagent zone of the filter paper.
REQUIREMENTS 5 Note the change in colour if any within 10 seconds.
I Reagents and glass wares

Fresh reagent: Tetramethyl – p-phenylene diamine
66 Oxidase Test
QUALITY CONTROL Table 21-1 List of oxidase positive and negative bacteria
Positive control: Pseudomonas aeruginosa (oxidase positive Oxidase positive bacteria

bacteria). Gram negative rods
Negative control: Escherichia coli (oxidase negative bacteria). 1. Pseudomonas spp(except Ps. cepacia).
2. Vibrio spp.
3. Aeromonas spp.
OBSERVATIONS 4. Camphylobacter spp.
5. Plesiomonas spp.
Direct plate technique 6. Flavobacterium spp.
In a positive test, bacterial colonies on the plate develop a 7. Alcaligenes spp.
deep blue colour at the site of inoculation within 10 seconds. 8. Haemophilus spp.
In a negative test the colour of the colonies remain unchanged. 9. Moraxella spp.
Indirect filter paper strip procedure 10. Chromobacterium spp.
In a positive test, a deep blue colour develops at the site of 11. Bordetella spp(except B.parapertusis)
smear in the filter paper, within 10 seconds (Fig. 21-1). 12. Brucella spp (except B.canis)
In a negative test the colour of the smear in the zone of the 13. Eikinella spp.
filter paper remain unchanged. 14. Cardiobacterium spp.
15. Achromobacter spp.
16. Pasteurella multocida
Gram negative cocci
Bacterial colonies having cytochrome oxidase activity develop 1. Neisseria spp.
a deep blue colour at the inoculation site within 10 seconds. In 2. Branhamella spp.
filter paper test, deep blue colour develops at the site of smear
within 10 seconds. It means bacteria possesses the enzyme Oxidative negative bacteria
oxidase, hence is oxidase positive. 1. All genera in family Enterobacteriaceae
2.Acinetobacter calcoaceticus
3. Bordetella parapertusis
4.Brucella canis
5.Francisella tularensis
6.Gardnerella vaginalis
FIGURE 21-1 Slide oxidase test
VIVA
1 What is the principle of oxidase test?
2 How is oxidase test done?
3 What are the precautions to be observed while doing oxidase test?

Ans: a.Fresh reagent must be used.
b. Iron wire loops should not be used.
c. Nichrome wire loops should not be used.
d. Colonies should not be picked up from selective media.
e. Results must be observed within 10 seconds.
4 Name oxidase positive bacteria.
5 Name oxidase negative bacteria.
6 What is chemical name of the oxidase reagent?

KEY FACTS
1 The dye, p-phenylene diamine dihydrochloride is the substitute for oxygen as artificial election acceptors.
2 In reduced state the dye is colourless and in oxidized state deep purple in colour.
3 Colour change must be noted within 10 seconds.
4 Tetramethyl derivative of p-phenylene diamine is recommended because the reagent is i more stable in storage, ii more
sensitive to the detection of cytochrome oxidase, and iii. less toxic than dimethyl derivative.
5 Nichrome or stainless steel inoculating loops or wires should not be used for performing the test because surface oxidation
products formed during the process of sterilization by flaming may result in false positive reaction.
6 Always freshly prepared reagent should be used.
7 Colonies for testing should not be taken from blood agar.
FURTHER READINGS
2002.
68
LESSON
Coagulase Test
22
LEARNING OBJECTIVES by tube coagulase test. In this method, a suspension of coagulase
producing staphylococci is prepared in plasma in a test tube,
After completing this practical you will be able to: and incubated at 37°C for 3-6 hours. In a positive test, the enzyme
1 Demonstrate the presence of coagulase, an intracellular coagulase secreted by S. aureus is liberated to the medium, which
enzyme in the bacteria, especially Staphylococcus aureus. reacts with fibrinogen to produce a visible fibrin clot.
2 Distinguish the bacteria based on the coagulase activity. List of coagulase positive bacteria is presented in the
table 22-1.
INTRODUCTION
REQUIREMENTS
The enzyme, coagulase, produced by a few Staphylococcus
species, is a key feature of pathogenic Staphylococcus. The I Reagents and lab wares
enzyme causes coagulation of blood, allowing the organism to Rabbit plasma with EDTA anticoagulant, saline, glass slides,
“wall” its infection off from the host’s protective mechanisms test tubes, glass rod/platinum loop/plastic loop and other
rather effectively. standard lab wares.
Coagulase is a protein having a prothrombin-like activity
capable of converting fibrinogen into fibrin, which results in II Specimen
the formation of visible clot. In the laboratory, the coagulase Pure growth of S. aureus from solid media preferably from non-
test is used to identify S. aureus and differentiate it from the blood agar plates (Examples: nutrient agar, Muller-Hinton agar).
other species of coagulase-negative Staphylococcus.
PROCEDURE
PRINCIPLE
Slide test
S. aureus produces the enzyme coagulase in 2 forms: a. bound 1 Take a clean glass slide.
coagulase and b. free coagulase. 2 Mark it into two halves by a glass marking pencil.
3 Add two drops of sterile saline on two halves of the glass
Bound coagulase slides.
4 Pick up the colonies of S. aureus to be tested from agar
Bound coagulase is also known as clumping factor. It is bound to culture and gently emulsify with drops of saline.
the bacterial cell wall and is not present in culture filtrates. Presence 5 Add a drop of undiluted plasma to the bacterial suspension
of this enzyme is tested by slide coagulase test. Fibrin strands are and mix with a wooden applicator sticks.
formed between the bacterial cells when suspended in plasma 6 Place another drop of saline in other half of the slide as a control.
(fibrinogen), causing them to clump into visible aggregates. 7 Rock the slide, back and froth, and observing for the prompt
clumping of the bacterial suspension within 10-15 seconds.
Free coagulase
Tube test
Free coagulase is a thrombin-like substance present in S. aureus 1 Take 0.5 ml of rabbit plasma (diluted 1 in 5 with saline) in a
culture filtrates (Box 22 -1). Presence of free coagulase is tested test tube.
2 Add approximately 5 drops (250 µl) of overnight broth culture

or small amount of the colony growth of S. aureus to the
diluted plasma in the test tube.
3 Incubate the tube at 37°C for 4 hours.
4 Observe for clot formation by gently tilting the tube.
5 If no clot is observed at that time, reincubate the tube at
room temperature and read again after 18 hours. POSITIVE
QUALITY CONTROL
Positive control: S. aureus (Coagulase positive bacteria). NEGATIVE

Negative control: S. epidermidis (Coagulase negative bacteria).
Coagulability of plasma may be tested by adding one drop
of 5% calcium chloride to 0.5 ml of the reconstituted plasma. A FIGURE 22-1 Tube coagulase test.
clot should form within 10 to 15 seconds.
organisms. The test is considered negative if no agglutination

OBSERVATION is observed after 2 minutes. All strains that are coagulase
positive can be reported as S. aureus. All strains producing
negative slide tests must be tested with the tube coagulase
In a positive slide test, prompt clumping of the organism shows test.
the presence of the bound coagulase. The tube coagulase test is considered positive if any degree of
In a positive tube test, the plasma in the tube clots and does clotting is noted.
not flow when the tube is inverted (Fig. 22-1).
Note: On continued incubation, the clot may be lysed by
fibrinolysin secreted by some strains. Table 22-1 List of coagulase positive bacteria
1 Staphylococcus aureus.
RESULTS AND INTERPRETATION 2 Staphylococcus schleiferi
4 Staphylococcus felis
5 Staphylococcus lutrae
In slide test, Positive reaction will be detected within 10–15 6 Staphylococcus intermedius
seconds of mixing the plasma with the suspension by the 7 Staphylococcus hyicus
formation of a white precipitate and agglutination of the 8 Peptostreptococcus hydrogenalis
BOX 22-1 FREE COAGULASE
Free coagulase is an extracellular enzyme produced by Staphylococcus aureus. It activates a coagulase reacting factor (CRF)
normally present in the plasma to clot by the conversion of fibrinogen to fibrin. Coagulase does not clot plasma of guinea pigs
because they lack CRF. There are 8 antigenic types of coagulase (designated as A to H).Most human strains of S. aureus
produce coagulase A.
All coagulase producing staphylococci are S. aureus and as a result coagulase production is considered the best laboratory
evidence for the potential pathogenicity of Staphylococcus.
Coagulase may act to coat the bacterial cells with fibrin rendering them resistant to opsonization and phagocytosis.
KEY FACTS
1 Two types of coagulase are produced by S. aureus. These are bound coagulase, and tube coagulase.
2 Bound coagulase is tested by slide coagulase test.
3 Free coagulase is tested by tube coagulase test.
4 In the tube coagulase test, on continued incubation, the clot already formed may be lysed by fibrinolysin secreted by some strains.
5 Rabbit plasma with EDTA is used in both tests.
6 Pooled human plasma can be used after checking with a standard strain of S. aureus.
70 Coagulase Test
VIVA
1 What is the principle of slide coagulase test?

2 What is the principle of tube coagulase test?
3 What is the positive control in the test?
4 What is the negative control in the test?
5 How will you interpret the slide coagulase test?
6 How will you interpret the tube coagulase test?
7 Give examples of coagulase positive bacteria.
FURTHER READINGS
2002.
LESSON
Urease Test
23
Inoculating wire, Christensen’s urea agar, and 12 × 100 mm test
After completing this practical you will be able to: tubes.
1 Demonstrate the presence of urease, an intracellular enzyme III Specimen

in the bacteria. Pure growth of Proteus mirabilis from solid media preferably
2 Distinguish the bacteria based on the urease activity. from non-blood agar plates (Examples: nutrient agar, Muller-
Hinton agar) is tested.
INTRODUCTION
PROCEDURE
Certain bacteria and fungi possess the enzyme urease that
hydrolyzes urea releasing ammonia into the medium. This 1 Pick up the colonies of P. mirabilis from the culture on
produces a change in the pH of the medium that can be detected nutrient agar.
by the color change in the indicator dye. This test can be used 2 Inoculate Christensen’s urea agar slope with these bacterial
to differentiate different groups of bacteria and fungi. colonies.
3 Incubate the tube at 37°C for 18 hours.
4 Observe any change of colour in the inoculated medium.
PRINCIPLE
Urea is a diamide of carbonic acid. Urease, the enzyme produced BOX 23-1 CHRISTENSEN’S UREA AGAR
by the bacteria and fungi, hydrolyses urea and releases ammonia
and carbon dioxide. Ammonia reacts in solution to form Composition
ammonium carbonate, which is alkaline leading to an increase Peptone – 0.1 gm.
in pH of the medium. Phenol red that is incorporated in the Glucose – 0.1 gm.
medium changes its color from yellow to red in alkaline pH, NaCl – 0.5 gm.
thus indicating the presence of urease activity. The composition Monopotassium phosphate – 0.2 gm.
and preparation of Christensen’s urea agar is described in the Phenol red (1.2%) – 1.0 ml.
box 23-1. Agar – 2 gm.
List of urease producing microorganisms is summarized in Distilled water – 100 ml.
the Table 23-1. pH – 6.8.
Preparation
REQUIREMENTS Prepare the base, sterilize by autoclaving at 121°C for 15
min. Cool to 50°C in a waterbath and then add 5 ml of filter
I Equipments sterilized 40% urea solution. Mix, distribute in 2–4 ml amounts
Incubator. 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.
72 Urease Test
Table 23-1 Urease producing bacteria and fungi
Urease producing bacteria
Strong (or) most rapid urease producers
Brucella species
Helicobacter pylori
Rapid urease producers
Proteus species
Morganella species
Slow urease producers
Klebsiella species
Enterobacter species
Urease producing fungi NEGATIVE POSITIVE
Cryptococcus neoformans
Trichophyton mentagrophytes
FIGURE 23-1 Urease negative and positive test.
QUALITY CONTROL
The uninoculated medium is colour less. In a positive test, after
Positive control: P. mirabilis (urease positive bacteria). incubation, the colour of the medium changes to purple pink.
Negative control: Escherichia coli (urease negative bacteria).
An uninoculated medium is incubated along with the test to
compare the colour change. RESULTS AND INTERPRETATION
Positive reaction is detected after 18 hours of incubation. When

OBSERVATION positive, the color of the medium changes to purple pink
(P. mirabilis).The test is considered negative if no colour
change of the medium is observed (E. coli) (Fig. 23-1).
Examine the medium after four hours and after overnight
P. mirabilis tested is a urease producing bacteria. E. coli
incubation.The test should not be considered negative till after
does not produce the enzyme urease.
four days of incubation.
KEY FACTS
1 Certain bacteria and fungi possess the enzyme urease that hydrolyzes urea releasing ammonia into the medium.
2 Phenol red that is incorporated in the medium changes its color from yellow to red in alkaline pH, thus indicating the
presence of urease activity.
3 Control strains should be used for interpretation of results.
VIVA
1. Describe principle of the urease test.

2. List different media used for testing urease activity of the microorganism and how do you interpret the results?
Ans. Christensen’s urea agar is a solid medium whereas Stuart’s broth is a liquid medium used for testing the urease activity
of the bacteria.
In Christensen’s agar, purple pink colour is seen throughout the medium which indicates alkalinization and urea hydrolysis.
In Stuart’s broth, purple pink colour throughout the medium indicates alkalinization and urea hydrolysis.
3. List the compositions of the Christensen’s urea agar medium.
4. Name the slow, rapid, and most rapid urease producing bacteria.
5. List urease producing microorganisms.
6. List an important use of urease test in mycology.
Ans: Urease test is used in mycology for identification of urease positive Cryptococcus neoformans.
FURTHER READINGS
2002.
74
LESSON
Indole Test
24
LEARNING OBJECTIVES Kovac’s reagent consists of para-dimethyl amino
benzaldehyde, 5.0 gm; isoamyl alcohol, 75.0 ml; and
After completing this practical you will be able to: concentrated hydrochloric acid, 25.0 ml.
1 Determine the ability of bacteria to degrade the amino acid Ehrlich’s reagent consists of p-dimethyl amino
tryptophan. benzaldehyde, 4.0 gm; absolute ethyl alcohol, 380.0 ml; and
2 Distinguish the bacteria based on the indole activity. concentrated hydrochloric acid, 80.0 ml.
III Specimen
24 hours to 48 hours peptone water culture of Escherichia coli
INTRODUCTION
incubated at 37°C.
Tryptophan is an essential amino acid that can undergo
oxidation by enzymatic activities of some bacteria. Conversion PROCEDURE
of tryptophan into metabolic products is mediated by the
enzyme tryptophanase. The metabolic end products are indole, 1 Take 0.5 ml of 24 hours to 48 hours peptone water cultures of
skatole and indole acetic acid. The ability to hydrolyse E. coli in a small test tube.
tryptophan with the production of indole is not a characteristic 2 Add 0.2 ml of Kovac’s reagent to the peptone water and shake.
of all bacteria.Only some bacteria produce indole. 3 Allow it to stand for few minutes and read the result.
PRINCIPLE QUALITY CONTROL
Tryptophan is an amino acid.This is present in peptone water Positive control: E. coli (indole positive bacteria).
of the culture medium, and when acted upon by the enzyme Negative control: Klebsiella pneumoniae (indole negative
tryptophanase, it is converted into indole, skatole and indole bacteria).
acetic acid. The indole reacts with aldehydes to produce a red
coloured product. The aldehyde used in the test is para dimethyl
amino benzaldehyde.
OBSERVATION
List of indole positive and negative bacteria are presented
in the Table 24-1 In a positive test, a red-violet ring develops within minutes on
addition of Kovac’s reagent. In a negative test a yellow ring
appears.
REQUIREMENTS
I Equipments
Incubator. Positive indole test is indicated by the appearance of red-violet
ring on adding the reagent. Negative reaction is indicated by
II Reagents and lab wares developing a yellow ring (Fig. 24-1). E. coli colonies tested are
Peptone water / tryptone broth, Kovac’s reagent, or Ehrlich’s an indole producing bacteria. K.pneumoniae does not produce
reagent, glass tubes and inoculating wire. the indole
Table 24-1 List of indole positive and negative bacteria
Indole positive bacteria Indole negative bacteria
1. Escherichia coli 1. Escherichia vulnaris

2. Klebsiella oxytoca 2. Klebsiella pneumoniae
3. Proteus vulgaris 3. Proteus mirabilis
4. Morganella morganii 4. Salmonella Typhi
5. Providencia rettgeri 5. Shigella sonnei
6. Aeromonas hydrophila
7. Pasteurella multocida
8. Vibrio cholerae
NEGATIVE POSITIVE
9. Falvobacterium
10. Plesiomonas shigelloides
FIGURE 24-1 Indole negative and positive test.
KEY FACTS
1 End product of tryptophan metabolism by tryptophanase is indole, skatole, and indole acetic acid.
2 Indole reacts with p-dimethyl amino benzaldehyde to form Quinoidal red-violet compound.
VIVA
1 What are the end products of tryptophan metabolism?

2 What is the principle behind the indole production test?
3 What are the reagents used in this test?
4 What are the constituents of Kovac’s reagent?
5 What are the constituents of Ehrlich’s reagent?
6 How to test indole production by adding Ehrlich reagent.
Ans.Take 0.5 ml of culture broth and mix with equal volumes of Ehrlich’s reagent.Shake and allow it to stand for few minutes
then observe the result. Positive reaction is indicated by development of red-violet ring and negative by yellow ring.
This can be also tested in another method by adding 0.5-1 ml of xylene or ether to the culture broth and shaking it well.
Then 0.5 ml of Ehrlich’s reagent is added and the result read.
FURTHER READINGS
2 Forbes BA, Sahm DF and Weissfeld AS (Eds). Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby
Company, St. Louis) 2002.
4 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC (Eds). Color Atlas and Textbook of Diagnostic
Microbiology. 5th ed. (Lippincott Williams and Wilkins, USA). 1997.
76
LESSON
Methyl Red Test
25
LEARNING OBJECTIVES The methyl red test is a quantitative test for acid production,
requiring positive organisms to produce strong acids (lactic
After completing this practical you will be able to: acid, acetic acid, formic acid) from glucose through the mixed
acid fermentation pathway. Because many species of the
1 Determine the ability of bacteria to oxidise glucose with the Enterobacteriaceae may produce sufficient quantities of strong
production of high concentrations of acidic end products acids that can be detected by methyl red indicator during the
by methyl red test. initial phase of incubation, only organisms that can maintain
2 Differentiate between all glucose oxidizing enteric bacteria this low pH after prolonged incubation (48–72 hours)
particularly Escherichia coli and Enterobacter aerogenes. overcoming the pH buffering system of the medium can be
called methyl red positive.
List of MR positive and negative bacteria is presented in
INTRODUCTION the table 25-1.
All enteric bacteria ferment glucose with the production of

organic acids and energy.The hexose monosaccharide glucose REQUIREMENTS
is the major substrate oxidized by the enteric bacteria. The end
products of this process vary depending on the specific I Equipments
enzymatic pathways present in the bacteria. Incubator.
In MR test the methyl red is the pH indicator. The methyl
red detects the presence of large concentrations of acidic end II Reagents and lab wares
products.This test is of value to differentiate between E. coli Inoculating wire.
and E. aerogenes, and other members of the family Methyl red test broth. It consists of poly peptone, 7 gm;
Enterobacteriacae. Both these bacteria initially produce organic glucose, 5 gm; dipotassium phosphate, 5 gm; and distilled water,
acidic products during the early period of incubation. The low 1l at a pH of 6.9.
acidic pH (4) is stabilized and maintained by E. coli at the end Methyl red indicator. It consists of methyl red, 0.1 g in 300
of incubation. During the later period of incubation, E. ml of 95% ethyl alcohol.
aerogenes enzymatically converts these acids to non-acidic
end products such as 2, 3 – butanediol and acetoin (acetyl III Specimen
methyl carbinol) resulting in an elevated pH of 6. Culture of E. coli, E. aerogenes and Klebsiella pneumoniae in
glucose phosphate medium incubated at 30°C for five days.
PRINCIPLE
PROCEDURE
Methyl red is a pH indicator with a range between 6.0 (yellow)
and 4.4 (red). The pH at which methyl red detects acid is 1 Take 0.5 ml of broth cultures of E. coli in a small test tube.
considerably lower than the pH of other indicators used in 2 Add five drops of 0.04% solution of methyl red directly to
bacteriologic culture media. Thus to produce a colour change, the broth culture and mix well.
the test organism must produce large quantities of acid from 3 Note any change in the colour of medium at once.
the carbohydrate substrate being used.
Table25-1 List of MR positive and negative bacteria RESULTS AND INTERPRETATION

MR positive bacteria MR negative bacteria The development of a stable red colour in the surface of the
medium indicates sufficient acid production to lower the pH to
1. E. coli 1. K. pneumoniae
2. K. ozaenae 2. Enterobacter spp 4.4 and constitutes a positive test. Because other organisms
3. K. rhinoscleromatis may produce smaller quantities of acid from the test substrate,
4. K. ornitholytica an intermediate orange colour between yellow and red may
5. Edwardsielleae develop. This does not indicate a positive test. Yellow colour
6. Salmonellae indicates a negative test (Fig. 25-1).
7. Citrobacter
8. Proteae
9. Yersinia
QUALITY CONTROL
Positive control: E. coli

Negative control: E. aerogenes
OBSERVATION INDOLE MR VP CITRATE
Look for the development of stable red colour on adding methyl FIGURE 25-1 IMViC Test.
red indicator.
KEY FACTS
1 MR test detects the products of stable high concentration of acidic end products.
2 The test differentiates between E. coli and E. aerogenes.
3 Methyl red is the indicator used in the test.
4 Methyl red is yellow in alkaline pH and red in acidic pH.
5 Development of red colour on addition of methyl red to broth culture of bacteria is considered positive.
VIVA
1 What are the group of organisms that can be differentiated by MR test?

2 What is the principle of MR Test?
3 What are positive and negative controls used in the test?
4 How long you have to incubate the test and why?
Ans: The broth is incubated for 48-72 hr, because the organisms have to produce sufficient quantities of acid and it has to
be maintained in the same acidic pH which turns the methyl red indicator to red colour.
5 What is the indicator used in this test?
FURTHER READINGS
2002.
78
LESSON
Voges-Proskauer Test
26
LEARNING OBJECTIVES small amount of acetyl methyl carbinol present in the medium
is converted to diacetyl, which reacts with the peptone of the
After completing this practical you will be able to: broth to produce a red colour.
List of VP positive and negative bacteria is presented in the
table 26-1.
1 Perform Voges-Proskauer test.
REQUIREMENTS
INTRODUCTION
I Equipments
Voges-Proskauer is a double eponym, named after two Incubator.
microbiologists working at the beginning of the 20th century.
They first observed the red colour reaction produced by II Reagents and lab wares
appropriate culture media after treatment with potassium
hydroxide. It was later discovered that the active product in Inoculating loop.
the medium formed by bacterial metabolism is acetyl methyl VP broth. It consists of polypeptone, 7 gm; glucose,5 gm;
carbinol, a product of the butylene glycol pathway. dipotassium phosphate, 5 gm and distilled water, 1 litre at a pH
of 6.9.
5% a naphthol. It consists of a naphthol, 5 gm; and absolute
PRINCIPLE ethyl alcohol, 100 ml. It serves as the colour intensifier.
40% potassium hydroxide. It consists of 40 gm potassium
The Voges-Proskauer test determines the capability of some hydroxide in 100 ml distilled water. It serves as the oxidising
bacteria to produce non-acidic or neutral end products such as agent.
acetyl methyl carbinol from the organic acids produced as a
result of glucose metabolism. Pyruvic acid, the pivotal III Specimen
compound formed in the fermentative degradation of glucose Culture of Escherichia coli, Enterobacter aerogenes and
is further metabolised through various metabolic pathways, Klebsiella pneumoniae in glucose phosphate medium
depending on the enzyme systems possessed by different incubated at 30°C for five days or 37°C for 48 hours.
bacteria.
One such pathway results in the production of acetoin
(acetyl methyl carbinol), a neutral-reacting end product. Enteric PROCEDURE
bacteria such as members of the Klebsiella-Enterobacter-
Hafnia-Serratia group produce acetoin as the chief end 1 Take 1 ml of broth cultures of E. coli in a small test tube.
products of glucose metabolism and form smaller quantities of 2 First add 40% KOH and then add 0.6 ml of a 5% solution of
mixed acids. a naphthol in ethanol to the broth culture and shake gently.
The test depends on the production of acetyl methyl It is essential that the reagents are added in this order.
carbinol from pyruvic acid, as an intermediate product in its 3 Note any change in the colour of medium within 2-5 minutes.
conversion to 2: 3 butylene glycol. In the presence of
atmospheric oxygen and alkali (40% potassium hydroxide), the
QUALITY CONTROL hour.The test should not be read after standing for over 1 hour
because negative VP test may produce a copper-like colour,
Positive control: E. aerogenes. leading to a false positive interpretation.
Negative control: E. coli.
Table 26-1 VP positive and negative bacteria

OBSERVATIONS
VP positive bacteria VP negative bacteria
Look for the development of pink colour 15 minutes or more
after addition of the reagents. 1. Klebsiella pneumoniae 1. Escherichia coli
2. Enterobacter cloacae 2. Edwardsiella tarda
3. Cedicia netri 3. Salmonellae
4. Ewingella americana 4. Proteae
5. Serratia marcescens 5. Yersinieae
6. Aeromonas sobria
A positive test is represented by the development of a pink
7. Vibrio cholerae
colour 15 minutes or more after addition of the reagents,
8. Chryseomonas luteola
deepening to magenta or crimson in half an hour.This indicates
9. Flavimonas oryzihabitans
the presence of diacetyl, the oxidation product of acetoin. A
10. Sphingomonas paucinobilix
negative test is indicated by colour less reaction for half an
KEY FACTS
1 The VP test shows the presence of non acidic neutral end product acetyl methyl carbinol.
2 A positive test is represented by the development of a pink colour 15 minutes or more after addition of the reagents,
deepening to magenta or crimson in half an hour.
3 A negative test is indicated by colour less reaction for half an hour.
4 Some times prolonged incubation of cultures is required before doing the test.
VIVA
1 What is the principle of VP test?

2 What are the positive and negative controls used in the VP test?
3 Name some VP positive bacteria?
4 Describe the biochemical reaction in the VP test.
FURTHER READINGS
2002.
80
LESSON
Citrate Utilisation Test
27
After completing this practical you will be able to: I Equipments

1 Differentiate certain enteric organisms on the basis of their Incubator.
ability to utilize citrate as a sole source of carbon.
II Reagents and lab wares
Inoculating loop, Simmon’s citrate medium (It consists of
INTRODUCTION ammonium dihydrogen phosphate, 1 gm; dipotassium
phosphate, 1 gm; sodium chloride, 5 gm; sodium citrate, 2 gm;
Sodium citrate is a salt of citric acid, a simple organic compound magnesium sulfate, 0.20 gm; agar, 15 g; bromo thymol blue,
produced as one of the metabolites in the tricarboxylic acid 0.08 gm and distilled water 1 litre) pH adjusted to 6.9. The
cycle of the bacteria. Some bacteria can also obtain energy by medium is poured into a tube on a slant.
using citrate as the sole source of carbon. Hence, any medium
used to demonstrate citrate utilisation by test bacteria must be III Specimen
devoid of protein and carbohydrates as sources of carbon. Culture of Escherichia coli, Enterobacter aerogenes and
Klebsiella pneumoniae in glucose phosphate medium
incubated at 37°C for 48 hours.
PRINCIPLE
In the absence of fermentable glucose or lactose, some bacteria PROCEDURE

are capable of using citrate as a sole source of carbon for their
energy. This ability depends on the presence of the enzyme, a 1 Using sterile technique, inoculate each bacteria into its
citrate permease that facilitates the transport of citrate in the cell. appropriately labeled tube by means of a stab and streak
Citrate is the first major intermediate in the Krebs cycle and inoculation.
is produced by the condensation of active acetyl with oxaloacetic 2 Incubate all cultures for 24 hours to 48 hours at 37°C.
acid. Citrate is acted on by the enzyme citrase which produces
oxaloacetic acid and acetate. These products are then
Table 27-1 Differences between Simmon’s citrate and
enzymatically converted to pyruvic acid and carbon dioxide
Koser’s citrate
(CO2). During this reaction the medium becomes alkaline
because the CO2 that is generated combines with sodium and
Simmon’s citrate Koser’s citrate
water to form sodium carbonate, an alkaline product. The
presence of sodium carbonate changes the indicator, bromo It is a slant (solid medium) It is a broth (liquid medium)
thymol blue present in the medium from green at pH 6.9 to deep It contains agar It contains no agar
Prussian blue at pH 7.6. This medium contains It does not contain any
Simmon’s citrate and Koser’s citrate are two examples of bromothymol blue as indicator indicator
different types of citrate media used in the test. Differences Positive test is indicated by Positive test is by observing
between the two media are summarized in the table 27-1. growth on the medium and the turbidity in the medium
List of citrate positive and negative bacteria are presented change in the colour of
in the table 27-2. the medium.
QUALITY CONTROL RESULTS AND INTERPRETATION
Positive control: E. aerogenes. A positive test is represented by the development of a deep

Negative control: E. coli. blue colour within 24 hours to 48 hours, indicating that the test
organism has been able to utilize the citrate contained in the
medium, with the production of alkaline products. A negative
OBSERVATIONS test is indicated by no change of colour of the citrate medium
(Fig. 27-1).
Look for the development of deep blue colour within 24-48
hours of incubation of the inoculated tube.
Table 27-2 List of citrate positive and negative bacteria
Citrate positive bacteria Citrate negative bacteria
1. Klebsiella pneumoniae 1. Escherichia coli

2. Citrobacter diversus 2. Salmonella Typhi
3. Enterobacter cloacae 3. Salmonella Paratyphi A
4. Serratia marcescens 4. Shigella species
5. Providencia alcalifaecians 5. Yersinia enterocolitica
6. Euringella americana 6. Edwardsiella tarda NEGATIVE POSITIVE
7. Acroncobacter oxylosoxidans 7. Vibrio holisae
8. Vibrio vulnificus
FIGURE 27-1 Citrate negative and positive test.
KEY FACTS
1 Simmon’s citrate medium contains sodium citrate which acts as a sole source of carbon.
2 Bromothymol blue is the indicator used in Simmon’s citrate medium.
3 Development of deep blue colour is taken as positive.
4 A negative test is indicated by no change of colour of the citrate medium.
VIVA
1 What are the constituents of Simmon’s citrate medium?
2 What is the principle behind citrate utilisation test?
3 What is the positive control and negative controls used in the citrate utilisation test?
4 What are the organisms that are citrate utilisation positive and negative?
5 How will you interpret the results of citrate utilisation test?
Ans: A positive test is represented by the development of a deep blue colour within 24 hours to 48 hours indicating that the
test organisms has been able to utilize citrate contained in the medium, with the production of alkaline products.
6 Mention different types of citrate utilization tests.
Ans: Simmon’s citrate utilization test and Koser’s citrate utilization test.
7 List differences between Koser’s citrate and Simmon’s citrate media.
FURTHER READINGS
2002.
82
LESSON
Triple Sugar Iron (TSI)
28 Agar Test
LEARNING OBJECTIVES to yellow and the whole medium appears yellow in colour. After
further incubation as the glucose is fully exhausted, the bacteria
After completing this practical you will be able to: begin to oxidatively degrade the amino acid present in the
medium. Since oxygen is exposed only to the slant portion,
1 Differentiate among the members of the family oxidative degradation occurs only in the slant portion. This
Enterobacteriaceae by the TSI test. oxidative degradation results in production of alkali products,
which reverts the colour of the slant to red colour. In the deeper
part of the tube, amino acid degradation is insufficient to
INTRODUCTION overcome the acid formed, so medium in the butt part remains
yellow in colour.
The triple sugar iron (TSI) agar is an example of a composite If the TSI medium is inoculated with lactose fermenting
medium used widely for the identification of bacterial organism, then even after the glucose is completely used up in
isolates.This medium is convenient and economical, because first 8–12 hours, fermentation continues as the organism is able
as a single composite medium different properties of the bacteria to use lactose which is present in concentration 10 times that of
which otherwise would have required the use of many separate glucose. So the acid production continues to occur even after
media could be used. 18–24 hours and both the slant and the butt appear yellow.
The TSI agar is designed to differentiate among different For the detection of H2S which is a colourless gas, medium
groups or genera of the family Enterobacteriaceae. The latter must include an indicator to detect the H2 S. Sodium thiosulfate
are the Gram Negative bacilli capable of fermenting glucose is the source of sulfur atoms. Ferrous sulfate is the indicator
with the production of acid. The differentiation can be made on used for the detection of the H2S which is indicated by the
the basis of differences in carbohydrate fermentation and production of insoluble black precipitate.
hydrogen sulfide production by various intestinal bacteria.
The TSI agar has glucose, lactose, and sucrose as the sources
of carbohydrates. The slant contains lactose and sucrose in the REQUIREMENTS
concentration of 1% and glucose in the concentration of
0.1%.Phenol red is the acid base indicator incorporated in the I Equipments
medium.This indicator helps to detect carbohydrate fermentation Incubator.
that is indicated by a change in colour of the medium from orange
red to yellow in the presence of acid. II Reagents and lab wares
Inoculating loop and triple sugar iron agar (It contains beef
extract, 3g; yeast extract, 3g; peptone, 15g; proteose peptone,
PRINCIPLE 5g; lactose, 10g; sucrose, 10 gm; glucose, 1 gm; ferrous sulfate,
0.2 gm; sodium chloride, 5g; sodium thiosulfate, 0.3g; agar, 12
The TSI agar is distributed in the tube which contains a slant gm; phenol red, 0.024 g; and distilled water to equal 1 litre) at
and a butt.TSI medium indicates whether the bacteria ferments pH 7.4.
glucose only, or lactose and sucrose also with or without
production of gas. The medium can detect production of III Specimen
hydrogen sulphide (H2S) as well as other bacteria which utilizes Culture of test organisms such as Escherichia coli, Proteus
only glucose (but not lactose or sucrose). Due to the acid mirabilis and Klebsiella pneumoniae in glucose phosphate
production, the colour of the phenol red (indicator) is changed medium incubated at 37°C for 48 hours.
PROCEDURE
1 Using sterile technique, inoculate each bacterial colony into

its appropriately labeled tube by means of a stab and streak
inoculation.
2 Incubate all cultures for 18 hours to 24 hours at 37°C.
QUALITY CONTROL
1 Alkaline slant /alkaline butt (K/K reaction): Pseudomonas

aeruginosa.
2 Alkaline slant /acidic butt (K/A reaction): Shigella, Vibrio.
3 Alkaline slant /acidic butt /production of H2 S (K/A reaction
and positive for H2 S): Salmonella spp, Citrobacter spp, A+/A- Un inoculated K-/K- K+/A+
Proteus spp.
4 Acidic slant /acidic butt (A/A reaction): E. coli, Klebsiella FIGURE 28-1 TSI reactions.
spp, Enterobacter spp.
5 Acid butt/acid slant, H2 S positive: Citrobacter.
2 Alkaline slant /acidic butt (K/A reaction): Glucose is

OBSERVATIONS fermented. Lactose and sucrose is not fermented. It indicates
that the organism is a non -lactose fermenter. Example:
Look for the colour change in the slant and butt after 18–24 Shigella, Vibrio.
hours incubation and also look for the development of black 3 Alkaline slant /acidic butt /black precipitate of H2S (K/A
precipitate to indicate H2S production (Fig.28-1). reaction and positive for H2S): Glucose is fermented.
Lactose and sucrose are not fermented. This is
characteristic of non -lactose fermenting, hydrogen sulfide
producing bacteria. Example: Salmonella spp, Citrobacter
spp, Proteus spp.
4 Acidic slant /acidic butt (A/A reaction): All the sugars
1 Alkaline slant /alkaline butt (K/K reaction): This shows no
(glucose, lactose, and sucrose) are fermented. This is
carbohydrate fermentation. This indicates that the bacteria
characteristic of lactose fermenting coliforms. Example E.
are non-fermented. Example: P. aeruginosa.
coli, Klebsiella spp, Enterobacter spp.
VIVA
1. What are the constituents of TSI medium?

2. What is the source of sulfur?
3. What is the source of nutrition?
Ans: Sucrose, lactose, glucose and peptone
4. What are the sugars used and at what concentration they are used in the TSI agar?
5. What is the principle behind this test?
6. What is the H2S indicator?
7. When the reading of TSI test is taken and why?
Ans: The readings of TSI test is taken only after 18-24 hrs.
The reason behind this is that the glucose concentration is 10 times less than that of lactose and sucrose. Glucose
fermenters break down glucose first then lactose. If the organism is non lactose or sucrose fermenter, the organisms
metabolise peptones aerobically for their energy, producing alkaline end products. In the first 6-8 hours whole of the tube
will be acidic and will be yellow in colour.
Production of alkaline end products results in the change of pH alkaline side and pink colour develops in the slant. So the
pH of the slant is alkaline after 8 hours. So the results are read after 24 hours.
84 Triple Sugar Iron (TSI) Agar Test
KEY FACTS
1 Beef extract, yeast extract, peptone, and proteose peptone makes the medium nutritionally rich.
2 Glucose and lactose; and sucrose are added in the ratio of 1: 10 in the TSI agar.
3 Oxidative degradation of amino acids in the medium occurs only in the slant.
4 Ferrous sulfate is the indicator for the production of the hydrogen sulfide.
5 Sodium thiosulfate is the source of sulfur.
6 Phenol red is the indicator used to detect acid /alkaline changes in the medium.
FURTHER READINGS
2 Forbes BA, Sahm DF and Weissfeld AS (Eds). Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby
Company, St. Louis) 2002.
LESSON
Hydrogen Sulfide Test
29
LEARNING OBJECTIVES reduction of inorganic sulfur compounds such as the
thiosulfates (S2O3), sulfates (SO4) or sulfites (SO3). The medium
After completing this practical you will be able to: contains sodium thiosulfate, which are reduced to sulfite by
certain microorganisms with the liberation of hydrogen sulfide.
1 Determine the ability of certain bacteria to produce hydrogen The sulfur atoms act as hydrogen acceptors during oxidation
sulfide from substrates such as the sulfur containing amino of the inorganic compound. Liberated H2S combines with
acids or inorganic sulfur compounds. indicator like ferrous sulfate (Fe SO4) to produce black insoluble
precipitate (ferrous sulfide).
List of media used for detecting production of hydrogen
INTRODUCTION sulphide is presented in the box 29-1.
List of bacteria producing hydrogen sulphide is presented
Some bacteria liberate sulfur from sulfur containing amino acids in the table 29-1.
or other sulfur containing compounds. The sulfur is used as
final hydrogen acceptor leading to the formation of hydrogen
sulphide (H2S). Sulfur containing amino acids such as REQUIREMENTS
methionine, cystine, cysteine or inorganic compounds such as
this sulphates, etc. should be present in the medium to detect I Equipments
the presence of the H2 S. H2S being a gas, will escape from the Incubator.
medium. So, the indicators such as heavy metal ions are added
to the medium, which support the growth of the organism. II Reagents and lab wares
Bacteria which are tested for the production of H2 S must contain Sulphur containing medium such as Kligler iron agar, TSI agar
enzyme system which release sulfide from the sulfur source. or lead acetate agar, etc. and inoculating loop.
Sulfides combine with hydrogen ion to form H2 S. H2 S combines
with heavy metals to form insoluble black precipitate. III Specimen
Soy broth cultures of test bacteria such as Enterobacter
aerogenes, Proteus vulgaris and Salmonella Typhimurium
PRINCIPLE incubated at 37°C for 24- 48 hours.
There are two major fermentative pathways by which hydrogen

sulfide is produced by bacteria. PROCEDURE
Pathway 1: Gaseous H2S may be produced by the reduction
(hydrogenation) of organic sulfur present in the amino acid 1 Using sterile technique, pick up the organisms from the top
cysteine, which is the source of sulfur in the medium. This of a single colony from primary isolation plate or from pure
amino acid in the presence of the enzyme, a cysteine growth with a straight wire.
desulfurase, loses the sulfur atom and is then reduced by the 2 Inoculate by stabbing down the center of agar butt carefully
addition of hydrogen from water to form hydrogen gas. and then streak the surface of the slant.
Pathway 2: Gaseous H2S may also be produced by the 3 Incubate all cultures for 18 hours to 24 hours at 37°C.
86 Hydrogen Sulphide Test
BOX 29-1 LIST OF MEDIA USED FOR Table 29-1 List of H2S positive bacteria
DETECTING PRODUCTION OF
1 Citrobacter freundii
HYDROGEN SULFIDE
2 Salmonella Arizona
List of media 3 Salmonellae spp (except S. Paratyphi A)
1. Bismuth sulfite agar 4 Proteus mirabilis
2. Citrate sulfide agar 5 Proteus vulgaris
3. Deoxycholate citrate agar 6 Edwardsiella tarda
4. Lysine iron agar. 7 Edwardsiella hoshinae
5. Kligler iron agar 8 Shewanella putrefaciens
6. TSI agar 9 Campylobacter sputorum
7. Lead acetate agar 10 Brucella abortus
8. Salmonella-Shigella agar 11 Brucella suis.
9. Sulphur indole motility medium 12 Erysiphilothrix rhusiopathiae
10. Xylose lysine deoxycholate agar
11. Hektoen enteric agar
12. Peptone water, lead acetate paper inserts. RESULTS AND INTERPRETATION
Note: Source of sulfur and H2S indicators are different for
each medium. Black coloration along the streak line or throughout the medium
indicates H2S production. If black colour is not produced then
Sources of sulfur H2S is not produced (Fig. 29-1).
1. Sulfite
2. Peptone
3. Sodium thiosulfate
H2S indicator
1. Ferrous sulfate
2. Ferric ammonium citrate
3. Lead acetate
4. Peptonised iron.
QUALITY CONTROL
Positive control: P. vulgaris, S. Typhimurium.

Negative control: E. aerogenes.
OBSERVATIONS NEGATIVE POSITIVE
Look for the development of black coloured streak line after

18–24 hours incubation. FIGURE 29-1 TSI agar showing absence and presence of H2S.
VIVA
1 What are the conditions required for the production and demonstration of H2S by bacteria?
2 What are the sequence of events occurring in the production and detection of H2S?
3 What is the source of sulfur in the media?
4 What are the indicators of H2S production?
5 What are the sulfur containing amino acids?
6 How will you pick up the colonies for inoculation?
7 What are the media that can be used in H2S production detection?
8 Name some H2S producing bacteria.
KEY FACTS
1 Requirements to demonstrate H2S production.

• A source of sulfur in the medium.
• A H2S indicator in the medium.
• Medium must support the growth of bacteria being tested.
• The bacteria must possess the enzyme system to produce H2S.
2 First step in the production of H2S is release of sulfide ions from sulfur containing amino acids or thiosulfates by bacterial
enzymes.
3 Second step is the coupling of sulfide to H+ ions to form H2S.
4 Third step is H2S reacting with heavy metals like iron, bismuth, lead to produce insoluble heavy metal sulfides that appear
black precipitate.
5 Cysteine, cystine, methionine are some sulfur containing amino acids.
6 Only tops of the colonies growing on selective media must be touched, since inhibited flora may still be present and viable.
These may interfere with the reaction. Otherwise single colony of purified culture must be used.
7 Sources of sulfur in the medium may be sulfite, sodium thiosulfate, or peptone.
8 Different H2S indicators used in various media are ferrous sulfate, ferric ammonium citrate, lead acetate and peptonised iron.
FURTHER READINGS
2002.
88
LESSON
Nitrate Reduction Test
30
LEARNING OBJECTIVES extract, 3 gm; peptone,5 gm; potassium nitrate,1 gm; agar,12
gm, and distilled water, 1 L).
After completing this practical you will be able to: Reagent A (a naphthylamine, 5 g; acetic acid (5N) 30%, 1 L).
1 Determine the ability of certain bacteria to reduce nitrates Reagent B (sulphanilic acid, 8 g; acetic acid (5N) 30%, 1 L).
(NO3–) to nitrites (NO2–) or beyond the nitrite stage. Inoculating loop.
III Specimen
INTRODUCTION Cultures of test bacteria such as Escherichia coli, Acinetobacter
baumannii, etc. in a broth containing 1% potassium nitrate
Nitrates serve as a source of nitrogen for many bacteria. They broth (KNO3) incubated at 37°C for 5 days.
can also act as final electron acceptor. Many bacteria can be
differentiated and are identified by their capacity to reduce
nitrates to nitrites. Most of the bacteria belonging to the family PROCEDURE
Enterobacteriaceae reduce nitrates. This character is also useful
for the identification of species in the genera Neisseria, 1 Mix an equal volume (0.5 ml) of reagent A with 0.5 ml of
Haemophilus and Branhamella. Some Pseudomonas and non- reagent B just before use.
fermenters reduce nitrate to nitrite and further down to N2 and 2 Add 0.1 ml of the test reagent to 1 ml of the culture broth of
molecular nitrogen. This is called denitrification. the bacteria to be tested.
3 Observe for any change of colour immediately within few
minutes.
PRINCIPLE
Bacteria demonstrating nitrate reduction have the capability of ex- QUALITY CONTROL
tracting oxygen from nitrates to form nitrite and other reduction
products. The chemical reaction is NO3–+ 2e– + 2H+®NO2– + H2O. Positive control: E. coli.
The presence of nitrites in the test medium is detected by Negative control: A. baumannii.
the addition of a-naphthylamine and sulphanilic acid, with the
formation of a red diagonium dye, p-sulfobenzena-azo-a-
naphthylamine. OBSERVATION
Observe for the development of red colour immediately within

REQUIREMENTS few minutes of adding the reagents.
I Equipments
Incubator.
II Reagents and lab wares The development of a red colour within 30 seconds. after adding
1% potassium nitrate broth (KNO3) or nitrate agar slant (beef the test reagents indicates the presence of nitrites and
represents positive reaction (Fig. 30-1).
If no colour develops after adding the test reagents, it is

taken as negative test. It might have been reduced to products
other than nitrites such as molecular nitrogen, nitric oxide or
nitrous oxide. In this case, the reaction may show a false negative
reading. Thus it is necessary to add a small quantity of zinc
dust to all negative reactions.
Zinc dusts reduce nitrates to nitrites, and the development
of a red colour after adding zinc dust indicates the presence of
residual nitrates and confirms a true negative reaction. NEGATIVE POSITIVE
FIGURE 30-1 Nitrate reduction test.
KEY FACTS
1 Nitrates are reduced to nitrites by some organisms.

2 Two reagents used in the test are: a naphthylamine and sulphanilic acid.
3 Zinc dust is added to detect all negative reactions.
VIVA
1 What is the principle behind nitrate reduction test?

2 What are the ingredients present in nitrate broth?
3 What are reagents added in the reactions?
4 What is the positive and negative control for nitrate reduction test?
5 What is the purpose of adding zinc dust to all negative reaction?
Ans: Negative reaction in nitrate reduction test may be of 2 types. One is the true negative reaction in which nitrates are not
reduced to nitrites or in false negative reactions the nitrates are reduced to products other than nitrites such as N2, NO or
N02. The reagents can detect only the presence of nitrites. Zinc ions reduces nitrates to nitrites and the development of a red
colour after adding zinc dust, indicates the presence of residual nitrates and confirms a true negative reaction.
6 How will you avoid false negative reaction? Explain.
FURTHER READINGS
2002.
90
UNIT
V
Antimicrobial
Sensitivity Tests
Lesson 31 Kirby-Bauer Method

Lesson 32 Stoke’s Method
Lesson 33 Agar Dilution Method
Lesson 34 Broth Dilution Method
Lesson 35 Epsilometer Test (E-test)
92
LESSON
Kirby–Bauer Method
31
LEARNING OBJECTIVES II Reagents and lab wares
0.5 McFarland standard, Mueller Hinton agar plates (pH 7.2-
After completing this practical you will be able to: 7.4), peptone water , filter paper discs impregnated with
appropriate concentration of antibiotics, sterile cotton swabs,
1 Determine antibacterial sensitivity of bacterial isolates by millimeter ruler, forceps and inoculating wire.
Kirby –Bauer disc diffusion method. Preparation of 0.5 McFarland standard: Solution A is
prepared by adding barium chloride (BaCl 2, 2H2O) to 100ml
distilled water. Solution B is prepared by adding 1ml of
INTRODUCTION sulphuric acid (H2S04 (0.36N) to 100 ml of distilled water. Then
0.5 ml of solution A is added to 99.5 ml of solution B, mixed well
Due to emergence of many antibiotic resistant strains of and distributed in test tubes with a screw cap. The cap is closed
bacteria, antimicrobial susceptibility testing is done in order to tightly to avoid evaporation. The mixture is stored in the dark.
determine which antimicrobial agent to use against a specific The solution is agitated vigorously before using it.
strain of bacteria. The available chemotherapeutic agents vary
in their scope of antimicrobial activity. Some have a limited III Specimens
spectrum while others have a wide spectrum of activities against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC
bacteria. The bacterial strains isolated from clinical samples 25922, Enterococcus faecalis ATCC 29212, and Pseudomonas
should be tested for antimicrobial sensitivity because it gives aeruginosa ATCC 27853.
the clinician an idea as to what antimicrobial therapy should be Preparation of suspension of bacteria: Approximately, 4-5
started to the patients (Box 31-1). well isolated colonies of the bacterial strain to be tested are
inoculated into 5 ml of peptone water, and is incubated at 37 °C
for 3-4 hours. The turbidity of the suspension is adjusted to
PRINCIPLE match 0.5 McFarland standards. If the density is more it is
diluted with sterile saline. The comparison is made against a
Kirby – Bauer method is a method of determination of antibiotic white back ground with a contrasting black line.
sensitivity of the bacteria by disc diffusion method. In this
method, a standard suspension of bacteria to be tested are
inoculated on the surface of Mueller Hinton agar plates. Filter PROCEDURE
paper discs containing specific concentration of antimicrobial
agents are pressed on to the surface and incubated at 35°C 1 After standardisation of bacterial suspension, immerse a
overnight (18-24 hr.). After incubation, the zone of inhibition sterile cotton swab in it and rotate the swab several times
of growth of bacteria around each disc is measured and the with firm pressure on the inside wall of the tube to remove
susceptibility is determined. excess fluid.
2 Prepare a Mueller Hinton agar (MHA) plate (pH 7.2-7.4) with
a depth of 4 mm.
REQUIREMENTS 3 Inoculate the dried surface of the MHA agar plate by
streaking the swab three times over the entire agar surface.
I Equipments It is streaked in three directions by rotating the plate 60°
Incubator. after each streak.
4 Place the appropriate antimicrobial impregnated discs on the

surface of the agar using sterile forceps.
5 Gently press each disc onto the agar to provide uniform
contact. Do not move the disc once it has contacted the agar Each antibiotics produces a specific zone size for each bacteria
because some of the antibiotics diffuse almost immediately tested. Depending on the zone size, the bacteria are classified
Discs must be placed in such a way that they are at least 20 as follows (Fig. 31-1):
mm from one another. Sensitive (S): Infection treatable with normal dosage of the
Note: 6 antibiotic discs may be put in an 85 mm plate. antibiotic.
6 Invert the plates and incubate at 35 °C -37°C for 16-18 hr. Intermediate (I): Infection may respond to therapy with higher
dosage
Resistant (R): Unlikely to respond to the antibiotic at the usual
dosage.
QUALITY CONTROL
S. aureus ATCC 25923, E. coli ATCC 25922, E. faecalis ATCC

29212, and P. aeruginosa ATCC 27853 should be tested
periodically.
OBSERVATIONS
1 Examine the plates for the presence and size of inhibitory

zones.
2 The diameter of the inhibitory zone including the diameter of
the disc is measured by using a millimeter scale upto the
nearest millimeter.
3 All measurements are made with unaided eye while viewing
the back of the petri dish with reflected light against a black
non-reflecting background.
4 Measure the inhibitory zones for each antimicrobial agent,
FIGURE 31-1 Kirby-Baur method of antibiotic susceptibility testing.
compare with the standard Kirby-Bauer’s chart and interpret
the zone of inhibition as sensitive, intermediate or resistant.
BOX 31-1 GLOSSARY OF TERMS
Antibiotics: It is a substance produced by microorganisms or a similar substance produced wholly or partially by chemical
synthesis that inhibits the growth or causes death of other organisms in low concentrations.
Antimicrobial agent: It is a chemical substance inhibiting the growth or causing death of microorganism.
Bacteriostatic drug: Certain antimicrobial agents inhibit the growth by preventing the multiplication of organisms. They do
not cause death. These are called bacteriostatic agents. eg. tetracycline, erythromycin, and sulphonamides.
Bactericidal drug: The drugs that cause irreversible damage to bacteria resulting in death are called bactericidal drugs.
e.g. aminoglycosides, penicillin and quinolones.
KEY FACTS
1 Depth of agar in medium should be 4 mm as some antibiotics show decreased zone size with increased depth while others
show slight increase.
2 Standardization of the bacterial inoculum is important. It should be such that it gives rise to a semi confluent growth, as
growth denser than this or lighter than this give problem while reading zone size.
3 Proper storage of the antimicrobial discs, so that they retain their potency.
94 Kirby-Bauer Method
VIVA
1 Define a) antibiotics, b) antimicrobial agent, c) bacteriostatic drug and d) bactericidal drug.

2 What is the importance of antimicrobial susceptibility testing?
3 Why is the standardization of pH of medium important in disc diffusion test?
Ans: Standardization of pH of the medium is important because some antibiotics show increased or decreased zone sizes
depending on pH.
Antibiotics stimulated by fall in pH i.e. larger zone sizes – Tetracycline, methicillin, and novobiocin.
Antibiotics stimulated by an alkaline pH – Aminoglycosides and macrolides such as erythromycin.
4 How are antimicrobial discs prepared?
Ans:
1 Discs are made from Whatman filter paper No. 1 using standard stationary paper punch.
2 Discs are arranged separately in a Petri dish and sterilized in hot air oven at 160°C for 1 hour.
3 A 27 SWG needle, bevel cut off is used to drop the antibiotics. It is attached to a tuberculin syringe fitted with a teat. This
delivers 140 drops/ ml.
4 A 6 mm disc is able to completely absorb the fluid volume (7 µl).
5 Each disc is impregnated with a drop of the solution and allowed to dry in the incubator at 37°C for 30 mins.
6 Any disc that is not to be used on the day of preparation is stored at -20 °C to 8 °C.
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 94-96, 1995.
3 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th
Edition. 1997. pp. 1395. Lippincott Williams and Wilkins.
4 Lalitha M.K. Manual on Antimicrobial Susceptibility Testing. Christian Medical College, Vellore, 2004, pp 43.
5 WHO. Guidelines on Standard Operating Procedures for Microbiology. Blood safety and Clinical Technology. Chapter 7: Antimicrobial
Susceptibility Testing.
LESSON
The Stokes Method
32
LEARNING OBJECTIVES III Specimens
Control strains: Staphylococcus aureus NCTC 6571,
After completing this practical you will be able to: Escherichia coli NCTC 10414 and Pseudomonas aeruginosa
1 Determine antibacterial sensitivity of bacterial isolates by NCTC 10662.
Stokes method. Preparation of suspension of bacteria: Approximately, 4-
5 well isolated colonies of the bacterial strain to be tested are
transferred to Tryptic soy broth or BHI broth. The turbidity
INTRODUCTION of the suspension is adjusted to match 0.5 McFarland
standards.
Stokes method is an example of the disc diffusion test and is
another method used for routine antibiotic sensitivity testing of
bacterial strains. The method makes use of in-built controls against PROCEDURE
many variables and therefore provides dependable results. A set
of standard strains are used as control strains depending on the 1 The inoculation plates are dried with lids open so that there
bacterium to be tested. The control strains are Escherechia coli are no droplets of moisture on the surface.
NCTC 10414 for testing coliform bacilli from urinary tract. 2 The control culture is applied in two bands on either side of
Pseutomonas aeruginosa NCTC 10662 against aminoglycosides. the plate leaving a central band uninoculated with the help
Kirby-Bauer and Stokes methods are compared in the of sterile swab.
table 32-1. 3 The test organism is applied in the central portion without
touching the either sides.
4 Antibiotic discs are applied with forceps on the line between
PRINCIPLE the test and control organisms and pressed gently to ensure
even contact with the medium. There should be a minimum
In this test antibiotic discs are applied between the standard and distance of 2 cm between two discs. Four discs can be
test inocula, so that zones of inhibition formed around each disc accommodated on an 85 mm circular plate.
are composed of standard and test bacteria. The diffusion of 5 For inoculation, a rotatory plating method can also be used
antibiotic takes place and thus the susceptibility of those wherein the control strain is applied on the outer periphery
organisms to the antibiotic are known by measuring zone size. and the test strain is applied in the central portion. In such a
method 6 discs can be put on an 85 mm circular plate.
6 The plates are then incubated overnight at 35°C –37°C.
REQUIREMENTS
I Equipments QUALITY CONTROL

Incubator.
S. aureus NCTC 6571, E. coli NCTC 10414 and P. aeruginosa
II Reagents and lab wares NCTC 10662.
0.5 McFarland standards, Mueller-Hinton agar plates (pH 7.2-
7.4), antibiotics discs, sterile cotton swabs, Millimeter ruler and
sterile forceps.
96 The Stoke’s Method
OBSERVATIONS
Zone sizes are measured from the edge of the discs to the edge
of the zone. Comparison of the zones of inhibition between the
standard and test bacteria indicates the sensitivity of the test
bacteria. If the test zones are obviously larger than the control
or give no zone of inhibition at all; there is no need to perform
any measurement with calipers or a millimeter scale.
Each zone size is interpreted as follows (Fig. 32-1):

Sensitive: Zone size equal to wider than or not more than 3 mm
smaller than control.
Intermediate: Zone size greater than 2 mm, but smaller than the
FIGURE 32-1 Stokes method.
control to more than 3 mm.
Resistant: Zone size 2 mm or less.
Table32-1 Comparison of Kirby-Bauer and Stokes methods
Kirby-Bauer Method Stokes Method
1. Test and control strains have 1. Test and control strains tested on the same
to be tested on separate plates. 2. 4 discs on 85mm plate and 6 discs by rotatory method.
2. 6 discs applied on 85mm plate. 3. Zone diameter measured from edge of disc to edge of zone
3. Zone diameter is measured including disc diameter. of inhibitation the disc.
KEY FACTS
1 It is better to apply the control organisms followed by test organisms.

2 Penicillinase producing Staphylococci, showing heaped up, clearly defined zone edges should be reported resistant
irrespective of zone size.
3 The advantage of Stoke’s method is that both control and test organisms can be tested on the same plate under similar
conditions and difference between zone sizes can be directly measured.
VIVA
1 How is the zone size measured in Stokes method?

2 How many discs can be applied on a plate?
Ans: A total of 4 discs can be accommodated on an 85 mm circular plate. If rotatory plating method is used 6 discs can be
accommodated on an 85mm circular plate. There should be a minimum distance of 2 cm between 2 discs.
3 Compare and contrast Kirby-Bauer and Stokes methods.
FURTHER READINGS
LESSON
Agar Dilution Method
33
0.5 McFarland standard, sterile Mueller Hinton agar (pH 7.2-
After completing this practical you will be able to: 7.4), sterile Mueller Hinton broth, antibiotic powder, sterile test
1 Determine antibacterial sensitivity of bacterial isolates by tubes, pipettes, screw capped flat bottomed bottles (25 ml
agar dilution method. capacity) and Petri dishes (90 mm diameter).
These also include sterile saline (0.85 %) and stock solution
of antibiotic.
INTRODUCTION Preparation of stock solutions of antibiotics: The required
dilutions of the antibiotics are made as per the table 33-1. Prepare
Agar dilution is a quantitative method for determining the a stock solution containing 2000 µg / ml of the antibiotic to be
minimum inhibitory concentration of the antibiotics against tested. For example weigh 200 mg of the antibiotic powder and
bacteria to be tested. It is mainly useful in testing isolates from dissolve in 5 ml of distilled water / appropriate solvent. Mix 0.5
serious infections like bacterial endocarditis or to verify ml of this solution with 9.5 ml distilled water (working solution
equivocal results (e.g. intermediate susceptibility of contains antibiotics at a strength of 200 µg / ml-solution A)
ciprofloxacin against Salmonella Typhi).
Diffusion tests used to determine the susceptibility of III Specimens
organisms isolated from clinical specimens have their own Preparation of suspension of bacteria: Approximately, 4-5 well-
limitations, when equivocal results are obtained, or in prolonged isolated colonies of the bacterial strain to be tested are
serious infections eg. bacterial endocarditis. In these cases, the transferred to Tryptic soy broth or BHI broth. The turbidity of
quantitation of antibiotic against pathogen needs to be more the suspension is adjusted to match 0.5 McFarland standards
precise So when in doubt about the sensitivity of pathogen the (106 organisms/ml).
way to a precise assessment is to determine the minimum inhibitory
concentration (MIC) of the antibiotic to the organisms concerned.
PROCEDURE
PRINCIPLE Preparation of the agar plate with different concentration of

the antibiotics
In agar dilution method, serial dilutions of the antibiotics are 1 Dispense 2 ml of the diluted antibiotic solution into each of
prepared in agar and poured into petri dishes. The dilutions are the marked sterile screw capped bottle.
made in a small volume of water and added to agar which has Note: It is advisable to start wilh the highest dilution so that
been melted and cooled to not more than 60°C. Agar dilutions single pipette can be used to dispense all the dilutions prepared.
methods have the advantage that it is possible to test a number 2 Sterile Muller-Hinton agar is cooled and maintained at 50°C
of organisms on each plate containing an antibiotics solution. – 55°C in a water bath (Table 33-1).
3 Pour this medium (18 ml) into the screw capped bottle
containing the different concentration of antibiotic, shake
REQUIREMENTS well and pour into sterile petri dish.
Note: By this method, exact volume of medium (22.6 ml) is
I Equipments delivered into the screw capped bottles without the danger
Water bath. of the molten agar jellifying during transfer into dilution of
the antibiotic (Table 33-1).
98 Agar Dilution Method
4 Keep the poured plates at 4°C for setting.

5 After the plates have set, dry the plates well in an incubator
QUALITY CONTROL
at 37°C for 30-60 mins. The plates must be dry before
performing the test. Staphylococcus aureus ATCC 25923, Escherichia coli ATCC
25922, Enterococcus faecalis ATCC 29212, and Pseudomonas
aeruginosa ATCC 27853 are used as control strains.
Test procedure
1 A grid is marked on the bottom of the plates containing OBSERVATIONS

antibiotics.
Note: 20 – 25 strains can be tested in plate including the Read the plates for presence or absence of growth. Check the
control. control plate for growth. Control plate must show confluent or
2 A loopful of inoculating loop is calibrated to deliver 0.001 – near confluent growth. Read the test plate. The concentration
0.002 ml (1-2 µl) of the culture. at which growth is completely inhibited is considered as the
3 Inoculate the culture on the surface of the medium, indicated minimum inhibitory concentration (MIC).
by the square marked below. In each case 104 bacteria is The organisms are reported sensitive, intermediate or
delivered to a spot 5 – 8 mm in diameter. resistant by comparing the test MIC values with that given in
Note: Inoculation is done starting with the plates containing the NCCLS table.
highest dilution of the antibiotic.
4 Inoculate a control plate without antibiotics simultaneously
as control. RESULTS AND INTERPRETATION
5 Allow the drops to dry and incubate the plates without
inverting at 37°C for 16 –18 hours. The highest dilution of antibiotic showing more than 99%
inhibition of growth of bacteria is considered as the minimum
inhibitory concentration (MIC) of the bacteria.
Table 33.1 System for preparing dilutions for agar dilution method
Antibiotic Solution + Sterile Water (Vol) = Intermediate conc Final conc at 1:10 in
(µg / ml) in tubes agar plates (µg / ml)
Volume µg/ml
6.4 2000 - A 3.6 1280 - B 128

(Stock)
2 1280 - B 2 640 - C 64
1 1280 - B 3 320 - D32
1 1280 - B 7 160 - E 16
2 160 - E 2 80 - F 8
1 160 - E 3 40 - G 4
1 160 - E 7 20 - H 2
2 20 - H 2 10 - I 1
1 20 - H 3 5 - J 0.5
1 20 - H 7 2.5 - K 0.25
KEY FACTS
1 Agar dilution method is a quantitative method for determining the minimum inhibitory concentration (MIC) of the antibiotic
against bacteria to be tested.
2 The method is carried out on Muller Hinton agar.
3 It is mainly useful in testing isolates from serious infections like bacterial endocarditis or to verify equivocal results (e.g.
intermediate susceptibility of ciprofloxacin against Salmonella Typhi).
4 Selective media should not be used for performing agar dilution method.
5 Electrolyte deficient media also should not be used because it will give false results due to variations in the salt content on
action of many antibiotics.
VIVA
1 What is the break point of an antimicrobial agent?

Ans: Break point of an antimicrobial agent is defined as the concentration of that agent that can be achieved in the serum
with optimal therapy.
2 What is the automated method used to inoculate the organisms?
Ans: Steer’s Replicator is used as the automated method to inoculate the organisms.
3 Mention the merits and demerits of broth dilution method.
Ans: Merit of the test is that it is possible to test many organisms on each plate. Demerit is that it is a cumbersome procedure.
FURTHER READINGS
100
LESSON
Broth Dilution Method
34
0.5 McFarland standard, sterile Mueller Hinton broth, antibiotic
After completing this practical you will be able to: powder, sterile test tubes. sterile pipettes of 10ml, 5 ml, 2 ml and
1 ml, sterile capped tubes and test tube rack.
1 Determine antibacterial sensitivity of bacterial isolates by These also include stock solution of antibiotic.
broth dilution method. Preparation of stock solutions of antibiotics: The required
dilutions of the antibiotics are made as per the table 34-1. Prepare
a stock solution containing 2000 µg / ml of the antibiotic to be
INTRODUCTION tested. For example weigh 200 mg of the antibiotic powder and
dissolve in 5 ml of distilled water / appropriate solvent. Mix 0.5
Broth dilution is also known as tube dilution method. It is ml of this solution with 9.5 ml distilled water (stock solution
another quantitative method for determining the minimum contains antibiotics at a strength of 200 µg / ml-solution A)
inhibitory concentration (MIC) of the antibiotic against a
bacteria to be tested. In this method, serial dilutions of the III Specimens
antibiotics are taken in test tubes and a standardise suspension Preparation of suspension of bacteria: Approximately, 4-5 well
of the bacterium is inoculated. After incubating overnight , the isolated colonies of the bacterial strain to be tested are trans-
MIC of the antibiotics is determined by observing the lowest ferred to Tryptic are soy broth or BHI broth. The turbidity of
concentration of antibiotics that inhibits growth of the bacteria. the suspension is adjusted to match 0.5 McFarland standards.
The minimum bactericidal concentration (MBC) can also be
estimated by this method by subculturing from the lowest
concentration of drug that kills the bacteria. PROCEDURE
1 Serial dilutions of the antimicrobial agent are made in broth

PRINCIPLE and are kept in test tubes.
2 The last tube is kept free of antibiotic and serves as a growth
A stock solution of antimicrobial agent to be tested is prepared. control.
Two fold dilutions of this solution is prepared in suitable broth. 3 Arrange the test tubes in a rack.
A standard suspension of the organism is inoculated into the 4 Standardised suspension of the microorganisms to be tested
medium with one antimicrobial agent -free medium as control. is inoculated into the tubes.
The inoculated media are inoculated at 35-37°C for 18-24 hr. 5 Tubes are incubated at 35-37°C for 18 hours.
and examined for growth. MIC is taken as the lowest
concentration of antimicrobial agent which completely inhibits
the growth. QUALITY CONTROL
Staphylococcus aureus ATCC 25923, Escherichia coli ATCC

REQUIREMENTS 25922, Enterococcus faecalis ATCC 29212, and Pseudomonas
aeruginosa ATCC 27853 are used as control strains.
I Equipments
Water bath.
The tubes not showing visible growth are subcultured on

OBSERVATIONS solid medium and incubated at 37°C overnight.
At the end of the incubation period the tubes are examined for
turbidity. Cloudiness indicates that bacterial growth has not been Table 34-1 Preparation of stock dilutions of the antibiotic stock
inhibited by the concentration of antibiotic present in the medium. solutions
Stock dilutions of the antibiotic stock solutions can be prepared

RESULTS AND INTERPRETATION using the formula:
Minimum inhibitory concentration (MIC) is defined as the 1000 x V X C =W

P
highest dilution which inhibits growth judged by lack of
turbidity in the tube. The main advantage of the broth dilution Where:
method for MIC determination is that it can readily be converted P =Potency given by manufacturer
to determine the minimum bactericidal concentration (MBC) V= Volume (ml) require
also. The highest dilution showing at least 99% inhibition is C= Final concentration of solution (per ml)
taken as MBC. W= Weight of antimicrobial to be dissolved in volume V
KEY FACTS
1 Minimum inhibitory concentration (MIC) is defined as the highest dilution which inhibits growth of the bacteria. It is noted
by lack of turbidity in the tube.
2 Because very faint turbidity may be given by the inoculum itself, the inoculated tube kept in the refrigerator overnight may
be used as the standard for the determination of complete inhibition,
3 Standard strain of known MIC should be tested as control to check the reagents and conditions.
VIVA
1. What is a bacteriostatic agent? Give examples.

Ans: Certain antimicrobial agents inhibit the growth by preventing the multiplication of organisms. They do not cause
death. These are called bacteriostatic agents. e.g. tetracycline, erythromycin, sulphonamides, etc.
2. What is a bactericidal agent? Give examples.
Ans: The drugs that cause irreversible damage to bacteria resulting in death are called bactericidal drugs. e.g. aminoglycosides,
penicillin and quinolones.
3. What is minimum inhibitory concentration (MIC)?
4. What is minimum bactericidal concentration (MBC)?
Ans: The lowest concentration of the antimicrobial agent that allows < 0.1 % of the original inoculum to survive is called the
minimum bactericidal concentration (MBC).
5. Mention merits and demerits of broth dilution agar method.
Ans:
Merits
1 Simple procedure for testing a small number of isolates, even single isolates.
2 Same tubes can be used for MBC tests also.
Demerits
There may be non specific turbidity due to the inoculum itself.
FURTHER READINGS
102
LESSON
Epsilometer Test (E-test)
35
After completing this practical you will be able to: I Reagents and lab wares
1 Determine antibacterial sensitivity of bacterial isolates by Commercially available E-test strips, 0.5 McFarland standards,
Epsilometer test (E-test), an automated system for measuring sterile Mueller Hinton agar plates (150 or 90 mm with a depth of
the minimum inhibitory concentration (MIC) of the bacteria. 4 mm). In a 90 mm plate, a single antibiotic strip can be tested.
In a 150 mm plate, at least 4 antibiotic strips can be tested.
These also include foreceps, 0.85% saline for inoculum
INTRODUCTION preparation, and sterile swabs
The E-test is an automated system for measuring the MIC of II Specimens

the bacterial isolate. It is a very simple test to perform MIC of Preparation of suspension of bacteria: Inoculate peptone water
the bacterial isolate as compared to the other techniques like with test organism and incubate at 37 °C for 3-4 hours. The
broth and agar dilution methods which are technically turbidity of the suspension is adjusted to match 0.5 McFarland
cumbersome. An elliptical zone of growth inhibition is seen standards.
around the strip after incubation. The MIC is read from the
scale at the intersection of the zone with the strip. It is easy to
interpret the result of MIC. PROCEDURE
Opening an E-test package

PRINCIPLE
1 Remove the package stored at – 20°C or - 70°C.
2 Equilibrate at room temperature. This takes approximately 30
The E-test is based on the principle of disc diffusion where the
minutes if stored at – 20° C and approximately one hour if
antibiotic diffuses into the medium when the strip is placed on
stored at – 70° C. Ensure all the moisture has evaporated
the medium.
before opening.
The E-test is a plastic strip (5 x 50 mm; antibiotic carrier)
3 Inspect the package for holes or cracks. Do not use if damaged.
with a continuous gradient of antibiotic immobilized on one
4 Cut along the broken line at the top of a blister. Do not cut in
side and MIC interpretative scale corresponding to 15 two fold
between the blisters.
MIC dilutions on the other side. A predefined antibiotic
5 Tip the strips out of the opening slightly and take them out
gradient is immobilized on the surface opposite the MIC scale.
with forceps.
When transferred to the agar, the continuous antibiotic gradient
6 If strips stick together, twist them apart with your fingers.
established under the strip remains stable over a period
7 Touch only the handle, i.e., the area labeled E.
covering the critical times of most microorganisms subjected
8 Place the strips to be used into a dry clean petri dish.
to susceptibility testing. This method produces a means for
producing MIC data in those situations in which the level of
resistance can be clinically important. e.g. penicillin or Application of strips
cephalosporins against Streptococcus pneumoniae. 1 Apply E-test strips with forceps. Ensure the MIC scale is
facing upwards i.e. towards the opening of the plate.
2 Ensure that the agar surface is dry before swabbing it. Dip a
swab in the inoculum, remove excess fluid and swab the
entire agar surface evenly in 3 directions.
3 Allow the agar surface to dry for 10 minutes to 15 minutes on
the bench or in the incubator.
4 Open the E-test package and place the strips in a dry petri dish.
5 Apply the strips to the agar surface with a forceps. Always
apply the strip with the MIC scale facing the opening of the
plate. Do not apply it upside down.
Note: Be firm when applying the strip. Once applied, do not
move the strip.
6 Use templates to position 4 to 6 strips on a 150 mm plate or
one to two strips on a 90 mm plate. FIGURE 35-1 E-Test.
7 Place the handle of the strip closest to the rim of the plate.
Note: Always store unused strips in airtight containers at -
20°C or -70°C. RESULTS AND INTERPRETATION
8 Incubate at 37°C for 18-24hours.
Read plates after the recommended incubation period only if
sufficient growth is seen and the inhibition eclipse is clearly
QUALITY CONTROL visible.
Read the MIC where the ellipse intersects the scale.
Package labels for each antibiotic will carry performance and Always read the end point at complete inhibition of all
reproducibility data. NCCLS QC ranges and interpretive guidelines. growth including hazes and isolated colonies.
Since E- test comprises a continuous gradient, MIC values
in between two – fold dilutions can be obtained.
Always round up these values to the next two-fold dilution
OBSERVATIONS
before interpretation. For example: If ampicillin breakpoints are
given as S=1, I = 2, R=4 µg/ml, then an E-test MIC of 1.5 µg/ml
After incubation an elliptical zone of growth inhibition is seen
is rounded up to 2 µg/ml and the category reported as
around the strip. The MIC is read from the scale at the
Intermediate (I)
intersection of the zone with the strip (Fig. 35-1).
KEY FACTS
1 The E-test strips have to be placed in proper orientation. Placing strips upside down on the agar will alter the results.
2 Diffusion of antibiotics begins immediately after placement of the strips, which cannot be moved once it has touched the agar.
3 Read plates after the recommended incubation period only if sufficient growth is seen and the inhibition eclipse is clearly
visible.
4 Read the MIC where the ellipse intersects the scale.
5 Always store unused strips in airtight containers at -20°C or -70°C.
VIVA
1 What is the principle of the E-test?

2 What is the advantage of E-test?
3 How is the result for MIC interpreted?
FURTHER READINGS
104
UNIT
VI
Immunology
Introduction
Lesson 36 Bacterial Agglutination Test
Lesson 37 Blood Grouping
Lesson 38 Latex Agglutination Test
Lesson 39 Co-agglutination Test
Lesson 40 Widal Test
Lesson 41 Weil Felix Test
Lesson 42 Anti-Streptolysin O (ASLO) Test
Lesson 43 VDRL Test
Lesson 44 Radial Immunodiffusion Test
Lesson 45 Immunoelectrophoresis Test
Lesson 46 Counter-current Immunoelectrophoresis Test
Lesson 47 Indirect Haemagglutination Test
Lesson 48 Immunofluorescence Test
Lesson 49 Enzyme-Linked Immunosorbent Assay
106
Introduction
Serological tests are widely used for diagnosis of many infectious diseases including bacterial, viral, fungal and parasitic. These
tests may be agglutination, precipitation, neutralization, etc. with a variation in their sensitivity and specificity. List of few
common serological tests are mentioned in the table.
Table Applications of various tests used in a microbiology laboratory
TYPE OF THE TEST NAME OF THE TEST APPLICATION
Slide agglutination tests. Bacterial agglutination test. For confirmation of identification of the bacterial
isolates by using specific antisera.
Shigella agglutination.
Salmonella agglutination.
Vibrio agglutination.
Streptococcus Lance field’s grouping.
Blood grouping. For blood group antigen detection A, B, AB and O.
Widal test. For antibody detection against
Salmonella Typhi
S. Paratyphi A
S. Paratyphi B
S. Paratyphi C.
Tube agglutination tests. Standard agglutination test. For antibody detection against
Brucella species.
Cold agglutination test. For antibody detection against
Mycoplasma pneumoniae.
Indirect haemagglutination test. For antibody detection in:
Amoebiasis
Lymphatic filariasis
Echinococcosis
Toxoplasmosis
Rickettsial infection.
Passive agglutination tests. Latex agglutination test. For antigen detection in infections caused by:
Streptococcus pneumoniae
Cryptococcus neoformans
Echinococcus granulosus.
Co-agglutination test. For antigen detection in:
Cryptococcosis
Echinococcosis
Lymphatic filariasis
For heterophile antibody detection in

Scrub typhus
Heterophile agglutination tests. Weil-Felix test. Endemic typhus.
Epidemic typhus.
Paul-Bunnel test. For heterophile antibody detection in
Infectious mononucleosis (EBV).
Neutralization test ASLO test. For detection of antibodies (Anti-streptolysin) in

acute rheumatic fever.
Precipitation tests Ring test. For antigen detection in infections caused by:
Brucella (Milk ring test)
B.anthracis (Ascoli’s test)
Tube test. Toxoid precipitation for diphtheria.
VDRL test. Slide flocculation test for demonstration of reaginic
antibodies in syphilis.
Gel diffusion test. For detection of:
a-Fetoprotein
N. meningitidis antigen
HBs antigen.
Immunofluorescence test Direct immunofluorescence test. For antigen detection in infections caused by:
Respiratory syncytial virus
Measles
Mumps
Rabies
Influenza
Indirect immunofluorescence test. For antibody detection in:
Toxoplasmosis
Amoebiasis.
108
LESSON
Bacterial Agglutination
36 Test
LEARNING OBJECTIVES only small quantities of culture are available. The slide
agglutination tests have many uses. They are used for
After completing this practical you will be able to: confirmatory identification of Salmonella, Shigella and Vibrio
isolates, identification of Bordetella pertussis and typing of
1 Demonstrate the application of slide agglutination reaction streptococci (e.g. Streptococcus Lance field’s grouping) and
for the identification of bacteria. pneumococci. The slide agglutination test is rapid and
convenient.
INTRODUCTION
PRINCIPLE
Agglutination is an example of antigen-antibody reaction in
which antigen is particulate or insoluble in nature. The soluble In the slide agglutination test a drop of the appropriate specific
antigens can be tested in agglutination reaction by coating antiserum is added to a smooth, uniform suspension of a
them with carrier particles such as red blood cells, bacteria or bacterial isolate from the clinical specimens resulting in
inorganic particles such as the latex. When a particulate antigen agglutination. A positive reaction is identified by the clumping
is mixed with its antibody in the presence of electrolytes at a together of bacteria and clearing of the drop. The reaction is
suitable temperature and pH, the particles are clumped or facilitated by mixing of the bacterial colony and the antiserum
agglutinated. with a loop. The agglutination reaction occurs instantly or
Antibodies cause agglutination by binding to antigens on within seconds.
the particles; they help to neutralize the slight negative charge
that particles in solution normally carry (the zeta potential).
This allows the particles to approach each other. IgM REQUIREMENTS
immunoglobulins actually function as a bridge between two
particles, with one of the five subunits binding to one particle, I Reagents and glass wares
and another subunit binding to another particle. As in the case Glass slides, bacteriological loop, glass marking pencil, saline
with precipitation reaction, more quantity of antibody can (0.85%), specific antiserum against the bacterium(e.g., antiserum
actually inhibit agglutination. In this case, each particle will be against Salmonella Typhi, Shigella flexneri or Vibrio cholerae)
completely covered with antibodies, and they will not clump to be tested.
together. This phenomenon is known as prozone reaction.
Not all antibodies can agglutinate particles. Antibodies, which II Specimen
cannot cause agglutination, are called “incomplete” antibodies. Pure 24 hour growth of bacteria (e.g., Salmonella Typhi, Shigella
This is probably because of restricted movement in the hinge flexneri or Vibrio cholerae) from solid media preferably from
region of the immunoglobulin. IgG4 antibodies are an example non-blood agar plates (Examples : nutrient agar, Muller-Hinton
of incomplete antibodies. agar).
The slide agglutination is a frequently used procedure for
the identification of many bacterial isolates from clinical
specimens. This method is also used for blood grouping and PROCEDURE
cross matching. The test is performed on a glass slide, hence
called slide agglutination test. This method is useful where 1 Take a clean glass slide.
2 Mark it into two halves by a glass marking pencil and label the test reagent as compared to control reagent is indicative of
them as test and control. a positive reaction. Absence of reaction with a test reagent
3 Place a drop of saline in both the halves. indicates a negative reaction irrespective of any reaction with
4 Pick up the colonies of Salmonella to be tested from agar control organism.
culture and gently emulsify with drops of saline in both the
halves by loop.
5 Add a drop of specific antisera to the bacterial suspension RESULTS AND INTERPRETATION
in the half labeled as test and mix.
6 Place another drop of saline in the half of the slide labeled as Positive agglutination: Clumping in the test half and no clumping
control, and mix. in the control half. It identifies specific bacteria (Fig. 36-1).
7 Gently rock the slide, back and froth for 2 minutes. Negative agglutination: No clumping either in the test half or
8 Observe the clumping of the bacterial suspension in the test. control half. It shows either antiserum is not good or bacteria
and the antisera are not specific to each other.
Auto agglutination: Clumping in both test and control halves.
QUALITY CONTROL Test is fallacious, hence discard the result.
On the same slide a control consisting of the bacterial

suspension in saline without the antisera is used to ensure
that bacteria is not auto agglutinable.
OBSERVATIONS
Observe the test mixture for agglutination by naked eye

POSITIVE NEGATIVE
observation, but sometimes it may require confirmation under
the microscope. A markedly stronger agglutination reaction in FIGURE 36-1 Slide agglutination test.
KEY FACTS
1 Fresh young cultures are always used for the test.

2 Check for the auto agglutination of the test organism.
3 On the same slide a control consisting of the bacterial suspension in saline without the antisera is used to ensure that
bacteria are not auto agglutinable.
VIVA
1 Discuss application of slide bacterial agglutination tests.

2 How do you check for the auto agglutination of the test organism?
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworth, London, 94-96, 1995.
Edition. Lippincott Williams and Wilkins. 1997; pp. 1395.
3 Stites DP. Terr AI and Parslow TG. Medical Immunology. 10th Edition. 2001. pp. 902.
110
LESSON
Blood Grouping
37
After completing this practical you will be able to: 1 Take three different slides and label the slides as A, B, and D.
1 Demonstrate blood grouping by agglutination reaction. 2 Clean middle finger of the left hand with the spirit and allow
it to dry.
3 Prick the finger with sterile lancet.
INTRODUCTION 4 Collect 3 drops of blood on three different slides labeled as
A, B, and D.
The ABO system contains four blood groups and is determined 5 Add a drop of antiserum A to A and anti B to B anti D to D.
by the presence or absence of two distinct antigens, A and B, on 6 Mix with an applicator stick. Mix the samples on the slide by
the surface of erythrocytes. Red cells of group A carry antigen A, gentle rocking for about two minutes.
cells of group B antigen B and cells of group AB have both A and 7 Examine each zone for agglutination of RBCs.
B antigens, while group O cells have neither A nor B antigen. The 8 Record result of the test immediately before the drop dries out.
four groups are also distinguished by the presence or absence of
two distinct isoantibodies in the serum. The serum contains the
isoantibodies specific for the antigen that is absent on the red cell. QUALITY CONTROL
Blood group antigens are inherited according to Mendelian
laws. Their synthesis is determined by allelomorphic genes A, B and Blood group antisera (Anti-A, anti-B, anti-D or Rh antiserum)
O. Genes A and B give rise to the corresponding antigens, but O is must be checked with known blood before to test.
an amorph and does not produce any antigen. Group O is the
commonest group and group AB is the rarest. In India the distribu-
tion is O – 40%, A – 22%, B – 33%, and AB – 5%. O group population OBSERVATIONS
is called universal donors and AB is called universal recipients.
Observe the test mixture for clumping.
Check for autoagglutination of test RBCs.
PRINCIPLE
When a drop of anti A / anti B or anti Rh antibody is added to RESULTS AND INTERPRETATION
a drop of blood, the antibody binds with its specific antigen
present on the RBCs and causes agglutination of the RBCs.
A clear agglutination indicates positive result.
If the test sample is agglutinated with anti A antibody then
REQUIREMENTS the blood is Group A.
If the test sample is agglutinated with anti B antibody then
the blood is Group B.
I Reagents and lab wares
If the test sample is agglutinated with anti A and anti B
Blood group antisera (Anti-A, anti-B, anti-D or Rh antiserum),
antibodies both, then the blood is Group AB.
glazed white ceramic slide, applicator stick and lancet.
If the test sample is not agglutinated with anti A and anti B
II Specimen antibodies, then the blood is Group O.
Blood with anticoagulant or finger prick blood.
KEY FACTS
1 Autoagglutination of RBCs during the test procedure should be checked.

2 Record result of the test immediately before the drop dries out.
VIVA
1 What exactly does it tell you about a person’s blood cells if you know that they are of blood type A, B, AB, or O?
Ans: It shows the presence or absence of two distinct antigens, A and B, on the surface of erythrocytes.
2 Explain what blood types a person of type A, B, AB, or O can receive and why?
Ans :Red cells of group A carry antigen A, cells of group B carry antigen B and cells of group AB have both A and B
antigens, while group O cells have neither A nor B antigen. The serum contains the isoantibodies specific for the antigen
that is absent on the red cell. Immune isoantibodies may develop following ABO incompatible transfusion. Red cells also
carry another antigen that reacts with rabbit serum to Rhesus monkey erythrocytes called Rhesus or Rh factor. Rh-negative
population contains anti Rh antibodies in their blood. Hence a person should not receive blood, which contains isoantibodies
to own RBC. O group population is called universal donors since their blood does not contain any blood group antigens (A,
B and AB). AB group is called universal recipients due to absence of isoantibodies.
FURTHER READINGS
112
LESSON
Latex Agglutination Test
38
LEARNING OBJECTIVES of the latex particles and form visible agglutination or clumping
of the particles.
1 Demonstrate the application of latex agglutination test for
detection of soluble antigen. REQUIREMENTS
I Reagents and lab wares

INTRODUCTION Hydatid antibody-coated latex bead, glass slides, and
applicator stick.
Latex agglutination test is an example of slide agglutination
test in which polystyrene spheres such as latex particles have II Specimen
been used as carrier particles for coating with antigens. In this Serum sample to be tested.
test both soluble proteins and non-protein antigens can be
bound on the surface of latex beads, which then can be used to
detect antibodies by demonstrating the agglutination of the PROCEDURE
antigen-carrying latex particles. The latex agglutination test is
used for demonstration of antibodies on a variety of bacterial, 1 Take a clean glass slide.
parasitic and fungal infections. The LAT are widely used for 2 Mark it into two halves by a glass marking pencil and label
diagnosis of typhoid fever, syphilis, infectious mononucleosis, them as test and control.
amoebiasis and hydatid diseases. 3 Add a drop of test serum in the test half, and a drop of saline
The LAT can also be used for detection of soluble antigen in the control half.
by using latex particles coated with the specific antibodies. 4 Add a drop of hydatid antibody-coated latex reagent to the
The LAT is used to demonstrate rheumatoid factor (RF) in the serum in the half labeled as test and mix.
serum, Streptococcus pneumoniae and Haemophilus 5 Place another drop of hydatid antibody-coated latex reagent
influenzae and cryptococcal antigen in the cerebrospinal fluid in half of the slide labeled as control, and mix.
for diagnosis of meningitis, Clostridium difficile antigens in 6 Gently rock the slide, back and froth for 2 minutes.
the stool for diarrhoea and hydatid antigen in the serum for 7 Observe the clumping of the latex reagent in the test.
diagnosis of hydatid disease.
In this exercise you will perform the LAT for detection of
antigen in the serum for diagnosis of hydatid disease. QUALITY CONTROL
The LAT is performed with a known positive and negative

PRINCIPLE hydatid sera, every time the test is performed with the test sera.
In the LAT for detection of hydatid antigen in the serum, the

latex particles are coated with specific polyclonal hydatid OBSERVATIONS
antibodies. Then hydatid antibody coated latex particles are
mixed with serum to be tested for the antigen, If serum contain Observe the test half for agglutination of latex reagent.
antigen, then the latter combines with the antibodies on surface Observe the control half for autoagglutination.
Agglutination of latex reagents with the sera, and absence of

any agglutination in the control half indicates the LAT to be
positive and shows the presence of hydatid antigen in the NEGATIVE POSITIVE
serum (Fig. 38-1).
Absence of agglutination either in the test half or in the
control half indicates the test to be negative and shows the
absence of hydatid antigen in the serum.
FIGURE 38-1 Latex agglutination test.
KEY FACTS
1 Latex agglutination test is an example of slide agglutination test.

2 The latex agglutination test is used for demonstration of antibodies on a variety of bacterial, parasitic and fungal infections.
3 The LAT can also be used for detection of soluble antigen by latex particles coated with the specific antibodies.
VIVA
1 List application of the LAT for diagnosis of infectious diseases by demonstration of antibodies in the serum.
2 List application of the LAT for diagnosis of infectious diseases by demonstration of antigens in the serum.
FURTHER READINGS
114
LESSON
Co-Agglutination Test
39
After completing this practical you will be able to: 1 Take a clean glass slide.
1 Demonstrate the application of Co-agglutination test for 2 Mark it into two halves by a glass marking pencil and label
detection of soluble antigen. them as test and control.
3 Add a drop of test CSF in the test half, and a drop of saline
in the control half.
INTRODUCTION Note: The CSF specimen has to be absorbed with stabilized
SAPA cells (not coated with specific pneumococcal antibody)
The Co-agglutination (Co-A) test is a simple slide agglutination to prevent nonspecific agglutination with human IgG.
test. The test is being used for detection of specific antigen in 4 Add a drop of pneumococcal antibody-coated Co-A reagent
the serum, cerebrospinal fluid (CSF), urine and other body fluids. to the serum in the half labeled as test and mix.
Commercial Co-A kits are now available for detection of 5 Place another drop of pneumococcal antibody-coated Co-A
haemophilus, meningococcal and pneumococcal antigens in reagent in the other half of the slide labeled as control, and mix.
the CSF, and salmonella antigen in the serum. The kits are also 6 Gently rock the slide, back and froth for 2 minutes.
available for identification of Neisseria gonorrhoeae and sero 7 Observe the agglutination of the bacteria in the test.
grouping of Staphylococcus aureus.
In this exercise you will perform the Co-A for detection of
pneumococcal antigen in the CSF for diagnosis of meningitis. QUALITY CONTROL
PRINCIPLE The Co-A is performed with a known pneumococcal antigen

positive and negative CSF, every time the test is performed
Co- agglutination test means cocci- mediated agglutination. In with the test sera.
this test certain strains of S. aureus organisms (Cowan’s I strain)
containing a large amount of an antibody binding Protein A
OBSERVATIONS
(SAPA) in their cell walls is utilized as a carrier particle. These
cocci on mixing with the specific antibodies (raised against
Observe the test half for agglutination of Co-A reagent.
Streptococcus pneumoniae to be detected in the CSF) bind IgG
Observe the control half for auto agglutination.
non-specifically through the Fc region leaving specific Fab sites
free. The subsequent reaction of Fab with pneumococcal antigen
in the test CSF is visualized by clumping.
REQUIREMENTS Agglutination of the Co-A reagents with the CSF , and absence
of any agglutination in the control half indicates the Co-A to
I Reagents and lab wares be positive and shows the presence of pneumococcal antigen
Co-agglutination reagent, glass slides, and applicator stick. in the CSF.
Absence of agglutination either in the test half or in the
II Specimen control half indicates the test to be negative and shows the
CSF sample to be tested. absence of pneumococcal antigen in the CSF.
KEY FACTS
1 The Co-agglutination test is an example of slide agglutination test.

2 S. aureus organisms (Cowan’s I strain) containing a large amount of an antibody binding Protein A (SAPA) in their cell walls
is utilized as a carrier particle.
3 The Co-A test is used for demonstration of antigens in the serum, urine and CSF in a variety of infections.
4 The commercial Co-A reagents are available for identification of N. gonorrhoeae and serogrouping of S. pyogenes A, B, C,
D or G.
5 The reagents are also available commercially for identification of meningococcal, pneumococcal and haemophilus antigen
in the CSF for diagnosis of meningitis.
VIVA
1 Explain the principle of the Co-agglutination test.

2 What is the strain used in Co-agglutination test?
3 List the uses of Co-agglutination test.
FURTHER READINGS
116
LESSON
Widal Test
40

Water bath (Fig. 40-1).
1 Perform Widal test for diagnosis of enteric fever.
II Reagents and glass wares
Small test tubes, test tube racks, and pipettes.
INTRODUCTION Saline (0.85 % NaCl), and salmonella antigen suspensions.
These antigens can be prepared in the laboratory or
Widal test is an example of tube agglutination test used widely commercially prepared suspensions are available from the
for diagnosis of enteric fever. Enteric fever is caused by Central Research Institute, Kasauli, (Himachal Pradesh). The
Salmonella Typhi, and by Salmonella Paratyphi A, B and C. In antigens are stored at 4 °C before use.
India enteric fever is caused commonly by S. Typhi, and by
S. Paratyphi A, rarely by S. Paratyphi B. In Widal test serum of III Specimen
the patients is tested for antibodies against H and O antigens Serum sample to be tested
of S. Typhi, and S. Paratyphi A and B. Note: Serum samples may be stored at 4 °C for 10-15 days
before testing. They may be stored at –20 °C for the period
beyond 2 weeks.
PRINCIPLE
Widal test detects antibodies in serum against H and O sus- PROCEDURE

pensions of S. Typhi and against H suspension of S. Paratyphi
A and S. Paratyphi B. Since S. Paratyphi C infections are not Preparation of master dilution of the serum
found in India, serum antibodies against this species are not
tested. 1 Add 2.3 ml of saline in a test tube.
Since “O” antigens are related to each other, the O antigen 2 Add 0.2 ml of serum to the test tube, which makes the serum
of S. Typhi is only tested, while H antigens of S. Typhi, dilution as 1: 12.5.
S. Paratyphi A and S. Paratyphi B are tested separately. In this 3 Patient’s serum is tested in a series of dilutions against each
test the serum to be tested are diluted in series in test tubes of the different antigen suspensions.
and antigen suspensions of salmonella are added to it. Four
rows of such dilutions are prepared and to that antigen
suspensions of O antigen of S. Typhi and H antigens of S. Performance of the test
Typhi, S. Paratyphi A and S. Paratyphi B are added. The test is
then incubated in water bath at 37°C and is checked for 1 Arrange 4 rows of test tubes, each row containing 7 test tubes.
agglutination by examining the bottom of the tubes for sediment. 2 Label the row 1 as TO, 2 as TH, 3 as AH and 4 as BH.
The titre of patient serum is calculated for each salmonella 3 Add 0.2 ml of diluted serum from master dilution into the first
suspension as the highest dilution producing visible and second tubes in all rows of the tubes, TO, TH, AH and BH.
agglutination. 4 Add 0.2 ml of saline to all the tubes from 2nd to 7th in all the
rows including the 7th control tube.
5 In the row labeled as TO, mix and transfer 0.2 ml of serum The last tube showing agglutination is considered as the
from 2nd tube to the 3rd, then from 3rd to 4th, and so on end point.
through the 6th tube. Discard 0.2 ml from the 6th tube. The reciprocal of the dilution is considered as the titre (e.g.,
6 Follow the same dilutions in the rows labeled as TH, AH If the dilution of the last tube showing agglutination is 1: 200,
and BH. then the titre is 200).
7 Add 0.2 ml of TO antigens to tubes from 1st to 7th in the
row TO.
8 Add 0.2 ml of TH antigens to tubes from 1st to 7th in the RESULTS AND INTERPRETATION
row TH.
9 Add 0.2 ml of AH antigens to tubes from 1st to 7th in the 1 A progressive rise in the titer between first and third week
row AH. after onset of fever is highly significant.
10 Add 0.2 ml of BH antigens to tubes from 1st to 7th in the 2 A positive or a negative result in a single test is not significant.
row BH. 3 Since the antibodies are detected only after 7 days to 10
Note: The final dilution of the sera after addition of antigen days of illness, test should be done later.
are 1:25, 1;50, 1;100, 1:200, 1:400, 1:800. 4 The serum of some uninfected subjects causes agglutinations
11 Incubate the racks in water bath at 37 °C for 18 hrs. at dilutions of about 1:50, so titers are considered significant
when agglutination occurs in serum dilution above 100.
5 H agglutinins tend to persist longer than O agglutinins.
QUALITY CONTROL 6 O antibody titre rise indicates recent infection.
7 Persons immunized with TAB vaccine may show high titers
7th tube in each row acts as a negative antigen control. of antibodies to all the antigens and so only a marked rise in
The same dilutions of a known positive serum is tested with titer is considered significant.
TO, TH, AH and BH antigens. 8 Early treatment with antibiotics will alter antibody response.
OBSERVATIONS
Observe the test tubes after 18 hours of incubation in waterbath

at 56°C for agglutination and note the result.
The results are read by viewing the tubes under good light
against a dark background with the aid of a magnifying lens.
In case of H agglutinins large loose, cotton and wooly clumps
are formed and with O agglutinins only small granules are formed
at the bottom of the tube. If necessary, the tubes can be gently
rotated, to swirl up granules from the deposit. The titer of the
serum is the highest dilution of serum giving visible
agglutination.
No agglutination is seen as a small compact deposit (button)
formation. FIGURE 40-1 Water bath.
VIVA
1. What are the advantages and disadvantages of Widal test?

Ans:
Advantages:
Simple and inexpensive test.
Sensitive test for the diagnosis of typhoid in endemic areas.
Useful for cases in children, who have a low prevalence of pre-existing antibody.
Disadvantages:
Less specific.
A positive or a negative result in a single test is not significant.
The antibodies are detected only after 7 to 10 days of illness.
The serum of some uninfected subjects causes agglutinations (False positive reactions).
Persons immunized with TAB vaccine may show high titers of antibodies to all the antigens.
Cases treated early with chloramphenicol may show a poor agglutinin response.
118 Widal Test
KEY FACTS
1 In India enteric fever is caused commonly by S. Typhi, and S. Paratyphi A, rarely by S. Paratyphi B.
2 Since “ O” antigens are related to each other, the O antigen of S. Typhi is only tested, while H antigens of S. Typhi,
S. Paratyphi A and S. Paratyphi B are tested separately.
3 The last tube showing agglutination is considered as the end point.
4 The reciprocal of the dilution is considered as the titre(e.g., If the dilution of the last tube showing agglutination is 1: 200,
then the titre is 200).
FURTHER READINGS
LESSON
Weil-Felix Test
41
Small test tubes, test tube racks, and pipettes.
After completing this practical you will be able to: Saline (0.85 % NaCl), and P. vulgaris OX-19, OX-2 and
P. mirabilis OX-K antigens. The antigens are stored at 4°C
1 Demonstrate the cross-reacting antibodies to Rickettsial before use.
antigens using Proteus strains OX-19, OX-2 of Proteus
vulgaris and OX-K of P. mirabilis by Weil-Felix test. III Specimen
Serum sample to be tested.
INTRODUCTION
PROCEDURE
Weil-Felix is a serological test used for diagnosis of Rickettsial
infection by demonstration of antibodies in the serum. In this Preparation of master dilution of the serum
test, certain strains of Proteus are used instead of specific
rickettsial pathogens as antigens. This test was developed 1 Add 2.7 ml of saline in a test tube.
from the observation that certain strains of Proteus isolated 2 Add 0.3 ml of serum to the test tube which makes the master
from the urine of patients with epidemic typhus were aggluti- serum dilution as 1: 10.
nated by the sera of the typhus patients. This test has been
used for presumptive serological evidence of Rickettsial Performance of the test
disease.
1 Arrange 3 rows of test tubes, each row containing 7 test tubes.
2 Add 1 ml of saline to all the tubes.
PRINCIPLE 3 Add 1 ml of diluted serum from master dilution into the first
tubes in all the three rows of the tubes.
It is an agglutination test for cross-reacting antibodies. It is 4 Make doubling dilutions in the first row from the first
based on the principle that many patients infected with one of tube(serum dilution 1:20) and discard 1 ml from the 6th tube
the Rickettsia produce antibodies that can agglutinate certain (serum dilution 1:640).
strains of bacteria of genus Proteus because of the presence Note: Leave the 7th tube as control without serum.
of a common alkali stable carbohydrate antigen. 5 In the same way, make doubling dilutions in the second and
Rickettsia prowazaki and R. mooseri share alkali stable third row tubes.
carbohydrate antigens with P. vulgaris OX 19, R. tsutsugamushi 6 Add 0.5 ml of concentrated P. vulgaris OX-19 in to all 7
with P. mirabilis OX K and R.rickettsi, and R.conori with tubes in the first row of tubes.
P. vulgaris OX 2. 7 Add 0.5 ml of concentrated P. vulgaris OX-2 in to all 7 tubes
in the second row of tubes.
8 Add 0.5 ml drop of concentrated P. mirabilis OX-K in to all
REQUIREMENTS 7 tubes in the third row of tubes.
9 Incubate the racks in water bath at 37°C for 2 hr., followed by
I Equipments reincubating at 4°C for 18 hours.
Water bath.
120 Weil Felix Test
The last tube showing agglutination is considered as the

QUALITY CONTROL end point.
The reciprocal of the dilution is considered as the titre(e.g.,
7th tube in each row acts as a negative antigen control. If the dilution of the last tube showing agglutination is 1: 640,
The same dilutions of a known positive serum are tested with then the titre is 640).
Proteus OX 19, OX 2 and OX K antigens.

OBSERVATIONS
A serum titer of 1: 80 and above is considered significant titer
Observe the test tubes after overnight incubation for but four fold or greater increase of antibody between acute
agglutination and note the result. and convalescent sera is considered diagnostic.
The results are read by viewing the tubes under good light Agglutination with Proteus OX 19 and OX 2 is suggestive
against a dark background with the aid of a magnifying lens. of spotted fever.
Complete agglutination is demonstrated by complete Agglutination with Proteus OX 2 is suggestive of murine typhus.
clearing of the supernatant fluid and the formation of white Agglutination with Proteus OX K is suggestive of scrub typhus.
flocculent masses in the bottom of tubes
KEY FACTS
1 Since the Weil-Felix antigens also react with Proteus antibodies, false positive reactions may occur in urinary tract infections
with Proteus, in leptospirosis, in Borrelia infections and in severe liver disease.
2 Proper precautions should be taken for standardization of antigens. It should not be standardized against sera from rabbits
immunized with homologous strain of Proteus species, but with sera derived from patients infected with rickettsiae.
3. The test is not helpful for the detection of antibodies in rickettsial pox, trench fever or Q fever as these persons do not
develop Proteus agglutinins.
VIVA
1 What are heterophile antibodies?

Ans: Antibodies produced in certain clinical conditions have the ability to cross react with more than one antigen called
heterophile antibodies
2 What is the principle of Weil-Felix reaction?
3 List other tests detecting heterophilic antibodies
Ans: a. Streptococcus MG agglutination test for the diagnosis of atypical pneumonia. b. Paul-Bunnel test for the diagnosis
of Epstein-Bar virus infections and c. cold agglutination test for the diagnosis of primary atypical pneumonia.
FURTHER READINGS
LESSON
Anti-Streptolysin O (ASLO)
42 Test

Antigen (Streptolysin O), phosphate buffered saline pH 6.3-
After completing this practical you will be able to: 6.5, 2.5% RBC’s in Alsever’s solution (human blood group
1 Demonstrate neutralizing antibodies to Streptococcus O or rabbit erythrocytes), microtitre plates, pipette, test tubes.
pyogenes haemolysin O antigen. III Specimen
Serum.
INTRODUCTION
PROCEDURE
Str. pyogenes produces several exotoxins and enzymes, which Serum dilution
contribute to its virulence. Streptococci produce two
haemolysins, namely streptolysin O and streptolysin S. 1 The initial serom dilutions of 1:10, 1:60 and 1:85 are prepared in
Streptolysin O is oxygen labile and heat labile. It is inactive test tubes. Subsequent dilutions are carried out in microtitre plates.
in the oxidized form but may be reactivated by treatment with 2 Both serum and buffer should be placed at room temperature
mild reducing agents. It is lethal on intravenous injection into when preparing dilution.
animals and has a specific cardiotoxic and leucotoxic activity. 3 Standard serum of know titer is included in each days run to
Streptolysin O is antigenic and elicits the production of serve as positive control.
specific antibodies against the antigen in an infected human
host. This antibody is known as antistreptolysin, it regularly Test procedure
appears in sera following streptococcal infection. Hence,
1 Label microtiter plate so that each specimen is alligned in
demonstration of antistreptolysin in the serum is an indirect
two rows (1:60 row and 1:85 row) with six wells per row.
indicator of infection. Many methods are available for detection
2 Prepare serial two fold dilutions through sixth well. Serum
of antistreptolysin in the serum. Streptolysin S is not antigenic,
dilutions for each serum sample are first row - 1:60, 1:120,
hence do not elicit production of any antibodies.
1:240, 1:480, 1:960, 1:1920; second row - 1:85, 1:170, 1:340,
1:680, 1:1360, 1:2760.
3 Add 0.025ml of antigen to all tubes, gently agitate, and
PRINCIPLE incubate at 37°C for 15 minutes.
4 Add 0.025 ml of cold 2.5% red blood cells to all wells and
When streptolysin O in its reduced form is added to red blood incubate in water bath at 37° C for one hour.
cells hemolysis occurs. If a patient’s serum contains
antistreptolysin O antibodies, antigen-antibody reaction takes
place and no hemolysis takes place. QUALITY CONTROL
A cell control, antigen control and a standard serum control
always should be used with the test.
REQUIREMENTS
I Equipments
OBSERVATIONS
Water bath. Observe the microtitre plates for presence or absence of hemolysis.
122 Anti-Streptolysin
The titer of ASLO is the highest serum dilution causing no

RESULTS AND INTERPRETATION hemolysis.
Significant titer is considered to be 200 or more.
Reading is taken by observing for button formation and
hemolysis in microtitre plate.
KEY FACTS
1 Streptolysin O is antigenic and elicits the production of specific antibodies against the antigen in an infected human host.
2 Streptolysin S is not antigenic; hence do not elicit production of any antibodies.
VIVA
1 Name the hemolysins produced by Streptococci.

2 Explain the principle of the LAT test.
FURTHER READINGS
LESSON
VDRL Test
43
LEARNING OBJECTIVES test. When cardiolipin antigen combines with reaginic antibodies
in patient’s serum, it forms visible floccules. This test is also
After completing this practical you will be able to: used to detect antibodies in the CSF.
The biological false positive reaction is a noted problem
1 Perform the VDRL test. associated with the VDRL test (Box 43-2).The advantages and
2 Demonstrate the presence of reaginic antibodies in patient’s disadvantages of the VDRL test are listed in the table 43-1.
serum.
REQUIREMENTS
INTRODUCTION
I Equipments
When soluble antigen and specific antibodies in the serum are Water bath and VDRL shaker.
combined in the right proportions, they will bind to each other
to form an insoluble complex which results in visible II Reagents and glass wares
precipitations. The precipitates settle at the bottom of the VDRL antigen (It contains cardiolipin, 0.3%, lecithin, 0.24%
container. The precipitation reaction can be demonstrated in and cholesterol, 0.9%), buffered saline solution, saline, glass
the liquid medium as well as in the solid media such as in gels. stoppered bottles, VDRL glass slides (2”X3”) with depressions
The process of precipitation in the gel can be hastened by of 14 mm diameter each, tubes, pipettes and racks.
migration of antigens and antibodies under an electric field. Preparation of buffered saline solution: This is prepared by
Temperature, pH and concentration of sodium chloride in the mixing 0.5 ml of formaldehyde, 3.037 gm of disodium hydrogen
medium influence the reaction of precipitation. phosphate (Na 2HPO 4, 2H 2O), 0.170 gm of potassium
Often the precipitate fails to settle down at the bottom of dihydrogen phosphate (KH2PO4), and 10 gm of sodium chloride
the container but remains suspended as floccules, such reaction (NaCl), in 1000 ml of distilled water. pH is adjusted to 6.0+ 0.1.
is known as flocculation. Venereal Disease Research Laboratory Each antigen pack usually consists of 10 ampoules of 5 ml
(VDRL) test is an example of flocculation test to detect reaginic buffered saline solution.
antibodies in serum of patients suffering from syphilis. Preparation of un buffered saline solution: This is prepared
VDRL –ELISA is a modification of the VDRL test (Box 43-1). by adding 1 gm of sodium chloride to 100 ml of distilled water.
III Specimen
Serum and CSF.
PRINCIPLE
PROCEDURE
The VDRL slide flocculation test is a simple, rapid convenient
and economical test for serodiagnosis of syphilis. This is an Preparation of serum
example of standard test of syphilis in which cardiolipin antigen
is used to detect non-specific reaginic antibodies in the serum. 1 Inactivate the serum in water bath at 56°C for 30 minutes.
Cardiolipin antigen is an alcoholic extract of beef heart tissue Note: After removal from the water bath, all the sera are
to which cholesterol and lecithin are added. This is a non- examined and those found to contain particulate debris are
treponemal test because no treponemal antigen is used in this recentrifuged. The quantity of serum used in each test is 0.05 ml.
124 VDRL Test
Preparation of antigen emulsion Quantitative serum test
1 Pipette 0.4 ml of buffered saline to the bottom of a 1 oz reagent Quantitative test is performed on all reactive serum samples
bottle with flat or concave inner bottom surface. and on all samples showing weakly reactive or rough in the
2 Add 0.5 ml of antigen, drawn from an ampoule in to a 1.0 ml qualitative test. Different dilutions of the serum are prepared in
pipette graduated to tip, directly on to the saline while rotating tubes as successive twofold dilutions (in the range of 1:2, 1:4,
the bottle on a flat surface. The antigen is added drop by 1:8, 1:16, etc.) with buffered saline solutions. Each dilution of the
drop but rapidly so that it takes approximately 6 seconds to serum is treated as an individual serum. These serum dilutions
complete the delivery. are tested as described above, in qualitative serum test.
3 Blow the last drop of antigen and continue rotation of the
bottle for 10 more seconds. Quality control
4 Then add 4.1 ml of buffered saline from a 5.0 pipette stopper
Known reactive (with known titre) and non-reactive sera are
the bottle and shake it vigorously for approximately 10 seconds.
included.
Note: Temperature of the buffered saline solutions and
antigen should be preferably between 23°C to 29°C during
the preparation of antigen emulsion. OBSERVATIONS
Maturation of the antigen: It increases the sensitiveness and Observe for the formation of floccules immediately after rotation
this is almost complete in 15 minutes to 30 minutes. The antigen under a microscope with low power objective (10 x
emulsion prepared ought to be used during the same day. magnification). The antigen particles are seen as small fusiform
Preliminary testing of antigen emulsion: Each batch of prepared needles, which remain more or less evenly, disperse in a non-
antigen emulsion should first be examined by testing known reactive serum, whereas these aggregate into clumps in reactive
reactive and non-reactive sera. This test should present sera. Zone reactions are recognized by the irregular clumping,
typically reactive and non-reactive results, and the number which are not compact. In such cases the results are reported
and distribution of antigen particles per microscopic field in on the basis of quantitative reaction done on the same serum.
the negative sera should be optimum.
Qualitative serum test RESULTS AND INTERPRETATION

1 Pipette 0.05 ml inactivated serum into the cavity of a VDRL 1 Qualitative serum test
slide.
Note: Slide must be thoroughly cleaned and then well dried The results are reported on the basis of quantitative reaction
before use. done on the same serum.
2 One drop (1/60 ml) antigen emulsion is added on to the serum. No clumps or very slight roughness: Non-reactive.
The drop of antigen is transferred from an 18 gauze needle Small clumps: Weakly reactive.
cut across and fitted to 1 ml syringe. Medium and large clumps: Reactive.
3 Rotate the slide for 4 minutes (180 rotations per minute).
Note: If rotated by hand on a flat surface this movement 2 Quantitative serum test
should roughly circumscribe a 2 inch diameter circle 120 times
per minute. Results are reported in terms of the highest dilution of the
4 Read the result under 10 x objectives of the microscope. serum that produces a definite reaction.
Table 43-1 Advantages and disadvantages of VDRL test
Advantages Disadvantages
It is a simple and rapid diagnostic test. It is a non-specific test.

It helps in presumptive diagnosis of syphilis. It shows biological false positive reactions.
It can be used as a prognostic test. This test alone is not confirmative.
It can be used as qualitative and quantitative tests. Improper temperature of the laboratory, specimens or reagents
It is an inexpensive test. contributes to the error in the test.
It is reproducible.
BOX 43-1 VDRL –ELISA
An automated VDRL-ELISA test has been developed using cardiolipin antigen, which can measure IgG and IgM antibodies
separately and is suitable for large scale testing of sera. The newest of the nontreponemal tests, VISUWELL Reagin, is based
on the Pedersen method. In the indirect ELISA procedure, VDRL antigen coats the wells of a microtiter plate. This test has a
sensitivity of 97% in untreated syphilis and specificity of 97%. The reactivity of this test disappears with treatment of the
patient. The disadvantage of this test is the inability to quantitate the reactivity of a patient’s serum to an end point titre in
order to assess the efficacy of treatment. Several hundred sera can be tested by VISUWELL Reagin test in a day.
BOX 43-2 BIOLOGICAL FALSE POSITIVE REACTIONS OF VDRL TEST
Biological false positive (BFP) reactions are defined as positive reactions obtained in tests using cardiolipin antigen, with
negative results in specific treponemal tests, in the absence of past or present treponemal infections, and not caused by
technical faults.
As cardiolipin antigen is present both in T. pallidum and in mammalian tissues, reaginic antibodies may be induced by
treponemal or host tissue antigens. This accounts for BFP reactions. They represent the nontreponemal cardiolipin antibody
responses. BFP reactions may occur in about one percent of normal sera. Clinically, BFP reactions may be classified as acute
or chronic.
Acute BFP reactions last only for a few weeks or months and are usually associated with acute infections, injuries or
inflammatory conditions. Chronic BFP reactions persist for longer than six months and are typically seen in SLE and other
collagen diseases. Leprosy, malaria, relapsing fever, infectious mononucleosis, hepatitis and tropical eosinophilia are examples
of other conditions associated with BFP reactions.
KEY FACTS
1 VDRL is an example of slide flocculation test and is an example of standard test of syphilis.
2 Cardiolipin antigen is used to detect non-specific reaginic antibodies in the serum.
3 Maturation of the antigen increases sensitiveness of the test.
4 Observe for the formation of floccules immediately after adding the antigen to the serum in a VDRL slide.
5 Quantitative test is performed on all reactive serum samples and on all samples showing weakly reactive or rough in the
qualitative test.
6 Zone reactions are recognized by the irregular clumping, which are not compact.
7 VDRL test shows biological false positives in a variety of conditions.
VIVA
1 What is the principle of the VDRL test?

2 What is the antigen used in the VDRL test?
3 What are the advantages and disadvantages of the VDRL test?
4 List the biological false positive conditions shown by the VDRL test.
FURTHER READINGS
126
LESSON
Radial
44 Immunodiffusion Test
LEARNING OBJECTIVES Analytical balance, water bath, dark ground illuminating box,
moist chamber, microscope slides, glass plates, beakers,
After completing this practical you will be able to: syringes, micropipettes, Pasteur pipettes, measuring cylinder,
1 Quantitate the protein antigens in a test serum by single measuring template and test tubes.
radial immuno diffusion.
III Specimen
Serum.
INTRODUCTION
Radial immunodiffusion is similar to the double diffusion PROCEDURE

method. In this test, antiserum is incorporated into the agar gel
during its preparation. As in the double diffusion method, holes Preparation of antibody containing gels
are cut in the agar, and antigen is placed in the well. As the
antigen diffuses out of the well, it will form complexes with the 1 Make 2% agar solution in barbitone buffer.
incorporated antibodies, which are visualized as a ring. The 2 Boil the mixture till the gel particles are dissolved fully and
size of the ring is proportional to the amount of antigen in the allow it to cool down to 45-50°C .
well. By constructing and using a standard curve, the amount 3 Mix the appropriate quantity of the gel with the pre-warmed
of antigen in each well can be quantitated. monospecific antibody (10% of the antibody is used).
Uses of gel diffusion tests are mentioned in the table 44-1. 4 Then mix and pour the gel on microscopic slide and allow
setting.
PRINCIPLE
Calibration of reference graph
When the radial diffusion of the soluble antigens from the
cylindrical wells occurs in to the antibody incorporated gel, then 1 A set of five standards (5, 6, 7, 10 and 15 md/dl) are selected
circular precipitates develop. If the antibody is evenly distributed and applied separately to the cylindrical wells using a suitable
in the uniformly thick gel and well size and volume of the antigen template.
is kept constant then the diameter of the precipitate is directly 2 Keep the gel in a moist chamber at 4°C .
proportional to the antigenic concentration. This technique helps 3 Measure the diameters of the circular precipitates under
in the quantification of human serum proteins feasible even in a oblique illumination after 24 hours by using the special
moderately equipped laboratory. measuring template.
4 Plot the graph between square of the diameters on the
ordinate and the corresponding standard antigenic
REQUIREMENTS concentrations on abscissa using a linear graph paper.
Note: A straight line is obtained by joining at least three
I Equipments reference points, which indicates accuracy of the graph.
Electrophoresis power supply. Alternatively, graph may be plotted between diameter of the
II Reagents and glass wares ring and the antigenic concentration and in that case a curved
Barbitone buffer, purified agar, Bacto agar, amido black, acetic line with a convexity upwards is obtained; a straight line or a
acid and physiological saline. line with concavity upwards indicates error in the technique.
Testing unknown serum samples RESULTS AND INTERPRETATION

1 Dilute the test sample in a ratio of 1 in 15 with physiological saline.
2 Punch out a total of 9 wells (2mm wide) in the gel using the 1 The antigen concentration is determined by taking the
suitable template. intercepts on the graph. The final value of the serum antigen
Note: At least one check standard is employed on each level is derived by multiplying with the dilution factor (15).
occasion to test the reproducibility of the procedure while 2 The slide may be stained and preserved in a plastic bag for a
the remaining wells are charged with the test samples. permanent record.
3 Transfer the gel to the moist chamber at 4°C.
4 Observe the gel after 24 hours under oblique illumination for
Table 44-1 Uses of gel diffusion tests
ring precipitates.
5 Measure diameter of the ring and record it. 1 Screening sera for antibodies to influenza viruses.
2 Diagnosis of small pox.
3 Elek’s test for toxigenicity in diphtheria bacilli.
QUALITY CONTROL 4 Testing for normal and abnormal proteins in serum and in
urine.
Known positive and negative samples. 5 Detection of a Fetoprotein antigen in serum.
6 Detection of specific antigens of cryptococci and
meningococci in cerebrospinal fluid.
OBSERVATIONS 7 Purification and identification of proteins and nucleic acids.
Observe the gels for precipitin lines after 24 hours under oblique
illumination for ring precipitates.
KEY FACTS
1 Circular well with a clean edge is very important for a well-defined ring precipitate.
2 Since there is a batch-to-batch variation the reference graph should be plotted for every new batch.
3 The slope of the calibration graph is influenced by antibody concentration in the gel.
4 The size of the ring is proportional to the amount of antigen in the well.
VIVA
1 What is the principle of gel diffusion test?
2 What are the advantages of gel diffusion?
Ans. The advantages are as follows:
a The reaction is visible as distinct band of precipitation.
b Precipitin band is stable and can be stained for preservation.
c The number of different antigens in the reacting mixture can be readily observed.
3 Classify gel diffusion methods?
Ans.
Immunodiffusion
Single diffusion in one dimension (Oudin procedure).
Double diffusion in one dimension (Oakley Fulthorpe procedure).
Single diffusion in two dimensions (Radial immunodiffusion).
Double diffusion in two dimensions (Ouchterlony procedure).
Immunoelectrophoresis.
Electroimmunodiffusion
Countercurrentimmunoelectrophoresis.
Rocket electrophoresis.
3 What are the uses of gel diffusion test?
FURTHER READINGS
128
LESSON
Immunoelectrophoresis
45 Test

Gel punch , glass plates, glass slides (3” x 2”) and standard
After completing this practical you will be able to: laboratory wares.
1 Demonstrate different antigens in antigen mixtures by Buffer (pH 8.6), purified agar or agarose, amido black, acetic
immunoelectrophoresis. acid, paper wicks and physiological saline.
III Specimen
INTRODUCTION Patients serum. Human serum (antigen) and antihuman serum
(antibodies)
When mixtures, such as those in body fluids, are being analyzed
in diffusion experiments like the double-diffusion system, it is
difficult to distinguish the individual antigenic components PROCEDURE
from each other. Immunoelectrophoresis in principle is a method
of combination of electrophoresis and diffusion. By these 1 Take a clean glass slide, To coat the slide place two to
methods antigens present in a mixture are separated from each three drops of the molten agar and spread over the surface
other by agar gel electrophoresis. Subsequently these antigenic of the slide.
components react specifically with their antibodies to form 2 Put the coated slide on a horizontal work bench.
antigen-antibody precipitates that are formed in the gel. 3 Pipette 6 ml of agar to the slide, spread it uniformly and
Immunoelectrophoresis is useful for demonstration of normal allow it to set at room temperature, and then at 4°C in a
and abnormal serum proteins such as the myeloma proteins. refrigerator for 1 hour to set completely.
4 Remove the slide from the refrigerator.
5 Place the slide on a graph paper. Punch two wells at about
PRINCIPLE one third of its length. Suck out the agar plugs from wells
and discard.
Immunoelectrophoresis techniques is carried out on a glass Note: These two wells should be 2 mm in diameter and I
slide layered with semisolid gel. An aliquot of the antigen cm apart.
mixture is kept in the well cut out of the gel. Electrophoresis is 6 Fill the two wells with the serum.
carried out for one hour under electric current, this procedure Note: the serum is mixed with the indicator dye (bromthymol
permits separation of proteins. A rectangular trough is then blue)with a pipette.
cut parallel to the line of these separated proteins. Antiserum 7 Keep the slide in the electrophoresis chamber. Connect it
is placed in this trough. Diffusion is allowed to occur for 18-24 to the buffer with Whatman 3 filter paper strips.
hours. The antiserum will diffuse laterally and react with the Note: The slide should be kept in such a way that the well
separated components. Individual precipitation lines develop containing antigen will be near the cathode. This will make
with each separated components of the antigen mixture. the components move toward the anode.
8 Fill the electrophoresis chamber with the buffer.
9 Connect it to the power supply and run the electrophoresis
REQUIREMENTS for 60-90 min at 10-15 mA/slide till the bromthymol blue
(indicator dye) spreads to the end of the slide.
I Equipments 10 Remove the slides after turning off the power. Cut a trough
Electrophoresis power supply, and electrophoresis chamber. between the two wells running parallel to the direction ofthe run.
11 Remove the agar and fill the trough with the antiserum.
12 Keep the slide in the moist chamber and allow the diffusion OBSERVATIONS
to take place at 4 °C for 24 hours.
13 Remove the slides and observe for the lines of precipitation. Observe the gels for precipitin lines after 24 hours at 4°C.
14 Wash the slide and stain the slide.
QUALITY CONTROL
1 The antigen components in the mixture is noted by observing
Known positive and negative samples are used in the test. different lines of precipitations.
2 The slide may be stained and preserved in a plastic bag for a
permanent record.
KEY FACTS
1. Immunoelectrophoresis is useful for demonstration of normal and abnormal serum proteins such as the myeloma proteins.
2. Keep the slide in the electrophoresis chamber in such a way that the well containing antigen will be near the cathode. This will
make the components move toward the anode.
3. Observe the gels for precipitin lines after 24 hours at 4°C.
VIVA
1 What is the principle of immunoelectrophoresis?

2 What are the advantages of immunoelectrophoresis?
3 List the applications of immunoelectrophoresis test.
FURTHER READINGS
130
LESSON
Counter-current
46 Immunoelectrophoresis
Test
LEARNING OBJECTIVES the cathodic side and the well containing antibodies will be on
the anodic side, so that during electrophoresis antibodies move
After completing this practical you will be able to: towards the cathode and antigens move towards the anode. A
1 Perform counter-current immunoelectrophoresis (CIEP). line of precipitation visible to the naked eye is formed at a point
2 Demonstrate antibodies or antigens in serum and other body between the antigen and antibodies. The test takes 30 minutes.
fluids by counter-current immunoelectrophoresis (CIEP). The lines of precipitation in the slide can be stained by Amido
black or 1% Coomassie brilliant blue stains.
In this chapter the CIEP to detect hydatid antigen in the
INTRODUCTION serum will be described.
Counter-current immunoelectrophoresis (CIEP) is a highly

specific test used for demonstration of either antibodies or REQUIREMENTS
antigens in the serum, CSF, urine and other body fluids. This
method was used earlier for diagnosis of many infectious I Equipment
diseases by demonstration of antigens or antibodies. The test Electrophoresis power supply and electrophoresis chamber.
is used for detection of hepatitis B antigen and antibodies in
the serum, cryptococcal antigen in the CSF, hydatid antigens II Reagents and glass wares
and antibodies in the serum, and amoebic antigens and Glass plates, glass slides (7.5 cm by 2.5 cm), template, gel punch
antibodies in the serum. The CIEP is also used to detect hydatid and standard laboratory wares.
and filarial antigens in the urine specimens. The CIEP test has Veronal buffer, 0.075 M (pH 8.6), normal saline, purified agar or
the advantages of being simple, and rapid test, the result can agarose, amido black,7% acetic acid and Whatman filter paper No.3.
be obtained within 30-45 minutes of performing the test. The
test is also highly specific test. Disadvantages of the test is III Specimen
that it is a low sensitive test. Patients test serum, hydatid antigen, polyclonal hydatid
The supporting medium, antiserum and its dilutions, control antibodies raised in the rabbits
sera, buffer pH, voltage of the current, staining of the slides Preparation of Veronal buffer 0.075 M (pH 8.6): The buffer is
and other variables if not properly standardised may affect prepared by mixing dimethyl barbituric acid, 276 gm in 1000 ml
final result of the test. of distilled water and dissolving it by heating. This is followed
by adding 15.45 gm sodium dimethyl barbiturate and 1 gm of
sodium azide to the solution. The pH is adjusted to 8.6.
PRINCIPLE Preparation of agarose: This is prepared by adding 1gm
agarose to 10 ml buffer and 90 ml distilled water. The agarose is
The CIEP test is based on the movement of the antibodies melted by keeping it in a boiling water bath.
toward the cathode while the antigens move in the opposite
directions (toward the anode) in a layer of agarose on a glass
slide under electric current. The test is carried out on glass PROCEDURE
slides layered with the agarose with the wells cut out off the
agarose. One well is filled with antigen while the other well is 1 Take a clean glass slide, To coat the slide place two to
filled with antibody. The slides are kept in an electrophoresis three drops of the molten agar and spread over the surface
tank in such a way that the wells containing antigen will be on of the slide.
2 Put the coated slide on a horizontal work bench. 15 After staining, the lines of precipitation stains dark blue.
3 Pipette 2.5 ml of 1% agar to the slide, spread it uniformly
and allow it to set at room temperature, and then at 4°C in a
refrigerator for 1 hour to set completely. QUALITY CONTROL
4 Remove the slide from the refrigerator.
5 Place the slide on a template. Punch out parallel rows of Known hydatid antigen-positive and - negative serum samples.
wells 4mm in diameter on the slides at distance of 3 mm With every slide known hydatid antigen is placed in the wells
from each other on the cathodic side and hydatid antibodies in the wells on the
Note: Nine pairs of wells are punched out on each slide. anodic side as control.
6 Fill each well with 10µl of the patients serum to be tested
and polyclonal hydatid antibodies.
7 Keep the slide in the electrophoresis chamber. Connect it OBSERVATIONS
to the buffer with Whatman 3 filter paper strips.
Note: The slide should be kept in such a way that the well Observe the lines of precipitation after 30 min.
containing test sera will be near the cathode and the wells First observe for the lines of precipitation in between the wells
containing the hydatid antibodies will be near the anodic side. containing known hydatid antigen and known hydatid
8 Fill the electrophoresis chamber with the buffer. antibodies (Control row).
9 Connect it to the power supply and run electrophoresis for Then observe for the lines of precipitation in the test sera.
30 min at 10-15 mA/slide.
10 Remove the slides after turning of the power off.
11 Remove the slides and observe for the lines of precipitation.
Note: The slides can be kept at 4 °C for 24 hr and the result
read and stained.
1 The line of precipitation is present in the control row : The
12 To stain the slides wash the slides first in saline for several
test is performed correctly.
hours. Remove the salts by washing the slides several times
2 Line of precipitation is formed between the test serum and
in distilled water for one hour.
hydatid antibodies wells: It indicates the patient serum is
13 Stain the slides by immersing the slides in 1% Amido black
positive for hydatid antigen by the CIEP test.
in 7% acetic acid for 15-30 minutes.
3 No line of precipitation is formed between the test serum
14 Destain the slides with 7% acetic acid till the background
and hydatid antibodies wells: It indicates the patient serum
stain is removed.
is negative for hydatid antigen by the CIEP test.
KEY FACTS
1 The CIEP test is based on the movement of the antibodies toward the cathode while the antigens move in the opposite
directions (toward the anode) in a layer of agarose on a glass slide under electric current.
2 The CIEP test has the advantages of being simple, and rapid test, the result can be obtained within 30-45 minutes of
performing the test.
3 After staining by Amido black, the lines of precipitation stains dark blue.
VIVA
1 What is the principle of counter current electrophoresis CIEP test?

2 What are the advantages of CIEP test?
3 What are the disadvantages of CIEP test?
4 What are the dyes used for staining the slide after electrophoresis?
5 List the applications of CIEP test.
FURTHER READINGS
132
LESSON
Indirect
47 Haemagglutination Test
LEARNING OBJECTIVES to the patients sera to demonstrate specific antibodies. The

haemagglutination pattern of the antigen sensitized-RBCs with
After completing this practical you will be able to: the sera are noted. Formation of button with test sera indicate
that agglutination of RBCS has not occurred. Matt formation
1 Perform indirect haemagglutination test. with test sera indicates that agglutination of RBCS has taken
2 Demonstrate antibodies in serum by the indirect place.
haemagglutination (IHA) test. In IHA for hydatid disease, the chick RBCs are sensitized
with hydatid antigen (fluid obtained from human hydatid cyst).
The haemagglutination pattern of chick RBCs are observed
INTRODUCTION after 30-45 min of the test.
Indirect haemagglutination (IHA) test is a technically simple

and inexpensive technique using antigen sensitized - REQUIREMENTS
erythrocytes for demonstration of serum antibodies in a variety
of parasitic diseases. The test has been proved useful for I Lab wares
diagnosis of various parasitic diseases viz., schistosomiasis, Microtitre plates, droppers (25µl) and diluters (25µl).
echinococcosis, leishmaniasis, malaria amoebic liver abscesses.
Erythrocytes from different sources have been used by various II Reagents
workers in the IHA test. Frequently and widely used Hydatid antigen, double aldehyde stabilized chick RBCs,
erythrocytes are from sheep. Other sources are from human of phosphate buffer saline (pH7.2), phosphate buffer saline
‘O’ blood group, turkey, goose, bovine and swine Avian cells (pH6.4) and diluent (PBS pH 7.2 with 0.1% bovine serum
are usually recommended because being nucleated, they are albumin).
heavier, they settle quickly, so results of hemagglutinated chick Chick RBCs are stabilized by double aldehyde stabilization
cells could be observed within 30-45 min of agglutination. The method.
IHA using the chick cells have been used in the IHA for Preparation of hydatid antigen: Hydatid cysts removed
demonstration of specific serum antibodies in the diagnosis of surgically from humans or animals (cattle) are the source of
amoebiasis, echinococcosis and filariasis. hydatid fluid. The intact cyst should be collected as such
Reverse passive haemagglutination test is a different test without any rupture and sent to the laboratory immediately.
used to detect antigen in the serum and other body fluids The hydatid cyst should not be preserved in the formalin. The
(Table 47-1). fluid is aspirated aseptically by syringe from an intact hydatid
In this chapter the IHA method for demonstration of hydatid cyst. This hydatid fluid is checked microscopically for the
antibodies in the serum for diagnosis of hydatid disease will be presence of any scolices or hooklets. The fluid obtained is
described. sterilized by method of filtration using a Seitz filter or a membrane
filter. The fluid is then checked for sterility by inoculating in a
blood agar and McConkey agar and incubating aerobically.
PRINCIPLE The protein content of the fluid is estimated by autoanalyser.
The antigens is stored in -20ºC in 1ml aliquots.
This is based on the principle that the red blood cells act as The optimum sensitizing dose (OSD) of the antigen is
carrier particles for the antigen (against which antibodies will determined for each dilution of stabilized chick cells by chequer
be demonstrated). The RBCs sensitized with antigen are added board titration against the known positive control and negative
control. The lowest amount of antigen which shows the

maximum heamagglutination with the known positive control
QUALITY CONTROL
and negative reaction with the negative control is taken as
OSD of the antigen for that batch. Known hydatid antibody-positive and - negative serum
samples.
III Specimen With every test, known hydatid antibody positive and
Serum, positive control serum and negative control serum. negative serum samples are tested in the microtiter plate.
A well containing only sensitized chick cells (no sera is
added to this well) is used as cell control.
PROCEDURE
OBSERVATIONS
Sensitisation of chick RBCs with OSD of the antigen.
Formation of button in the cells with test sera indicate that
1 Take 0.1ml of packed DAS chick cells in a centrifuge tube
agglutination of RBCs has not occurred
and wash with PBS 7.2 (twice) and centrifuge.
Matt formation with test sera indicates that agglutination
2 Wash with PBS 6.4 and centrifuge.
of RBCs has taken place.
3 Add 0.1ml of this packed chick cells to 0.9ml of optimum
Matt formation in the cell control well rules out non-specific
sensitizing dose (OSD) of the hydatid antigen.
agglutination of RBCs.
4 Keep the chick cells in water bath for 5 minutes at 50°C with
intermittent shaking at the end of each minute.
5 Keep the cells at 4°C overnight.
6 Next day mix the cells and keep the cells again in waterbath
at 50°C for 10 minutes.
7 Wash twice with PBS 7.2 and once with 0.1% bovine albumin Formation of button in the cells with test sera indicate that
(prepared in PBS 7.2). agglutination of RBCs has not occurred. The serum showing a
8 Make 1% suspension with 0.1% BSA. titre of 1:128 and below is considered negative for hydatid
9 Store it at 4 °C until use. disease.
Matt formation with test sera indicates that agglutination
of RBCs has taken place. The serum showing a titre of 1: 128
Performance of the IHA test
and above is considered positive for hydatid disease.
Matt formation in the cell control well rules out non-specific
1 Inactivate the test serum at 56ºC for 30 minutes before starting
agglutination of RBCs.
the test.
2 Dispense a 25µl volume of the diluent (PBS pH 7.2 with 1%
bovine serum albumin) in each well of U bottomed microtitre
tray.
BOX 47-1 REVERSE PASSIVE
3 Add 25µl volume of the serum to the first well of the HAEMAGGLUTINATION TEST
appropriated row.
4 Dilute the sera upto the eleventh well leaving the last well as In this test, RBCs act as carrier particles for polyclonal and
a serum free control. monoclonal antibodies for demonstration of specific antigen
5 Add 25µl volume of the 1% chick cells sensitized with the in the serum, CSF and other body fluids. Reverse passive
OSD antigen to each well. haemagglutination test (RPHA) is used for demonstration
6 Agitate the plate gently for 2 min. of specific antigens for diagnosis of Hepatitis B infection,
7 Incubate the plates at room temperature for 30 min and note Japanese encephalitis, tuberculous meningitis and many
the pattern of haemagglutination. other infectious diseases.
KEY FACTS
1 The red blood cells from different sources have been used by various workers in the IHA test.
2 The red blood cells act as carrier particles for the antigen (against which antibodies will be demonstrated).
3 The RBCs sensitized with antigen are added to the patients sera to demonstrate specific antibodies in serum.
4 The IHA test is carried out in a microtiter plate.
5 The lowest amount of antigen which shows the maximum haemagglutination with the known positive control and negative
reaction with the negative control is taken as OSD of the antigen for that batch.
6 The serum showing a titre of 1: 128 and above is considered positive for hydatid disease.
134 Indirect Haemagglutination Test
VIVA
1 What is the principle of IHA test?

2 List different RBCs which can be used in the IHA test.
3 List the uses of IHA test.
4 What is reverse passive haemagglutination test?
FURTHER READINGS
LESSON
Immunofluorescence
48 Test
LEARNING OBJECTIVES In this experiment you will use the method to demonstrate
unknown bacterial antigens. The fluorescein labeled antibody
After completing this practical you will be able to: is tagged to alcohol or acetone fixed bacterial smear. This is
followed by washing the smear with physiologically buffered
1 Demonstrate bacterial antigen by immunofluorescence test. saline. During this step any uncombined fluorescent antibody
will be washed away. In a positive test, the final reaction will be
observed by a green fluorescence when observed under a
INTRODUCTION fluorescent microscope.
Advantages and disadvantages of the immunofluorescence
Immunofluorescence assays involve the use of either antigen test are summarized in the table 48-1.
or antibody labeled with a fluorescent substance. The
fluorescent dyes have the property of absorbing the light of
one wavelength (e.g., ultra violet light) and reflect back the REQUIREMENTS
light of a different wavelength (visible light). These reflected
lights are visualized by a fluorescent microscope under ultra I Equipments
violet radiation.. Fluorescein isothiocyanate (FITC) is the most Fluorescent microscope.
common dye used in the test. The dye emits a greenish/yellow
light. Rhodamine (red/orange), dansyl (yellow), and II Reagents and lab wares
phycoerythrin are the other dyes used in the test. Acetone, phosphate buffered saline (pH 7.2), buffered glycerol
Direct fluorescent antibody test is employed to detect the (pH 7.2): 10% glycerol in PBS, Evan’s blue counter stain
specific antigens of bacteria, viruses, parasites or other antigens (1:10000), and bovine serum albumin.
in the serum, CSF, urine, faeces, tissues and other body fluids. Glass slides, Petri dishes, U-shaped glass rods to fit into
This test is most frequently used to detect rabies virus antigen petri dishes, filter paper, and Coplin jar and glass marking pencil.
in the tissue collected from the skin on the nape of the neck or Commercially available fluorescent antibody Streptococcus
face. This is also used to detect Corynebacterium diphtheriae, group A and fluorescent antibody Enterococcus group D.
Neisseria gonorrhoeae, measles and mumps in various clinical
specimens.
III Specimen
Broth cultures of Group A Streptococcus pyogenes and Group
D Enterococcus faecalis incubated at 35°C for 24 hours.
PRINCIPLE
Unknown mixed broth cultures of Group A S. pyogenes/
Escherichia coli and Group D E.faecalis / E. Coli
In this test specific antibodies raised against the antigen to be
detected (e.g., anti-rabies antibodies to detect rabies antigen)
is labeled with a fluorescent dye, and is used to detect unknown
antigen (e.g., rabies antigen) in the specimen. If antigen is
PROCEDURE
present in the specimen, the same will bind the fluorescein 1 Take three clean glass slides.
labeled antibodies, and the antibody-bound fluorescein will 2 With glass marking pencil label first slide as S. pyogenes,
emit a fluorescence, which will be observed by a fluorescent and second slide as E. faecalis. Divide the third slide in
microscope using ultra violet radiations. half and label as mixed unknowns.
136 Radial Immunodiffusion Test
3 Prepare alcohol or acetone fixed smears of S. pyogenes on

the first slide, of E. faecalis on the second slide and of QUALITY CONTROL
mixed unknowns on the third slide.
4 Add one drop of fluorescent antibody Streptococcus group The slide with S. pyogenes and E. faecalis treated with
A on to the slide one and fluorescent antibody Enterococcus fluorescent antibody Streptococcus group A and fluorescent
group D to the second slide and spread gently over the antibody Enterococcus group D are used as known positive
surface of the smear. controls.
5 In the third slide, label one side FA-A and the other slide
FA-D and add one drop of fluorescent antibody Streptococcus
group A to the side FA-A and one drop of fluorescent antibody OBSERVATIONS
Enterococcus group D to the side FA-D.
6 Allow to spread gently over the surface of the smear. The slides are observed for the absence or presence of
7 Place the prepared slides on the U-shaped glass rod kept in fluorescence in all the slides.
a petri dish and incubate at 25°C for 30 minutes.
8 Remove the slides from the petri dish and wash with 1% First slide with S. pyogenes : Positive for fluorescence.
buffered saline to remove away excess antibody. Second slide with E. faecalis : Positive for fluorescence.
9 Put the slides in a Coplin jar containing 1% buffered saline Third slide, the side labeled FA-A : Negative for fluorescence.
for 10 minutes at 25°C. Third slide, the side labeled FA-D : Positive for fluorescence.
10 Blot dries the slides with paper.
11 Add one drop of buffered glycerol to each slide and cover
with a cover slip. RESULTS AND INTERPRETATION
Note: 1-10 µg/ml of phenylene diamine could be added to
the mounting medium to prevent the fading of fluorescence The unknown specimen contains the bacteria E. faecalis but
of fluorescein. does not contain S. pyogenes.
12 Examine under a fluorescent microscope.
Table 48-1 Advantages and disadvantages of the immunofluorescence test
More sensitive. Difficult to quantitate the test.

More specific when fluorescent dye is labeled to Requires UV radiation for visualization of the test result.
monoclonal antibodies. Requires expertise personnel.
It can avoid the danger of radiation-based hazards. Fluorescent dye fades faster.
Rapid method. Improper washing steps cause a very high background.
Useful for the identification of viruses. False positive reactions may occur.
Requires expensive equipment.
KEY FACTS
1 Immunofluorescence assays involve the use of either antigen or antibody labeled with a fluorescent substance.
2 Direct fluorescent antibody test is employed to detect the specific antigens.
3 Slides should be cleaned before use.
4 The smears should not be allowed to dry at any stage.
5 To minimize non-specific binding of proteins to cells or tubes, all dilutions and washings should be done in PBS containing
0.1 – 1% BSA.
6 1-10 µg/ml of phenylene diamine could be added to the mounting medium to prevent the fading of fluorescence of fluorescein.
VIVA
1 What is the principle of the direct fluorescent antibody test?

2 What are the uses of direct fluorescent antibody test?
3 Give examples of fluorescent dyes.
4 What are the advantages and disadvantages of the direct fluorescent antibody test?
FURTHER READINGS
138
LESSON
Enzyme-linked
49 Immunosorbent Assay
LEARNING OBJECTIVES PRINCIPLE
After completing this practical you will be able to: 1. The sandwich ELISA for antigen: This method to detect
antigen is very sensitive. The walls of the microtiter plates are
1 Know the importance of various steps involved in performing coated with specific antibody against the antigen to be
enzyme-linked immunosorbent assay (ELISA). detected. The specimen such as serum to be tested are added
2 Perform the ELISA test and interpret the results. to the coated wells. If antigen is present in the serum, then it
will combine with the coated antibody. Antibody conjugated
with enzyme is added to detect the antigen-antibody reactions.
INTRODUCTION The conjugated antibody combines with antibody that has
combined with the antigen. A substrate is added to detect the
Enzyme-linked immunosorbent assay (ELISA) is a widely used binding of conjugated antibody to antigen-antibody complex.
method for demonstration of specific antigens or antibodies in In a positive test, the positive reaction is tested by the change
the serum and other body fluids. They are used extensively for of colour, which can be read by spectrophotometer or ELISA
diagnosis of a variety of human diseases including many viral reader.
(AIDS, hepatitis, rubella, respiratory syncytial infection, etc.), 2. The indirect ELISA for antibodies: This is a frequently used
parasitic (amoebiasis, cysticercosis, leishmaniasis, lymphatic method to detect antibodies. Polystyrene tubes or wells in
filariasis, etc.), bacterial (syphilis, brucellosis, salmonellosis, polystyrene plates are coated with the antigen solution. An
etc.) and fungal (histoplasmosis, aspergillosis, etc.). The aliquot of the serum to be tested is added to each tube and is
principle of ELISA in essence is similar to that of the incubated, followed by washing. To detect the reaction a goat
fluorescence, the only difference being that an enzyme is used antihuman immunoglobulin antibody conjugated with enzyme
instead of fluorescent dye. The most commonly used enzymes is added. The enzyme conjugated antihuman immunoglobulin
are alkaline phosphatase, horseradish peroxidase and ß- binds to the antibody. A substrate is added to detect the binding
galactosidase. Enzyme activity is generally determined by the and in a positive reaction the enzyme acts on the substrate to
change of colour, which can be read by spectrophotometer or produce a change of colour.
ELISA reader. Then the enzyme-linked antiglobulin is added. If antibodies
ELISA is a heterogenous enzyme assay that uses solid are present in the serum, they will have bound to the antigen,
phases e.g. plastic tubes, polyvinyl chloride plates, beads, which has bound to the plate, and now the antiglobulin will
microplate, and membranes such as cellulose acetate or cellulose bind to the primary antibodies. Add the substrate for the
nitrate. The enzyme immunoassay can be broadly of three types: enzyme. If the reaction occurs (for example, if the solution
a. the sandwich ELISA for antigen, b. the indirect ELISA for changes color), this means that the enzyme-linked antiglobulin
antibodies, and c. competitive ELISA for antibodies. was bound, which means that the serum did, in fact, contain
Depending on the nature of the antigen used, the ELISA the antibodies of interest. The color change can be quantified
can also be classified as 1st, 2nd and 3rd generation ELISA as using a spectrophotometer.
used in HIV infections (Box 49-1). 3. Competitive ELISA for antibodies: This involves the use of
Enzymes and substrates used in the ELISA are listed in the two specific antibodies. One antibody is conjugated with enzyme;
table 49-1. where as the other antibody is present in the serum to be tested.
Competition occurs between these two antibodies for the same
antigen, hence is called the competitive ELISA. This is used for
demonstration of HIV antibodies in the serum.
The Dot ELISA is a rapid visually read microassay form of 7 Add 100 µl of conjugate to each well and incubate at 37°C
the ELISA (Box 49-2). The advantages and disadvantages of for 1 hour.
ELISA tests are mentioned in the table 49-2. 8 Wash the wells thrice and add 100 µl of substrate solution
In this experiment indirect ELISA to detect antibodies and and incubate for 30 minutes at 37°C in dark.
sandwich ELISA to detect antigen will be described. 9 Stop the reaction with 50µl of 3N sulphuric acid (H2SO4) to
each well.
10 Read the results with naked eye or at 492 nm in an ELISA reader.
INDIRECT ELISA TO DETECT ANTIBODIES
QUALITY CONTROL
REQUIREMENTS
Appropriate controls, such as controls with positive and
negative sera, buffer control, conjugate control and substrate
I Equipments control should be tested along with every plate.
Micro titer plate, ELISA reader and ELISA washer.
II Reagents and glass wares OBSERVATIONS

Carbonate buffer (pH 9.6), washing buffer (PBS-Tween 20),
conjugate, citric acid phosphate buffer (0.1M) pH 5.0, substrate, Observe the microtiter plate for the development of colour.
4% bovine serum albumin (BSA) in PBS-tween 20 and 3N Observe control (negative, positive and blank) wells.
H2SO4, micro pipettes, tips, glass beakers, and filter paper.
1 Carbonate buffer (pH 9.6) RESULTS AND INTERPRETATION

Solution A: 5.3 g sodium carbonate (Na2CO3) is dissolved in
one liter of double distilled water. Development of yellow colour indicates the test serum contains
Solution B: 1.2 g sodium bicarbonate (NaHCO3) is dissolved antibodies to tested antigen. No colour is negative reaction
in one litre of double distilled water. (Fig. 49-1).
Mix 16 ml of solution A with 34 ml of solution B and make
volume to 100 ml with distilled water and adjust pH to 9.6.
2 Washing buffer (PBS-Tween 20): Phosphate buffered saline SANDWICH ELISA TO DETECT ANTIGEN
(0.01M) pH 7.2 with 0.05% tween 20.
3 Conjugate: Goat antimouse immunoglobulin conjugated with
horse radish peroxidase.
4 Citric acid phosphate buffer (0.1M) pH 5.0: Weigh and dissolve
REQUIREMENTS
7.3 g of citric acid and 11.86 g of disodium hydrogen phosphate
(Na2HPO4) in one liter double distilled water and adjust pH to 5.0. I Equipments
5 Substrate: Dissolve 8 mg of Orthophenylene diamine in 15 Micro titer plate, ELISA reader and ELISA washer.
ml of citrate buffer. Then add and mix 15 ml of Hydrogen
peroxide just before to use. II Reagents and glass wares
Carbonate buffer, blocking solution, washing buffer, monoclonal
III Specimen antibodies (1-10 µg/ml in carbonate buffer) and substrate solution,
Serum, CSF, tear, etc. micropipettes, tips, glass beakers, and filter paper.
III Specimen
PROCEDURE Serum, CSF, urine etc.
1 Make up antigen to optimal dilution in carbonate buffer and

add 100 µl per well in 96 well microtiter plate and incubate at PROCEDURE
37°C for 3 hours or at 4°C overnight.
2 Decant the antigen solution and wash the plates thrice with 1 Coat the ELISA plates with monoclonal antibody (mAb) in
washing buffer. Remove all residual fluid. carbonate buffer by adding 100µl to each well, and incubate
3 Add 100 µl of 4% BSA in washing buffer to each well and overnight at 4°C .
incubate at 37°C for 1 hour. 2 Remove monoclonal antibody from ELISA plate and add 100µl
4 Wash thrice with wash buffer and remove all residual fluid. of blocking solution. Incubate for 30 minutes at 37°C. Wash
5 Add 100 ul of diluted serum samples and incubate for 1 hour the plates thrice with washing buffer. Remove all residual fluid.
at 37°C . 3 Add 100µl of 4% BSA in washing buffer to each well and
6 Wash the wells with washing buffer and remove all residual fluid. incubate at 37°C for 1 hour.
140 Enzyme-linked Immunosorbent Assay
4 Wash thrice with wash buffer and remove all residual fluid.
5 Add 100µl of diluted serum samples and incubate for 1 RESULTS AND INTERPRETATIONS
hour at 37°C.
6 Wash the wells with washing buffer and remove all residual Development of yellow colour indicates the test serum contains
fluid. antigens to tested antibody. Development of no colour
7 Add 100µl of conjugate to each well and incubate at 37°C inducates negative reaction.
for 1 hour.
8 Wash the wells thrice and add 100µl of substrate solution
and incubate for 30 minutes at 37°C in dark.
9 Stop the reaction with 50µl of 3N H2S04 to each well.
10 Read the results with naked eye or at 492 nm in an ELISA reader.
QUALITY CONTROL
Appropriate controls, such as controls with positive and

negative sera, buffer control, conjugate control and substrate
control should be tested along with every plate.
OBSERVATIONS
Observe the microtiter plate for the development of colour. FIGURE 49-1 ELISA test.
Observe control (negative, positive and blank) wells.
BOX 49-1 1ST, 2ND AND 3RD GENERATION ELISA
1st generation ELISA: It is an ELISA in which crude antigen is used for the detection of antibodies.
2nd generation ELISA: It is an ELISA in which semi-purified antigen is used for the detection of antibodies.
3rd generation ELISA: It is an ELISA in which recombinant antigens or synthetic peptides are used for the detection of antibodies.
BOX 49-2 DOT ELISA
Dot ELISA is a rapid visually read microassay. The principle is similar to that of ELISA where enzyme is used as a marker or
label to detect the binding of antigen and antibody. The test is performed on a nitrocellulose membrane. Enzyme converts
colourless substrate to a coloured product, which indicates the presence of antigen-antibody binding. When the test serum
is layered on to the membrane, specific antibodies if present, will bind to corresponding dot of the antigen. Addition of a
labeled serum antibody (conjugate) and the subsequent development of the colour allow the detection of the presence of
antibodies based on the pattern of the antigen sites. Dot-ELISA can be used for the detection of antibodies as well as antigen.
Table49-1 Enzymes and substrates used in the ELISA
Enzymes Substrates
Horseradish peroxidase. Hydrogen peroxide (H2O2).

Alkaline phosphatase. Nicotinamide adenine dinucleotide phosphate (NADP+).
b-D galactosidase, 2-nitrophenyl-b-D-galactoside.
Glucose 6-phosphate dehydrogenase (G6PD). Glucose-6 phosphate.
Acetylcholinesterase. Acetylcholine.
Table 49-2 Advantages and disadvantages of ELISA test
Highly sensitive Requires specialized expert personnel.

Specific Requires expressive laboratory equipment.
Rapid Enzymes require cold environment for storage.
KEY FACTS
1 Preliminary chequer board titrations are carried out for standardizing antigen and antibody concentration.
2 Read all plates at a standard time after addition of the substrate because colour is not stable.
3 Substrate should be made just before use as it decomposes spontaneously and is unstable.
4 OPD should be stored at 4°C in the dark. It is carcinogenic.
VIVA
1 Expand ELISA.
Ans. Enzyme-linked immunosorbent assay.
2 What is the principle of enzyme immuno assay?
3 What are different steps in the ELISA procedure?
Ans.
a Coating solid surface with antigen/antibody.
b Binding antibody/antigen from test sample.
c Binding conjugate.
d Addition of substrate.
e Observe the colour change and read the O.D. values.
4 What are various types of the ELISA?
Ans. Competitive ELISA, sandwich ELISA, indirect ELISA, membrane bound ELISA, cassette ELISA, biotin-avidin ELISA,
protein-A ELISA, etc.
5 What are the enzymes and substrates used in the ELISA?
6 What do you understand by the first, second and third generation ELISA?
7 What are the advantages and disadvantages of the ELISA?
FURTHER READINGS
142
UNIT
VII
Microbial Genetics and
Molecular Techniques
Introduction
Lesson 50 Isolation of Plasmids
Lesson 51 Polyacrylamide Gel Electrophoresis
Lesson 52 Isolation of Antibiotic Resistant Mutant
Lesson 53 Bacterial Conjugation
144
Introduction
The objectives of the study are to demonstrate the genetic FREQUENTLY USED TERMINOLOGY IN
methods such as isolation of plasmids, polyacrylamide gel GENETICS
electrophoresis (PAGE), isolation of antibiotic resistant mutants
and conjugation in bacterial systems.
Phenotype: Phenotype refers to “the observable outward
appearance of an organism, which is controlled by the
Genetic information is stored in DNA of the bacteria as a
genotype and its interaction with the environment”.
sequence of nucleotide bases (adenine, cytosine, guanine,
Genotype: Genotype refers to “the genetic makeup of an
thymine, abbreviated as A, C, G, T respectively) read sequentially
individual organism”.
in a 5' to 3' direction (or in RNA, with uracil, abbreviated U,
Transcription: The “central dogma” of molecular biology
replacing thymine). The most common form of DNA (present
describes the transcription of genetic information from a
in all cellular genomes, as well as many viral genomes) is double
DNA nucleotide triplet code to an RNA triplet code, followed
stranded. The 5' to 3' polarity of the two strands is opposite,
by translation to specific amino acid sequences in protein.
and they are held together by hydrogen bonding between
Prototrophs: A wild type strain that has minimal
nucleotide base pairs, A to T and G to C. The sense strand
requirements for exogenously supplied nutrients is referred
carries the coded genetic information. The antisense strand
to as a prototroph.
consists of a complementary sequence of bases oriented in the
Auxotrophs: A mutant strain that has lost the ability to
opposite 5' to 3' direction. During DNA replication, the two
synthesize its own supply of a particular nutrient, such as
strands separate and each is used as a template for synthesis
histidine or adenine or thiamine is called an auxotroph.
of a new complementary strand. This allows genetic information
Recombinant DNA technology: Recombinant DNA in this
to be replicated with a high level of precision. Because
context refers to the creation of a new combination of DNA
replication is bidirectional, new DNA can only be synthesized
segments that are not found together naturally. Such
in a 5' to 3' direction, the overall pattern of replication is rather
technology is now widely used in many practical applications
complex.
ranging from basic research on control of gene expression
to forensic medicine to biotechnology.
Genetic information is transcribed from DNA to RNA, with the
Restriction endonucleases: An endonuclease is an enzyme
antisense strand of the DNA serving as a template for
that can cleave the phosphodiester bonds of a nucleic acid at
synthesis of RNA with the same base sequence (5' to 3') as the
an internal site (as opposed to cleavage by an exonuclease,
sense strand of the double helical DNA, except that uracil (U)
which can only remove nucleotides from one of the ends of a
replaces thymine (T). Genetic information contained in
nucleic acid). Example: Eco RI G|AATTC.
messenger RNA (mRNA) is translated into a sequence of amino
Hybridization probes: Complementary strands of DNA, RNA,
acids in a polypeptide chain during protein synthesis
or DNA plus RNA hybridize readily to form double stranded
(translation). A redundant nucleotide triplet code, read 5' to 3'
helical structures when placed under suitable annealing
on the mRNA (and on the sense strand of the DNA), specifies
conditions. This property is used extensively in molecular
the amino acid sequence of the protein, read from N-terminal to
genetics to identify specific nucleic acid sequences. A probe
C-terminal.
consisting of radioactively labeled DNA (or RNA) is hybridized
to denatured DNA (or naturally single-stranded RNA)
The study of genetic materials helps to a). demonstrate mutations,
immobilized on a support, such as a nitrocellulose membrane.
b). study the gene transmission among progeny or generations,
Hybridization is normally done at a temperature about 25°C
c) treat genetic disorders, and d) in diagnosis and research.
below the melting (denaturation) temperature for the DNA.
LESSON
Isolation of Plasmids
50
Ethidium bromide solution, Luria-Bertani medium,
After completing this practical you will be able to: micropipettes, Eppendorf tubes, and Bunsen burner.
1 Isolate the plasmid from bacteria. Preparation of ethidium bromide stock solution: The solution
is prepared by dissolving 10 mg of ethidium bromide powder in
1 ml of double distilled water.
INTRODUCTION Preparation of ethidium bromide working solution: The
solution is prepared bu adding 10µl ethidium bromide stock
Bacterial plasmids are small, circular, extra chromosomal DNAs solution in 100 ml of agarose solution.
that are capable of autonomous replication within bacterial cells. Preparation of Luria-Bertani medium (LB medium): The
Plasmids have their own origins of replication, such that they medium is prepared by adding the following ingredients: Bacto-
can multiply independently within the bacterial cells. They tryptone, 10 g; Bacto yeast extract, 5 g; sodium chloride (NaCl),
usually contain only a few thousand base pairs of DNA and 10 g; and distilled water, 1000 ml. The pH is adjusted to 7.0.
carry only a few genes, often for antibiotic resistance. Plasmids III Specimen
that carry appropriate genes are capable of making bacteria Bacterial strain: Plasmid containing strain PHSV 106 or
resistant to antibiotics(Box 50-1), which makes it possible to equivalent strain is grown in a medium containing 20µg/ml
select for bacteria that have taken up the plasmids. ampicillin. The medium contains the following solutions:
Solution A
PRINCIPLE 50 mM glucose.
25 mM Tris chloride (ph 8).
This method utilizes the molecular characteristics of covalently 10mM EDTA (pH 8).
closed circular deoxyribonucleic acid (DNA) that is released Solution B
from cells under conditions that denature DNA by using alkaline 0.2 N sodium hydroxide (NaOH).
sodium dodecyl sulfate. Under these conditions, chromosomal 1% SDS.
DNA concentrations are reduced or eliminated. The DNA is
freed of RNA and proteins by RNase and protease treatments. Solution C
Just before extraction, the plasmid DNA is released from the 5M potassium acetate, 60 ml.
folded chromosomal complex by a shearing step or by RNase Glacial acetic acid, 11.5 ml.
treatment. The plasmid DNA can be resolved as covalently Distilled water, 28.5 ml.
closed circular molecules by centrifugation in ethanol
containing ethidium bromide. The clarified extract is used
directly for electrophoretic analysis.
PROCEDURE
Extraction of plasmid
REQUIREMENTS
1 Inoculate a single bacterial colony into 2 ml of LB medium
I Equipments containing antibiotic ampicillin in a 15 ml test tube and
Centrifuge and inoculating chamber. incubate over night with vigorous shaking.
146 Isolation of Plasmids
2 Take 1.5 ml of culture into a Microfuge tube and centrifuge 6 Remove the gel carefully and place on UV transilluminator
at 12, 000 g for 2 min at 4°C and discard the supernatant. and examine for the bands.
3 Resuspend the pellet in 100 µl of ice-cold solution A and
vortex vigorously.
4 Add 200 µl of solution B and mix the contents by inverting QUALITY CONTROL
the tube rapidly several times.
5 Add 150 µl of ice-cold solution C and mix the contents by A parallel procedure should be carried out using standard strain
inverting the tube. of bacteria.
6 Store the tube in ice for 5 minutes. Molecular weight marker must be used in the analysis.
7 Centrifuge at 12, 000 g for 5 minutes at 4°C and transfer the
supernatant to a fresh tube.
8 Precipitate the DNA with two volumes of 100% ethanol at OBSERVATIONS
room temperature. Mix well and allow it to stand for 2
minutes. Observe the stained gel under UV illumination for DNA band.
9 Centrifuge at 12000 g for 5 minutes at 4°C and remove Observe the bands of molecular weight marker.
supernatant.
10 Resuspend the pellet in 50 µl of TE (pH 8) containing
RNAse (20 µg/ml).
11 Run in agarose gel containing ethidium bromide and view
under UV transilluminator.
Plasmid appears as a sharp orange band.
Undigested RNA appears as hazy opaque area along the lanes.
Electrophoresis on agarose gel
1 Prepare 1% agarose in 1 X TE buffer, melt in a boiling water

bath and add 1 µl of ethidium bromide. BOX 50-1 ROLE OF PLASMIDS IN
2 Pour 20 ml -25 ml of molten agarose in the gel chamber after DRUG RESISTANCE
placing the comb and allow it to set.
3 Place the gel in electrophoresis tank, remove the comb and Bacterial plasmids, covalently closed circular DNAs, have
pour the TE buffer until it fully covers the gel surface. their own origins of replication and are capable of
4 Load the wells with 5 µl plasmid preparation and 2 µl autonomous replication. They usually contain only a few
bromophenol blue tracking dye and a molecular weight thousand base pairs of DNA and carry only a few genes,
marker in other well. often for antibiotic resistance. Plasmids that carry
5 Run electrophoresis at 50-70 V/gel till the tracking dye reaches appropriate genes are capable of making bacteria resistant
the end. to antibiotics, which makes it possible to select for bacteria
that have taken up the plasmids.
KEY FACTS
1 Plasmid is small, circular, extra chromosomal DNA that is capable of autonomous replication within bacterial cells.
2 Care must be taken at every step of the procedure for extraction of the plasmid DNA and electrophoresis on agarose gel.
3 Care must be taken while decanting the supernatant after centrifugation.
VIVA
1 What are plasmids?

2 What are the properties and functions of plasmids?
Ans. Plasmids code for synthesis of a few proteins not coded for by the nucleoid. For example, R-plasmids, found in some
Gram-negative bacteria, often have genes coding for both production of a conjugation pilus and multiple antibiotic resistance.
Through a process called conjugation, the conjugation pilus enables the bacterium to transfer a copy of the R-plasmids to
other bacteria, making them also multiple antibiotics resistant and able to produce a conjugation pilus. In addition, some
exotoxins, such as the tetanus exotoxin and Escherichia coli enterotoxin are also coded for by plasmids.
3 How do you extract plasmids from bacteria?
FURTHER READINGS
1 Collier L, Balows A, Sussman M. Topley and Wilson’s Microbiology and Microbial Infections. 9th Edition. Systemic Bacteriology. Volume
2.. Arnold publishers. pp. 2002, pp 1501.
2 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995. pp 94-96.
3 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. 1996. pp. 921.
148
LESSON
Polyacrylamide
51 Gel Electrophoresis

1 Prepare agarose gel. Minigel/maxigel apparatus, power supply (capacity 200V,
2 Assemble the electrophoresis apparatus and power pack. 500mA), boiling water bath, microcentrifuge and rocking/
3 Analyse the protein profile from the desired source, by rotatory shaker.
separating and characterizing based on the molecular weight
of the protein. II Reagents and lab wares
Micropipette or Hamilton syringes (50µl and 100µl).
INTRODUCTION 1 Stock solutions

1 2M Tris-Hcl (pH 8.8) : 100ml.
Sodium dodecyl sulphate polyacrylamide gel electrophoresis 2 1M Tris-Hcl (pH 6.8): 100ml.
(SDS-PAGE) is used in a wide variety of applications for 3 10 %(w/v) SDS (store at room temperature).
characterizing proteins. Few examples include analysis of 4 50% (v/v) glycerol: 100ml.
antigen (protein) fractions from different sources for 5 1 %(w/v) bromophenol blue-10ml. (solution filtered after
immunological and molecular biological studies, taxonomical preparation to remove aggregated dye).
applications by analysis of whole cell /cell envelope protein of 6 N, N, N’, N’-tetramethylene-ethylenediamine (TEMED).
the organism, and so on. Other types of electrophoresis in gels 7 2-mercaptoethanol or, dithiothreitol.
are listed in the table 51-1. 8 Glycine.
Advantages and disadvantages of PAGE are mentioned in
the box 51-1. 2 Working solutions
Acrylamide stock (30%) :100ml.
Acrylamide : 29.2g.
PRINCIPLE Bisacrylamide : 0.8g.
Add distilled water to make 100ml and stir until completely
Electrophoresis is the migration of charged molecules in dissolved.
solution in response to an electric field. Their rate of migration
depends on the strength of the field, on the net charge, size 3 Separating gel buffer (4x) : 100ml.
and shape of the molecules and also on the ionic strength, 75ml 2M Tris-Hcl (pH 8.8) : 1.5M.
viscosity and temperature of the medium in which the molecules 4ml 10% SDS : 0.4%.
are moving. Polyacrylamide acts as a support matrix for running 21ml distilled water
the sample.
Sodium dodecyl sulphate (SDS) is an anionic detergent 4 Stacking gel buffer (4x) : 100ml(Table 51-2).
which denatures proteins by “wrapping around” the 50ml 1M Tris-HCl (pH 6.8) : 0.5M.
polypeptide backbone - and SDS binds to proteins fairly 4ml 10% SDS : 0.4%.
specifically; hence SDS confers a negative charge to the 46ml distilled water
polypeptide in proportion to its length. The disulphide bridges
in proteins are reduced with 2-mercaptoethanol. 5 10% Ammonium persulfate (APS) : 5ml.
0.5g APS dissolved in 5ml distilled water.
6 Electrophoresis / Running Buffer (1x) : 1000ml. 11 Connect the electrodes to the power pack and make pre-
3g Tris : 25mM. run at a constant current of 20mA for 10-15minutes.
14.4g glycine : 192mM. 12 Switch off power supply and load the wells with your protein
1g SDS : 0.1%. samples (10-50µl) diluted in sample buffer along with a
Distilled water to make 1000ml molecular weight marker (5-10µl) using a micropipette/
syringe.
7 Sample buffer : 10ml. 13 Run the electrophoresis at a constant current of 20mA till
0.6ml 1M Tris-HCl (pH 6.8) : 60mM. the dye front reaches the separating gel and then increases
5ml 50% glycerol : 25%. it to 25mA.Stop the run when the dye front reaches the
2ml 10% SDS : 2%. bottom of the gel.
0.5ml 2-mercaptoethanol : 14.4mM. 14 Now remove the side spacers from the glass plates and
1ml 1% Bromophenol blue : 0.1%. carefully scoop the gel using a spatula. The gel may be
0.9ml distilled water stained and destained for visualization or, used as such in
blotting experiments without staining.
8 Staining solution : 1000ml.
15 The gel may be stained using the Coomassie staining
1.0g Coomassie blue R-250.
solution for 1-2hrs and then destained using the destaining
450ml methanol.
solution for overnight in a rocking shaker. An alternate
450ml distilled w.ater
silver staining procedure too can be followed who is
100ml glacial acetic acid.
sensitive but a little expensive.
9 Destaining solution : 1000ml.
100ml methanol.
100ml glacial acetic acid. QUALITY CONTROL
800ml distilled water.
Known protein ladder is loaded in each run.
III Specimen
Protein suspension to be separated.
OBSERVATIONS
PROCEDURE Observe the control protein bands.
Observe and analyze the test protein bands.
1 Mix protein sample (20µl) with sample buffer (5µl) in an
Eppendorf tube and heat it at 75-100°C for 2- 10 minutes
depending on the nature of the sample. RESULTS AND INTERPRETATION
2 Assemble the clean glass plates by placing the spacers-
two on either sides, or one along the bottom edge and fix 1 The protein bands separated according to the molecular
the whole assembly tightly with clamps or gel casting stand. weight and charge are visualized directly.
3 Seal the glass plate assembly with 2% agar or, melted wax 2 Check out for the desired bands by comparing with a
on all three sides leaving the topside so that the assembly standard marker.
is leak proof. 3 Interpret the desired band based on experimental and
4 Prepare and cast the separating gel using a small beaker. literature analysis by either visually or using a gel
5 Mix the components of the separating gel mixture without documentation system with appropriate software.
TEMED and de aerate. 4 The bands can also be transferred by blotting and confirmed
6 Add TEMED finally to the mixture and pour immediately by coupling with appropriate antisera containing
between glass plates upto 2cm below the notch and allow complimentary antibodies for the antigen (electro-immuno
the gel to polymerize for 30-60 minutes at room temperature. transfer blot).
7 Overlay the acrylamide with n-butanol, which helps to keep
the gel surface flat. After polymerization remove the butanol
and rinse with water. Table51-1 List of other types of electrophoresis in gels
8 Prepare the stacking gel and cast over the separating gel as
mentioned above and insert the comb. Electroimmunodiffusion.
9 Allow to polymerize for 15-30minutes. Then remove the Counterimmunoelectrophoresis.
comb carefully and rinse the wells with distilled water. Rocket electrophoresis.
10 Now remove the casted gel plate from the gel casting stand/ Southern blotting.
clamps and detach the bottom spacer. Place the gel assembly Northern blotting.
into electrophoresis chamber with the notched plates facing
inside and fill the upper and lower tanks with running buffer.
Note: Remove air bubbles in the wells if any, using a syringe.
150 Polyacrylamide Gel Electrophoresis
Table 51-2 Calculation for X % separating or, stacking gel
The gel concentration percentage, i.e.: whether 7.5, 10, 12.5 or 15 %, depends on ones experimental need and the desired quantity of stock
solutions to cast the gel are calculated using the following formula.
Acrylamide stock x /3 ml
Stacking / separating gel buffer 2.5ml
Distilled water (7.5-x /3) ml
10% APS 50µl
TEMED 5µl (10µl if x < 8%)
Total volume 10 ml
10% Separation gel 5% Stacking gel
Acrylamide stock 3.3 ml. Acrylamide stock 1.6 ml. Separating gel buffer 2.5ml Stacking gel buffer 2.5ml
Distilled water 4.2ml. Distilled water 5.9ml.
10% APS 50µl. 10% APS 50µl.
TEMED 5µl. TEMED 5µl.
Total volume 10 ml. Total volume 10 ml.
BOX 51-1 ADVANTAGES AND DISADVANTAGES OF PAGE
Advantages
Efficient tool for characterizing proteins.
Highly sensitive.
Rapid method.
Protein bands are stable and can be stained for preservation.
The number of different antigens can be readily observed.
Disadvantages
Chemicals are neurotoxic.
Very expensive.
Trained professional and well equipped laboratory is required.
VIVA
1 What is the principle of electrophoresis?

2 What are the applications of PAGE in microbiology?
Ans.SDS-PAGE can be used a) in a wide variety of applications for characterizing proteins, b) in the diagnosis of infectious
diseases, c) in analysis of antigen (protein) fractions, from different sources for immunological and molecular biological
studies, d) in purification of antigen (protein) fractions, and e) in taxonomical applications by analysis of whole cell /cell
envelope protein of the organism and categorizing accordingly.
3 Write a brief note on electro immuno transfer blot.
Ans. Electro-immuno transfer blot (EITB), also known as Western blot is an immunological technique, which is used to
detect a protein immobilized on a matrix.
Prior to immunoblot, the protein of interest from a complex mixture is separated based on their molecular weight by sodium
dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE). EITB combines the resolution of gel electrophoresis
with the specificity of immunochemical detection. The limit of detection by EITB is of the order 10 picogram with horse
radish peroxidase labeling. Since the loading capacities of the gels are limited, an antigen cannot be detected when its
concentration falls below 1 ng/sample.
The EITB technique involves six steps:
1 Preparation of antigen sample.
2 Resolution of the sample by gel electrophoresis.
3 Transfer of the separated polypeptides to membrane support.
4 Blocking of nonspecific binding sites on the membrane.
5 Addition of antibody.
6 Detection.
4 What is the action of sodium dodecyl sulphate on proteins?
KEY FACTS
1 Care must be taken for spillage or direct exposure to reagent while casting a gel.
2 Avoid introducing air bubbles in to the gel.
3 Always freshly prepared buffers and reagents should be used.
4 Observe for complete circuit of electricity (use sufficient levels of buffer) throughout the procedure.
FURTHER READINGS
2.. Arnold publishers. pp. 2002, pp 1501.
152
LESSON
Isolation of Antibiotic
52 Resistant Mutant
LEARNING OBJECTIVES method, colonies appearing in a region of high streptomycin

concentration are indicative of streptomycin-resistant mutants.
After completing this practical you will be able to: Different types of mutations and their role in microbial
infections are summarized in the table 52-1.
1 Isolate the antibiotic resistant mutants from a prototrophic
bacterial population.
REQUIREMENTS
INTRODUCTION I Equipments
Micro centrifuge.
Mutation is any change in genetic information relative to a reference
“wild-type” genome, including changes that affect expression of II Reagents and lab wares
genes without altering their coding sequences and changes that Eppendorf tubes, micropipettes, two 10 ml trypticase soy agar
do not cause any detectable phenotypic difference (silent in 20 ml test tubes and streptomycin solution (10 mg per 100 ml
mutations). In a complex organism, mutation can occur at many distilled water).
different structural levels and can be classified in many different
ways. Mutations are mainly classified into two types: Point III Specimen
mutations and large-scale mutations (Box 52-1).Mechanisms of Pure growth of Escherichia coli from solid media preferably
mutations are many (Box 52-2). from non-blood agar plates (Examples: nutrient agar, Muller-
Hinton agar), to be tested for antibiotic resistance is used as
The importance of mutation specimen.
Genes are stable repositories of the information needed for

synthesis of all of the RNA and proteins in a living organism. PROCEDURE
Survival and stability of each species is dependent on faithful
replication of genetic information for use by each new 1 Melt two trypticase soy agar tubes in a water bath and cool
generation. However, a low level of mutational change is highly to 45°C.
desirable. Over an extended period of time, mutational changes 2 Place a glass rod under one end of a sterile Petri dish, pour
provide the ability for species to adapt to changing conditions the molten agar until it cover the entire bottom surface and
and challenges. Hence, mutational changes serve as the raw allow to solidify in the same position.
material for selective survival and the evolution of more 3 Add 0.1 ml of the streptomycin solution to a second tube of
advanced and efficient species, as well as the development of molten trypticase soy agar tube using a sterile pipette and
biological diversity (Box 52-3). mix gently.
4 Place the dish in horizontal position and pour the molten
agar medium containing streptomycin medium until it fully
PRINCIPLE covers the gradient agar layer and allow to solidify.
5 Add 0.2 ml of E. coli test culture with a sterile pipette and
After inoculation of the prototrophic test culture in to trypticase spread the culture with a sterile bent rod on entire agar
soy agar medium containing streptomycin by spread plate surface.
6 Incubate the plate in an inverted position for 48 hours at

Table 52-1 Different types of mutations and their role in
37°C. microbial infections
7 Select one or two isolated colonies from the middle of the
streptomycin concentration gradient with a sterile loop and Type of mutation Role in microbial infections
streak toward the high concentration end of the plate.
8 Incubate the plate for 24-48 hours at 37°C. Point mutations Avian sarcoma/leukemia virus
Large scale mutations HIV virus
Leishmania donovani
Neisseria gonorrhoea
QUALITY CONTROL MRSA
Gyrase risistance gene in
Salmonella Typhi
The parent strains (streptomycin sensitive strains) are included
as controls.
OBSERVATIONS 1 Presence of colonies after first incubation indicates that the

strain is resistant to streptomycin.
2 Sensitive strains do not produce colonies after first
Observe the colonies after first and second incubation steps. incubation.
3 Presence of colonies after second incubation denotes the
resistant strains.
BOX 52-1 POINT MUTATIONS AND LARGER SCALE MUTATIONS
In classical genetics, a point mutation was originally defined as a change in an inherited trait that was not accompanied by any
chromosomal change that could be seen with a light microscope. Point mutation can result in missense (amino acid substitution),
nonsense (insertion of a stop codon), or frameshift (either positive or negative).
Missense mutations
Most base pair substitutions change the amino acid specified by the codon in which they occur. Such mutations are described
as missense mutations because they cause an amino acid substitution in the coded protein. Depending on the nature of the
amino acid substitution and its location within the protein, missense mutations may have a variety of effects, ranging from
complete loss of biological activity to reduced activity or temperature-sensitive activity or no functional effect at all.
Nonsense mutations
Base pair substitutions that generate an in-frame stop codon within a previously functional protein coding sequence cause
premature termination of translation of the protein in question and are referred to as nonsense mutations.
Silent mutations
In some cases, base pair substitutions generate a different codon for the same amino acid, with no biological effect whatsoever.
This is most likely to happen in the third position (wobble base) of redundant codons for the same amino acid. Such changes
are considered to be mutations because they alter the genetic code. However, because they have no phenotypic effect, even
at the level of protein amino acid sequence, they are called silent mutations.
Frameshift mutations
Addition or deletion of a single base pair in the middle of a coding sequence will result in out-of-frame translation of all of the
downstream codons, and thus result in a completely different amino acid sequence, which is often prematurely truncated by
stop codons (UAG, UAA, UGA) generated by reading the coding sequence out-of-frame. Such mutations, which are a special
subclass of point mutations, are referred to as frameshift mutations. Deletion of a single base pair results in moving ahead one
base in all of the downstream codons, and is often referred to as a positive frameshift. Addition of one base pair (or loss of two
base pairs) shifts the reading frame behind by one base, and is often referred to as a negative frameshift.
Large scale mutations

Mutations that involve larger changes in chromosomes, including deletions, duplications, inversions, translocations from one
chromosome to another, and extra or missing chromosomes are large-scale mutations.
154 Isolation of Antibiotic Resistant Mutant
BOX 52-2 MECHANISMS OF MUTATION
Tautomerization
Spontaneous mutations that involve base pair substitutions are caused primarily by configurational changes within the
individual bases that result in mispairing. These changes, which are called tautomeric shifts, involve momentary expression of
rare alternative molecular configurations that exist in equilibrium with the more common forms. Specifically, proton shifts can
convert the amino groups in adenine and cytosine to imino groups, and the keto groups in guanine and thymine to enol
groups.
Transitions
A tautomeric shift in any of the four DNA bases can lead to mispairing of A to C or G to T. The tautomeric state can occur either
in the template base or the incoming base. During the next round of DNA synthesis, the mispaired base will pair with its normal
partner, resulting in a transition, in which an AT base pair replaces a GC or a GC replaces an AT, with no change in the purine:
pyrimidine polarity of the base pair. Transitions are the most common type of mutation resulting from spontaneous mispairing
due to tautomerization.
Transversions
To achieve a transversion, in which the positions of purine and pyrimidine are reversed in the DNA double helix, two events
are thought to be involved, tautomerization of one of the bases and rotation of the other to yield a purine: purine pairing. The
frequency of spontaneous transversions, which is lower than that of transitions, appears to be consistent with this interpretation.
A second possible mechanism for transversions is the formation of an apurinic site, which can result in replacement of the
original purine with any of the four bases.
BOX 52-3 THE IMPORTANCE OF MUTATION
Genes are stable repositories of the information needed for synthesis of all of the RNA and proteins in a living organism.
Survival and stability of each species is dependent on faithful replication of genetic information for use by each new generation.
However, a low level of mutational change is highly desirable. Over an extended period of time, mutational changes provide the
ability for species to adapt to changing conditions and challenges, and thus serve as the raw material for selective survival and
the evolution of more advanced and efficient species, as well as the development of biological diversity.
KEY FACTS
1 Properly mix streptomycin solution to molten trypticase soy agar.

2 Sufficiently cool the molten agar before adding antibiotic solution.
3 Presence of colonies after second incubation denotes the resistant strains.
VIVA
1 Define and classify mutations.

2 Write a short note on point mutations.
3 What are the mechanisms of mutations?
Ans. Tautomerization, transitions, and transversions.
4 Explain the mechanism of isolation of antibiotic resistant mutant strains.
5 What are different types of mutations and their role in microbial infections?
FURTHER READING
2. Arnold publishers. pp. 2002, pp 1501.
LESSON
Bacterial Conjugation
53
LEARNING OBJECTIVES Differences between F+ strain and Hfr strain are listed in the
Table 53-1.
1 Demonstrate genetic recombination in bacteria by PRINCIPLE

conjugation.
After inoculation and incubation of F– and Hfr strains together
in a minimal growth medium containing thiamine and streptomycin,
INTRODUCTION threonine and leucine, recombinant strains of the bacteria only
grow. Thiamine acts as a nutrient supplement to thiamine negative
Bacterial conjugation can be viewed as a primitive form of sex, recombinant cells. Streptomycin inhibits the growth of parental
in which a cell from a donor strain injects DNA into a recipient streptomycin sensitive Hfr strains. Parental F– strains cannot
cell, where it can undergo recombination and become part of grow due to the lack of threonine and leucine.
the recipient’s genome. Donor strains contain an additional Mechanisms of DNA transfer are highlighted in the Box 53-1.
genetic element, called the fertility factor (F), usually in the
form of a plasmid. Cells that are receptive to conjugation lack
the F factor and are sometimes designated F –. During REQUIREMENTS
conjugation of an F + cell with an F –, the frequency of
transformation for any genes other than the F factor is very I Equipments
low. However, if it does happen, it is due to random chromosomal Incubator.
integration of the F factor.
II Reagents and glass wares
Hfr strains Bend glass rod, sterile pipettes, and test tubes, 24 hour nutrient
broth cultures of streptomycin resistant F– Escherichia coli
strain which require threonine, leucine and thiamine, and
In certain F+ strains, the F factor has become integrated into
streptomycin sensitive Hfr E. coli strain, nutrient agar plates
the bacterial chromosome. When this happens, transfer of
and 95% ethanol.
chromosomal DNA from the donor to the recipient F– cell begins
adjacent to the integrated F factor and can progress around
III Specimen
the entire bacterial chromosome if the process is not interrupted.
F– E. coli culture.
Bacterial strains with integrated F factors are referred to as Hfr
strains (“high frequency of recombination”).
PROCEDURE
F’ factors and sexduction
1 Take nutrient agar medium.
Sometimes an integrated F+ factor from an Hfr strain will escape 2 Add 1 ml of F– E. coli culture and 0.3 ml of Hfr E. coli cultures
from the bacterial chromosome carrying a few chromosomal into a sterile test tube.
genes with it in its circular plasmid DNA. Such a plasmid is 3 Incubate for 30 min at 37°C.
called an F-prime (F’) factor. The bacterial genes carried on the 4 Vigorously agitate the mixed culture to terminate the genetic
F’ plasmid are easily transmitted into F– cells by conjugation. transfer.
156 Bacterial Conjugation
5 Add 0.1 ml of the mixed culture to a minimal growth medium

containing streptomycin and thiamin and spread over the OBSERVATIONS
entire surface agar surface.
6 Incubate the plates for 48 hours at 37°C. Observe the culture plates for colonies.
Prepare control plates of parental Hfr and F– and aseptically add 0.1 Presence of colonies in the test medium indicates the transfer
ml of each strain to its agar plate and spread with a bent gloss rod. of genetic material.
BOX 53-1 MECHANISMS OF DNA TRANSFER
The DNA transfer that occurs in conjugation begins as a single strand break in the donor chromosome (or plasmid), with only
one strand transferred through the F– pilus to the recipient cell. The single strand left behind in the donor and the one that is
transferred to the recipient are both converted into double strands, restoring the donor chromosome and generating double
stranded donor DNA in the recipient. As a result of receiving new DNA from the donor, the recipient is temporarily partially
diploid. Recombination can then integrate parts of the transferred DNA into the recipient genome. Any DNA that is not
integrated is soon destroyed.
Transformation
Transformation refers to the ability of extracellular DNA to enter a bacterial cell and recombine with the bacterial genome,
thereby giving the bacterial cell new genetic properties.
Transduction
Transduction refers to a genetic exchange in which bacteriophages carry bacterial genes from one bacteria cell to another.
There are two classes of transduction, specialized and generalized. In specialized transduction, a lysogenic phage undergoes
recombination with the host genome and later when it is excised to become an independent phage genome, it carries one or
more host genes with it, which can be transduced into a new host cell and recombined into the genome of that cell. In
generalized transduction, fragments of host DNA are mistakenly packaged into a bacteriophage in place of its own DNA.
Table53-1 Differences between F+ strain and Hfr strain
F+ strain Hfr strain
1. F factor usually present in the form of a plasmid. 1. F factor has become integrated into the bacterial chromosome.
2. Low frequency of recombination. 2. High frequency of recombination.
3. Only F factor transfer occurs in conjugation. 3. Transfer of chromosomal DNA from the donor to the
recipient F– cell begins adjacent to the integrated F factor
and can progress around the entire bacterial chromosome if
the process is not interrupted.
KEY FACTS
1 Young cultures of bacterial strains must be used.

2 Mix the culture properly to terminate the genetic transfer.
3 Avoid contamination.
VIVA
1 Define bacterial recombination.

2 What are the methods of transfer of genetic material in bacteria?
3 How do you demonstrate bacterial conjugation?
4 What are the applications of bacterial recombination?
Ans: Applications of bacterial recombination include:
a Production of recombinant antigens for diagnosis.
b Production of recombinant vaccine proteins.
c Gene therapy.
d Transfer of antibiotic resistance, and
e Production of recombinant strains of bacteria with required characteristics.
5. Differentiate between F+ and Hfr strains.
6. Discuss the role of conjugation in acquiring drug resistance.
Ans: Plasmids carry only a few genes, often for antibiotic resistance. Plasmids that carry appropriate genes are capable of
making bacteria resistant to antibiotics. The plasmid is infectious and can transfer itself by conjugation. The plasmid (F
factor) is present as an independent double stranded ring of DNA distinct from chromosome. During conjugation there is
a break in one of the two strands of the plasmid. This strand passes in to the recipient cell. Donor cell retains one strand.
Donor and recipient cells synthesize complimentary strands to form intact duplexes and recipient cell becomes a potential
donor cell. The F1 factor is formed by the excision of the F factor from the chromosome along with a segment of the
chromosome. The release of F factor occurs by breakage and reunion at a point different from the point of plasmid insertion.
This results in an interchange of plasmid and chromosomal DNA segments.
FURTHER READINGS
2. Arnold publishers. pp. 2002, pp 1501.
158
UNIT
VIII
Bacteriology
Lesson 54 Normal Microbial Flora of the Mouth
Lesson 55 Normal Microbial Flora of the Throat
Lesson 56 Normal Microbial Flora of the Skin
Lesson 57 Identification of Staphylococcus aureus
Lesson 58 Identification of Streptococcus pneumoniae
Lesson 59 Identification of b-haemolytic Streptococci
Lesson 60 Identification of Corynebacterium diphtheriae
Lesson 61 Identification of Lactose Fermenting Enterobacteriaceae
Lesson 62 Identification of Vibrio cholerae
Lesson 63 Identification of Pseudomonas aeruginosa

160
LESSON
Normal Microbial Flora
54 of the Mouth

Petri dishes, sample collection bottle, glass slides, pencil,
After completing this practical you will be able to: inoculation loop, and burner, Gram stain reagents (methyl violet,
1 Demonstrate various microorganisms which are present as iodine, 95% alcohol and dilute carbol fuchsin), filter paper
part of the normal flora in the mouth. containing 1% tetramethyl paraphenylene diamine
dihydrochloride, two blood agar and chocolate agar plates.
INTRODUCTION III Specimen

Saliva.
Normal flora usually denotes to microorganisms, which inhabit
the skin and mucous membrane of the normal human body.
Within a few hours of birth, normal oral and nasopharyngeal PROCEDURE
flora appear. The normal microbial flora offers many benefits to
the human (Box 54-1). 1 Collect the saliva into a sterile sample collection bottle.
There are many microorganisms which are present as part of 2 Inoculate the saliva onto blood agar and chocolate agar
the normal flora of the mouth and teeth. These are Streptococcus plates.
viridans, Streptococcus mutans, nonpathogenic Neisseria, 3 Incubate the chocolate agar plate in CO2 incubator for 48
Staphylococcus, Fusiform species, anaerobic spirochaetes, etc. hours at 37°C.
In infant at the time of birth, the mouth contains usually the 4 Incubate one blood agar plate aerobically for 48 hours
organisms that are present in mother’s vagina (e.g., micrococci, at 37°C.
streptococci, coliform bacilli, Döderlein’s bacilli, etc.), as they
acquire these organisms during their birth through the vaginal
canal. These organisms however diminish in number during first QUALITY CONTROL
week after birth and are replaced by the normal bacterial flora
present in the mouth of the mother. 1 Test all agar plates for sterility before inoculation.
Some of these organisms can cause opportunistic infections. 2 Incubate uninoculated blood agar and chocolate agar plates
Many of these bacteria can be demonstrated in the saliva. along with the inoculated ones.
PRINCIPLE OBSERVATIONS
Saliva is collected from the mouth and inoculated onto the 1 Look for the presence of haemolysis on blood agar plate by
surface of the agar plates. The colonies grown on these plates viewing the plate under transmitted light.
are studied further for their identification. 2 Observe all the colonies on both plates.
3 Record your observations.
4 Perform Gram stain. Observe and record morphology of the
REQUIREMENTS bacteria.
I Equipments
Microscope and CO2 incubator.
RESULTS AND INTERPRETATION BOX 54-1 BENEFICIAL EFFECTS OF

NORMAL FLORA
1 On blood agar streptococci produce pinpoint a-hemolytic
colonies where as staphylococci produce pinhead colonies 1 They suppress the colonization of the body by pathogens.
with b - haemolysis. 2 Colicins produced by some bacteria lyse some pathogenic
2 On Gram stain, streptococci are identified based on the bacteria.
arrangement, they are Gram positive cocci arranged in chains. 3 Antibodies produced against some commensals show
Staphylococci are arranged in clusters. cross reaction with some pathogens, thereby enhancing
3 On chocolate agar, colonies grown are tested for the presence immune status of the host.
of the enzyme oxidase (refer chapter 21). Neisseria are oxidase 4 Endotoxins produced by some bacteria facilitate
positive. They turn the oxidase paper into deep purple in color complement mediated defence system of the humans.
when they are streaked on the surface of the filter paper.
KEY FACTS
1 There are many microorganisms which are present as part of the normal flora in the mouth and teeth.
2 Saliva sample should be collected in a sterile bottle.
3 Saliva sample should be processed immediately after collection.
VIVA
1 What are the microorganisms present in the mouth as normal flora?
FURTHER READINGS
3 Jawetz, Melnick and Adelberg. Medical Microbiology. 23rd Edition. McGaw Hill. 2003.
4 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. 1996.
5 Murray PR, Rosenthal KS, Kobayashi GS and Pfaller ME. Medical Microbiology. 5th Edition. Mosby Inc. 2002.
162
LESSON
55 of the Throat
LEARNING OBJECTIVES III Specimen

Throat swab.
1 Demonstrate various microorganisms which are present as
part of the normal flora in the throat and respiratory tract. PROCEDURE
1 Place a tongue depressor on the extended tongue and collect

INTRODUCTION the specimen by using a sterile cotton swab, by rotating the
cotton swab gently over pharyngeal tonsils.
Many microorganisms are present in the throat and upper 2 Emulsify the throat swab in sterile saline and mix to form a
respiratory tract. These are a-hemolytic streptococci, uniform suspension.
Streptococcus pneumoniae, Staphylococcus, Branhamella, 3 Inoculate the specimen onto blood agar, chocolate agar and
Neisseria, Haemophilus, diphtheroids and spirochaetes. potassium tellurite agar plates.
a- haemolytic streptococci are present in the upper respiratory 4 Incubate the chocolate agar plate in CO2 incubator for 48
tract within 12 hours of the birth. They continue to remain as hours at 37°C.
part of important microbial flora through out life. The microbial 5 Incubate one blood agar plate and potassium tellurite agar
flora present in the pharynx and trachea are similar to that plates aerobically for 48 hours at 37°C.
present in the mouth.
OBSERVATIONS
PRINCIPLE
1 Look for the presence of hemolysis on blood agar plate by
Specimen taken from the throat is inoculated onto the surface viewing the plate under transmitted light.
of the media plates. The colonies grown on these plates are 2 Perform oxidase test for the colonies grown on chocolate agar.
studied further for their identification. 3 Look for the presence of black colored colonies on potassium
tellurite agar.
REQUIREMENTS 5 Perform Gram stain. Observe and record morphology of the
bacteria.
I Equipments
Microscope and CO2 incubator.
RESULTS AND INTERPRETATIONS
Bunsen burner, tongue depressor, sterile cotton swabs, glass 1 On blood agar, streptococci produce pinpoint a-hemolytic
slides and pencil, Gram staining reagents (methyl violet, Iodine, colonies where as staphylococci produce pinhead colonies
95% alcohol and dilute control fuchsin), filter paper containing with b-hemolysis.
1% tetramethyl paraphenyline diamine dihydrochloride, two 2 On Gram stain, streptococci are identified based on the
blood agar plates, one potassium tellurite agar plate, and one arrangement, they are Gram positive cocci arranged in chains.
5ml sterile saline tube. Staphylococci are arranged in clusters.
3 On chocolate agar, colonies grown are tested for the presence paper. Oxidase negative bacteria do not produce color when
of the enzyme oxidase (refer chapter 21). Neisseria are oxidase the colonies are streaked on oxidase paper.
positive. They turn the oxidase paper into deep purple in 4 Diphtheroids when grown on potassium tellurite agar
color when they are streaked on the surface of the filter produce pinpoint black colored colonies.
KEY FACTS
1 There are many microorganisms which are present as part of the normal flora in the throat and upper respiratory tract.
2 Throat swab should be collected in a sterile bottle.
3 Throat swab should be processed immediately after collection.
VIVA
1 In which respect, the normal microbial flora of human is helpful?

Ans. The normal microbial flora of human can reduce the infections by competing with pathogens for nutrients and binding
receptors. Some organisms also form symbiotic association with the host.
FURTHER READINGS
164
LESSON
56 of the Skin
LEARNING OBJECTIVES dextrose agar plate, lactophenol cotton blue and reagents for
Gram stain.
1 Demonstrate various microorganisms which are present as III Specimen
part of the normal flora on the skin. Skin swab.
INTRODUCTION PROCEDURE
Skin as part of its normal flora, normally harbours many bacteria. 1 Collect the swab from surface of the skin by rubbing the
It contains 102 to 104 organisms per cm2. Staphylococcus cotton swab after moistening the swab in sterile saline.
epidermidis and diphtheroids are predominant bacteria. The 2 Place the swab in a tube containing sterile saline and mix it.
microbial flora present in the pharynx and trachea are similar to 3 Inoculate a loopful suspension on one plate each of blood
that present in the mouth. Streptococcus viridans, micrococci, agar, mannitol salt agar and Sabouraud’s dextrose agar
peptostreptococci, propionibacterium, b-haemolytic 4 Incubate the Sabouraud’s agar plate for 48 hours at 25°C
streptococci, enterococci, diphtheroids, Candida species, and the remaining plates for 48 hours at 37°C.
Malasezia furfur , etc. are the other microorganisms.
OBSERVATIONS
PRINCIPLE
1 Look for the presence of hemolysis on blood agar by viewing
Swab is collected from surface of the skin and is inoculated on the plates under transmitted light.
agar plates and incubated. The colonies are studied further for 2 Look for the presence of mold like or moist growth on
their identification. Sabouraud’s agar plate.
3 Look for the presence of yellow color of the medium
surrounding colonies grown on mannitol salt agar medium.
REQUIREMENTS Yellow color colony is suggestive of S. aureus.
I Equipments
Microscope and incubator. RESULTS AND INTERPRETATION
II Reagents 1 Perform Gram stain of the colonies.

Bunsen burner, sterile cotton swabs, glass slides and pencil, 2 Perform LPCB stained smears of colonies grown on
blood agar plate, mannitol salt agar plate, Sabouraud’s Sabouraud’s dextrose agar.
VIVA
1 What is the role of resident flora of skin?

KEY FACTS
1 Skin as part of its normal flora, normally harbours many bacteria.

2 The normal resident flora of skin can reduce the infections by competing with pathogens for binding receptors.
FURTHER READINGS
166
LESSON
Identification of
57 Staphylococcus aureus
LEARNING OBJECTIVES
demonstrate the presence of Gram positive cocci arranged in
After completing this practical you will be able to: clusters along with pus cells (Fig. 57-1).
1 Know about the clinical importance of staphylococci.
2 Know the procedures used to identify Staphylococcus 2 Culture
aureus and differentiate it from other staphylococcal species.
Clinical specimens are inoculated onto nutrient agar, blood
agar and MacConkey agar plates and kept for aerobic incuba-
INTRODUCTION tion at 37°C. S. aureus produces opaque, circular colonies with
butyrous consistency. Golden yellow pigment is demonstrated
Staphylococcus species are Gram positive spherical shaped on nutrient agar (Fig. 57-2). On blood agar, they produce b-
bacteria measuring 0.8 µm to 1 µm in size arranged in clusters. haemolytic golden yellow or white colonies (Fig. 57-3). On
They are catalase positive, non-spore forming bacteria MacConkey agar they produce minute pink colored colonies.
Staphylococci are part of the indigenous flora of skin surfaces,
mucous membranes and upper respiratory tract. They usually 3 Coagulase test
cause suppurative lesions such as pustule, furuncle or carbuncle
and can also involve muscles and bones. They are less Coagulase test is most important test used for identification of
commonly associated with systemic infection. However they S. aureus (refer chapter 22).
produce a variety of exotoxins causing various toxin-mediated
diseases such as food poisoning, toxic shock syndrome and 4 Deoxyribonuclease test
staphylococcal scalded skin syndrome.
Laboratory tests for differentiation of staphylococcal Coagulase positive S. aureus also produce the enzyme
species are listed in the table 57-1. deoxyribonuclease enzyme. Detection of the presence of the
enzyme, deoxyribonuclease is used to reconfirm the
identification of S. aureus.
SPECIMENS The test organism is streaked onto the DNA agar medium
(0.2% DNA). Then the DNA plate is incubated overnight at
Clinical specimens like pus swab, and wound swab. 37°C. After incubation, 3.6% of hydrochloric acid (1N HCl) is
In this chapter the following tests will be described which added to the medium to determine the DNase activity. DNase
are employed routinely for identification of S. aureus. positive S. aureus hydrolyze the DNA resulting in production
of halo around the growth. Absence of halo around the growth
indicates DNase-negative S. aureus which are also coagulase
TESTS FOR THE IDENTIFICATION OF negative.
STAPHYLOCOCCUS AUREUS
5 Mannitol salt agar
1 Direct examination This is a selective medium used to isolate staphylococci from
clinical specimens. It contains nutrient agar with 1% mannitol,
Gram stain and 7.5% sodium chloride. Phenol red is used as an indicator.
Gram stain of smears of clinical specimens is carried out to The medium is used to test mannitol fermenting ability of
S. aureus. It also tests ability of S. aureus to tolerate the high The plate is then incubated overnight at 37°C. The presence of
salt concentration in the medium and grow readily. zone of inhibition (17 mm) around the disc indicates novobiocin-
S. aureus produces yellow colonies on the medium while sensitive S. aureus and S. epidermidis and no zone of inhibition
other staphylococcal species do not. The yellow colour is seen around the disc indicates novobiocin-resistant S. saprophyticus.
due to the fermentation of mannitol with production of acid by
S. aureus, thereby reducing pH of the medium to the acidic. In
acidic pH the indicator, phenol red, produces yellow colour.
6 Novobiocin sensitivity
Novobiocin sensitivity testing is used to detect sensitivity or

resistance of S. aureus to the antibiotic, novobiocin. This test is
used to differentiate S. aureus and S. epidermidis from S.
saprophyticus. Both S. aureus and S. epidermidis are novobiocin-
sensitive where as S. saprophyticus is novobiocin-resistant.
In this test, staphylococci strain to be tested is inoculated
onto the surface of a Mueller Hinton agar plate, followed by
application of a 5µg novobiocin disc on the surface of the agar. FIGURE 57-1 Pus smear showing Gram positive cocci in clusters, x 1000.
Table 57-1 Laboratory tests for differentiation of staphylococcal species
Test S. aureus S. epidermidis S. saprophyticus
Growth on
Mannitol salt agar + - -
Colonial pigmentation Golden yellow White White
Coagulase test + - -
DNAase test + - -
Hemolysis on blood agar Beta - -
Novobiocin sensitivity Sensitive Sensitive Resistant
FIGURE 57-2 Staphylococcus aureus colonies on nutrient agar. FIGURE 57-3 Staphylococcus aureus colonies on blood agar.
168 Identification of Staphylococcus aureus
KEY FACTS
1 Yellow color pigmentation can also be produced by micrococci which can be distinguished from staphylococci by various
biochemical tests such as modified oxidase and sensitivity for novobiocin, bacitracin and furazolidone.
2 Some coagulase negative staphylococci can produce beta hemolysis on blood agar similar to coagulase positive
staphylococci, hence should not be confused with that of S. aureus.
3 Some species of staphylococci produce only bound coagulase (clumping factor) such as S.hydenensis and S.schleifer
VIVA
1 What are the tests to identify S. aureus?

2 List novobiocin-sensitive staphylococci.
FURTHER READINGS
LESSON
Identification of
58 Streptococcus pneumoniae
LEARNING OBJECTIVES small, slightly elongated cocci arranged in pairs (diplococci)

with the broad ends in apposition. Each coccus has one end
After completing this practical you will be able to: pointed and other end broad or rounded (lanceolate shaped).
Gram stain of the CSF specimen from a case of meningitis
1 Know about the clinical importance of pneumococci. shows both intracellular as well as extra cellular cocci.
2 Know the procedures used to identify Streptococcus
pneumoniae and differentiate it from other alpha hemolytic 2 Culture
streptococci.
The specimens are inoculated onto blood agar and kept for
aerobic incubation at 37°C for 24 hours in the presence of C02.
INTRODUCTION S.pneumoniae produces small, shiny dome-shaped and
translucent colonies which are surrounded by alpha hemolysis
S. pneumoniae are Gram positive cocci arranged in pairs (Fig. 58-1). On prolonged incubation, these colonies may show a
and individual coccus is lanceolate shaped. They are depressed center with an elevated rim (Draughtsman’s colonies).
encapsulated and are an important cause of lobar This is due to the autolysis of the bacteria in old colony.
pneumonia. They also cause meningitis and other infections
such as otitis media, conjunctivitis, peritonitis, sinusitis and 3 Bile solubility test
suppurative arthritis.
S. pneumoniae undergo autolysis in the presence of surface
active agents such as sodium deoxycholate whereas other alpha
SPECIMENS hemolytic streptococci are bile insoluble hence is not lysed by
these bile salts.
Clinical specimens such as sputum, pleural exudate, CSF, Label two brain heart infusion broth tubes, one as S.
blood, etc. pneumoniae (positive control) and the other tube as S. mitis
S. pneumoniae produce a haemolysis on blood agar. S. (negative control). Add loopful of test organisms in the broth
pneumoniae can be differentiated from other alpha hemolytic to give a heavy suspension. Then add 0.5 ml of sodium
streptococci by many tests (Table 58-1). deoxycholate solution into each tube. Incubate the tubes in a
In this chapter the following tests will be described which water bath at 37°C for one hour.
are employed routinely for identification of S.pneumoniae. Clearing of turbidity indicates positive test whereas
persistence of turbidity indicates negative test. S. pneumoniae
is bile solubility positive.
TESTS FOR THE IDENTIFICATION OF
STREPTOCOCCUS PNEUMONIAE 4 Optochin test
1 Direct examination S. pneumoniae are sensitive to Optochin (ethyl hydrocupreine

hydrochloride) and produce a zone of inhibition measuring 15
mm and more. Other alpha hemolytic streptococci are resistant
Gram stain to Optochin which produce a zone of inhibition of less than 15
Gram stain of smears from clinical specimens is carried out to
mm (Fig. 58-2).
demonstrate the presence of Gram positive cocci. These are
170 Identification of Streptococcus pneumoniae
A blood agar plate is divided into two halves by glass marking 5 Inulin fermentation
pencil. One half is labeled as S. pneumoniae while the other half
is labeled as S. mitis. Using a sterile cotton swab, test strains are S. pneumoniae is capable of fermenting the sugar, inulin whereas
inoculated onto the surface of blood agar, followed by application other alpha hemolytic streptococci do not ferment inulin.
of 0.05 units Optochin disc over the inoculated surface by using The test strain is inoculated into the inulin sugar medium
forceps. The plate is incubated aerobically at 37°C under 10% (serum sugar) and is incubated over night at 37°C. Positive test
CO2 environment for 24 hours. Development of zone of inhibition is indicated by change of color of the media from red to yellow.
of 15 mm and more shows the test to be positive. Zone of Negative test is indicated by no color change, and medium
inhibition less than 15 mm shows the test to be negative. continues to appear as red.
FIGURE 58-1 Streptococcus pneumoniae on blood agar. FIGURE 58-2 Optochin sensitivity test.
Table58-1 Differences between Streptococcus pneumoniae and Streptococcus mitis
Tests Streptococcus pneumoniae Streptococcus mitis
1. Bile solubility + -
2. Optochin sensitivity + -
3. Inulin fermentation + -
4. Quellung reaction + -
5. Mouse virulence + -
KEY FACTS
1 Streptococcus pneumoniae are Gram positive cocci arranged in pairs or as short chains and individual coccus is lanceolate
shaped.
2 Gram stain of the CSF from a case of meningitis shows both intracellular as well as extra cellular cocci.
3 Str. pneumoniae is bile solubility test positive.
4 Str. pneumoniae are sensitive to Optochin.
5 Str. pneumoniae is capable of fermenting the sugar, inulin whereas other alpha hemolytic streptococci do not ferment inulin.
VIVA
1 How do you demonstrate capsules of S. pneumoniae?

Ans. India ink preparation.
2 What is the mechanism of autolysis?
Ans. Intracellular autolytic enzyme mediated.
3 What is the strength of the routinely used Optochin disc?
4 What are the rapid diagnostic methods for pneumococcal meningitis?
Ans. Capsular antigen detection in the CSF by latex agglutination and CIEP.
FURTHER READINGS
172
LESSON
Identification of
b-haemolytic Streptococci
59
LEARNING OBJECTIVES streptococci cause neonatal infections such as septicemia and
meningitis. Group D streptococci may cause urinary tract
After completing this practical you will be able to: infections and wound infections.
1 Know about clinical significance of b-haemolytic

streptococci. SPECIMENS
2 Perform important tests to identify group A streptococci,
group B streptococci and group D streptococci. Throat swab.
In this chapter the following tests will be described which
are employed routinely for identification of S. pyogenes.
INTRODUCTION
All members of b-haemolytic streptococci are Gram positive TESTS FOR IDENTIFICATION OF
and fastidious. They are cocci arranged in chains. They appear STREPTOCOCCUS PYOGENES
as circular translucent pinpoint colonies on blood agar.
Streptococci, both aerobic and anaerobic are classified on the 1 Direct examination
basis of their haemolysis on blood agar. On blood agar three
types of haemolysis are observed. These are:
Gram stain
a) a hemolysis: It is an incomplete form of hemolysis where a Gram stain of smears from clinical specimens like throat swab is
green zone is produced around the colonies. carried out to demonstrate the presence of Gram positive cocci
b) b hemolysis: It is a complete form of hemolysis. This appears arranged in short chains (Fig. 59-1).
as a clear zone around the colonies. b-haemolytic streptococci
are frequently associated with pathogenicity, and 2 Culture
c) g haemolysis: It is described as absence of any hemolysis
around the colony. g-haemolytic streptococci are avirulent. The specimens are inoculated onto 5% sheep blood agar and
kept for aerobic incubation at 37°C for 24 hours in the presence
b-haemolytic streptococci produce a clear zone of of C02. Str.pyogenes produces pinpoint colonies which are
haemolysis on the blood agar surrounding the colonies, within surrounded by a large zone of b-hemolysis (Fig. 59-2).
which erythrocytes are completely lysed. The lysis is caused
by two haemolysins, streptolysin O and streptolysin S. 3 Bacitracin sensitivity test
b-haemolytic streptococci on basis of their carbohydrate C
antigen are classified in to 20 groups (from A to V, excepting I Bacitracin test is a frequently used test to identify and
and J). This classification is known as Lancefield classification. differentiate group A streptococci from non- Group A
Group A streptococci, also known as Streptococcus streptococci. Bacitracin disc when placed on the surface of
pyogenes is the important species causing most human blood agar plate streaked with test organism inhibits its growth
infections. S. pyogenes causes a wide variety of suppurative and forms a zone of inhibition.
infections of the respiratory tract, skin, genital and other deep The test organism is streaked on to the surface of the blood
infections. It also causes non-suppurative infections such as agar. Then a bacitracin having strength of 0.04 units is placed
rheumatic fever and acute glomerulonephritis. Group B on the inoculated plate. The plate is incubated at 37°C for
overnight. Positive test is indicated by a zone of inhibition when reacts with iron salts in the medium causes brown to
around the bacitracin disc. The negative test is indicated by no black discolouration of medium following incubation.
zone of inhibition around the bacitracin disc. Test strain is inoculated onto the surface of the bile aesculin
Most strains of S. pyogenes are bacitracin sensitive. agar and kept for aerobic incubation at 37°C for 24–48 hr. Positive
test is indicated by change of the brown colour of the medium
4 CAMP (Christie, Atkins and Munch-Peterson) in to black. Negative test is indicated by no brown or black
test discoloration of medium.
CAMP (Christie, Atkins and Munch-Peterson) test is a test

used to identify Group B streptococci. CAMP substance is a
peptide produced by group B streptococci which acts
synergistically with the b-hemolysis produced by some strains
of Staphylococcus aureus enhancing the effect of haemolysis.
The test is performed by inoculating S. aureus strain as a
straight line on the surface of the sheep blood agar. Known
positive control (Group B streptococci), negative control (group
A streptococci) and test strains are also inoculated as a straight
lines parallel to S. aureus streak line leaving 1 cm space. The
plate is incubated aerobically at 37°C overnight in 5–10% CO2
environment. Positive test is indicated by an arrow head zone of
hemolysis near the S. aureus growth. Negative test is indicated
by the absence of arrow head zone of hemolysis (Fig. 59-3).
5 Bile aesculin test
This test is carried out to identify Group D streptococci. The

FIGURE 59-2 Beta hemolytic streptococci colonies on blood agar.
cocci hydrolyse aesculin into 6, 7 dihydroxy-coumarin which
FIGURE 59-1 Streptococci in short chains, x 1000. FIGURE 59-3 CAMP Test.
VIVA
1 What are different types of haemolysis produced by streptococci?

2 What is the principle of the bacitracin sensitivity test?
3 What is the principle of the CAMP (Christie, Atkins and Munch-Peterson) test?
4 What is the principle of the bile aesculin test?
174 Identification of b-haemolytic streptococci
KEY FACTS
1 b-haemolytic streptococci produce a clear zone of haemolysis on the blood agar surrounding the colonies, within which
erythrocytes are completely lysed.
2 Bacitracin test is a frequently used test to identify and differentiate group A streptococci from non- Group A streptococci.
3 Blood agar plate for CAMP test should be incubated in the environment of 5–10% CO2.
4 CAMP test should be done with parallel positive and negative control strains.
5 Bile aesculin test is carried out to identify Group D streptococci, heat resistant test and salt tolerance test.
FURTHER READINGS
5 Murray PR, Rosenthal KS, Kobayashi GS and Pfaller ME. Medical Microbiology. 5th Edition. Mosby Inc. 2002
LESSON
Identification of
60 Corynebacterium diphtheriae
LEARNING OBJECTIVES Albert’s stain

Albert’s stain is done to detect the presence of metachromatic
After completing this practical you will be able to granules in C. diphtheriae.
1 Know about the clinical importance of Corynebacterium The specimen is collected from the surface of the
diphtheriae. pseudomembrane by a sterile cotton swab. The swab containing
2 Know the procedures used to identify C. diphtheriae and the specimen is rolled on the surface of the clean glass slide.
differentiate it from other non pathogenic Corynebacterium The smear is heat fixed and Albert’s stain is carried out (refer
species. chapter 9). The smear is examined under oil immersion (100 x)
objective (Fig. 60-1).Presumptive identification of C. diphtheriae
is made based on the presence of metachromatic granules by
INTRODUCTION Albert’s stain
C. diphtheriae are thin, non-sporing, non-capsulated and non- 2 Culture

motile Gram positive bacilli which measure approximately 3 µm
× 0.3 µm in size. These bacilli have typical club-shaped swellings Loeffler’s serum slope and potassium tellurite agar are the two
at one or both ends of bacilli. They are arranged in angled pairs media frequently used for culture of C. diphtheriae.
or in angular fashion which resemble Chinese letters. These Loeffler’s serum slope is inoculated with the throat swab
bacilli have metachromatic granules towards the end of the and incubated at 37°C for 6 hours. C. diphtheriae produces
logarithmic growth period. These granules are the storage minute smooth colonies after 6 hours of incubation (Fig. 60-2).
depots of polymetaphosphate. Colonies grown on this medium are stained by Albert’s staining
C. diphtheriae causes natural infection only in man. They for demonstrating metachromatic granules.
produce diphtheriae toxin that causes local tissue necrosis Similarly, the swab specimen is inoculated on the surface of
which results in the formation of grayish white membrane at Macleod’s potassium tellurite agar media (0.04%) and is
the affected site. Commonest sites affected are tonsils, posterior incubated at 37°C for up to 48 hours. C. diphtheriae reduces
pharyngeal wall, nasal passages, larynx and trachea. Though potassium tellurite into metallic tellurium which results in
diphtheria is a disease of respiratory tract this can also affect production of black colored colonies on the medium (Fig. 60-3).
skin, conjunctiva, ear, etc.
3 Biochemical tests
SPECIMENS
The following biochemical tests are carried out:
a. Fermentation of serum sugars (glucose, maltose, sucrose,
Throat swab.
lactose, mannitol, and trehalose) for the detection of acid.
In this chapter the following tests will be described which
The colonies grown on blood agar are inoculated on to
are employed routinely for identification of C. diphtheriae.
serum sugars and gelatin, and incubated at 37°C for 24 hours.
The colour change in the serum sugar media is looked for.
TESTS FOR THE IDENTIFICATION OF Positive test is indicated by the change of media to yellow
CORYNEBACTERIUM DIPHTHERIAE colour due to production of acid. Negative test is indicated by
the persistence of red colour, because no acid is produced.
1 Direct examination C. diphtheriae ferments glucose, maltose and sucrose with
176 Identification of Corynebacterium diphtheriae
production of acid only. They do not ferment lactose, mannitol,

and trehalose.
b. Other biochemical tests which are used primarily to
differentiate C. diphtheriae from other Corynebacterium spp.
include urea hydrolysis and gelatin hydrolysis.
Positive urea hydrolysis test is indicated by change in colour
of the medium into red. Negative urea hydrolysis test is
indicated by no colour change.
Positive gelatin hydrolysis test is indicated by free flowing
of medium on inverting the gelatin tube. No free flowing on
inversion of tube indicates negative test.
C. diphtheriae do not hydrolyse urea and gelatin whereas
non-pathogenic Corynebacterium species such as C. ulcerans
hydrolyses urea. FIGURE 60-2 Loeffler’s serum slope.
FIGURE 60-1 Albert’s stain showing volutin granules, x 1000.
FIGURE 60-3 Black coloured colonies on potassium tellurite agar.
KEY FACTS
1 C. diphtheriae causes natural infection only in man.
2 Toxin detection is important to prove the role of C. diphtheriae in causation of disease than just identification of
C. diphtheriae.
3 Throat swab should be processed immediately without delay. If any delay is anticipated Amie’s transport medium can be
used to transport the specimen.
4 Presumptive identification of C. diphtheriae is made based on the presence of metachromatic granules by Albert’s stain.
5 Loeffler’s serum slope and potassium tellurite agar are the two media frequently used for culture of C. diphtheriae.
6 C. diphtheriae ferments glucose, maltose and sucrose but do not ferment lactose, mannitol and trehalose.
7 C. diphtheriae do not hydrolyse urea and gelatin whereas non-pathogenic C. ulcerans hydrolyses urea.
VIVA
1 Name other staining methods used to demonstrate volutin granules.
Ans. Neisser’s and Ponder’s stains.
2 Why serum sugars are used for testing acid production by C. diphtheriae?
Ans. These organisms are fastidious. They cannot grow in ordinary sugar media.
3 Name other organisms, which have metachromatic granules.
Ans. Bordetella and Brucella.
FURTHER READINGS
178
LESSON
Identification of
61 Lactose Fermenting
Enterobacteriaceae
LEARNING OBJECTIVES Gram’s stain

Gram stain is indicated in wound infection where pus ells are
After completing this practical you will be able to detected along with Gram negative bacilli. Both centrifuged
1 Know the procedures used to identify lactose fermenting and un centrifuged urine are subjected for microscopic
Enterobacteriaceae and differentiate it from other members examination for the detection of pus cells.
of the family Enterobacteriaceae.
2 Perform the tests to identify Escherichia coli and Klebsiella 2 Culture
species. Stool specimen is inoculated onto MacConkey agar and CLED
media and wound swabs are processed using blood agar and
MacConkey agar. Inoculated plates are kept for aerobic
INTRODUCTION incubation at 37 °C for 24 hours. Pink colored lactose fermenting
colonies of E.coli and K.pneumoniae appear on MacConkey
Enterobacteriaceae are the part of the normal intestinal flora of agar plates (Fig. 61-1 and 61-2). On CLED medium E.coli produces
animals and humans. They are aerobes and facultative lactose fermenting yellow colonies (Fig. 61-3). These are subjected
anaerobes. They are non-fastidious and reduce nitrate to to testing by various biochemical tests to identify the bacteria.
nitrites. They are catalase positive and oxidase negative. Based
on their action on lactose they are classified into lactose 3 Biochemical tests
fermenters and non-lactose fermenters. Many of commensal
Biochemical tests and other tests, as summarized in the tables
floras of the intestine are lactose fermenters. Late lactose
are performed to identify E. coli (Fig. 61-4) and Klebsiella
fermenting bacteria are called as para colon bacilli and with the
species (Fig. 61-5).
exception of Shigella sonnei all others are commensals.
These organisms cause gastrointestinal infection, urinary 4 Antibiotic susceptibility testing
tract infection, pneumonia and septicaemia. Lactose fermenters
such as E. coli, Enterobacter, Klebsiella, Citrobacter and Antibiotic susceptibility testing is carried out for the identified
enterotoxigenic E. coli cause watery diarrhoea. bacteria which provide the important information of the
The tests used for identification of E. coli and Klebsiella susceptibility pattern of the organism.
species are listed in the boxes 61-1 and 61-2 respectively.
SPECIMENS
Stool, urine, wound swab, sputum, etc.
In this chapter, following tests will be described which are
routinely used for identification of E.coli and Klebsiella spp.
TESTS FOR IDENTIFICATION OF E. COLI

AND KLEBSIELLA SPP.
FIGURE 61-1 Mc Conkey agar with lactose fermenting pink colonies
1 Direct examination of Escherichia coli.
+ + – –
FIGURE 61-2 Mc Conkey agar with lactose fermenting pink mucoid FIGURE 61-4 IMViC test for Escherichia coli.
colonies of Klebsiella pneumoniae.
– – + +
FIGURE 61-3 CLED Medium with lactose fermenting yellow FIGURE 61-5 IMViC test for Klebsiella pneumoniae.
colonies of Escherichia coli.
BOX 61-1 IDENTIFICATION OF BOX 61-2 IDENTIFICATION OF

ESCHERICHIA COLI KLEBSIELLA SPECIES
Gram stain Gram stain

Gram negative bacilli. Short thick gram negative bacilli.
Motility by hanging drop preparation method Motility by hanging drop preparation method
Motile. Non motile.
Biochemical tests Biochemical tests
Catalase test: Positive. Catalase test: Positive.
Oxidase test: Negative. Oxidase test: Negative.
Nitrate reduction test: Reduced to Nitrite. Nitrate reduction test: Reduced to Nitrite.
Kligler’s iron agar medium: Acid/Acid. No H2S gas is produced. Kligler’s iron agar medium: Acid/Acid. gas is present.
Fermentation of sugars (glucose, lactose and mannitol): Acid Fermentation of sugars (glucose, lactose and mannitol): Acid
and gas. and gas.
Methyl red test: Positive. Methyl red test: Negative.
Voges-Proskauer test: Negative. Voges-Proskauer test: Positive.
Indole test: Positive. Indole test: Negative.
Citrate utilization test: Negative. Citrate utilization test: Positive.
Urease test: Negative. Urease test: Positive (only K. pneumoniae).
Lysine decarboxylation test: Positive. Lysine decarboxylation test: Positive.
180 Identification of Lactose Fermenting Enterobacteriacae
KEY FACTS
1 Enterobacteriaceae is the part of the normal intestinal flora of animals and humans.
2 Lactose fermenters such as Enterobacter, Klebsiella and Citrobacter and enterotoxigenic E. coli causes watery diarrhoea.
3 Antibiotic susceptibility testing should be done for the identified organism which will give the important information of the
susceptibility pattern of the organism.
VIVA
1 Name the indicator used in MacConkey agar medium?

Ans. Neutral red.
2 Why some bacteria are late lactose fermenters?
Ans. They lack the enzyme lactose permease.
3 What is the color of the colonies of lactose fermenting organism on CLED medium?
Ans. Yellow colour.
4 Lactose fermenting colonies grown on MacConkey agar plate are not picked up for oxidase test why?
Ans. Acidic pH of the colonies will give false positive reactions.
5 Differentiate between E. coli and Klebsiella species.
FURTHER READINGS
LESSON
Identification of
62 Vibrio cholerae
LEARNING OBJECTIVES the corners of cover slip. A loopful of stool specimen is

collected by a heat sterilised wire loop and transferred it on to
After completing this practical you will be able to the middle of the cover slip. This is placed over a clean cavity
slide in such a way that the drop hangs from the cover slip to
1 Know about the clinical importance of Vibrio cholerae. cavity of the slide. This preparation is observed first under 10x
2 Perform the tests to identify V. cholerae and differentiate it followed by 40x, for the presence of characteristic darting
from other Vibrio species. motility of V.chloerae.
Observation of darting motility in stool sample helps in
presumptive diagnosis of cholera, which should be confirmed
INTRODUCTION by biochemical and serologic methods.
V. cholerae are short curved or straight Gram negative bacilli 2 Culture

measuring about 1.5 µ × 0.2 to 0.4 µ. These organisms are
actively motile by means of a single sheathed polar flagellum. Alkaline peptone water is used as enrichment medium.
They are strongly aerobic and they grow in alkaline pH (7.4 to DCA and TCBS are employed as the selective media for
9.6) media. They cause cholera, a toxin mediated disease. Cholera V. cholerae.
occurs in many forms sporadic, endemic, epidemic or pandemic. A loopful of stool is inoculated into alkaline peptone water
Cholera is an exclusively human disease. Infections are spread and incubated at 37°C for 6 hours. Then this is sub cultured on
by feco-oral transmission. to TCBS medium, and incubated at TCBS at 37°C overnight.
List of tests for identification of V.cholerae is summarized After overnight incubation the presence of yellow colonies on
in the box 62-1. TCBS medium and non lactose fermenting colorless colonies
on DCA is looked for. The biochemical tests of these colonies
are performed for identification of V. cholerae.
SPECIMENS
3 Biochemical tests
Stool (rice watery stool) and rectal swab.
In this chapter the following tests will be described which Biochemical tests such as lysine and ornithine decarboxylase
are employed routinely for identification of V. cholerae. and arginine dihydrolase (Fig. 62-1) and other tests performed
to identify V. cholerae are summarized in the box 62-1.
Biochemical tests like Voges Proskauer, polymyxin-B (50 U)
TESTS FOR IDENTIFICATION OF sensitivity (Fig. 62-2), sheep RBC hemolysis and chick cell
VIBRIO CHOLERAE agglutination are done further for the differentiation of two
biotypes Classical and Eltor (Table 62-1).
1 Direct examination
4 Antibiotic susceptibility testing
Hanging drop preparation Antibiotic susceptibility testing is carried out for the identified
This test is carried out to demonstrate motility of V.chloerae,
bacteria which provide the important information of the
which are actively motile.
susceptibility pattern of the organism.
A clean cover slip is taken and vaseline is applied over all
182 Identification of Vibrio cholerae
BOX 62-1 IDENTIFICATION OF

VIBRIO CHOLERAE
Gram stain
Gram negative curved or straight rods.
Motility by hanging drop preparation method

Actively motile.
Biochemical tests
Catalase test: Positive. + + –
Oxidase test: Positive. FIGURE 62-1 Lysine and ornithine decarboxylase and arginine
dihydrolase tests for Vibrio cholerae.
Nitrate reduction test: Reduced to Nitrite.
Kligler’s iron agar medium – K/A.
Fermentation of sugars (glucose, sucrose and mannose
fermented: Acid only, lactose not fermented).
Indole test: Positive.
Citrate utilization test: Positive.
Urease test: Negative.
Lysine decarboxylation test: Positive.
Ornithine decarboxylation test: Positive.
Arginine dihydrolase test: Negative.
Confirmation of V. cholerae isolates is done by serotyping
with specific O antisera (slide agglutination test) using
O1 Ogawa and, O1 Inaba antisera. FIGURE 62-2 Polymyxin B sensitivity of Vibrio cholerae.
Table62-1 Differences between V. cholerae biotype classical and V. cholerae biotype El Tor
Test V. cholerae biotype classical V. cholerae biotype El Tor
Voges-Proskauer (VP) test - +

Sheep RBCs haemolysis - +
Chick RBCs agglutination - +
Polymyxin B sensitivity + -
Susceptibility to
Mukherjee’s phage IV + -
Sensitivity to
Vibriostatic (O/129) agent + -
Susceptibility to group V phage - +
KEY FACTS
1 Subculture from the incubated alkaline peptone water to be done within 6 hours of incubation.
2 Looking for the presence of darting motility is not recommended for the diagnosis of cholera because this type of motility can
also be observed in other bacteria.
3 Biochemical tests like Voges Proskauer, polymyxin B sensitivity, sheep RBC hemolysis, chick cell agglutination are usually
carried out to differentiate between V. cholerae biotypes Classical and Eltor.
VIVA
1 What are non agglutinable vibrios?

2 Mention the application of vibriostatic O/129 reagent.
3 Why yellow colonies are produced on TCBS while culturing V. cholerae?
Ans. Sucrose fermentation degreases pH, which converts bromo thymol blue to yellow colour.
4 What is the chemical agent used for string test for V. cholerae?
Ans. Sodium deoxycholate (0.5 %).
5 How the haemodigestion of V. cholerae differs from hemolysis of other bacteria?
Ans. Haemodigestion is not toxin mediated. It is mediated by peroxides.
FURTHER READINGS
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11th ed. (The CV Mosby Company, St. Louis) 2002.
184
LESSON
Identification of
63 Pseudomonas aeruginosa
LEARNING OBJECTIVES 2 Culture
After completing this practical you will be able to Pus swab is inoculated on to blood agar, MacConkey agar and
1 Know the clinical importance of Pseudomonas aeruginosa. nutrient agar and the plates are incubated at 37°C overnight.
2 Perform the tests to identify P. aeruginosa. Moist hemolytic colonies are produced on blood agar (Fig. 63-1).
Greenish blue pigmented colonies are produced on nutrient
agar (Fig. 63-2). P. aeruginosa colonies produce earthy or grape
INTRODUCTION juice like odour.
P. aeruginosa are non-fermentative slender Gram negative 3 Oxidase test

bacilli that measure about 1.5 µm to 3µm by 0.5 µm in size. They
are obligate aerobes. They grow at a temperature range of 6°C
to 42°C. They produce opaque irregular colonies with earthy P. aeruginosa is an oxidase positive bacterium. It produces
smell and form non-lactose fermenting colonies on MacConkey the enzyme cytochrome oxidase. The enzyme in the presence
agar. In liquid media they form a surface pellicle. P. aeruginosa of oxygen oxidizes 1% tetramethyl paraphenylene diamine
produces pigments such as pyocyanin, pyoverdin and dihydrochloride and produces a deep purple colour. A filter
pyorubrin. They utilise carbohydrates oxidatively. paper strip impregnated with 1% tetramethyl paraphenylene
Pseudomonas causes infection of burns and wounds and is a diamine dihydrochloride is taken. By a platinum wire, the
common cause of nosocomial infections. colonies on nutrient agar are picked up and streaked on the
The tests for identification of P. aeruginosa are mentioned surface of the filter paper. Positive test is indicated by
in the box 63-1. appearance of deep purple color, and negative test by no color
change (refer chapter 21). Pseudomonas and other non-
fermentative Gram negative bacilli are oxidase positive whereas
SPECIMENS all Enterobacteriaceae bacteria are oxidase negative.
Pus swab.
4 Biochemical tests
In this chapter the following tests will be described which are
employed routinely for identification of P. aeruginosa. They are non-fermenters. They break down glucose oxidatively
with production of acid only (Fig. 63-3).
TESTS FOR IDENTIFICATION OF 5 Antibiotic susceptibility testing

PSEUDOMONAS AERUGINOSA
1 Direct examination Antibiotic susceptibility testing is carried out for the identified
bacteria which provide the important information of the
susceptibility pattern of the organism (refer chapter 31).
Gram stain
Gram stain of the pus swab reveal thick Gram negative bacilli
measuring 1.5–3 µm by 0.5 µm in size.
BOX 63-1 IDENTIFICATION OF

PSEUDOMONAS AERUGINOSA
Gram stain
Gram negative bacilli.
Motility
Motile.
Biochemical tests
Growth at 42°C: Positive.
Catalase test: Positive.
Oxidase test: Positive.
Nitrate reduction test: Reduced to nitrite.
Kligler’s iron agar median: K/K
Oxidative breakdown of sugars glucose: acid only, lactose,
mannitol, sucrose, maltose: No acid.
Indole test: Negative.
FIGURE 63-2 Pseudomonas colonies on nutrient agar.
Citrate utilization test: Positive.
Urease test: Variable.
Lysine decarboxylation test: Negative.
Arginine dihydrolase test: Positive.
– +
FIGURE 63-1 Non hemolytic Pseudomonas colonies on blood agar. FIGURE 63-3 OF test showing oxidative utilization of glucose.
KEY FACTS
1 P. aeruginosa are non-fermentative slender Gram negative bacilli.
2 They utilise carbohydrates oxidatively.
3 P. aeruginosa produces greenish blue pigmented colonies on nutrient agar.
4 P. aeruginosa is an oxidase positive bacteria.
VIVA
1 Name the selective media used for the isolation of P. aeruginosa.
Ans. Cetrimide agar.
2 Name different pigments produced by P. aeruginosa?
FURTHER READINGS
186
UNIT
IX
Parasitology
Introduction
Lesson 64 Saline Wet Mount of Stool
Lesson 65 Iodine Wet Mount of Stool
Lesson 66 LPCB Wet Mount of Stool
Lesson 67 Acid-fast Staining of Stool Smears
Lesson 68 Leishman’s Staining of Peripheral Blood Smears
Lesson 69 Concentration of Stool for Parasites
Lesson 70 Culture of Stool for Entamoeba histolytica

188
Introduction
Parasitology traditionally includes the study of three major groups of animal parasites: parasitic protozoa, parasitic helminths
(worms), and those arthropods that directly cause disease or act as vectors of various pathogens. Infections of humans caused
by parasites number in the billions and range from relatively innocuous to fatal. The diseases caused by parasites constitute
major human health problems throughout the world.
The incidence of many parasitic diseases (e.g. schistosomiasis, malaria) have increased rather than decreased in recent years.
Other parasitic illnesses have increased in importance as a result of the AIDS epidemic (e.g., cryptosporidiosis, Pneumocystis
carinii pneumonia, and strongyloidiasis). The unicellular parasites (protozoa) and multicellular parasites (helminths, arthropods)
are antigenically and biochemically complex, as are their life histories and the pathogenesis of the diseases they cause. The basis
for effective treatment and cure of a patient is the rapid diagnosis of the disease and its causative agent, which is based on the
analysis of the clinical symptoms in combination with laboratory tests. As we are in the 21st century, clinicians are becoming
increasingly able to diagnose and treat diseases at the molecular level.
During past few years, there has been an increased awareness of the importance of trained and qualified personnel to perform
diagnostic procedures. It becomes even more important to provide well-written lab protocols and to standardize test methods for
consistency. Therapy based on patient history and symptoms is not generally recommended in cases of parasitic infections.
Thus understanding of characteristics of any parasitic infection and the use of appropriate diagnostic procedures accompanied
by a complete understanding of the limitations of each procedure become very important.
LESSON
Saline Wet Mount
64 of Stool

Microscopic slides, cover slips, physiological saline, and
After completing this practical you will be able to: applicator stick
1 Perform saline wet mount preparation of faeces for Preparation of physiological saline: Normal saline is also
demonstration of intestinal parasites. called physiological saline. It is 0.85% sodium chloride in
2 Identify intestinal protozoal cysts, trophozoites, helminthic distilled water. It is prepared by weighing 8.5 gm of sodium
eggs and larva based on the recognition of specific chloride and dissolving it in 1000 ml of distilled water.
morphological characters, in the stool specimen.
III Specimen
Fresh stool specimen is required for stool microscopy.
INTRODUCTION
Stool microscopy is an easy and rapid method employed for PROCEDURE

detection of intestinal ova and cysts with the aid of wet mount
preparations. The wet mount preparations most commonly used 1 Take a clean glass slide.
for detection are the i) saline wet mount preparation and ii) 2 With help of a glass dropper, put a drop of saline on the
iodine wet mount preparation. Of late, lacto-phenol cotton blue glass slide.
(LPCB) wet mount preparation is also used. The wet mount 3 With the help of an applicator stick, take a small portion of
preparations of stool specimen are screened first under the low the stool specimen (match stick head size) and emulsify it in
power objective (10x) and then under high power (40x). the drop of saline.
The saline preparation is mainly used to a. demonstrate the 4 Put a cover slip over the saline suspension of the stool.
presence of trophozoites by demonstrating the motility and b 5 Examine the preparation first under the low power (10x) and
differentiate between bile stained and non bile-stained eggs. then under high power (40x) objective of the microscope.
6 Record the findings with description of the morphological
characteristics.
PRINCIPLE
QUALITY CONTROL
The saline wet mount preparation is prepared by mixing a small
quantity of faeces with physiological saline. The saline wet A saline and iodine wet mount preparation of known positive
mount is a colourless preparation that highlights the staining stool specimen for protozoal cysts, larva and helminthic eggs are
property of the egg, whether bile or non-bile stained, detects prepared. These preparations are observed and compared with the
motility of trophozoites, and facilitates demonstration of saline and iodine wet mount preparations of the test stool specimen
chromatoidal bodies in the cyst. for various morphological forms of the parasite and identified.
REQUIREMENT OBSERVATIONS
I Equipments 1 A 10–15 µm sized round refractile, colourless structure seen
Compound light microscope. with four nuclei, though not distinct.
190 Saline Wet Mount of Stool
2 A 15–30µm sized, round refractile, colourless structure seen 3 Cyst of Giardia intestinalis (Fig. 64-2).
with at least eight nuclei, though not distinct. 4 Hook worm egg (Fig. 64-3).
3 A 6–10µm sized oval shaped colourless structure seen with 5 Fertilized egg of Ascaris lumbricoides (Fig. 64-4).
distinct wall and faint axostyle. 6 Egg of Trichuris trichiura. (Fig. 64-5).
4 A 60µm × 40µm sized, oval shaped, non-bile stained,
colourless structure seen with transparent hyaline shell
membrane and segmented ovum with blastomeres. A clear
space is visible between the thin hyaline shell membrane
and blastomeres.
5 A 60–75µm × 40–50µm sized, round oval shaped, bile stained
structure seen with thick translucent shell with an
albuminous coat. It contains a very large conspicuous
unsegmented ovum with a clear space at each pole.
6 A 25µm × 50µm sized, bile coloured, and barrel shaped
structure seen with visible mucus plugs at each pole. It has a
double layered egg shell enclosing a visible unsegmented ovum.
1 Cyst of Entamoeba histolytica/dispar (Fig. 64-1).

2 Cyst of Entamoeba coli. FIGURE 64 -3 Hook worm egg, x 400.
FIGURE 64 -1 Cyst of Entamoeba histolytica/dispar, x 400. FIGURE 64 -4 Fertilized egg of Ascaris lumbricoides, x 100.
FIGURE 64 -2 Cyst of Giardia intestinalis, x 400. FIGURE 64 -5 Eggs of Trichuris trichiura, x 400.
KEY FACTS
1 A stool wet mount preparation should always first be screened under the low power objective (10x) and then under the high
power (40x) for identification.
2 A wet mount preparation should neither be too thick nor thin. The preparation should be such that, a printed letter should
be read through it. The wet mount preparation must not overflow.
3 Trophozoites and larvae are visualised best in the saline wet mount.
4 Bile staining property of the parasitic egg can be appreciated.
VIVA
1 List the normal constituents of stool.

Ans. The normal constituents of stool are yeast cells, cotton fiber, starch cell, potato parenchymal cell, plant epidermal hairs,
fungal spores, muscle fibres, vascular structure of plants, pollen, stone cell, plant cells, oil drops, rice starch, potato starch,
corn starch, leucocytes, moulds and bacteria.
2 What are the different wet mount preparations of stool?
Ans. Different wet mount preparations of stool are saline wet mount, iodine wet mount, LPCB wet mount, and buffered
methylene blue.
3 What are the uses of saline wet mount?
FURTHER READINGS
1 Garcia LS. Diagnostic Medical Parasitology. ASM press, Washington D.C. 4th Edition. 2003.
2 Parija SC. Textbook of Medical Parasitology. All India Publishers and Distributors. 3rd Edition. 2006.
3 Parija SC. Stool Microscopy. BPKIHS, Dharan, Nepal, 1998.
192
LESSON
Iodine Wet Mount
65 of Stool

Microscopic slides, cover slips, applicator stick and Dobell
After completing this practical you will be able to: and O’Connor’s iodine.
1 Perform saline wet mount preparation of faeces for Preparation of Dobell’s iodine: Dobell’s iodine is used for
demonstration of intestinal parasites. making iodine wet mount of stool. It contains iodine and
2 Identify intestinal protozoan cysts, trophozoites, helminthic potassium iodide in distilled water. It is prepared by weighing
eggs and larva based on the recognition of specific 2 grams of iodine crystals and dissolving it in 100 ml of distilled
morphological characters, in the stool specimen by iodine water. Then 4 grams of potassium iodide is weighed and added
wet mount. to the solution prepared. It is mixed well and Dobell’s iodine
solution is prepared.

Iodine wet mount is prepared by using a drop of saline for
preparation of wet mount of stool. Different types of iodine
solution are used. These are Dobell and O’Connor’s iodine, PROCEDURE
Lugol’s’ iodine and D’ Antonie’s iodine. Iodine wet mount is
mainly used for protozoal cysts. The preparation clearly 1 Take a clean glass slide.
demonstrate the presence of nuclei in protozoan cyst as brown 2 With the help of a glass dropper, put a drop of iodine on the
dots, and also demonstrates the presence of glycogen mass in glass slide.
protozoan cyst. 3 With the help of an applicator stick, take a small portion of
the stool specimen (match stick head size) and emulsify it in
the drop of iodine.
PRINCIPLE 4 Put a cover slip over the iodine suspension of the stool.
5 Examine the preparation first under the low power (10x) and
The iodine wet mount preparation is a brown coloured preparation then under high power (40x) objective of the microscope.
that highlights the presence of pale refractile nuclei, yellowish 6 Record the findings with description of the morphological
cytoplasm and brown glycogen material within the cysts. Bile characteristics.
staining property of the helminthic eggs cannot be appreciated
in the iodine preparation, as it is already coloured. The motility
of trophozoites is also inhibited in the iodine wet mount. QUALITY CONTROL
List of bile-stained and non-bile stained eggs are provided
in the table 65-1. A saline and iodine wet mount preparation of known positive
stool specimen for protozoal cysts, larva and helminthic eggs
are prepared. These preparations are observed and compared
REQUIREMENTS with the saline and iodine wet mount preparation of the test
stool specimen for various morphological forms of the parasite
I Equipments and identified.
OBSERVATIONS
1 A 10–15µm sized, round, yellow coloured, structure seen

with brown coloured 1–4 nuclei and glycogen mass.
2 A 15–30µm sized, round, yellow coloured structure seen with
brown coloured 1–8 nuclei and diffuse glycogen mass.
3 A 6–10µm sized oval shaped brown coloured structure seen
with visible axostyle and distinct cyst wall surrounded by a
halo.
4 A 60µm × 40µm sized, oval shaped, yellow coloured structure
seen with lightly stained shell membrane and segmented
ovum with light yellow stained blastomeres. A clear space is
visible between the stained shell membrane and blastomeres.
5 A 60–75µm×40–50µm sized, round oval shaped, yellow
coloured structure seen with yellow stained outer corticated
thick cell wall. The unsegmented ovum and also the space FIGURE 65-2 Cyst of Giardia intestinalis, x 400.
between the shell and ovum at each pole are stained yellow.
6 A 25µm × 50µm sized yellow coloured, barrel-shaped structure
seen with lightly stained mucus plugs at each pole. The egg
shell is stained brown and encloses the light yellow stained
unsegmented ovum.

2 Cyst of Entamoeba coli.
3 Cyst of Giardia intestinalis (Fig. 65-2).
4 Hook worm egg (Fig. 65-3).
5 Fertilized egg of Ascaris lumbricoides (Fig. 65-4).
6 Eggs of Trichuris trichiura (Fig. 65-5).
FIGURE 65-3 Hook worm egg, x 400.
Table 65-1 List of bile-stained and non-bile stained eggs
Bile stained eggs

Ascaris lumbricoides, Trichuris trichiura, Taenia species,
Echinococcus species, Diphyllobothrium latum,
Schistosoma mansoni, Schistosoma japonicum,
Schistosoma hematobium, Fasciola hepatica, Fasciolopsis
buski, Paragonimus westermani, and Clonorchis sinensis.
Non-bile stained eggs

Hook worm, Enterobius vermicularis and Hymenolepis
FIGURE 65- 1 Cyst of Entamoeba histolytica/dispar, x 400. nana.
194 Iodine Wet Mount of Stool
FIGURE 65- 4 Fertilized egg of Ascaris lumbricoides, x 400. FIGURE 65 –5 Eggs of Trichuris trichiura, x 400.
KEY FACTS
1 A stool wet mount should always be screened immediately.

2 A wet mount preparation should neither be too thick or thin.
3 Iodine wet mount kills both living trophozoites of protozoa and larvae of worms.
4 Bile staining property of the parasitic egg cannot be appreciated in the iodine preparation as it is already coloured.
5 Iodine solution should always be handled with care, as it proves injurious if inhaled or comes in contact with eyes.
VIVA
1 What are the uses of iodine wet mount?

2 What are the different types of iodine solution that can be used for stool wet mount preparation?
3 List the advantages and disadvantages of iodine wet mount.
Ans.
Advantages
a The number of nuclei present in the protozoan cyst can be clearly distinguished.
b It also demonstrates the yellowish cytoplasm and brown glycogen mass present in the cysts.
Disadvantages
a Trophozoites are killed by iodine, hence cannot be demonstrated in iodine wet maint.
b The bile staining property of the helminthic eggs cannot be made out.
c Chromatoidal bars in protozoan cysts are not clearly demonstrable.
4 List bile stained eggs.

5 List non-bile stained eggs.
FURTHER READINGS
LESSON
LPCB Wet Mount
66 of Stool

1 Perform lacto-phenol cotton blue (LPCB) wet mount II Reagents and glass wares
preparation of faeces for demonstration of intestinal Microscopic slides, cover slips, applicator stick and
parasites. lactophenol cotton blue (LPCB) reagent.
2 Identify intestinal protozoal cysts, trophozoites, helminthic Preparation of LPCB solution: Weigh 20 gm phenol crystals,
eggs and larva based on the recognition of specific and transfer it to a beaker (100 ml capacity). Measure 20 ml of
morphological characters, in the stool specimen by the LPCB distilled water in a measuring cylinder and add to the phenol,
wet mount and mix it well. Measure 20 ml of lactic acid. Then transfer to
the bottle. Measure 40 ml of glycerol and then transfer to the
bottle. Mix it well. Dissolve the solution by heating gently over
INTRODUCTION a spirit flame. Then allow it to be cooled. Weigh 0.05 gm of
cotton blue. Add this to the solution and mix it well. Transfer
Lacto-phenol cotton blue (LPCB) is a staining reagent which is the LPCB solution to a clean, leak proof, brown bottle. Label
extensively used in the examination of clinical specimens for the bottle and mark it. For daily use, students can transfer
demonstration of fungi and fungal elements. Of late, the LPCB about 10 ml of LPCB solution to a small brown dropper bottle
has also been used in the direct wet mount preparation of stool or insert a dropping pipette through the cap of a small brown
specimens for demonstration of larvae, ova and cysts of the bottle.
intestinal parasites.
III Specimen
PRINCIPLE
The LPCB is a combined fixative, staining and clearing agent. It PROCEDURE

contains cotton blue which stains both the helminthic ova and
1 Take a clean glass slide.
protozoal cysts deep blue. It contains phenol and lactic acid
2 With help of a glass dropper, put a drop of LPCB on the
which clears faecal debris. Glycerol in the LPCB provides a
glass slide.
semi-permanent preparation. Therefore, in the wet mount
3 With the help of an applicator stick, take a small portion of
preparation of LPCB, blue-colour stained cysts of intestinal
the stool specimen (match stick head size) and emulsify it in
protozoa and ova of eggs could easily be detected and identified.
the drop of LPCB.
Helminthic ova are stained such a deep blue that it is difficult
4 Put a cover slip over the LPCB suspension of the stool.
to miss them even during screening with a low power objective.
5 Examine the preparation first under the low power (10x) and
Additional advantage of the LPCB is that it can also detect
then under high power (40x) objective of the microscope.
blue coloured Cyclospora and Isospora, the intestinal coccidian
Note: Examine at least 30 minutes after preparation of the
parasites, in the stool. Vegetable cells, mucus, muscle fibres
wet mount.
and other artifacts are clearly stained with LPCB stain. They
6 Record the findings with description of the morphological
are still recognisable as artifacts when the LPCB stain is used.
characteristics.
196 LPCB Wet Mount of Stool
5 Fertilized egg of Ascaris lumbricoides (Fig. 66-4).

QUALITY CONTROL 6 Egg of Trichuris trichiura.
A LPCB wet mount preparation of known positive stool
specimen for protozoal cysts, larva and helminthic eggs are
prepared. These preparations are observed and compared with
the LPCB wet mount preparation of the test stool specimen for
various morphological forms of the parasite and identified.
OBSERVATION
1 A 10–15 µm sized, round, deep blue coloured, structure seen

with blue coloured 1–4 nuclei and glycogen mass.
2 A 15–30 µm sized, round, deep blue coloured structure seen
with blue coloured 1–8 nuclei and diffuse glycogen mass.
3 A 6–10 µm sized oval shaped deep blue coloured structure
seen with visible axostyle and distinct cyst wall surrounded
by a halo.
4 A 60µm × 40µm sized, oval shaped, deep blue coloured
structure seen with lightly stained shell membrane and FIGURE 66 - 2 Cyst of Giardia intestinalis, x 400.
segmented ovum with deep blue stained blastomeres. A clear
space is visible between the stained shell membrane and
blastomeres.
5 A 60–75 µm × 40–50µm sized, round oval shaped, deep blue
coloured structure seen with blue stained outer corticated
thick cell wall. The unsegmented ovum and also the space
between the shell and ovum at each pole are stained blue.
6 A 25µm × 50µm sized blue coloured, barrel -shaped structure
seen with lightly stained mucus plugs at each pole. The egg
shell is stained blue and encloses the blue stained
unsegmented ovum.

2 Cyst of Entamoeba coli.
3 Cyst of Giardia intestinalis (Fig. 66-2). FIGURE 66 - 3 Hook worm egg, x 400.
4 Hook worm egg (Fig. 66-3).
FIGURE 66 - 1 Cyst of E. histolytica / dispar, x 400. FIGURE 66 - 4 Fertilzed egg of Ascaris lumbricoides, x 100.
KEY FACTS
1 LPCB wet mount of stool is always examined at least 30 minutes after preparation of the wet mount.
2 A wet mount preparation should neither be too thick nor thin.
3 LPCB kills the trophozoites of Entamoeba, Giardia and Trichomonas, hence can not be demonstrated by LPCB.
4 In LPCB preparation, both bile-stained and non bile-stained helminthic eggs are stained blue.
VIVA
1 What are the uses of LPCB mount?

2 List the advantages and disadvantages of the iodine wet mount.
FURTHER READINGS
198
LESSON
Acid-fast Staining
67 of Stool Smears

Inoculation loop, carbol fuchsin, 5% aqueous sulphuric acid
After completing this practical you will be able to: (decolouriser), and 0.3% methylene blue (counter stain).
1 Perform modified acid fast staining of faeces for
demonstration of intestinal coccidian parasites. III Specimen
2 Detect and recognize the presence of acid fast oocysts in Stool specimen.
the stool smear stained by acid-fast method.
PROCEDURE
INTRODUCTION
1 Make a smear of stool on a clean glass slide.
Intestinal coccidian parasites, namely Cryptosporidium parvum, 2 Heat fix the smears by heating at 70°C for 10 minutes.
Cyclospora cayetanensis and Isospora belli cause infection of 3 Put the smears on a slide rack and flood the smear with
the gastrointestinal tract of humans. The infection causes carbol fuchsin.
diarrhoea, which may be serious in the immunocompromised 4 Heat the slides from below intermittently by Bunsen flame
patients. Oocysts, the diagnostic morphological form of the until the steam rises. Do not allow the stain to dry on the
parasite, are usually excreted in the human faeces. These oocysts slide, and if necessary add more carbol fuchsin to cover
are demonstrated by the modified acid fast staining technique the smear. The slide is allowed to stain for 9 minutes.
as pink-coloured acid fast structures against a blue background. 5 Wash the smears with tap or distilled water.
6 Cover the smear with 5% aqueous sulphuric acid, as
decolouriser, for 30 seconds.
PRINCIPLE 7 Wash the slides with water to remove all traces of acid.
8 Cover the smear with methylene blue, as counter stain, for
The principle of modified acid fast staining is based on the fact 1 minute.
that the oocyst of these coccidian parasites are acid fast and 9 Rinse the smears again under tap water and air dry it.
retain the basic dye (dilute carbol fuchsin)appearing pink. They 10 Observe the smear first under low power (10x) objective,
do not get decolourised with the acid alcohol and hence do not and then under oil immersion (100x) objective.
take up the counter stain methylene blue. The stool material in Note: The smear should be examined following a zig-zag pattern
the background gets decolourised easily and take up the for at least 10–30 minutes, before declaring the smear negatives.
counter stain appearing blue. Both hot and cool modified acid 11 Record the observations in the note book. Findings are
stains have been used with equal sensitivity. Acid fast parasites recorded, together with grading of the positive smear.
and parasitic components are listed in the box 67-1.
In this chapter hot modified acid fast technique will be described.
QUALITY CONTROL
A known positive control stool smear stained by the modified

REQUIREMENTS
acid fast staining method containing the pink coloured acid
fast oocysts of C. parvum. The stained smear is compared with
I Equipments
the control smear for appropriate oocyst morphology and
Compound light microscope and Bunsen flame.
staining appearance.
BOX 67-1 ACID FAST PARASITES AND RESULTS AND INTERPRETATION

PARASITIC COMPONENTS
1 The acid fast structure is oocyst of C. parvum (Fig. 67-1).
The intestinal coccidian parasites 2 The acid fast structure is the oocyst of I. belli (Fig. 67-2).
Cryptosporidium parvum. 3 The acid fast structure is the oocyst of C. cayetanensis
Cyclospora cayetanensis. (Fig. 67-3).
Isospora belli. Note: Cyclospora oocysts are variably acid fast.
The non- intestinal coccidian parasites
Toxoplasma gondii.
Sarcocystis hominis.
Other parasitic components
Scolices of Echinococcus granulosus.
Spores of Microsporidia.
Eggs of Taenia saginata.
OBSERVATIONS
1 Red coloured spherical acid fast structure measuring 4-6µm

diameter seen against a blue background of stool smear.
2 Red coloured elliptical shaped acid fast structure measuring
20µ m x 10µm seen against a blue background of stool smear.
3 Red coloured, round to ovoid shaped acid fast structure
measuring 8µm–10µm diameter seen against a blue
FIGURE 67- 2 Oocyst of I. belli, x 1000.
background of stool smear. Some structures are acid fast
while others are not.
FIGURE 67- 1 Oocyst of C. parvum, x 1000. FIGURE 67- 3 Oocyst of C. cayatenensis, x 1000.
KEY FACTS
1 Modified acid fast staining is a widely used method for the demonstration of the oocysts of intestinal coccidian parasites
in stool.
2 It is an example of permanent staining of stool.
3 Stool specimens should be handled carefully, as infection is caused by ingestion of oocysts from hands.
4 Oocysts of Cryptosporidium and Isospora are consistently acid fast in nature while Cyclospora is variably acid fast.
5 Cyclospora oocysts are twice the size of the Cryptosporidium oocysts.
6 Cryptosporidium oocysts are to be distinguished from yeast cells which are non acid fast and show budding and variable
sizes.
200 Acid-fast Staining of Stool Smears
VIVA
1 List the intestinal and non intestinal coccidian parasites.

2 How are oocysts of Cryptosporidium differentiated from yeast cells in this staining technique?
Ans. The features that differentiate the oocyst of Cryptosporidium from that of yeast cells in modified acid-fast staining are
that oocyst of Cryptosporidium are acid-fast while that of the yeast cells are non-acid fast and are of variable sizes with
budding.
3 Name the coccidian parasite associated with traveler’s diarrhoea.
Ans. Cyclospora cayetanensis and Cryptosporidium parvum.
4 Name the stool concentration method for intestinal coccidian parasites?
Ans. Sheather’s sucrose floatation method.
5 List all acid fast parasitic components.
FURTHER READINGS
LESSON
Leishman’s Staining of
68 Peripheral Blood Smears
LEARNING OBJECTIVES Thin blood films are used primarily for identification of
species of malarial parasites. Thin blood films are of monolayer
After completing this practical you will be able to: thickness, allows better identification of species. The
disadvantage of thin blood smear is that as less amount of the
1 Perform Leishman’s staining of peripheral blood smear for blood is examined, the number of parasites per field too are
malaria parasites and microfilariae. much less than that of thick blood smear.
2 Detect and identify important parasites of blood such as The procedure followed for staining thick smears are
malarial parasites (various morphological forms) and essentially the same as for thin smears except that the initial
microfilaria of filarial worm. steps of fixation smears by methanol is not done. The absence
of fixation of the smears by methanol allows lysis of red blood
cells and dehaemoglobinisation by aqueous stain solution.
INTRODUCTION Thick blood smear is primarily used as a screening procedure.
It is a sensitive procedure for detection of parasites compared
Haemoparasites are found in the peripheral blood smears of to thin smear, as it allows examination of larger amount of blood.
many parasitic diseases. These include i). protozoal infections Disadvantage of thick blood smear is that species identification
such as malaria, babesiosis, Chagas’ disease, African of parasites cannot be made. Thick blood smears are several
trypanosomiasis and leishmaniasis; and ii) helminthic infections layers thick, allows only detection of parasites.
such as lymphatic filariasis, and loiasis. The parasites found in the peripheral blood smear are listed
Examination of permanent stained blood smears is essential in the box 68-1.
for detection and specific identification of blood parasites. Thick
and thin blood smears stained with Romanowsky’s stains are
the permanent staining methods widely used for demonstration REQUIREMENTS
of blood parasites. These blood smears, although simple and
easy to prepare require experienced eyes for the screening, I Equipments
detection and identification of these haemoparasites. Compound light microscope.
Microfilariae although can be identified in wet mount preparation
of fresh blood, on basis of their shape and motility, their correct II Reagents and lab wares
and accurate identification is possible only after permanent Bunsen flame and clean glass slides, Leishman’s stain, EDTA
staining of blood smear. For malarial parasites, blood films help anticoagulated blood, 70% ethyl alcohol, buffered distilled
quantitate parasitaemia, in addition to species identification. water and methyl alcohol.
Preparation of EDTA anticoagulated blood: It is prepared by
dissolving 5 grams of EDTA in 100 ml of distilled water. Then
PRINCIPLE 0.4 ml is aliquoted into tubes and the water is evaporated. Blood
can then be added to the anticoagulant or 20 mg of dry EDTA
As a rule thin blood smears are fixed by alcohol or other fixatives too can be added per tube. Then blood can be added to it.
before staining. Two types of stains are used. One type of stains Preparation of Leishman’s stain: Leishman’s stain is prepared
(e.g. Leishman’s, Wright’s, etc.) has both fixatives and staining by dissolving 0.15 gram of Leishman’s dry powder in 100 ml of
reagents. The other type of reagents (e.g. Giemsa, Field, JSB absolute methyl alcohol in a bottle. The bottle is shaken until
stains) has only staining reagents. Therefore, the thin films need the powder is dissolved and allowed to stand for 48 hours with
prior fixation before staining with any of these stains. frequent shaking in between.
202 Leishman’s staining of Peripheral Blood Smears
III Specimen b The thin film only must be fixed in absolute methanol before
Fresh blood obtained by finger prick (or) EDTA anticoagulated staining.
blood. c Both the thick and thin blood films should be stained
simultaneously.
PROCEDURE
Leishman’s staining
Preparation of thin blood smear
1 Flood the blood smear with 5–10 drops of the Leishman’s
1 Take an alcohol cleaned and grease free slide. stain and allow to stand for 2 minutes.
Note: If old slides are to be used, they should be first cleaned 2 After 2 minutes, dilute the stain by adding twice as many
with detergent and then with 70% ethyl alcohol. drops of buffered distilled water. Allow the solution to mix
2 Place a small drop of fresh blood on the clean microscopic well.
slide, about 1.5 cm from the end of the slide. 3 Allow the solution to stand for 15–20 minutes.
3 Touch the drop of blood with the edge of another slide and 4 Wash the slide with buffered distilled water, and rinse it dry.
allow the blood to spread along the edge. 5 Examine the stained slide under the oil immersion objective
4 Push the second slide or the spreader across at about a 30° (100 x) of the microscope.
angle, along the surface of the horizontal slide to the far end
forming a ‘tongue shaped’ thin film.
Note: The thin, feathered end should be at least 2 cm long, QUALITY CONTROL
and occupy the central area of the slide, with free margins on
both sides. A known positive control, Leishman stained thick and thin
5 Allow the thin blood film to dry, and then stain the smear blood films for malarial parasites and microfilaria, is compared
with Leishman’s stain. with the stained test blood films for similar morphology of the
7 First screen the film with the low-power objective of the parasites.
microscope for detection of microfilaria and examine at least
two hundred microscopic fields using 100 x objectives for
the presence of malarial parasites. OBSERVATIONS
Preparation of thick blood smear 1 The blood film shows the presence of RBC’s identified as
pale round cells with dense periphery and lighter core. Some
1 Place two or three small drops of fresh blood (without any of the normal sized RBC’s show blue ring-shaped parasite
anticoagulant) on a grease-free alcohol cleaned slide. cytoplasm surrounding a central vacuole with a red coloured
2 Spread and mix the drops of blood with the corner of another nucleus (1 chromatin dot) present at its centre. Often 2 or
slide, over an area of about 2 cm in diameter. The mixing is more ring forms of the parasite are found inside a single RBC.
continued for about 30 seconds to prevent formation of any
fibrin strands which may conceal the parasites in a stained a Neutrophils in the blood films are identified by their
smear. multilobed nucleus.
Note: If blood containing anticoagulant is used, 2 or 3 drops b Lymphocytes are identified as having big nucleus pushing
maybe spread over an area of about 1 cm in diameter. the cytoplasm to the periphery.
3 Allow the thick blood film to air-dry at room temperature in a c Monocytes have kidney shaped nucleus.
dust free area.
4 After drying, lyse the thick blood film with distilled water for 2 The blood film shows the presence of normal RBC’s,
dehaemoglobinisation. neutrophils, lymphocytes and monocytes. In addition, typical
5 After the step of dehaemoglobinisation, stain the film with crescent (banana) shaped structures are seen with dark blue
Leishman’s staining. cytoplasm, compact nucleus, pigment and central chromatin.
6 After staining, air-dry the film in a vertical position. These structures have rounded or pointed ends and are
7 Examine the stained smear under oil-immersion objective about one and half-time larger than the RBC.
(100 x) for detection of malarial parasites and microfilaria. 3 The blood film shows the presence of RBC’s, neutrophils,
lymphocytes and monocytes. In addition, some of the
Preparation of combined thick and thin films enlarged RBC’s show within it, large amoeboid coarse
haemazoin pigments occupying the entire RBC. 12 to 24 darkly
In field surveys, it may be helpful to prepare slides with both stained merozoites are found within the RBC’s.
the thick and thin film on the same slides. 4 The Leishman stained thick blood smear shows the presence
Note: With this type of preparation: of coiled structure with a sheath and the absence of nuclei in
a Sufficient time should be allowed for the thick portion of the tail end. In addition, RBC’s, neutrophils, lymphocytes
the smear to dry before staining. and monocytes are also seen.
BOX 68-1 THE PARASITES FOUND IN

THE PERIPHERAL BLOOD SMEAR
In RBC’s Plasmodium vivax.

Plasmodium falciparum.
Plasmodium malariae.
Plasmodium ovale.
Babesia species.
In leucocytes Amastigote forms of Leishmania species.

Amastigote forms of Trypanosoma species.
In plasma Microfilaria of Wuchereria bancrofti.

Microfilaria of Brugia malayi.
FIGURE 68-2 The blood film shows the presence gametocyte of
Microfilaria of Brugia timori.
Plasmodium falciparum, x1000.
Microfilaria of Loa loa.
Microfilaria of Mansonella perstans.
1 The blood film shows the presence of ring forms of

Plasmodium falciparum (Fig. 68-1).
2 The blood film shows the presence of gametocyte of
Plasmodium falciparum (Fig. 68-2).
3 Erythrocytic schizonts of Plasmodium vivax in the blood
films (Fig. 68-3).
4 Leishman stained thick blood smear with microfilaria of
FIGURE 68-3 Erythrocytic schizont stage of Plasmodium vivax in
Wuchereria bancrofti (Fig. 68-4). the blood films, x1000.
FIGURE 68-1 The blood film shows the presence of ring forms of FIGURE 68-4 Leishman stained thick blood smear with microfilaria
Plasmodium falciparum, x1000. of Wuchereria bancrofti, x1000.
KEY FACTS
1 Always thin blood films should be fixed by alcohol or other fixatives before staining.
2 The stains used for staining blood films are of two types: i) One type of stain which has both fixatives and staining reagents
e.g., Wright’s, Leishman stains, and ii) Other type of stain which has only staining reagents e.g. Giemsa, Field’s, Jaswant
Singh Bhattacharjee stain. Hence with the use of these stains, the thin films should be fixed before staining.
3 Thick blood films should not be fixed because fixation with methanol prevents lysis of RBC’s and dehaemoglobinisation.
4 Thick blood films helps in detection of microfilaria and malarial parasites. It does not serve the purpose of species identification.
5 Thin blood films allows identification of malarial parasite to the species level.
204 Leishman’s staining of Peripheral Blood Smears
VIVA
1 Name different Romanowsky’s stains.

Ans. Different Romanowsky’s stains are Leishman’s stain, Wright’s stain, Jenner’s stain and Giemsa stain.
2 Name the parasites that can be found in the peripheral blood smear.
3 Name two intra-erythrocytic parasites.
Ans. Plasmodium and Babesia.
4 What are the clinical conditions in which examination of a peripheral blood smear may be helpful?
Ans. Clinical conditions in which examination of peripheral blood may be useful include:
Protozoal infections
Malaria.
Babesiosis.
Chagas disease.
African trypanosomiasis.
Leishmaniasis.
Helminthic infections
Lymphatic filariasis.
Loiasis.
5 What are differences between thick and thin smears?

Ans.
Thick smear: It allows examination of larger amount of blood.
It is several layers thick.
Smear prepared with 2-3 small drops of fresh blood spread over 2 cm diameters.
Thin smear: Allows examination of small amount of blood.

Made up of monolayer of RBCs.
Smear prepared with a small drop of blood spread along the edge.
FURTHER READINGS
LESSON
Concentration of
69 Stool for Parasites
LEARNING OBJECTIVES The principle of formalin ether sedimentation method is

based on recovering helminthic eggs and protozoal cysts in
After completing this practical you will be able to perform: the sediment of the faeces using centrifugation. In this method,
the stool sample to be tested is mixed with formalin ether in a
1 Saturated salt solution flotation method for concentration test tube and centrifuged. The eggs and cysts, because of the
of stool for parasitic ova and cysts. centrifugal force settle down at the bottom of the centrifuge
2 Formalin-ether sedimentation method for concentration of tube and form sediment. The concentrated parasites can be
stool for parasitic ova and cysts. demonstrated in the wet mount preparation of the sediment.
The advantages and disadvantages of both the salt solution
flotation method and formalin ether sedimentation methods
INTRODUCTION are summarized in the box 69-1.
Concentration of stool is recommended when direct examination

of stool wet mounts fail to demonstrate any parasites REQUIREMENTS
particularly when the number of parasites in stool specimens
are low. By concentration methods the helminthic ova and I Equipments
larvae, and protozoal cysts can be concentrated. However, Microscope, centrifuge and discarding jar.
trophozoites cannot be concentrated by any of the
concentration methods as they get killed.
Various concentration methods are available. Broadly, they II Reagents and lab wares
can be classified into two groups: flotation techniques and Microscope slides, broad cover slips, 30 ml glass vials, Pasteur
sedimentation techniques. These two approaches are based pipette, wooden spatula for both salt solution flotation and
on the principle of separating parasite from faecal debris and formalin-ether sedimentation procedure. In addition to the
other materials by their differences in the specific gravity. above, for formalin-ether method alone, centrifuge tubes, glass
In this chapter, the two commonly used concentration funnel, measuring cylinder, applicator sticks, surgical gauze
methods, i) Lane’s saturated salt solution floatation method and stopper are required.
and ii) formalin-ether sedimentation method will be described. Distilled water, sodium chloride powder (for salt solution
Other floatation methods for stool concentration are listed in method), 5%-10% formalin, ether, saline (for formalin-ether
the table 69-1. method)
Preparation of saturated sodium chloride solution: Saturated
sodium chloride solution is prepared by dissolving a spoonful
PRINCIPLE of sodium chloride in 100 ml of distilled water. The salt is
dissolved in water using a spatula or a shaker. After all the salt
In the standard salt solution flotation method, saturated salt has dissolved completely without a trace, more salt is added till
solution, a liquid with high specific gravity is used. This helps it remains undissolved. This makes saturated sodium chloride
in separation of protozoal cysts and helminthic eggs from faecal solution.
debris. In this method the eggs and cysts of many parasites
float because specific gravity of these eggs and cysts are less III Specimen
than specific gravity of the saturated salt solution. The faecal Stool.
debris are found at the bottom of the container.
206 Concentration of Stool for Parasites
11 Discard all the fluid into the discarding jar by one firm
PROCEDURE swing, leaving behind one or two drops of fluid with the
sediment.
Saturated salt solution flotation method 12 Mix the sediment with the fluid using an applicator stick.
Then with a Pasteur pipette, aspirate a little of the sediment
1 Take a walnut sized stool (approx. 1 gram) in a 30 ml glass fluid and examine by making the saline and iodine wet mount
vial. preparations.
2 Add a few ml of saturated sodium chloride solution to the 13 Examine the wet mount preparation first under low power
stool and mix it with a broomstick. objective (10 x) of the microscope for parasite eggs and cysts.
3 When the stool suspension is smooth, add a few more ml Then focus under the high power objective (40x) to study
of salt solution and mix. the morphology and identification of the egg and cysts.
Note: The steps 2 and 3 are repeated, until the container is
nearly full with continuous stirring.
4 Remove any coarse material found floating by a broomstick
5 Add more salt solution little by little using a Pasteur pipette, QUALITY CONTROL
until a convex meniscus is formed at the top of the container.
6 Then place a broad cover slip over the glass vial, so that it Wet mounts are prepared from a stool specimen known to be
is in contact with the top of the meniscus. positive for ova and cysts and compare with the wet mounts
7 Allow it to stand for 20 minutes to 30 minutes. obtained after the formol ether sedimentation technique for
8 Lift the cover slip from the meniscus with a steady but similar morphology of eggs and cysts.
rapid movement and place on a clean microscope slide.
9 Make a wet mount preparation with one or two drops
adherent to the cover slip.
10 Examine the wet mount preparation first under low power OBSERVATIONS
objective (10x) of the microscope for parasite eggs. Then
focus under the high power objective (40x) to study the 1 A 10-15µm sized spherical structure seen possessing 1-4
morphology and identification of the egg. nuclei.
2 A 15-30µm sized spherical structure seen possessing 1-8 nuclei.
Formalin –ether sedimentation method 3 A 6-10µm size oval ellipsoidal shaped structure seen with 4
nuclei remains of axoneme and flagella.
1 Take a half teaspoon of stool in a 15ml centrifuge tube
4 A 60µm × 40µm size oval shaped, non bile stained structure
containing 10 ml of 10% formalin, and allow it to stand for
seen with a thin transparent hyaline shell membrane
30 minutes.
enclosing 7-8 blastomeres, and clear space between the shell
2 Filter the faecal suspension through two layers of gauze in
membrane and blastomeres.
a funnel into a 15 ml centrifuge tube. Add saline to the tube
5 A 60-75µm × 40-50µm size rounded, bile stained structure
to bring the fluid level within several millimeters of the rim
seen with a thick translucent shell with an albuminous coat
of the tube.
containing a large conspicuous unsegmented ovum with a
3 Centrifuge the tube at 500 g for 10 min.
clear space at each pole.
4 Discard the supernatant. Suspend the sediment in saline,
nearly filling up to the brim of the tube.
5 Centrifuge the tube again for 10 min at 500 g.
6 Resuspend the sediment in 7 ml of 10% formalin, and 3 ml RESULTS AND INTERPRETATION
of ether.
7 Close the tube with a stopper and shake it well for 30
seconds. 1. Cyst of Entamoeba histolytica /dispar.
Note: Hold the tube in such a way that the stopper is held 2. Cyst of Entamoeba coli.
away from the face. 3. Cyst of Giardia intestinalis.
8 Remove the stopper carefully. 4. Egg of hook worm.
9 Centrifuge the tube at 500 g for 10 minutes. Allow the tube
to stand 5 min. 5. Fertilized egg of Ascaris lumbricoides.
Note: After centrifugation, four layers are formed namely,
i) first sediment layer at the bottom of the tube containing Table 69-1 Other floatation methods for concentration of
parasitic cysts or eggs, ii) second layer of formol saline, iii) stool
third layer of faecal debris on the top of the formol saline
layer and iv) lastly a top layer of ether. Zinc sulphate floatation method.
10 Remove the plug of faecal debris by piercing all around Magnesium sulphate floatation method.
with an applicator stick, taking care not to disrupt the debris. Sheather’s sucrose floatation method.
BOX 69-1 ADVANTAGES AND DISADVANTAGES OF

THE CONCENTRATION METHODS
Saturated salt floatation method

Advantages
Simple procedure.
Inexpensive method and easy to perform.
Good method for concentration of parasitic eggs.
Can be done at any level of the laboratory.
Least subjective to technical error.
Disadvantages
Not suitable for demonstration of unfertilized eggs of Ascaris, operculated eggs of trematodes, larvae of Strongyloides and
eggs of Taenia species.
High specific gravity of the fluid may cause distortion of the morphology of the parasitic eggs and cysts.
Formalin-ether sedimentation method
Advantages
More sensitive method.
Good method for both cysts and ova.
Morphology of eggs and cysts preserved.
Disadvantages
Technically cumbersome.
Ether is highly inflammable and not easily available.
KEY FACTS
1 Concentration of stool is the method of choice, when the number of parasites in the stool specimens are less and cannot be
demonstrated in stool wet mount preparation.
2 Trophozoites of protozoa cannot be concentrated by any of the concentration methods.
3 Stool concentration by salt floatation and formol ether sedimentation are the two reliable methods of concentration of
parasites in stool. It increases sensitivity of stool microscopy.
4 Salt floatation is a better method for concentration of helminthic eggs.
5 In formol ether procedure, formol saline is used to fix the cysts of protozoa and eggs, thus unaltering morphology of these
structures. These also kill the parasitic eggs and cysts. Ether is used to dissolve and extract the fat and other debris in the stool.
VIVA
1 What are the advantages of floatation method?

2 What are the disadvantages of floatation method?
3 What are the advantages of sedimentation method?
4 What are the disadvantages of sedimentation method?
5 Name the other floatation methods that can be employed as stool concentration method.
6 Which is the stool concentration method employed for recovery of coccidian parasites?
Ans. The stool concentration method employed for the recovery of coccidian parasites is the Sheather’s sucrose floatation method.
FURTHER READINGS
208
LESSON
Culture of Stool for
70 Entamoeba histolytica

Bacteriological incubator and inspissator.
1 Culture stool for Entamoeba histolytica.
Boeck and Drbohlav’s Locke-egg-serum (LES) medium, heat
INTRODUCTION inactivated bovine serum, screw cap tubes, and antibiotics
solution.
For the cultivation of Entamoeba histolytica various media
have been used. The media have been classified broadly into Preparation of polyxenic Boeck and Drbohlav’s medium:
two groups: polyxenic (polybacterial culture) and axenic
(bacteria –free culture) medium. The polyxenic media are Boeck 1 Break four eggs into a sterile flask containing glass beads,
and Drbohlav’s Locke-egg-serum (LES) medium, Balamuth’s, after that they are washed and shells are wiped dry with
Nelsons’ or Robinson’s medium. Boeck and Drbohlav’s Locke- 70% alcohol.
egg serum (LES) medium is a polyxenic medium commonly used 2 Then add 50 ml of Locke’s solution and shake the mixture
for culture and isolation of the amoebae from the stool until homogenous.
specimens for diagnostic purposes. Trophozoites of E. 3 Dispense the medium in tubes, such that a slant of 1 to 1.5
histolytica usually appear in large numbers within 48 hours of inches are produced at the bottom of the tube.
inoculation. 4 Plug the tubes and place the tubes in a slant position in an
inspissator at 70°C until the slant solidifies.
5 Autoclave the tubes at 15 lb pressure for 20 minutes.
PRINCIPLE 6 Prepare a mixture of 8 parts of sterile Locke’s solution with
1 part sterile inactivated bovine serum.
Polyxenic culture medium is usually composed of egg and 7 Sterilise the mixture by filtration and incubate at 37°C for 24
Locks’ solution mixture supplemented with serum, starch and to 48 hours as a sterility check before use.
bacterial flora, which provide nourishments for the growing 8 Cover the slants of the medium to a depth of 1 cm with 6 ml
parasites. This is used routinely for diagnosis of intestinal to 8 ml of the sterile lock’s solutions.
amoebiasis by isolation of the amoeba from the stool. 9 Add a loopful of sterile rice powder/starch to each tube
Axenic culture is a bacteria-free culture medium. This is along with loopful of Escherichia coli colony.
used to study i) the pathogenicity of amoebae, ii) testing anti- 10 Add antibiotics solution (penicillin, 1000 units/ml,
amoebic drugs in vitro, iii) immunological properties of amoebic streptomycin 2 mg/ml and acriflavine, 0.1 ml of 0.02%) to
antigen and iv) prepare axenic amoebic antigen for use in the the medium to inhibit the overgrowth of commensal bacteria
immunodiagnosis of amoebiasis. Axenic culture is not used for present in the stool.
diagnosis by isolation of amoebae from the stool.
In this chapter polyxenic culture of E.hsistolytica in Boeck III Specimen
and Drbohlav’s medium will be described. Stool in cases of suspected amoebic dysentery, and pus in
cases of amoebic liver abscess.
PROCEDURE QUALITY CONTROL
1 Inoculate a loopful of the stool sample or liver pus on to the A known positive culture of E. histolytica already maintained
slant of the two medium tubes. by serial sub cultivation in the culture medium.
2 Incubate the tubes in an incubator at 37°C for 24 hours to 48
hours. OBSERVATIONS
3 Observe the cultures regularly at 2, 3 and 4 days by examining
Presence of 8–30 µm structure, actively motile with hyaline,
0.1 ml of sediment under light microscope for characteristic
finger-shaped pseudopodia, clearly differentiated cytoplasm
motility of the trophozoites.
into ectoplasm and endoplasm, and cytoplasmic inclusions
Note: Although the initial culture may appear negative, such as red blood cells, leucocytes and tissue debris.
subcultures may reveal amoebae.
4 Examine the growth of amoebae by a wet mount preparation
of culture fluid. RESULTS AND INTERPRETATION
Trophozoites of E. histolytica is grown in the culture.
KEY FACTS
1 Cultivation of E. histolytica can be done using both polyxenic and axenic culture medium.
2 In polyxenic culture medium (polybacterial culture) bacterial flora serve as rich source of nutrients for the feeding amoebae.
3 In axenic culture medium (bacteria-free culture), addition of special vitamin mix provides a rich source of nutrition.
4 Axenic culture medium is not used routinely for culture and isolation of the amoebae from stools specimens.
5 Axenic culture medium is employed to study pathogenicity of amoeba, in vitro testing of efficacy of anti-amoebic drugs and
preparation of axenic amoebic antigen for immunodiagnosis of amoebiasis.
VIVA
1 Name the axenic culture medium used for culture of E. histolytica.

2 Define axenic and polyxenic culture medium.
3 What are the uses of axenic culture of E. histolytica?
4 List the polyxenic culture media and the uses of polyxenic culture of E. histolytica.
FURTHER READINGS
210
UNIT
X
Mycology
Introduction
Lesson 71 Cultivation of Fungi
Lesson 72 Gram’s Staining for Fungi
Lesson 73 Lactophenol Cotton Blue (LPCB) Wet Mount
Lesson 74 Potassium Hydroxide Wet Mount
Lesson 75 India Ink Preparation
Lesson 76 Slide Culture
Lesson 77 Germ Tube Test
Lesson 78 Urease Test
Lesson 79 Carbohydrate Assimilation Test
Lesson 80 Carbohydrate Fermentation Test
Lesson 81 Identification of Common Fungi
212
Introduction
The fungi are now recognized as significant causes of morbidity and mortality. They have emerged as important etiological
agents of opportunistic infections and full-fledged diseases. Today, the incidence of the fungal infections has increased
enormously, due to underlying predisposing factors such as immunocompromised situations. The prognosis among these
patients is very poor and therefore an early diagnosis and treatment is essential. The symbiotic relation of the fungi, like other
organisms, can be divided into three modes of their existence namely mutualism, commensalisms and parasitism.
The aged patients, whose life span has been extended by treatment of cancer or other debilitating diseases, are more susceptible
to secondary fungal infections than the young individuals. In addition many factors directly or indirectly decrease the accuracy
of data on fatal mycoses cases. The fungal infections are not usually transmitted sexually like those of the viral, bacterial or
parasitic diseases. Till date there is no ideal vaccine available to control any fungal infections. Simultaneously, the reported
deaths from the fungal infections have maintained an approximately constant numerical ratio to the general increase in the
population.
The nosocomial fungal infections have been recognized as a significant ground for adverse patient outcome and a major
public health problem. Nosocomial blood stream infections are particularly serious and there is evidence to suggest that these
infections are becoming more common. Nosocomial mycoses have developed especially in association with or as a consequence
of the extraordinary progress in the management of seriously ill patients. However, despite this increase, there has been
comparatively little progress in understanding the pathogenesis of nosocomial fungal infections or in their prevention, diagnosis
and treatment.
The fungal diseases can be classified according to the primary site of infection as follows:
1 Superficial mycoses: The infection is limited to the outer most layers of the skin and its appendages. The immune response is
rarely induced.
2 Cutaneous mycoses: The infection extends deeper into the epidermis and it also invades hair and nails. It evokes a high
inflammatory response in the host.
3 Subcutaneous mycoses: The infection is due to the pathogenic organism of low virulence and usually following traumatic
injury. It involves the dermis, subcutaneous tissues, muscles and fasciae.
4 Systemic mycoses: The infection originates primarily at one site and disseminate systemically to other body sites.
5 Besides these a fifth group opportunistic has come into focus because of increasing use of immunosuppressive therapy or
AIDS epidemic. These infectious agents are of low pathogenic potential and produce disease only under unusual circumstances,
mostly involving host debilitation.
Fungal infections can be diagnosed by their demonstration, isolation and final identification from clinical specimens. Diagnosis
of fungal infections is made by direct and indirect methods. Direct methods include the demonstration of fungi or their components
in body tissues or fluids. Wet mount of clinical specimens is a very useful method for demonstration of fungi in clinical
specimens. Various wet mount preparation used in a mycology laboratory include lactophenol cotton blue (LPCB) wet mount,
potassium hydroxide (KOH) wet mount and India ink preparation.
LESSON
Cultivation of Fungi
71
Skin and nail scrapings, hair, and exudate from lesions, urine,
After completing this practical you will be able to: sputum, bronchial washings, biopsied materials and CSF.
1 Select appropriate medium to grow fungi from clinical

specimens. PROCEDURE
2 Isolate fungi from clinical specimens in the laboratory.
1 Choose and take two appropriate sterile medium tubes and
label RT (room temperature) and 37°C.
INTRODUCTION 2 Take processed specimen and inoculate a loop full of
specimen in each tube.
Different kinds of media are available to grow and isolate 3 Incubate RT tube at room temperature and 37°C tubes at
different types of fungi. List of different media used for culture 37°C temperature.
of fungi are summarized in the table 71-1. Sabouraud’s dextrose
agar (SDA) (Fig. 71-1) is the most frequently used media in a
diagnostic mycology laboratory (Box 71-1). QUALITY CONTROL
Prepared media should be incubated and then used to ensure

PRINCIPLE sterility. Quality of sterilization should be checked periodically
and appropriate pH should be adjusted for best results. The
Clinical specimens should be processed promptly and plated control strains should be used to grow and see if growth is
on to isolation media as a means to recover fungi that may be perfect in prepared medium.
causing disease. Appropriate media and incubation
temperatures are selected to allow for the growth of pathogenic
and opportunistic yeasts and fungi. OBSERVATIONS
Observe both the test tubes and control tubes everyday for
REQUIREMENTS upto 30 days.
I Equipments 1 Observe for fungal colonies.

Water bath, autoclave/hot air oven for sterilization, pH meter 2 Tubes growing bacterial colonies are discarded.
or pH paper.
II Reagents and lab wares RESULTS AND INTERPRETATION

Glassware for preparation of media, tubes for aliquots of media,
Bunsen burner, inoculating loop, glass marking pencil, Growth of fungal colonies in the tube inoculated with test specimen
appropriate sterile media (Table 71-1). indicates the patient is infected with fungus (Fig. 71-1).
214 Cultivation of Fungi
Table 71-1 List of media used for fungal culture BOX 71-1 SABOURAUD’S DEXTROSE
AGAR
Sabouraud’s dextrose agar (SDA)
Sabouraud’s dextrose agar with antibiotics Sabouraud’s dextrose agar
Ascospore medium It consists of 40 gm of glucose / dextrose, 10 gm of
Bennett’s agar for Nocardia neopeptone and 35 gm of agar in 1000 ml of distilled water.
Blood agar base pH is 5.5 – 6.0.
Casein agar base for identification of Trichophyton species
Sabouraud’s dextrose agar with antibiotics
Casein agar for Nocardia It consists of 20 gm of glucose/dextrose, 10 gm of neopeptone
Casitone medium of Howell and Pine for Actinomyces and 20 gm of agar in 1000 ml of distilled water. pH is 7.0.
Carbohydrate broth for Candida Sterilised by autoclaving. It contains 40 mg of
Czapek – Dox medium chloramphenicol and 40 mg of cycloheximide (actidione).
Gelatin medium for Nocardia
Malt – Yeast extract agar (Wicker ham)
Medium 199 for Nocardia munutissima
Mohapatra and Pine medium for Nocardia
Potato glucose agar
Rice agar with Tween 80
Rice medium for Microsporum species
Salvin’s YP medium for yeast phase of Histoplasma
capsulatum
Tarshis’ penicillin blood Agar
Trypticase soy agar
Tyrosine agar
Xanthine Agar
FIGURE 71-1 SDA with Candida albicans colonies.
KEY FACTS
1 Choose appropriate medium based upon clinical history of the patient.
2 Always inoculate in duplicate tubes and incubate one at room temperature and another at 37°C.
3 Media should be sterile.
4 pH should be appropriate.
VIVA
1 What are the different types of media used in fungal culture?

2 What are the most commonly used media in laboratory for routine diagnosis of fungal infections?
Ans. Sabouraud’s dextrose agar (SDA) is the most frequently used media in a diagnostic mycology laboratory The other
commonly used media include carbohydrate broth, Czapek- Dox medium, gelatin medium, potato glucose agar, trypticase
soy agar and urea agar.
3 How do you sterilize different media used for culturing fungi?
Ans. Most media used for fungal culture are sterilized by autoclaving at 15 lbs for 15 mins.
4 How do you test for the proper working of different media?
Ans. The media that are prepared can be tested to see for its proper working by inoculating standard strains of appropriate
fungi and incubating them both at 37°C and at room temperature.
FURTHER READINGS
1 Collier L, Balows A, Sussman M. Topley and Wilson’s Microbiology and Microbial Infections. 9th Edition. Mycology. Volume 4. pp. 711.
Arnold publishers.
3 Jagadish Chander. A Text Book of Medical Mycology. Interprint. 2000.
LESSON
Gram’s Staining for Fungi
72
LEARNING OBJECTIVES Sabouraud’s dextrose agar and place on the clean slide with
a bacteriological loop.
After completing this practical you will be able to: 3 Then with circular movement of the loop, spread the cell
suspension into a thin area.
1 Stain fungal smears by Gram’s staining method. 4 Allow the smear to air dry.
5 Heat fix the smear while holding the slide at one end, and
INTRODUCTION two to three times.
Gram stain was devised by Christian Gram in 1884 as a method of II Staining Procedure
staining bacteria in tissues. Fungal material appears Gram positive.
1 Heat fixes the smear by passing the slide 2–3 times gently
over the flame with the smear side up.
PRINCIPLE 2 Cover the smear with the methyl violet. Allow it to stand
for one minute.
The Gram reaction is dependent on the permeability of the 3 Rinse the smear gently under tap water.
fungal cell wall, to the dye-iodine complex like those of bacteria 4 Cover the smear with Gram’s iodine and allow it to stand
(refer chapter 7). for one minute.
5 Rinse the smear again gently under tap water.
6 Decolourise the smear with 95% alcohol for 15 to 20 seconds.
REQUIREMENTS 7 Rinse the smear again gently under tap water.
8 Cover the smear with dilute carbol fuchsin for 30 seconds
I Equipments to 1 minute.
Compound light microscope. 9 Rinse the smear again gently under tap water and air dry it.
10 Observe the smear first under low power (10x) objective,
II Reagents and lab wares and then under oil immersion (100x) objective.
Bunsen flame, loop wire, methyl violet (basic dye), Gram’s iodine 11 Record the observations in the note book.
(mordant), 95% ethanol (decolourising agent), and 1% safranine
or dilute carbol fuchsin (counter stain).
QUALITY CONTROL
III Specimen
Candida albicans culture on Sabouraud’s dextrose agar. On the same slide, at one end a thin control smear of mixture of
Staphylococcus aureus (Gram positive bacteria) and
Escherichia coli (Gram negative bacteria) is made and at other
PROCEDURE end of the slide test smear is made. The slide with control and
test smears is stained by Gram’s staining. The appearance of
I Preparation of fungal smear: purple coloured Gram positive bacteria and pink coloured Gram
negative bacteria in the control smear indicates proper staining
1 Take clean, and grease free glass slides for making the smears. technique and stained test smear is compared with it.
2 Take one or two loopful of C. albicans culture on
216 Gram’s Staining for Fungi
OBSERVATION
Presence of Gram positive budding yeast cells.
The stained smear contains Gram positive yeast , C. albicans

(Fig. 72-1).
FIGURE 72-1 Gram stained smear of Candida albicans, x 1000.
KEY FACTS
1 Gram staining is a differential stain, which can also be used for detection of fungi in clinical specimens.
2 Gram staining gives preliminary indication of infection.
3 Tissue cells, leucocytes and the debris of inflammatory exudates all stain pink in Gram’s stained smears.
FURTHER READINGS
Arnold publishers.
LESSON
Lactophenol Cotton
73 Blue (LPCB)
Wet Mount

Glass Petri dishes, slide, cover slip, straight/bent wire and
After completing this practical you will be able to: needle, lactophenol cotton blue (LPCB) stain.
1 Prepare lactophenol cotton blue (LPCB) wet mount of fungal Preparation of Lactophenol cotton blue stain: Weigh and
colony. add 20 gm of phenol crystals, 20 ml of lactic acid, and 40 ml of
2 Demonstrate fungi and fungal elements under the microscope glycerol to 20 ml of distilled water. Dissolve the ingredients by
in LPCB wet mount preparation. heating the container in a hot water bath. Add 0.05 gram cotton
blue.

Rhizopous culture on Sabouraud’s dextrose agar.
Fungal infections can be diagnosed by their demonstration in
clinical specimens. LPCB staining wet mount is the most
commonly used method adopted in a mycology laboratory to PROCEDURE
identify filamentous fungi.
PRINCIPLE Scotch tape preparation
Identification of filamentous fungi is made by their characteristic 1 On a clean glass slide, place one drop of LPCB.
microscopic morphology such as shape, size, arrangement of 2 Touch the adhesive side of the tape of transparent scotch
spores and hyphae. There are three different preparations of tapes on the surface of the colony at a point intermediate
LPCB mounts as mentioned below: between its centre and periphery.
1 Scotch tape preparation. 3 Fix the adhesive side of the tape over an area on the glass
2. Tease mount preparation, and slide containing the LPCB.
3 Slide culture preparation. 4 Examine the preparation under 10x and 40x of a light microscope.
In case of scotch tape preparation, the fungal elements are

undisturbed whereas in tease mount preparation, the fungal
Tease mount preparation
elements are disturbed but finer details of the fungus can be
visualized. Slide culture (Ref. chapter 75) is the most advantageous
in that, the fungus as it grows on the medium can be visualized. 1 Place a drop of LPCB on a clean glass slide.
In this chapter, LPCB wet mount of both Scotch tape 2. Remove a small portion of the colony and the supporting
preparation and tease mount preparations will be described. agar at a point between the centre and periphery and place it
in the drop of LPCB.
3 With a needle, tease the fungal culture first and spread in
REQUIREMENTS the LPCB.
I Equipments 4 Examine microscopically after giving sufficient time for the
Microscope. structures to take up the stain, usually 30 mins.
218 Lactophenol Cotton Blue (LPCB) Wet Mount
types of morphological structures including hyphae and spores.

QUALITY CONTROL This should be thoroughly differentiated and the fungus
identified.
Preparation of lactophenol cotton blue should be done by Fast growing fungi in case of slide culture preparation will
heating the ingredients in a waterbath. give satisfactory results in 24–48 hr.
OBSERVATIONS
1 The stained preparation should be observed under 10x or

40x, for presence of mould.
2 Fungi appear as dark blue stained mycelium (Fig. 73-1).
The fungal element grown should be observed and results

interpreted depending on the morphology of the hyphae and
the spores.
Different fungi under LPCB wet mount will show different FIGURE 73-1 LPCB mount of fungi, x 400.
KEY FACTS
1 The fungal culture should be first is teased well and then spread in LPCB for better results.
VIVA
1 What is lactophenol cotton blue and how is it prepared?

2 What are three different methods of wet mounts used and their advantages in a lactophenol cotton blue preparation?
3 What are the functions of each component of lactophenol cotton blue stain?
Ans.
a Lactic acid: Helps in preserving the morphology of the fungal element.
b Phenol: Acts as disinfectant.
c Cotton blue: Stains the fungal elements.
d Glycerol: Hygroscopic agent. It prevents drying.
4 What is the other use of LPCB apart from examination of fungi?
Ans. LPCB is also used in wet mount of stool for intestinal parasites.
FURTHER READINGS
Arnold publishers.
LESSON
Potassium Hydroxide
74 Wet Mount
After completing this practical you will be able to: 1 Emulsify the specimen in a drop of 10% KOH on a
microscopic slide with the help of a loop.
1 Prepare potassium hydroxide (KOH) wet mount of clinical 2 Apply gentle heat by passing the slide over a Bunsen flame
specimens. for 3–4 times.
2 Demonstrate the presence of fungal elements in the given 3 Cover the smear with the cover slip.
clinical specimen by KOH wet mount preparation. 4 Leave it for 5–10 min.
5 Examine the slide under low (10x) and high power (40x)
magnifications
INTRODUCTION 6 Examine the slide for 15–20 min. for demonstration of shining
fungal elements.
The potassium hydroxide (KOH) wet mount preparation is very
useful for the presumptive diagnosis of the type of fungal
infection. The procedure also helps in the selection of appropriate QUALITY CONTROL
culture media for the isolation of etiological fungal agent.
1 10% KOH should be prepared at the right concentration.
2 Emulsification of specimen should be homogenous in KOH
PRINCIPLE solution.
The KOH clears out the background scales or cell membranes

that may be confused with fungal hyphal elements in OBSERVATIONS
microscopy of clinical specimens. Gentle heating also
accelerates clearing of artifacts. Shining fungal elements shall be observed in microscopy of
the clinical specimens.
REQUIREMENTS
I Equipments
Microscope. Different fungi will have different morphological forms (yeasts,
cells with pseudo hyphae, budding, septate, and aseptate
II Reagents and lab wares hyphae, granules, etc.) which can be clearly seen in a KOH wet
Glass Petri dishes, slide, cover slip, straight/bent wire, needle, mount.
Bunsen flame and 10% KOH. Interpretation of results should be done by critical analysis
of the type, size and color of fungal elements which will be
III Specimen different for different fungi.
Pus from draining sinuses, aspirate from nasal sinuses,
respiratory specimen, skin scrapings, nail scrapings, hair,
corneal scrapings, material from external ear, etc.
220 Potassium Hydroxide Wet Mount
KEY FACTS
1 The KOH clears out the background scales or cell membranes that may be confused with fungal hyphal elements in
microscopy of clinical specimens.
2 The KOH should be prepared at the right concentration (10%).
3 Emulsification of specimen should be homogenous.
VIVA
1 What percentage of KOH should be used?

2 What is the function of KOH?
3 How do you interpret results of a KOH wet mount?
FURTHER READINGS
Arnold publishers.
3 Jagadish Chander. A Text Book of Medical Mycology. Interprint. 2000. pp. 232.
LESSON
India Ink Preparation
75
After completing this practical you will be able to: 1 Put a drop of CSF on the microscopic slide.
2 Put a drop of India ink to the CSF on the microscopic slide.
1 Prepare India ink wet mount of cerebrospinal fluid (CSF). 3 Emulsify the specimen with India ink on the slide.
2 Demonstrate the presence of capsulated yeast, Cryptococcus 4 Place a cover slip over the preparation, taking care not to
neoformans in the CSF. trap air bubbles in the preparation.
5 Blot dry the excess fluid.
6 Examine the slide under low (10x) and high power (40x)
magnifications
INTRODUCTION
Capsule is a protective layer found around some bacteria and QUALITY CONTROL
some fungi like C. neoformans. Hence demonstration of capsule
by India ink preparation, especially in an emergency conditions, 1 India ink preparation in distilled water should be made exactly
in cerebrospinal fluid (CSF) is a very useful procedure for to 0.5%.
diagnosis of meningitis caused by C. neoformans. An early 2 Care should be taken not to trap air bubbles which will mimic
diagnosis will help for prompt treatment of the condition. capsules of yeast cells.
OBSERVATIONS
PRINCIPLE
The India ink preparation is observed under microscope and
India ink is used as a negative stain preparation. When used in noted for presence of clear halo around yeast cells (Fig. 75-1).
wet mount preparation of the CSF, the background appear black,
and the unstained capsule of C. neoformans appears as a white
halo around the yeast cells in microscopy. RESULTS AND INTERPRETATION
Since India ink stains the background and leaves a clear halo
around the cells, preparations with such appearance can be
REQUIREMENTS confirmed to have yeast cells with capsules and the organism
may be identified as C. neoformans.
I Equipments
Microscope.

Microscopic slide, cover slip, glassware, loop wire and 0.5%
India ink in distilled water.
III Specimen
Cerebrospinal fluid (CSF).
FIGURE 75-1 India ink preparation showing Cryptococcus
neoformans with capsule, x 400.
222 Indian Ink Wet Mount Preparation
KEY FACTS
A clear distinction should be made between capsules and air bubbles, trapped between the cover slip and slide.
VIVA
1 What is the percentage of India ink used?

2 What is the advantage of India ink preparation and how is it useful in emergency mycology laboratory?
Ans. India ink preparation is one of the best methods to demonstrate the presence of capsule in case of capsulated yeasts.
The demonstration of capsulated yeast like C. neoformans in CSF is significant in emergency mycology laboratory for
prompt administration of antifungal agents.
3 How to interpret results of India ink preparation?
Ans. The India ink preparation should be critically interpreted for the presence of a clear capsule around the yeast cell. This
should not be confused with any air bubble that might be present.
FURTHER READINGS
Arnold publishers.
LESSON
Slide Culture
76
LEARNING OBJECTIVES (d) Wrap the preparation in Kraft 20 paper for sterilizing in
hot air oven. Several of these setups should be kept ready
After completing this practical you will be able to: on hand.
1 Perform slide culture of the fungal preparation. 2 Sterile test tube with rimless mouth and an inside diameter of
2 Mount the cover slip after the growth of fungus. approximately 15 mm.
3 Demonstrate fungal morphology without disturbing the aerial 3 Standard laboratory glassware.
hyphae and conidiophores, if present.
II Reagents
Sterile distilled water and Petri dish containing Sabouraud’s
INTRODUCTION agar (or other medium of choice) to a depth of 2 mm.
Slide culture is a very useful technique to study undisturbed III Specimen

morphological details of fungi, particularly relationship between Fragments of mould to be cultured is used as the specimen.
reproductive structures like conidia. An entire fungal colony
can be demonstrated within a short period of time with the use
of minimum materials. PROCEDURE
1 From the Petri dish containing Sabouraud’s agar cut out one
PRINCIPLE square cm block of agar for each slide culture to be
inoculated.
Slide culture is a very useful technique in identification of the 2 With the flat side of a sterile bacteriological loop, or with a
type of fungi. The fungal element that is to be identified will spatula, place an agar block in the centre of the slide in the
produce characteristic hyphae and spores, when incubated on slide culture set up.
a suitable growth medium. This can be visualized undisturbed 3 With a probe, inoculate around the periphery of the agar
using this technique. The mould that is to be cultured is block, three to four fragments of the mold to be cultured.
inoculated onto a small piece of an agar below a cover slip. The 4 With forceps, the tips of which have been flamed, place the
whole setup is kept in a Petri dish with moisture. The cover slip cover slip on the agar block.
after incubation is lifted, stained and observed under a 5 With a pipette, thoroughly moisten, but not to excess, the
microscope for identification of the fungi. filter paper with sterile distilled water.
6 Incubate the slide culture at room temperature.
7 Remove the slide culture from the Petri dish and dry the
REQUIREMENTS bottom of the slide with a tissue.
8 When growth appears peneath the cover slip. Take a slide
I Equipments place a drop of LPCB, place the cover slip removed from the
1 Slide culture set (Fig. 76-1). block on the LPCB.
(a) Into a Petri dish, place one piece of filter paper slightly 9 Place the slide on the microscope stage and examine. The
smaller in diameter than the Petri dish. aerial hyphae including the conidiophores will be seen to
(b) Place a V-shaped glass rod on the filter paper. grow along the undersurface of the cover slip
(c) Place a 1 by 3 inch glass slide and a 22 mm square, No. 1
glass cover slip on top of the filter paper.
224 Slide Culture
1 All the materials should be sterilized and checked for sterility Small spore bearing fungi make beautiful permanent mounts.
before use. Some large spore bearing organisms like Microsporum gypsum
2 Distilled water to be used should be checked for sterility . do not stain as well. With the type of hyphae, arrangement of
3 Components of the medium used should be adjusted conidiophores, staining characters etc., the final interpretation
according to standard procedure. of the fungal type can be made.
OBSERVATIONS
Usually a minimum of 48 hr. is needed before a slide culture

shows growth of aerial hyphae. Thus, the culture may be
examined after 48 hours incubation and as frequently thereafter
as necessary.
FIGURE 76-1 Slide culture set.
KEY FACTS
1 The Petri dish chamber should be always moist.

2 Agar used should support growth of the suspected fungus.
3 Sterility should be maintained to the maximum.
VIVA
1 What are the requirements for slide culture?

Ans. The requirements for slide culture include Petri dish, piece of filter paper slightly smaller in diameter than the Petri dish,
V shaped glass rod, 1 by 3 inch glass slide, 22 mm square, No. 1 glass coverslip; Kraft 20 paper, sterile distilled water, sterile
test tube with rimless mouth and an inside diameter of approx. 15 mm, Sabouraud’s agar (or other medium of choice) to a
depth of 2 mm and standard laboratory glassware and.
2 When is slide culture used in routine diagnosis of fungal infections?
Ans.Slide culture can be used in routine diagnosis of fungal infections when examination of the entire fungal colony is
required without disturbing the aerial hyphae and conidiophores, if present.
Slide culture is a technique used to study an entire fungal colony within a short period of time with the use of minimum
materials.
3 What are the main advantages of slide culture?
Ans. Slide culture is a convenient method to demonstrate an entire colony without disturbing the aerial hyphae and
conidiophores of fungi such as Aspergillus species, Penicillium species etc.
FURTHER READINGS
Arnold publishers.
LESSON
Germ Tube Test
77
Standard laboratory glassware, and test tubes 12×75 mm,
After completing this practical you will be able to: human, foetal calf or rabbit serum.
1 Demonstrate the production of germ tube by Candida species.
III Specimens
24 hour culture of suspected fungal colony on Sabouraud’s
dextrose agar to be tested.
INTRODUCTION 24 hour culture of known strains of C. albicans and
C. parapsilosis colony on Sabouraud’s dextrose agar.
Candida species are usually found as normal flora of the oral
cavity and gastrointestinal tract of man. These may be isolated
from respiratory secretions, gastric washings, stool, vaginal PROCEDURE
secretions, urine, skin, nail, etc. Under normal conditions
Candida species are not pathogenic. However, in case of 1 Take three test tubes and label as 1, 2 and 3.
immunocompromised individuals, and certain other situations 2 Add 0.5ml. of serum to each of the test tube.
(Box 77-1), Candida species can cause a wide variety of 3 Take a half of a single colony to be tested by using a sterile
opportunistic infections. loop, and mix with serum in the test tube 1.
Germ tube test is a very simple and efficient test to distinguish 4 Similarly, take a half of C. albicans single colony by using a
pathogenic Candida from non-pathogenic ones. It is very widely sterile loop, and mix it with serum in the test tube 2.
used in diagnostic laboratories because of its reproducibility. 5 Similarly, take a half of C. parapsilosis single colony by using
a sterile loop, and mix it with serum in the test tube 3.
6 Incubate all the tubes at 37°C for a maximum of 1½ hrs.
PRINCIPLE 7 Place one drop of suspension from tube 1, 2, and 3 onto 3
different slides and place cover slips over the drops.
Germ tube is an initial hypha from a sprouting conidia, spore or 8 Examine the slide under low (10x) and high power (40x)
yeast. Formation of germ tube can be demonstrated by magnifications.
inoculating rabbit, fetal calf or human serum with a small quantity
of growth of Candida species. The suspension is then
incubated at 37°C for a minimum of 1½–2 hours, after which a
QUALITY CONTROL
drop is examined under the microscope for the germ tube. 1 Positive control for germ tubes: C. albicans.
Germ tubes are produced by C. albicans, C.stellatoidea 2 Negative control for germ tubes: C. parapsilosis.
and rarely C. tropicalis. At times, some strains of C. albicans 3 Human or rabbit serum should be checked for contamination
isolated from the patients with antifungal drugs or patients prior to use.
with cancer do not produce germ tubes. 4 Control organisms should be tested individually for
production of germ tubes.
REQUIREMENTS
OBSERVATIONS
I Equipments
Under the microscope, the whole field under the cover slip is
Microscope. examined for any cell showing production of germ tube (Fig. 77-1).
226 Germ Tube Test
Germ tubes are seen as long tube like projections extending

from yeast cells. This should be differentiated from
pseudohyphae (Table 77-1).
Tube 2 will show production of germ tube and Tube 3 will not.
The drop from tube 1 should be read and compared with these
controls.
Tube 2 contains C. albicans and hence shows germ tube
production while tube 3 does not show production of germ
tube since it contains C. parapsilosis. Tube 1 should be
interpreted with care by observing for the presence or absence
of germ tube and should be compared with tube 2 and tube 3. FIGURE 77-1 Germ tube test, x 400.
BOX 77-1 PREDISPOSING FACTORS Table 77-1 Differences between germ tubes and
FOR CANDIDIASIS pseudohyphae
Immunosuppression Germ tubes Pseudohyphae

Long term antibiotic therapy
Use of oral contraceptives No constriction at the site Constriction at the site of
Pregnancy of attachment. attachment.
Premature birth
Obesity Non-septate with Septate and not necessarily
Diabetes mellitus parallel sides. with parallel sides.
Immunocompromised status
KEY FACTS
1 Germ tube is a useful test to identify C. albicans and few other species which are pathogenic.
2 Rabbit, foetal calf or human serum can be used for demonstrating germ tube formation.
3 Germ tubes are produced by C. albicans, C.stellatoidea and rarely C. tropicalis.
4 At times, some strains of C. albicans isolated from the patients with antifungal drugs or patients with cancer do not produce
germ tubes.
5 C. tropicalis may show germ tube formation after 3 hours with a constriction at the base of the germ tube.
VIVA
1 What is a germ tube and how is it significant in routine identification of fungi?

2 Why is germ tube test important in a mycology laboratory?
3 What are the advantages of a germ tube test?
FURTHER READINGS
Arnold publishers.
LESSON
Urease Test
78
C. neoformans culture.
1 Demonstrate production of the enzyme urease by the fungus PROCEDURE

Cryptococcus neoformans.
1 Pick up one colony of C. neoformans.
2 Inoculate Christensen’s urea agar slope with these fungal
colonies.
INTRODUCTION 3 Incubate the tube at 37°C for 2 days.
4 Observe any change of colour in the inoculated medium.
Some fungi produce the enzyme urease that hydrolyses the
urea releasing ammonia into the medium. Ammonia in turn
produces a change in the pH of the medium that can be detected QUALITY CONTROL
by the colour change in the indicator dye. This test can be
used to differentiate different groups of fungi. Positive control: Trichophyton mentagrophytes (urease positive
fungi).
Negative control: Candida albicans and Trichophyton
rubrum (Urease negative fungi).
PRINCIPLE An un inoculated medium is incubated along with the test
to compare the colour change.
Urea is a diamide of carbonic acid. Urease, the enzyme produced
by the fungi, hydrolyses urea and releases ammonia and carbon
dioxide. Ammonia reacts in solution to form ammonium OBSERVATION
carbonate, which is alkaline, leading to an increase in the pH.
Examine after 2 days of incubation. The test should not be
Phenol red that is incorporated in the medium changes its colour
considered negative till after 2 days of incubation.
from yellow to red in alkaline pH, thus indicating the presence
The un inoculated medium is colour less. In a positive test,
of urease activity.
after incubation, the colour of the medium changes to purple pink.
REQUIREMENTS RESULTS AND INTERPRETATION
I Equipments Positive reaction is detected within 1 to 2 days of incubation.

Incubator. When positive, medium shows growth of the colony and
the color of the test medium as well as positive culture medium
II Reagents and lab wares changes to purple pink.
Inoculating wire, Bunsen flame, test tubes, fungi from culture No colour change is seen in negative culture medium. The
tube, and Christensen’s urea agar slope. test is considered negative if no colour change of the test
medium is observed.
228 Urease Test
KEY FACTS
1 Certain fungi possess the enzyme urease that hydrolyzes urea releasing ammonia into the medium.
2 Phenol red that is incorporated in the medium changes its color from yellow to red in alkaline pH, thus indicating the
presence of urease activity.
3 Always check the sterility of the slants before inoculation.
4 An un inoculated medium must be incubated along with the test.
5 Observe the growth of inoculum irrespective of change in colour.
VIVA
1 What is the medium used in urease test?

2 What is the principle of urease test?
3 Give examples of urease positive fungi?
FURTHER READINGS
Arnold publishers.
LESSON
Carbohydrate
79 Assimilation Test
After completing this practical you will be able to: 1 Pipette 2.0 ml. of sterile saline into a test tube.
2 With a sterile loop pick up few isolated colonies of the
1 Find out the pattern of assimilation of carbohydrates by organism from the SDA plate and emulsify to a turbidity
yeast and yeast-like fungi. equal to McFarland 4 units.
3 Cover the surface of yeast nitrogen base agar with the
suspension of the yeast cells.
4 Remove the excess fluid and allow the surface of the agar to dry.
INTRODUCTION
5 With sterile forceps place the carbohydrate disc onto the
surface of agar in such a way that at least 30 mm space is
Yeast and yeast-like fungi use carbohydrates as sources of present between each disc.
energy. Hence in this test, utilization of carbohydrate is used 6 Incubate the plate at 30°C or 37°C for 24–48 hours.
as a definitive diagnosis for yeast or yeast-like fungi. 7 At the end of the incubation period observe the plate for
growth around the disc.
PRINCIPLE
QUALITY CONTROL
Yeast and yeast-like fungi are identified by the pattern of
carbohydrate assimilation. They are inoculated on the 1 Carbohydrate discs and media should be checked using
carbohydrate-free yeast nitrogen base agar on which different standard control strains as follows:
filter paper discs containing various carbohydrates are placed. Candida albicans ATCC 14053
After incubation for appropriate time, growth around the discs C. guilliermondii ATCC 6260
is observed and the carbohydrate utilization pattern is assessed. C. pseudotropicalis ATCC 4135
2 Sterility of glassware and media should be ensured.
REQUIREMENTS OBSERVATION
I Equipments After incubation, growth of the fungi around the discs is
Incubator. observed and the carbohydrate utilization pattern is assessed.

Standard laboratory glassware and equipments, filter paper RESULTS AND INTERPRETATION
discs, McFarland standard, yeast nitrogen base agar (Table 79-1),
Growth of the fungi around each carbohydrate disc is observed
sterile saline, and filter paper discs containing different
and results are interpreted in such a way that if the fungus has
carbohydrates grown touching the edges of the disc, and that organism
assimilates that particular carbohydrate in the disc then it is
III Specimens considered positive. Similarly, if the fungus does not grow in
Pure growth of test fungi on Sabouraud’s dextrose agar (SDA) the proximity of a particular carbohydrate disc, it is considered
medium. negative for assimilation of that carbohydrate.
230 Carbohydrate Assimilation Test
Table 79-1 Preparation of Yeast Nitrogen Base
Composition Preparation
Boric acid – 500 µgm All the above ingradients are to be sterilized by filtration and dispersed
Copper sulphate – 40 µgm aseptically.
Potassium iodide – 100 µgm Add. 10 ml. of the above solution to 90ml of 2% molten agar and pour into
Ferric chloride – 200 µgm the plates.
Manganese sulphate – 400 µgm
Sodium molybdate – 200 µgm
Zinc sulphate – 400 µgm
Biotin – 2 µg
Calcium pantothenate – 400 µgm
Folic acid – 2 µg
Inositol – 2000 µgm
Niacin – 400 µgm
p-aminobenzoic acid – 200 µgm
Pyridoxine hydrochloride – 400 µgm
Riboflavin – 200 µg
Thiamine hydrochloride – 400 µgm
L-Histidine monohydrochloride – 10.0 milligram
DL-Methionine – 20 mg
DL-Tryptophan – 20 mg
Magnesium sulphate – 500 mg
Sodium chloride – 100 mg
Ammonium chloride – 5 g
Monopotassium phosphate – 1 g
Distilled water – 1000 ml
KEY FACTS
1 Turbidity using the organism should be done in such a way that it is equal to McFarland Standard 4 units.
2 Sterility should be maintained for medium and carbohydrate discs.
3 Growth of fungi near carbohydrate discs should be confirmed after thorough examination.
VIVA
1 Describe how carbohydrate medium is prepared.

Ans. The basic salt solution is prepared and 10 ml. of this solution is added to 90 ml of 2% molten agar and poured in plates.
2 How do you interpret carbohydrate assimilation test result?
FURTHER READINGS
Arnold publishers.
LESSON
Carbohydrate
80 Fermentation Test
LEARNING OBJECTIVES Preparation of indicator broth medium: The indicator broth

medium (IBM) contains peptone, 1.0 gm.; sodium chloride, 0.5
After completing this practical you will be able to: gm.; beef extract, 0.5 gm; bromocresol purple (0.4%),10 ml and
distilled water, 90.0 ml.
1 Demonstrate the ability of different yeast and yeast-like fungi Durham’s tube is placed in an inverted position in screw
to ferment various carbohydrates. capped test tubes. In each tube, fill 5–6 ml IBM just above the
Durham’s tube and sterilize at 121°C for 15 min. Aseptically,
0.3 ml of 20% filter sterilized carbohydrate solution (glucose,
lactose, sucrose, maltose, etc.) are added to the medium.
INTRODUCTION
III Specimen
Yeasts and yeast-like fungi use carbohydrates as sources of Pure growth of test fungus on Sabouraud’s dextrose agar (SDA)
energy. Fermentation of carbohydrate in the medium produces medium.
a color change and bubbles which can be used as a marker to
find out the type of yeast in culture.
PROCEDURE
PRINCIPLE
1 Take tubes with indicator broth medium with sugars.
Sometimes, a definite identification of the suspected fungi 2 With a sterile loop pick up few isolated colonies of the fungus
cannot be made using carbohydrate assimilation test. In such from the SDA plate and emulsify to a turbidity equal to
cases, carbohydrate fermentation tests are used as a McFarland 4 units.
supplement. Fermentation of carbohydrates produces a visible 3 Inoculate each sugar tube with 0.2 ml (5 drops) of culture
color change in the medium, and bubbles will be produced in suspension.
the Durham’s tube. This is an indicator of fermentation. 4 Incubate the tubes at 30°C or 37°C for 2-10 days.
Glucose, maltose, sucrose, lactose, galactose, and trehalose Note: Do not screw caps on tubes tightly.
are the sugars used in carbohydrate fermentation tests. 5 Observe the presence of air bubbles in Durham’s tubes.
REQUIREMENTS QUALITY CONTROL

I Equipments
1 Quality of sterile glassware must be checked for proper sterility.
Incubator.
2 Sterility of medium used should be confirmed before use.
II Reagents 3 Quality of medium used should be checked by growing
Standard laboratory glassware and equipments, McFarland known standard strains of fungi.
standard. Different carbohydrates, and indicator broth medium Candida albicans: Glucose, maltose positive.
(IBM). Candida kefyr: Glucose, sucrose positive.
232 Carbohydrate Fermentation Test
OBSERVATIONS RESULTS AND INTERPRETATION
After incubation, the Durham’s tubes are observed for presence Presence of bubbles or drop in the fluid level in Durham’s tube
of any gas bubbles. indicates fermentation of sugars. Development of yellow color
is not a reliable indicator of fermentation and is ignored.
KEY FACTS
1 Carbohydrate fermentation test is a supplementary test only when there is difficulty in making a definitive identification
using carbohydrate assimilation test.
2 Development of yellow color is not reliable indicator of fermentation and hence production of air bubble in Durham’s tube
is to be noted.
VIVA
1 What are the different carbohydrates used in carbohydrate fermentation test?

2 What percentage of carbohydrate should be incorporated in carbohydrate medium?
3 How do you interpret carbohydrate fermentation test?
4 What are the advantages of carbohydrate fermentation test over carbohydrate assimilation test?
FURTHER READINGS
Arnold publishers.
LESSON
Identification of
81 Common Fungi
After completing this practical you will be able to: Lactophenol cotton blue wet mount preparation of the fungus
(refer chapter 73).
1 Know important characteristic morphological features of
different fungi.
2 Identify a given fungus. QUALITY CONTROL
Quality of sterility of medium should be checked before use.

INTRODUCTION
Fungi cause varied infections in man, hence identification of

OBSERVATION
the fungi is a very important approach in patient management. The wet mount preparation should be visualized under low
Hence it is necessary to know different fungi causing infections power (10x) and high power (40x) objectives and the
in man and methods to identify them. morphology observed.
PRINCIPLE RESULTS AND INTERPRETATION

By microscopic observation, characters of the yeast, mold,
In a mycology laboratory, the fungi can be cultivated, and its
hyphae, conidia, etc. can be made out and used in classifying
colonial characteristics will throw some light on the type of
the type of fungus isolated.
fungus. Further, the fungal element can be stained and observed
Molds Rhizopus
under a microscope and presumptive identification can be done.
Mucor
Alternaria
Fusarium
REQUIREMENTS Aspergillus
Penicillium
I Equipments Cladosporium
BOD incubator and dissecting microscope. Cephalosporium
Trichophyton
Epidermophyton
II Reagents and lab wares Yeast Torulopsis
Standard laboratory glassware, Bunsen burner, hard lens, glass Candida
slides, cover slips, sterile cotton swab, glass marking pencil,
SDA medium, lactophenol cotton blue stain. Rhizopus
A Common laboratory contaminant.
III Specimen Colony morphology
Fungal culture grown on SDA’s medium. Rapidly growing white colored fungus swarms over entire plate
showing aerial mycelium cottony.
234 Identification of Common Fungi
Microscopy
Spores are oval, colorless and sometimes brown. Mycelium Aspergillus
non-septate, sporangiophores straight and terminate in black
sporangium containing sporangiospores and columella; root Usually infect plants and animals. More than 170 species are
like hyphae (rhizoids) penetrating the medium. known. Almost sixteen different species cause human disease.
They are:
Mucor
Aspergillus fumigatus
Common food contaminant. Aspergillus flavus
Aspergillus niger
Colony morphology
Resembles colony of Rhizopus. Aspergillus oryzae
Aspergillus amstelodami
Microscopy Aspergillus verscicolor
Oval spores, non-septate mycelium giving rise to single Aspergillus terreus
sporangiophores and terminate in globular sporangium Aspergillus nidulans
containing columella and sporangiospores. Aspergillus candidus
Aspergillus ustus
Alternaria Aspergillus carnens
Aspergillus arenaceus
Usually found on plants.
Aspergillus clavatus
Colony morphology Aspergillus caseillus
Grayish green or black colonies with grey edges and swarming Aspergillus restricutus
over entire plate with grayish white aerial hyphae.
The characteristics used to identify them are:
Microscopy A) Colour and shape of conidial head.
Conidia multi celled, pear-shaped and attached to single B) Number of sterigmata/phialids.
conidiophores arising from septate mycelium. C) Shape of vesicles.
D) Colour of conidiophore.
Fusarium E) Presence and absence of cleistothecia, and
F) Size and shape, and color of ascospore and conidia.
Usually found in soil.
Aspergillus fumigatus
Colony morphology
Woolly white colonies may change to pink, purple or yellow
Colonies
(Fig. 81-1).
Colonies are blue-green, velvety, surface powdery or granular,
sometimes radially folded (Fig. 81-2).
Microscopy
Conidia multi celled, oval or crescent shaped, attached to
conidiophores arising from a septate hypha. Conidial head
Columnar, compact.
Conidiophore
Smooth, terminate into club or flask shaped green.
Vesicle
Club or flask shaped, green.
Sterigmata
Produced on upper half of the vesicle, uniseriate, appears green
in color and crowded. Axis of sterigmata is roughly parallel to
that of conidiophore and conidia produced in chains on
sterigmata.
Conidia
FIGURE 81-1 SDA with colonies of Fusarium species.
Globose, green and echinulate.
FIGURE 81-2 SDA with colonies of Aspergillus fumigatus. FIGURE 81-4 SDA with colonies of Aspergillus flavus.
Aspergillus niger
Aspergillus flavus
Colonies
White, fluffy, reverse buff colored, covered with black spores Colonies
(Fig. 81-3). Rapidly growing, yellowish green and velvety colony (Fig. 81-4).
Conidial head Conidial head

large black to brownish, initially globose, become radiate then Radiate, loosely columnar.
splits into divergent spore columns.
Conidiophore
Conidiophore
hyaline, thick walled and roughened.
Variable in size, thick smooth walls, conidiophore hyaline and
brownish near the vesicle.
Vesicle
Vesicle Globose, sub globose or elliptical and sterigmata cover entire
Globose, concave under surface, brownish sterigmata produced surface or at least three fourth.
in two series, septate primary sterigmata and short secondary
sterigmata. Sterigmata
Monoseriate and biseriate sterigmata seen in same strain,
Conidia produced by small and large vesicles respectively.
Globose, echinulate.
Conidia
Conidia globose, subglobose or elliptical. Conidia echinulate
and appears yellow green.
Penicillium species
Colonies
Initially white and fluffy, later turns to green and blue green,
with radial folds.
Hyphae
Septate, hyaline.
Conidiophores
Long with branching phialids.
Phialids
FIGURE 81-3 SDA with colonies of Aspergillus niger.
Flask shaped, branched and gives rise to brush like appearance,
producing sterigmata.
236 Identification of Common Fungi
Sterigmata Macroconidia
Long with tapering end, producing chains of conidia. String-like, bean shaped, septate, with characteristic rat tail
appearance, rarely produced.
Conidia
Long chains of conidia, spherical or oval in shape. Microconidia
Tear-shaped, rarely produced. Swollen antler-like hyphae
Cladosporium produced rarely, may produce chains of chlamydospores.
Usually found on dead and decaying plants. Trichophyton violaceum
Colony Colony
Small, heaped, greenish black and powdery. Slow growth, port wine or deep violet colored, flat or heaped
up, surface waxy and glabrous, wrinkled loose pigment present.
Microscopy
Conidia develop at the end of complex conidiophores arising Macroconidia
from brown septate mycelium. Generally not present. Thick walled structures resembling
macroconidia of T. rubrum may be present.
Cephalosporium
Used in antibiotic production Microconidia
Generally not present, pyriform. Chains of chlamydospores
produced.
Colony
Rapidly growing compact, moist colonies, cottony, with gray
or rose colored aerial hyphae. Epidermophyton floccosum
Microscopy Colony
Velvety or powdery surface, surface folded in radiating furrows,
Single celled conical or elliptical conidia held together in clusters
reverse of colony yellow tan.
at the tips of conidiophores. Erect, unbranched conidiophores
arise from septate mycelium.
Macroconidia
Clavate, smooth, thick walled, septa 2–3, characteristics clusters
Trichophyton verrucosum of twos and threes.
Colony Microconidia
Slow grower, white glabrous heaped up colony, sometimes Not produced.
button like with velvety texture. No pigment produced on reverse.
Variants with yellow or grey white colonies, rugal folds seen. Chlamydospores
Produced, racquet hyphae and nodular bodies present.
KEY FACTS
1 Characters of aerial hyphae, septation, conidial shape, size and color are very important for identifying the fungus.
2 Colony characters, color, obverse and reverse should be kept in mind.
VIVA
1 What are the common fungi that cause disease in humans?

Ans. The common fungi that cause human infections include pathogenic species of
Curvularia
Rhizopus
Mucor
Alternaria
Fusarium
Aspergillus
Penicillium
Cladosporium
Cephalosporium
Trichophyton
Epidermophyton
Torulopsis
Candida
Absidia
Cryptococcus
Histoplasma
Sporothrix
Blastomyces, etc
2 How are culture and staining of fungal elements useful in characterizing fungi?
Ans. Different fungi have their own predilection to grow on different media depending upon the exact nutritional requirement.
Moreover different fungi produce a wide variety of morphological structures like conidiophores, conidia, sporangiophores,
sporangia, septate or aseptate hyphae, etc., which on staining can be visualized and the fungi identified.
3 What are different types of spores or conidia produced by different molds?
Ans. Different molds produce different types of spores and conidia, like chlamydospores, ascospores, microconidia,
macroconidia etc.
4 How is the colony morphology of fungi important in diagnosing fungal infections?
Ans. The colonies produced by many pathogenic fungi differ greatly from each other. Moreover production of pigments by
these colonies also vary greatly. Hence this helps for a preliminary identification of the type of fungus
FURTHER READINGS
Arnold publishers.
3 Jagadish Chander. A Text Book of Medical Mycology.. Interprint. 2000.
238
UNIT
XI
Virology
Lesson 82 Cultivation of Viruses in the Cell lines

Lesson 83 Cultivation of Viruses in
Embryonated Egg
240
LESSON
Cultivation of Viruses in
82 the Cell lines
LEARNING OBJECTIVES Many viruses kill the infected viral cells in which they grow
and bring about detectable changes in morphology of the cells.
After completing this practical you will be able to: These changes are collectively known as cytopathic effects.
Some viruses however do not produce any cytopathic effect
1 Know and understand the common cell lines used for routine (e.g., rubella virus).
maintenance of viruses in a virology laboratory. The most important precaution to be taken during
maintenance of cell lines is sterility. Contamination of cell lines
should be prevented and even cross contamination among cell
INTRODUCTION lines should be avoided.
Viruses are intracellular pathogens which are dependent on

host cell machinery for their multiplication and growth. Now a REQUIREMENTS
days monolayer cell cultures are mostly used in diagnostic and
research work in viral diseases. I Equipments
Cell culture is the most widely used system for cultivation Inverted microscope (Fig. 82-1), incubator (Fig. 82-2),
of viruses since it is more convenient method compared to the haemocytometer and biological safety cabinet.
other methods like egg inoculation and animal inoculation.
The cell cultures are used for a) isolation of viruses from II Reagents and lab wares
clinical specimens for diagnosis of viral diseases, b) biochemical Sterile glassware, pre-sterilized tissue culture plasticware (Fig.
studies of viral replication and c) production of viral antigens 82-3), Pasteur pipettes and measuring pipettes, membrane filter,
and vaccines. Depending upon the number of divisions which syringes, vials, discard jar, Eagle’s, minimum essential medium
a cell line undergoes in-vitro before dying, the cell lines have (MEM), sodium bicarbonate (NaHCO3), EDTA trypsin mixture,
been classified as primary, diploid and continuous cell lines. foetal calf serum (FCS), sterile double distilled water, virus
The most commonly used cell culture systems in most inoculum, spirit and sodium hypochlorite.
laboratories include chick embryo fibroblast cells, human amnion Monolayer of a cell culture in a culture flask is treated with
cells, Rhesus monkey kidney cells, HeLa (Human carcinoma of trypsin or versene to disperse cells.
cervix cell line), Hep 2 (Human epithelioma of larynx cell line), III Specimen
McCoy (Human synovial carcinoma cell line),Vero (Vervet Suspected virus infected specimen like the cerebrospinal fluid
monkey kidney cell line), and W1-38 (Human embryonic lung (CSF), stool, rectal swab, and throat swab.
cell line).
PRINCIPLE PROCEDURE
Viruses infect healthy cells grown in the laboratory. When 1 Discard the trypsin versene mixture and add a small amount
susceptible cells are used for inoculation of viruses, they show of MEM with 10% FCS to the monolayer of cells.
pathological changes and the viruses can be harvested from 2 Count the cells with the medium in a hemocytometer for
the cells for further tests. The growth of viruses in the cell lines appropriate splitting.
can be known by a) cytopathic effects, b) immunofluorescence, 3 Inoculate the cells into sterile flasks or tubes for viral
c) haemagglutination and haemadsorption, and d) interference. inoculation.
4 Fill the new flasks with MEM and incubate in horizontal

position.
5 Select a healthy monolayer, which is also confluent, for viral
inoculation.
6 Inoculate the monolayer of cells with virus using sterile
Pasture pipette, and incubate at 37°C.
7 Observe for the cytopathic effect (CPE) 7 days after
inoculation.
QUALITY CONTROL
FIGURE 82-1 Inverted microscope.
1 Sterility precautions should be taken perfectly.
2 Susceptible cells to be selected for the appropriate virus.
OBSERVATIONS
After incubation, the flasks are observed for confluency and

healthy monolayer of cells and virus infected cells are classified.
Viruses are known to produce cytopathic effects are
identified by observing the same in the infected cell lines.
Non-cytopathogenic viruses are identified by other methods
like immunofluorescence, haemagglutination and
FIGURE 82-1 Incubator.
haemadsorption, and interference
The cell lines are observed for any cytological alterations that
are diagnostic of viral infections (Table 82-1).
FIGURE 82-3 Cell culture bottle.
Table 82-1 The cell lines and indications for the viruses and cytopathic effects they produce
Type of viruses Susceptible cell line Cytopathic effect
Adenovirus Primary human embryonic kidney cells. Rounding and clustering of swollen cells.
HeLa cells.
Herpes simplex virus HEp 2 cells. Rounding and swelling of cells.
Varicella zoster virus Human kidney cells. Multinucleated giant cell formation.
Cytomegalovirus Human fibroblast cells. Giant cell formation.
Enterovirus Hep 2 cells. Rapid rounding of cells progressing to complete
Vero cells. cell destruction.
RD cells.
Paramyxovirus Primary human and monkey kidney cells. Syncytia formation.
LLC-MK2.
242 Cultivation of Viruses in the Cell lines
KEY FACTS
1 The most important aspect to be taken care of in cell culture is sterility. Hence precautions for sterility should be meticulously
followed.
2 Susceptible cells should be selected for the appropriate viral inoculation.
VIVA
1 Why is cell culture method, the most preferred method in a virology laboratory?
2 What precaution is to be taken during maintenances of cell lines?
3 What are the commonly used cell lines in virology laboratory?
FURTHER READINGS
1 Collier L, Balows A, Sussman M. Topley and Wilson’s; Microbiology and Microbial Infections. 9th Edition. Virology. Volume 1. Arnold
publishers.
2 Knipe DM, Howley PM. Field’s Virology. 4th Edition. Lippincott Williams and Wilkins. 2001.
3 Zuckerman AJ, Banatvala JE, Pattison JR, Griffiths PD and Schoub BD. Principles and Practice of Clinical Virology. 5th Edition. John
Willey and sons Ltd. 2004.
LESSON
Cultivation of Viruses in
83 Embryonated Egg
LEARNING OBJECTIVES Allantoic cavity is mainly employed for harvesting influenza

virus for vaccine production , and also for yellow fever (17D
After completing this practical you will be able to: strain) and rabies (Flury strain) vaccines.
CAM is mainly used for growing pox viruses. These produce
1 Know the routes of inoculation of eggs for propagation of characteristic visible lesions such as pocks. Pocks produced
viruses. by different viruses vary in their morphology.
INTRODUCTION REQUIREMENTS
Prior to 1950s the technique of propagation of viruses in I Equipments
embryonated eggs was popular because of non-availability of Egg holders, candling lamp and hole puncher,
cell culture techniques during those times. The eggs are used
because they are inexpensive, easily available and much simpler II Reagents and lab wares
to handle than animals. The viruses can grow in different Syringes (1 tuberculin syringe preloaded with 0.1 ml of 10-3
compartments of the egg . Since eggs lack a well developed dilution of NDV in GLB), gauze, pencils, gloves, sterile forceps,
defence mechanism of their own, they do not interfere with Pasteur pipette with bulb, screw capped sterile vials, egg, melted
growth of viruses. paraffin wax and 70% ethanol.
However, many viruses fail to grow on primary inoculation The eggs are candled to determine the position of the
into eggs. Nevertheless, the embryonated eggs are still used embryo and its viability. Since viruses need living tissues to
for the isolation of avian viruses, influenza viruses and also for replicate, candling is important. When candled, healthy embryo
vaccine production. has an orange color with evident vasculature. Dead embryo shows
clear yellow with no vasculature. Black, green or brown color
indicates contamination. Dead embryos are promptly discarded.
PRINCIPLE
III Specimen
Usually 8-11 days old chick embryos are used. Age of the egg Virus isolate or specimen suspected to contain virus.
chosen depends on route of inoculation described since various
membranes and their contents vary in size as embryo matures.
These eggs are inoculated by one of the following routes: PROCEDURE
yolk sac, amniotic sac, allantoic cavity and chorioallantoic
membrane (CAM). Any of the viruses or specimen suspected 1 Disinfect the egg shell.
to contain viruses are inoculated. After inoculation, eggs are 2 Drill the egg shell.
incubated for 2-9 days. Viral growth is recognized by death of 3 Inoculate the specimen in the embryonated egg through
embryo, formation of pocks, or haemagglutination property of appropriate route.
embryonic fluid. Note: Age of the embryonated egg is chosen depending on
Yolk sac is mainly used for culture of some viruses, certain bacteria the route of inoculation since various membranes and their
(Chlamydia and Rickettsia) and parasite (Toxoplasma gondii). contents vary in size as embryo matures (Table 83-1).
Amniotic sac is used for the primary isolation of influenza 4 After 2–5 days post injection, viral growth in the egg is
virus. recognized by death of embryo, pocks, or hemagglutination.
244 Cultivation of Viruses in Embryonated Egg
1 Before inoculation, the eggs are candled to determine the Viral growth in the inoculated egg is recognized by death of
position of the embryo and its viability. the embryo, pock formation in CAM (Fig. 83-1),
2 Age of the embryonated egg is chosen depending on the haemagglutination, etc.
route of inoculation since various membranes and their
contents vary in size as embryo matures.
3 Sterile precautions should be taken throughout the
procedure.
OBSERVATIONS
The inoculated part of embryonated egg is observed for

changes due to viral infection.
FIGURE 83-1 CAM showing pocks.
Table 83-1 The routes of inoculation of the egg and the viruses isolated
Route of inoculation Age of embryo Virus Use
Chorio allantoic membrane. 10-14 days. Variola virus. Isolation.

Herpes simplex virus. Isolation and typing.
Vaccinia virus. Vaccine titration.
Amniotic sac. 10-12 days. Influenza A virus. Isolation.

Mumps virus. Isolation.
Allantoic sac. 9-12 days. Mumps virus. HA antigen preparation.

Influenza A, Influenza B. Hybrid vaccine production.
Parainfluenza viruses. Isolation.
Yolk sac. 6-8 days. Flavi viruses. Isolation, vaccine production.
KEY FACTS
1 Usually 8-11 days old chick embryos are used for cultivation of viruses.
2 Since viruses need living tissues to replicate, candling of eggs is important.
VIVA
1 Why is the age of the embryo an important factor in virus inoculation?

2 What are some important parameters to be noted before passaging viruses in embryonated eggs?
3 What is the purpose of candling?
Ans. Since viruses need living tissues to replicate, candling is important. When candled, healthy embryo has an orange
color with evident vasculature, dead embryo show clear yellow colour with no vasculature, black, green or brown color
indicates contamination.
4 Would you expect a higher yield of virus from dying or dead embryo, why?
Ans. Higher yield of viruses cannot be expected from dying or dead embryo since viruses require healthy living tissues for
their multiplication.
FURTHER READINGS
1 Collier L, Balows A, Sussman M. Topley and Wilson’s; Microbiology and Microbial Infections. 9th Edition. Virology. Volume 1. Arnold
publishers.
2 Knipe DM, Howley PM. Field’s Virology. 4th Edition. Lippincott Williams and Wilkins. 2001.
3 Zuckerman AJ, Banatvala JE, Pattison JR, Griffiths PD and Schoub BD. Principles and Practice of Clinical Virology. 5th Edition. John
Willey and sons Ltd. 2004.
246
UNIT
XII
Microbiology of
Water, Milk and Air
Lesson 84 Microbiology of Water

Lesson 85 Microbiology of Milk
Lesson 86 Microbiology of Air
248
LESSON
Microbiology of Water
84
LEARNING OBJECTIVES The total coliform count is widely regarded as the most
reliable indicator of potable water quality. However, the
After completing this practical you will be able to: presence of coliforms not necessarily indicate faecal or sewage
contamination, because, these organisms are present in large
1 Know various methods used for testing potable water. quantities in the environment.
2 Test the quality of the potable water for drinking.
Faecal or thermotolerant coliforms
INTRODUCTION These satisfy the criteria for coliforms but have the additional
property of the ability to grow at a higher temperature 44°C.
Drinking water is acceptable and fit for drinking when it is
clear, colourless, odourless and without disagreeable taste. Faecal Escherichia coli
Microscopically it should be free from pathogenic organisms.
Natural sources of water usually contain some saprophytic These are defined as thermotolerant coliforms which ferment
bacteria, such as Pseudomonas, Serratia, Flavobacterium, lactose (or) mannitol at 44°C with the production of acid and
Chromobacterium, Acinetobacter, Alcaligenes, etc. These gas within 24 hours and also form indole from tryptophan at
saprophytes are harmless. 44°C. The presence of E. coli is considered as the contamination
Water gets contaminated by pathogens which are of water with feces of human or animal origin.
introduced into water by sewage pollution. A wide varieties of
diseases are transmitted by contaminated water. There are some Faecal streptococci
indicator organisms, whose presence indicates the
contamination of water with fecal matter. These are: These are catalase negative, Gram positive cocci present in the
intestinal tract of man and animals. These organisms have the
1 Coliforms. Lancefield group D antigen, hydrolyse aesculin and can grow
2 Fecal (thermotolerant) coliforms. at 45°C, in the presence of azide and 40% bile. Such organisms
3 Faecal Escherichia coli. which can survive 60°C for 30 min and can grow at 10°C at pH
4 Faecal Streptococci, and 9.6 and in 6.5% of sodium chloride (NaCl) belong to the genus
5 Clostridium perfringens. Enterococcus (Enterococcus faecalis and E. faecium) .
Clostridium perfringens
Coliforms
The presence of this organism in water in the absence of other
Coliforms are defined as members of the family Entero- indicators of contamination of water implies remote or
bacteriaeceae which grow in the presence of bile salts and intermittent fecal pollution.
produce acid and gas from lactose within 48 hours at 37°C. In Viruses in water are destroyed by chlorination. When the
order to include anaerogenic bacteria and those which are non- concentration of free residual chlorine is at least 0.5 mg per
lactose fermenters, it has been modified as the members of the litre, for a minimum contact period of 30 minutes at pH below 8
Enterobacteriaeceae capable of growing galactosidase at 37°C and a turbidity of 1 nephelometric turbidity unit or less, protozoa
that normally posses. such as Entamoeba histolytica, Giardia species and
Balantidium coli may be present in the drinking water. Coliforms Differential coliform test
are not the reliable indicator of protozoal contamination. This is called Eijkman test. This test is usually employed to
find out whether the coliform bacilli detected in the presumptive
Collection of water samples test are E. coli or not.
After the usual presumptive test, subcultures are made from
Water is collected in heat sterilised bottles containing a all the bottles showing acid and gas to fresh tubes of single
sufficient volume of sodium thiosulphate to neutralise the strength MacConkey medium already warmed to 37°C. They
bactericidal effect of any chlorine or chloramine in the water. are incubated at 44°C for 24 hours. Those showing gas in
For collection from tap, water is allowed to run to waste for Durham’s tubes contain E. coli. From the number of positive
2–3 min. before collecting it into the bottle. When collecting tubes obtained, results are read off the probability tables.
water samples from lakes or streams the bottle is opened at a Further confirmation of the presence of E. coli is done by
depth of about 30 cm with its mouth facing the current. At least testing for indole production and citrate utilization tests.
100 ml of water to be tested is collected in each bottle.
After collecting the water in the bottle, the bottle is stoppered, Membrane filtration method
and sent to the laboratory as quickly as possible within 6 hours A measured volume of water is filtered through a Millipore
keeping it in a cool container and protecting it from light. filter. All the bacteria present are retained on its surface. This is
then placed on suitable media and incubated at the appropriate
temperatures. Colonies on the surface of the membrane are
PRINCIPLE counted. After 18 hours of incubation the presumptive coliform
counts and detection of E. coli can be directly made.
The following tests can be done for bacteriological analysis of water:
1 Plate count.
Detection of faecal streptococci
2 Detection of coliform bacteria and E. coli. Subcultures are made into tubes containing 5 ml of glucose
a Presumptive coliform count: multiple tube technique. azide broth from the positive bottles in the above test.
b Differential coliform test. Streptococcus faecalis if present produces acid in the medium
c Membrane filtration method. within 18 hours at 45°C.
3 Detection of faecal streptococci.
4 Examination of Cl. perfringens, and Examination for Cl. perfringens
5 Test for pathogenic bacteria. Water is incubated in litmus milk medium at 37°C for 5 days, if
positive, stormy fermentation occurs.
Plate count
Examination for pathognic bactrial cells test
This consists of inoculating the nutrient agar with water to be For other special pathogens like Salmonella, Vibrio, etc.
tested and incubating the agar aerobically in parallel at 37°C corresponding special media are used.
for 1–2 days and at 22°C for 3 days. After incubation number of In this chapter Presumptive coliform test and diffrential
the colonies formed in the agar are counted. Those which grow coliform test will be discussed.
at 37°C are associated with organic material of human or animal
origin and those growing at a lower temperature are mainly
saprophytes that normally inhabit water, soil, and vegetables. REQUIREMENTS
The agar count at 22°C gives an indication of the amount of
decomposing organic matter in the water available for bacterial I Equipments
nutrition. Though these are non-pathogenic the greater the amount Bacteriological incubator, autoclave and water bath.
of organic matter present, the more likely is the water to be
contaminated with parasitic and potentially pathogenic organisms. II Reagents
The agar count at 37°C is a more important index of dangerous MacConkey broth, brilliant green bile broth
pollution. Preparation of MacConkey broth: This is prepared by mixing
pancreatic digest of gelatin, 20 grams; lactose, 10 grams; bile
salt, 5 grams; bromocresol purple, 0.01 grams and final pH (at
Detection of coliform bacteria and E.coli
25°C) is adjusted at 7.3 ± 0.2.
Preparation of double strength medium: It is prepared by
Presumptive coliform test – Multiple tube suspending 35 grams in 500 ml of distilled water and heated to
technique dissolve the medium completely. 50 ml and 10 ml of the media are
The test is called presumptive, because of the presumption dispensed into screw capped bottles with inverted Durham’s tubes
that the reactions are due to coliforms organisms. The count is and are sterilized by autoclaving at 15 lbs (121°C for 15 minutes).
made by adding varying quantities of water (0.1 ml–50 ml) to Preparation of single strength medium: It is prepared by
bile salt lactose peptone water with an indicator for acidity. suspending 3.5grams in 100 ml of distilled water. 5 ml quantities
Acid and gas formation indicate the growth of coliform bacilli. of the medium are dispensed in screw capped bottles with
250 Microbiology of Water
inverted Durham’s tubes and are sterilized by autoclaving at

15 lbs (121°C for 15 minutes).
OBSERVATIONS
Preparation of brilliant green bile broth (BGBB): This is
prepared by mixing peptic digest of animal tissue, 10 grams; Observe for the presence of gas and production of indole.
lactose, 10 grams; ox gall, 20 grams and brilliant green, 0.0133 Presence of gas in brilliant green bile broth and indole
gm and adjusting the final pH (at 25°C) to 7.2 ± 0.2. 4 grams of production at 44°C is indicative of the presence of E. coli. For
the medium is suspended in 100 ml distilled water and mixed interpretation refer MacGrady’s table.
well. The media is distributed in 5ml volumes in test tubes with
inverted Durham’s tubes and sterilized by autoclaving at 15
1bs pressure (121°C) for 15 minutes. RESULTS AND INTERPRETATION
III Specimen Presumptive coliform count

Water specimen to be tested. The no. of bottles showing acid and gas is counted and
compared with the MacGrady’s table.
Result is expressed as- coliforms most probable no (MPN)/
PROCEDURE 100ml.
e.g. for Presumptive coliform count
1 Collect 500 ml of water in a sterile bottle. No. of tubes giving positive reaction
2 Inoculate water sample immediately into the MacConkey
broth medium. 50 ml 10 ml 1 ml
3 Inoculate 50 ml of water into 50 ml double strength media (1 1 2 2
bottle) Result: Coliforms most probable no 10/100 ml.
4 Inoculate 10 ml of water into 10 ml double strength medium
(5 bottles). Differential coliform count
5 Inoculate 1 ml of water into 5ml single strength medium (5 The no of tubes showing positive results i.e. both gas
bottles). productions in BGBB and Indole test at 44°C is compared with
6 Incubate all the bottles at 37°C for 48 hours. MacGrady’s table. Result is expressed a Escherichia coli Most
7 Check for acid and gas after 24 hours and 48 hours. probable No (MPN) / 100 ml.
8 Any positives are inoculated into brilliant green bile broth Eg. for Differential coliform counts:
and peptone water. No. of tubes giving positive reaction.
9 Incubate at 44°C in a water bath overnight.
50 ml 10 ml 1 ml
1 1 1
QUALITY CONTROL Results: Escherichia coli MPN —— 5 /100 ml
Report: Unsatisfactory.
Sterility of media and strength of media should be properly Grades of the quality of drinking water supplied as
checked. determined by results of periodic Escherichia coli and coliform
counts is listed in the table 84-1.
Table 84-1 Grades of the Quality of Drinking Water Supplies

Determined by results of Periodic Escherichia coli and Coliform counts
Quality of supply Results from routine samples Tolerance

Coliform counts / 100 ml E. coli count / 100 ml
1 Excellent. 0 0 In all samples.

2 Satisfactory. 1–3 0 Provided that coliforms do not occur in
Consecutive samples or in more than
5% of samples.
3 Intermediate. 4–9 0
4 Unsatisfactory. 10 coliforms or 1 or more E. coli or any coliform In any sample.
present in consecutive samples, or presence of
any coliform organisms in more than 5% of routine
samples.
KEY FACTS
1 Drinking water is acceptable and fit for drinking when it is clear, colourless, and odourless and without disagreeable taste.
Microscopically it should be free from pathogenic organisms.
2 Coliforms are defined as the members of the family Enterobacteriaeceae which grow in the presence of bile salts and produce
acid and gas from lactose within 48 hours at 37°C.
3 They are indicator of faecal contamination.
4 Coliforms are not the reliable indicator of protozoal contamination.
5 Eijkman test is usually employed to find out whether the coliform bacilli detected in the presumptive test are E. coli or not.
VIVA
1 What are the common water-borne infections transmitted by contaminated water?

Ans. Typhoid fever, cholera, poliomyelitis, viral hepatitis, amoebiasis, giardiasis, cysticercosis, etc.
2 Mention the routine laboratory tests done for the analysis of water.
3 Which are the organisms which indicate fecal contamination of water?
FURTHER READINGS
1 Ananthanarayanan. R, Paniker. C.K, Ananthanarayanan and Paniker’s Textbook Of Microbiology; 7th Edition, Orient Longman, pp 603 –
609, 2005.
2 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. pp. 921, 1996.
252
LESSON
Microbiology of Milk
85
LEARNING OBJECTIVES number of viable bacteria in the milk. For un refrigerated milk
there is a consistent relationship between the bacterial count
After completing this practical you will be able to: and the dye reduction time.
1 Test the quality of the un refrigerated milk by methylene

blue test. REQUIREMENTS
I Equipments
INTRODUCTION Water bath.
Human infections may be caused by the ingestion of animal II Reagents and glass wares
milk which contains microorganisms derived either from the
animal (faeces), from the environment, or from milk handlers Sterile test tubes, sterile pipettes, sterile rubber stopper,
such as dairy workers. Certain precautions are taken to prevent methylene blue tablets and distilled water.
contamination. A wide number of bacteria are found in
contaminated milk (Table 85-1). III Specimen
The infections that can be transmitted from infected animals Milk to be tested.
to humans through contaminated milk are many. They include
tuberculosis, brucellosis, streptococcal and staphylococcal
infections, salmonellosis and Q fever. PROCEDURE
The routine bacteriological examination of milk to detect
bacterial contaminations can be done by the following methods: The time of reduction is affected by the temperature at which
the milk is held before testing. The test should not be done if
1 Viable count. the atmospheric shade temperature exceeds 21°C.
2 Test for coliform bacilli.
3 Methylene blue reduction test. 1 Prepare a 1 in 3,00,000 solution of methylene blue by
4 Phosphatase test. dissolving a standard methylene blue tablet in 200ml cold
5 Turbidity test, and sterile glass-distilled water and making upto 800ml with more
6 Examination for specific pathogens. of such water.
Note: This solution can be stored in dark in a refrigerator
Of these tests, methylene blue test is a rapid, simple, inexpensive and can be used for 2months.
and most widely used test for testing the milk. This method will 2 Mix thoroughly the milk sample.
be described in this chapter. 3 Aseptically transfer 10ml of sample of milk with a pipette to
a sterile test tube.
4 Add 1ml of methylene blue solution by a 1ml sterile pipette.
PRINCIPLE 5 Put a sterile rubber stopper to the test tube; invert it once or
twice to mix the contents.
Time taken for bacterial dehydrogenases to reduce methylene 6 Place these test tubes at 37°C in a thermostatically controlled
blue dye and decolourise it, is taken as an indicator of the water bath for 30 min.
Note: The water level must be above the top of the milk and
the bath covered with a lid to exclude the light.
If the dye is wholly decolourised or decolourised within 5mm

of the milk surface, the milk fails the test. It is considered as
QUALITY CONTROL
contaminated milk. The time for complete decolourisation need
to be recorded. Because, untreated milk is often considered
With each test the following milk specimens are incubated as
satisfactory if it fails to decolourize the dye in 30 minutes.
controls:
A. Add 1ml methylene blue to 10ml milk that has been held at
Table 85-1 List of bacteria that can be found in contaminated milk
100°C for 3 min, to inactivate its reducing system.
1 Streptococcus lactis
B. Add 10 ml milk to 1 ml of tap water. 2 Streptococcus faecalis
3 Achromobacter
4 Clostridium perfringens
OBSERVATIONS 5 Clostridium butyricum
6 Bacillus subtilis
After incubation, compare the test mixture with control A to 7 Bacillus cereus
see whether there is any decolourization in the former and with 8 Proteus vulgaris
control B to see whether decolourisation is complete.
10 Gaffkya tetragena
KEY FACTS
1 Tuberculosis, brucellosis, streptococcal and staphylococcal infections, salmonellosis and Q fever are some of the infections
transmitted by contaminated milk.
2 Methylene blue test is rapid, simple, inexpensive and most widely used test for testing the microbial contamination of the
milk.
3 The time of reduction is affected by the temperature at which the milk is held before testing. The methylene blue reduction
test should not be done if the atmospheric shade temperature exceeds 21°C.
VIVA
1 List common infections transmitted by contaminated milk.

2 What is the principle of methylene blue test?
3 List the routine tests used for the bacteriological examination of milk?
FURTHER READING
609, 2005.
254
LESSON
Microbiology of Air
86
These include Petri dishes containing nutrient agar media for
After completing this practical you will be able to identify: the growth of organisms.
1 Bacteria present in the air.
2 Organisms present in the air by settle plate method.
PROCEDURE
INTRODUCTION 1 Prepare nutrient agar media, pour it into plates and dry off
any surface moisture.
Demonstration and estimation of the number of bacteria carrying 2 Remove cover of the Petri dish in its chosen position for the
particles in air may be required in certain situations. For example, measured period of time, and then replace its lid.
in the premises where safe working depends on the bacterial 3 Incubate the plates aerobically for 24 hours at 37°C.
content in air being kept at a very low level and premises where 4 Count the colonies, preferably with the use of a plate microscope.
certain foods or pharmaceutical materials are prepared, the
monitoring of density of microbial pathogens in the air is a priority.
The number of bacteria in the air at any given time is dependent QUALITY CONTROL
on the number of persons present, the amount of their body
movements and the amount of disturbance of their clothing, etc. The Petri dish agar plates should remain open for a specified
The list of bacteria commonly found in the air are summarized and adequate time.
in the table 86-1. The infections that can be transmitted through
air include wound infections, tuberculosis, anthrax,
streptococcal and staphylococcal infections. OBSERVATIONS
Settle plate method and slit sampler method are the two
methods used for routine bacteriological examination of the Observe the colonies grown on the medium after incubation.
air. Settle plate method will be described in this chapter.

PRINCIPLE
Growth rate on the medium in a given time indicates the bacterial
In the settle plate method, petri dishes containing an agar load in a given area.
medium of known surface area are left open for a measured
period of time. Large bacteria carrying dust particles settle on
to the medium. The plates are incubated and a count of the Table86-1 List of bacteria commonly found in air
colonies formed shows the number of settled particles that
contained bacteria capable of growth on the medium.
2 Streptococcus pyogenes
3 Mycobacterium tuberculosis
REQUIREMENTS 4 Pseudomonas aeruginosa
5 Bacillus anthracis
I Equipments 6 Bacillus subtilis
Incubator and plate microscope. 7 Proteus vulgaris
KEY FACTS
1 The number of bacteria in the air at any given time is dependent on the number of persons present, the amount of their body
movements and the amount of disturbance of their clothing, etc.
2 Settle plate method and slit sampler method are the two methods used for routine bacteriological examination of the air.
VIVA
1 List the infections transmitted by air.

2 What are the methods used for the detection of microorganisms in air?
FURTHER READINGS
609, 2005.
256
UNIT
XIII
Animal Experiments
Lesson 87 Intravenous Inoculation into Mice Tail Vein

Lesson 88 Collection of Blood from the Marginal Ear
Vein of Rabbit
Lesson 89 Animals and their uses in the Laboratory
258
LESSON
Intravenous Inoculation
87 into Mice Tail Vein
After completing this practical you will be able to: Procedure for loading the syringe for injection
1 Inoculate the appropriate culture suspension into the tail 1 Open the culture tube near the flame.
vein of mouse. 2 Fill the syringe with the culture to be injected.
Note: The air is to be expelled by pushing the needle into the
cotton inserted into a sterile tube and the volume to be
INTRODUCTION adjusted
3 The needle should be recapped carefully and placed ready
Mouse is one of the most commonly used lab animal for different for use.
animal experiments. The suckling mouse is used for isolation
and cultivation of viruses like Coxsackie A and B viruses and Animal preparation for injection
arboviruses, and also for testing enterotoxins. The adult mouse
is used for isolation and cultivation of organisms such as 1 Remove the selected animal from the cage.
Mycobacterium leprae, Toxoplasma gondii, Cryptosporidium 2 Keep the animal on a rough surface such as over a mouse
parvum, etc. (Box 87-1). cage or a wire.
3 Lift the mouse by holding the tail halfway.
4 Insert the tail through the mesh of a wire basket and hold the
PRINCIPLE animal in such a way that the animal is inside the wire mesh
and tail outside.
Mouse is a very suitable laboratory animal for different types 5 Wipe clean the tail with spirit or antiseptic soaked cotton.
of pathogens. After inoculation of the animal with appropriate 6 Apply little xylol over the area of the vein, and allow it to act
specimens, the changes can then be observed in the inoculated for a minute so that the vein will be prominent.
mouse and result interpreted.
Injection of material
REQUIREMENTS 1 Hold the syringe and needle in such a way that animal should
be horizontal to the tail and vein.
I Equipments 2 Then insert the needle into the vein midway between base
Mice cage. and tip of tail.
Note: It should be ensured that the needle is inside the vein
II Reagents and lab wares by withdrawing some blood.
Discarding jar, Bunsen flame, 1 ml syringe, 26G needle, culture 3 Inject carefully the material (approx. 0.1 ml and 0.2 ml) into
tube, cotton, colour dye and other standard laboratory glassware. the tail vein.
Anaesthetic agent, and antiseptic. 4 Remove the needle, then place plain sterile cotton over the
prick and hold it firmly until bleeding stops.
III Specimen 5 Keep back the animal in the cage after labeling with colored
Laboratory bred mouse and culture material to be tested. dye.
6 Clean the table after performing the experiment and then

dispose off the needle into a discarding jar.
OBSERVATIONS
The inoculated mouse should be observed and handled with

care every day and any change in its health condition should
QUALITY CONTROL
be monitored.
1 The mouse has to be marked well before use for appropriate
selection of animal. RESULTS AND INTERPRETATION
2 The mouse, before inoculation should be tested for any
infection or other physiological changes. Depending on the type and size of inoculation, the animal will
show changes and typical characteristic health manifestations.
These are to be noted and interpreted.
BOX 87-1 USES OF MICE IN LABORATORY
Suckling mice
Less than 48 hours old weighing 0.5 to 1.0 gm.
1 For testing enterotoxins of enteropathogenic bacteria.

2 For isolation and cultivation of coxsackie A and B virus, and arbovirus.
Adult mice
1 For isolation and cultivation of
(a) Mycobacterium leprae – foot pad.

(b) Toxoplasma gondii – intraperitoneal.
(c) Cryptosporidium species - intestinal.
(d) Plasmodium berghei - tail.
2 For isolation of the causative agents namely
(a) Borrelia recurrentis.

(b) Francisella tularensis.
(c) Rickettsia species.
(d) Trypanosoma brucei.
(e) Chlamydia psittaci.
(f) Rabies virus.
(g) Histoplasma capsulatum.
3 For pathogenicity and virulence testing of
(a) Streptococcus pneumoniae.

(b) Listeria monocytogenes.
(c) Bacillus anthracis.
(d) Bordetella pertussis.
(e) Nocardia asteroides.
(f) Leishmania spices.
(g) Herpes simplex virus.
(h) Cryptococcus neoformans.
260 Intravenous Inoculation into Mice Tail Vein
KEY FACTS
1 The mouse to be inoculated should be first checked thoroughly for proper health conditions.
2 The area for inoculation should be disinfected before inoculation.
VIVA
1 What are the common uses of mice as laboratory animal?

2 What care should be taken during handling the mouse?
Ans. The mouse should be handled only with gloved hands. It should be handled gently and the handler should thoroughly
disinfect his hands after the inoculation.
3 What post inoculation care should be given for mice?
Ans. After inoculation, the mouse should be kept in an individual cage with proper labeling. The animal should be fed and
watered well. Its health should be monitored regularly.
FURTHER READINGS
LESSON
Collection of Blood
88 from the Marginal Ear
Vein of Rabbit
After completing this practical you will be able to: 1 Restrain the rabbit by gently wrapping it in a blanket.
2 Shave the hair from the rear margin of an ear. Smear the skin
1 Collect the blood from the ear marginal vein of rabbit. over the marginal ear vein with petroleum jelly to delay clotting.
3 Grasp the ear near its base so that the venous return is
impeded and the vein raised.
INTRODUCTION 4 With a scalpel blade make a diagonal cut across the vein.
Note: The cut should penetrate the skin and vein but not be
Rabbit is one of the most commonly used lab animal for different so deep as to sever the vein. The ear should not be released
experiments. Infant rabbits are used for the propagation of at this time.
Vibrio cholerae, and for testing of enterotoxins of 5 Blood will then start to flow. When the flow begins to recede,
enteropathogenic pathogens. Adult rabbits are used for the wiping the cut with cotton will restart it.
isolation of Listeria monocytogenes, Streptococcus 6 Collect the blood in a clean sterile test tube.
pneumoniae, etc. 7 After collection release pressure on the vein, cover the cut
Uses of rabbits are summarized in the box 88-1. with a small piece of cotton and press firmly.
8 Clean the ear with a sterile swab and return the rabbit to its
cage.
PRINCIPLE
Rabbit is widely employed for raising and production of QUALITY CONTROL

antibodies against specific antigens. Antigen is injected into
rabbit and antibodies are produced. These antibodies can be 1 The rabbit has to be marked well before use for ease in
obtained by drawing the blood from marginal ear vein of the identification.
rabbit. Blood samples can also be collected from central artery 2 The rabbit, before inoculation should be tested for any
of the ear or directly from the heart by cardiac puncture. infection or other physiological changes.
REQUIREMENTS OBSERVATIONS
I Laboratory wares The inoculated rabbit should be observed and handled with
Jar, Bunsen flame, test tube, cotton, colour dye, and sterile care every day and any change in its health condition should
scalpel. be monitored.
II Reagents
Antiseptic, and petroleum jelly. RESULTS AND INTERPRETATION
III Specimen Repeated samples of blood upto 50 ml can be collected at 2-4

Laboratory bred adult healthy rabbit weighing 1.5- 3 Kg. week intervals.
262 Collection of Blood from the Marginal Ear Vein of Rabbit
BOX 88-1 USE OF RABBITS IN LABORATORY
Infant rabbit
1. Animal model for Vibrio cholerae.
2. In enteropathogenic organisms, testing of enterotoxins can be done.
Adult rabbit
1. For testing virulence and pathogenicity of
(a) Listeria monocytogenes.
(b) Streptococcus pneumoniae.
(c) Nocardia spp.
(d) Mycobacterium tuberculosis.
(e) Herpes simplex virus.
(f) Candida albicans.
2. Other uses
(a) Differentiating M. tuberculosis and M. bovis.
(b) Sereny’s test.
(c) Rabbit ileal loop test (enterotoxin testing).
(d) Maintenance of Treponema pallidum.
(e) Increasing virulence of rabies virus and vaccinia virus.
(f) For collecting blood for antistreptolysin O test.
(g) To raise antibodies to antigen.
(h) For serum as source of complement for cytotoxic assays.
KEY FACTS
1 After cutting the ear and vein, the ear should not be released immediately.
2 Sudden movements or noise should not be made during the procedure.
3 Blood samples can also be collected from central artery of the ear or directly from the heart by cardiac puncture.
VIVA
1 What are the other routes for the collection of blood from rabbit?
FURTHER READINGS
LESSON
Animals and their uses
89 in the Laboratory
LEARNING OBJECTIVES After inoculation following care of the animals should be taken:
After completing this practical you will be able to: 1 Physical properties of the animal should be monitored
regularly.
1 Know the commonly used laboratory animals (mice, rabbit, 2 Health of the animal should be checked regularly.
guinea pig and other animals) for animal experiments. 3 Make sure that the animal is taking normal amount of food
2 Know the uses of these animals in the laboratory. and water as before to inoculation.
4 Take care of animal to prevent it from other infections.
5 Test results should be monitored and recorded regularly.
INTRODUCTION
The animal if dies due to the effect of the specimen inoculated,
Laboratory animals have been used for ages, in laboratories then the animal should be disinfected and incinerated with
for different purposes. These animals include mice, rabbits, proper care.
guinea pigs, hamsters, monkeys, etc. They are used for a wide
range of applications and form an integral part of research (Box
89-1). Animals such as these should be properly taken care of, REQUIREMENTS
and should be in accordance to the governing rule for animal
usage in laboratory. I Equipments
Different animals and their usage are listed in the table 89-1. Wire cage.
II Reagents and animals

Syringe with needle, wire mesh, hood with Bunsen flame,
PRINCIPLE
personnel protective equipment, masks, spirit/antiseptic and
soaked cotton.
The principle behind using of laboratory animals are that when
Animals such as mouse (Fig. 89-1), rabbit (Fig. 89-2) and
an experimental material is inoculated into a laboratory animal,
guinea pig (Fig. 89-3).
it shows pathological changes which can be later studied.
The routes of inoculation in various laboratory animals III Specimen
include subcutaneous, intradermal, intraperitoneal, intrathecal, Specimen to be inoculated may be toxins including endotoxin,
intravenous, intramuscular, intracerebral, etc. These different exotoxin and enterotoxin; bacterial, fungal or viral culture;
routes are selected depending on the nature of the inoculum pyrogen, etc.
and it is important because different organisms have different
tissue tropism.
The information that are to be recorded before and after PROCEDURE
inoculation of a lab animal include the number of the animal,
age of the animal, physiological condition of the animal, nature 1 Different routes of inoculation are followed for different types
of the specimen, date of inoculation of the specimen and route of inoculum and lab animals.
of inoculation. It should also include the changes noted in 2 Commonly used routes of inoculation include intravenous,
animals on a daily basis and the date of death of the animal if intraperitoneal, intra-abdominal, intracranial, intra-nasal,
the inoculated specimen proves fatal. intramuscular, intradermal, subcutaneous, etc.
264 Animals and their uses in the Laboratory
3 Whichever are the lab animal used and whichever route of

inoculation be the case the following general procedure can
be adopted.
a The animal be it mouse, rabbit or guinea pig, they are
properly labeled first before use.
b In case of mice, the mouse is held tightly in one hand with
the tail running between two fingers.
c In case of rabbit, the animal is kept inside a rabbit holding
box.
4 If box is unavailable, keep the rabbit on a towel and wrap it
completely leaving the head outside in such a way that the
animal does not move, and is comfortable. FIGURE 89-1 Mouse.
5 Guinea pigs, hamsters can also be held using a cloth wrapped
around them.
6 The syringe is loaded with the appropriate material and gently
introduced into the marked area of inoculation.
7 The area of prick can be later wiped with dry cotton and after
monitoring the animal for any adverse reaction, it can be left
in its cage.
QUALITY CONTROL
1 The laboratory animal’s health and physical condition
should be checked before using them.
2 The animal should be labeled properly before and after FIGURE 89-2 Rabbit.
inoculation.
OBSERVATIONS
The animal is marked or tagged with all necessary information

like the number, date of inoculation, route of administration,
type of inoculation etc. and kept in a place away from other animals.
Depending upon the changes that the animal shows, the results FIGURE 89-3 Guinea pig.
of the test shall be interpreted.
BOX 89-1 USES OF LABORATORY ANIMALS

To test endotoxin and exotoxin production by various bacteria.
To study immune response to an antigen.
To produce monoclonal and polyclonal antibodies against select antigens.
To study hypersensitivity by producing reactions due to introduction of allergens.
To produce complements.
To use the blood/serum of animals.
To test and producte vaccines.
To test for pyrogens.
To study changes in the condition of animals by inoculating test substances.
Table 89-1 Laboratory animals and their usage
Monkeys
Monkeys are primates and are extensively used in laboratories. Since they are genetically evolved animals, they are considered very suitable
for lab testing.
Uses
1 To raise antiserum.
2 For RBC for serological tests.
3 For testing virulence of Entamoeba histolytica.
4 Testing pathogenicity of polio virus.
5 Testing hemagglutinating property of measles virus.
6 Laboratory cultivation of hepatitis A virus and Plasmodium falciparum.
7 Animal model for influenza virus.
Hamsters
Uses
Infant hamsters
1 To test tumorigenicity of adeno viruses.
Adult hamster
1 For pathogenicity testing of
(a) Leishmania donovani.
(b) Entamoeba histolytica.
(c) Bacillus anthracis.
2 For isolation of
(a) Leptospira species.
(b) Leishmania species.
(c) Toxoplasma gondii.
3 Other uses
(a) Maintenance of virulence of Entamoeba histolytica.
(b) Drug efficiency testing against Leishmania donovani.
Guinea pig
Uses
1 For the isolation of
(a) Mycobacterium tuberculosis.
(b) Mycobacterium bovis.
(c) Yersinia pestis.
(d) Leptospira icterohaemorrhagiae.
(e) Brucella abortus.
(f) Chlamydia species.
(g) Toxoplasma gondii.
2 For toxigenicity testing of

(a) Corynebacterium diphtheriae.
(b) Clostridium perfringens.
(c) Clostridium tetani.
(d) Clostridium botulinum.
3 Pathogenicity testing of
(a) Listeria monocytogenes.
(b) Bacillus anthracis.
(c) Clostridium perfringens.
(d) Entamoeba histolytica.
(e) Cryptococcus neoformans.
4 Other uses
(a) For the preparation of complement.
(b) For the evaluation of bronchodilator compounds.
(c) Pathogenicity testing of M. tuberculosis (spleen enlargement, lesions in spleen and liver).
(d) Pyrogen testing.
(e) Sereny’s test.
266 Animals and their uses in the Laboratory
KEY FACTS
1 Animals should be properly taken care of, and should be in accordance to the governing rule of CPSEA for animal usage in laboratory.
2 The lab animal’s health and physiological condition should be checked before and after inoculation.
VIVA
1 What are the information to be recorded about an animal and inoculation before and after inoculation of test material?
2 What are the different routes of inoculation in lab animals and why are the different routes significant?
3 How should an inoculated animal be looked after and what precautions are to be taken?
FURTHER READINGS
UNIT
XIV
Medical Entomology
Lesson 90 Identification of Common Insects

268
LESSON
Identification of
90 Common Insects
LEARNING OBJECTIVES 2. Head has a long needle-like structure named proboscis, a

pair of palpi, each situated on either side of proboscis, a pair
After completing this practical you will be able to: of antennae or feelers.
3. Antennae are bushy in male.
1 Know important identifying features of common insects. 4. Thorax is large and rounded, bearing a pair of wings dorsally
and three pairs of legs ventrally.
5. Wings are characterized by a fringe of scales on the posterior
INTRODUCTION border.
6. Abdomen has 10 segments.
Insects are responsible for transmitting a wide variety of
infections to humans. Hence it is essential to know different Identifying features of Anopheles
insects causing infections in man and methods to identify
them. 1. All the general features of mosquitoes as mentioned above.
2. Other characteristic features are:
a). Dark (blackish) or white scales on the wing veins.
PRINCIPLE b). Palpi is as along as proboscis.
c). When anopheles is at rest, head, thorax, abdomen are at
The insects can be identified by observing the killed insect an angle of 45° to the resting surface.
under a dissecting microscope and observing their d). The middle lobe of tri-lobed salivary gland is short.
morphological features for specific identification.
Diseases transmitted
Malaria
REQUIREMENTS
Identifying features of Aedes
I Equipments and lab wares
1. All the general features of mosquitoes as mentioned above.
Standard dissecting microscope, glass slides, cover slips, sterile 2. Other characteristic features are:
cotton swab, glass marking pencil, etc. a). Characteristic of unspotted wings and white stripes on
a black body are present.
II Specimen b). Tip of abdomen is pointed.
Insect to be identified. c). Legs are striped or banded in nature. So they are called
as tiger mosquitoes.
d). Palpi are short in female.
MOSQUITOES e). When at rest, body shows a hunch back.
These are day bitters.
General features
1. Head is semi-globular in shape with a pair of large compound 1. Dengue.
eyes. 2. Dengue haemorrhagic fever.
3 Chikungunya fever. Diseases transmitted

4 Rift valley fever.
5 Yellow fever. 1 Typhoid and paratyphoid fever.
2 Diarrhea.
Identifying features of Culex 3 Dysentery.
4 Cholera.
1 Similar to that of Aedes aegypti, except the tip of abdomen is 5 Gastroenteritis.
blunt. 6 Amoebiasis.
2 These mosquitoes bite about midnight. 7 Helminthic infections.
8 Poliomyelitis.
Diseases transmitted 9 Conjunctivitis.
1 Bancroftian filariasis. 10 Trachoma.
2 Japanese encephalitis. 11 Anthrax.
3 West Nile fever. 12 Yaws.
SAND FLY ITCH MITE
General features The itch mite or Sarcoptes scabies var hominis belongs to the
class Arachnida.
1 2-4mm in length, smaller than mosquito.
2 Greyish brown having dark conspicuous eye and hairy body. Identifying features
3 Head contains a pair of long, slender, hairy antennae and a
proboscis with 16 segments. 1 It is 0.5mm in size, visible to naked eye.
4 Thorax bears a pair of wings and three pairs of legs. 2 Body is tortoise shaped, rounded above and flattened below.
5 Wings are upright and lanceolate shaped. The second 3 Body is hairy.
longitudinal vein on the wing branches twice, first branching 4 Has 2 pairs of legs in front and 2 pairs behind.
takes place in the middle of the wing. 5 Front legs have suckers at the end, and the hind legs end in
6 Legs are longer and hairy. long bristles.
7 Only females suck blood at night. 6 Males are smaller than the females.
8 Males have three pairs of clappers on the last abdominal 7 In females, the first 2 pairs of legs possess suckers. In males,
segment. the fourth pair of legs also have suckers.
9 They do not fly, they only hop about up to 3 feet. 8 Dorsal surface has backwardly pointed spines.
Diseases transmitted by sand fly are summarized in the
box 90-1. Diseases transmitted
1 Scabies (seven year itch), Norwegian itch of man.
2 Forage mite and house dust mite cause allergic respiratory
HOUSE FLIES distress.
Identifying features
TROMBICULID MITE
1 It is mouse gray in colour.
2 Body is divided in to head, thorax, and abdomen. It is also known as scrub typhus mite.
3 Head bears a pair of antennae, a pair of large compound
eyes and a refractive proboscis, for sucking liquid food. Eyes Identifying features
of the male are close together and those of the female are set 1 Adult mites are 1-2mm in length and are red in colour.
apart widely. 2 Covered dorsally and ventrally with numerous feathered hairs.
4 Thorax is marked with 2 to 4 dark longitudinal stripes, which 3 Has 4 pairs of legs.
is characteristic of the genus Musca. It bears a pair of wing 4 Body is constricted between the 3rd and 4th pairs of legs,
and three pairs of legs. giving a figure of eight appearance.
5 Each leg is provided with a pair of pads which enable the fly
to walk on highly polished surfaces. Diseases transmitted
6 Legs and body are covered with numerous short and stiff Rickettesia orientails (Orientia tsutsugamushi).
hairs called Tenent hairs which secrete a sticky substance. Trombicula dilensis is the vector in India.
7 Abdomen is segmented and shows dark and light markings. Trombicula akamush is the vector in Japan.
270 Identification of Common Insects
3 Laterally compressed, which is an adaptation to move freely

HARD TICK in hairs and feathers.
4 Contains a hard chitinous exoskeleton and backwardly
Identifying features directed strong bristles.
1 Sac-like body varying in size from 2mm to 10mm. 5 Demarcation into head, thorax and abdomen is not clear.
2 Dorsoventrally flattened, and oval in shape. 6 Head has simple eyes and short paired antennules.
3 Cannot distinctly be separated into head, thorax, and abdomen. 7 Mouth parts are conspicuously blood sucking and pointed
4 Contains 4 pairs of legs but no antennae. downwards.
5 Dorsal surface is covered by a chitinous shield, called scutum. 8 They do not have wings, but legs are highly developed,
6 Males have scutum on the entire dorsal surface, whereas which helps them to leap up to 10-15cms.
in female it is found in small portion. 9 Abdomen is bulky and has 10 segments.
7 It has anteriorly placed capitulum called head, which 10 Males have coiled genitalia.
projects forward beyond the scutum. 11 Females have a spermatheca at the posterior end and Genal
8 Spiracular openings are situated behind the basal segments combs are useful for species identification.
of the fourth pair of legs.
9 Spurs are present in coxal segments which assist in
LOUSE
classification of ticks.
10 Pulvilli are present.
It is also called as Pediculus humanus.
11 Only one nymphal stage is present.
12 Takes blood meal at both day and night time.
13 Remain ectoparasite on host for long time. Identifying features
14 Cannot resist starvation for a long time.
15 Life Span is 3 years. Female dies after laying eggs. 1 Measures 2-4mm in length.
Diseases transmitted by hard ticks are summarized in the box 90-2. 2 Grayish or reddish after blood meal.
3 Wingless ectoparasite and flattened dorsoventrally.
4 Head is narrower than thorax and has a pair of eyes.
SOFT TICK 5 Antenna in the larval stages is short having 3 segments
while in adult it has 5 segments.
Identifying features 6 Thorax has 6 legs, each having 5 segments.
7 Abdomen has 9 segments, it is pointed in males, the last
All features of hard ticks except segment has penis. In females, abdomen is bilobed.
8 The louse are of 3 different types:
1 Scutum is absent a Head louse – Pediculus capitus.
2 Capitulum lies ventrally, mouth parts are not visible from b Body louse – Pediculus corporis.
above. c Pubic louse – Phthirus pubis.
3 Spiracular openings lies behind the 3rd pair of coxal segments.
4 Coxal segments lack spurs. Diseases transmitted
5 Pulvilli are absent. 1 Louse borne epidemic typhus caused by Rickettsia
6 More than one nymphal stage is present. prowazaki.
7 Take blood meals only at night. 2 Epidemic relapsing fever caused by Borrelia recurrentis.
8 Remain ectoparasite only during their short feeding time. 3 Trench fever or 5 day fever caused by Bartonella quintana.
9 They can withstand starvation for one year.
10 Life span is 16 – 21 years.
CYCLOPS
Ornithodorus species is the vector for tick borne relapsing Identifying features
fever caused by Borrelia duttoni.
In India relapsing fever is transmitted by Ornithodorus 1 Pear-shaped.
tholozani, Ornithodorus lahorensis and Argas persicus. 2 Semitransparent.
3 Varies from 0.5 to 1mm in length.
4 Body shows anterior cephalothorax and posterior abdomen.
RAT FLEA 5 Cephalothorax has
a Pigmented eye.
Identifying features b Two pairs of antennae.
c Three pairs of jaws (1 pair mandibles and 1 pair maxillae).
1 Light to dark brown in colour. d Five pairs of legs which are swimming appendages. 5th
2 Oval in shape, 1-8mm in size. pair is vestigeal.
6 Abdomen has 5 segments. The last segment has two Diseases transmitted
feathered filaments. In females the first abdominal segment 1 Dracunculiasis. Mesocyclops act as intermediate host for
has got 2 ovisacs on both sides to carry the eggs. Dracunculosis medinensis.
7 Swims with characteristic jerky movements in water. Hence 2 Diphyllobothriasis. First intermediate host for
called as water fleas. They can be seen as small moving Diphyllobothrium latum.
specks in water. 3 They also act as vectors for Gnathostoma spinigerum and
Gnathostoma hispidium.
BOX 90-1 DISEASES TRANSMITTED BY SAND FLY
Diseases Species
1 Kala azar. Phlebotomus argentipes.

2 Oriental sore . Phlebotomus sergenti.
3 Sand fly fever. Phlebotomus papatasi and
Sergentomyia spp.
4 Oroya fever . Lutzomyia verrucarum.
(Bartonellosis, Carrion’s disease) Lutzomyia columbiana.
Lutzomyia peruensis.
BOX 90-2 DISEASES TRANSMITTED BY HARD TICKS
Diseases Species
1 Tick paralysis. Dermacentor, Ixodes, Haemophysalis.

2 Colorado tick fever. Dermacentor andersoni.
3 Kyasanur forest disease. Haemophysalis spinigera.
4 Crimean – congo hemorrhagic fever. Hyalomma marginatum, Dermacentor marginatus.
5 Russian spring summer encephalitis. Ixodes persculatuis, Haemophysalis concinna.
6 Tick borne encephalitis. Dermacentor marginatum.
7 Osmk hemorraghic fever. Dermacentor, Ixodes.
8 Lyme disease. Ixodes scapularis.
9 Rocky mountain Spotted fever. D. andersoni, D. variabilis.
10 Boutonneuse fever. Rhipicephalus sanguineus.
VIVA
1 List the diseases transmitted by mosquitoes.

2 List the identifying features of cyclops.
3 What are the identifying features of Anopheles mosquitoes?
4 What are the diseases transmitted by house fly?
FURTHER READINGS
272
UNIT
XV
Common Viva Spots
Lesson 91 Identification of Common Viva Spots

274
LESSON
Identification of
91 Common Viva Spots
LEARNING OBJECTIVES Preparation
After completing this practical you will be able to identify the Prepared by adding agar in nutrient broth and sterilized by
following common viva spots given in Microbiology autoclaving.
examination:
Modifications
1 Culture media.
2 Culture media with growth. 1 Semi-solid agar if concentration of agar is 0.2–0.5%.
3 Biochemical reactions. 2 Concentrated agar if agar concentration is 2–6%.
4 Specimens of parasites.
5 Glass wares. Uses
6 Instruments, and
7 Microscopic slides. 1 For growing of non fastidious bacteria.
2 Determination of antibiotic sensitivity.
3 Preparation of blood agar.
CULTURE MEDIA 4 Concentrated agar (3% and more) prevents swarming of
bacteria such as Proteus vulgaris, Clostridium tetani, etc.
NUTRIENT AGAR
BLOOD AGAR
Also referred as ‘Agar’ (Fig. 91-1).
The blood agar is an enriched medium as well as an indicator medium.
Composition The colour of the medium is red like that of blood (Fig. 91-2).
Nutrient broth : Peptone water. Composition

Meat extract : 1%.
Agar : 2–3%. Nutrient agar.
Sheep blood : 5–10%.
FIGURE 91-1 Nutrient agar. FIGURE 91-2 Blood agar.

Preparation
Prepared by adding sterile blood to sterile nutrient agar that

has been melted and cooled to 55°C. The blood agar is
sterilized by autoclaving. Concentration of blood vary from
5% to 50% for special purposes. 10% is most usual concentration
used. Either human or animal (sheep, rabbit, horse, etc) blood
may be used.
Uses
FIGURE 91-4 Mac Conkey agar.
1 For growth of most of the pathogenic bacteria.
2 Used in preparation of the potassium tellurite blood agar. Composition
1 Peptone : 20 g.
CHOCOLATE AGAR 2 Sodium taurocholate, commercial : 5 g.
3 Water : 1 litre.
The medium is so called because it is chocolate in colour. 4 Agar : 20 g.
Chocolate agar is an enriched medium (Fig. 91-3). 5 Neutral red solution, 2 percent
in 50 percent ethanol : 3.5 ml.
Composition 6 Lactose 10% aqueous solution.
Nutrient agar. Preparation

Heated 10% sterile blood (horse, sheep).
Dissolve peptone and taurocholate (bile salt) in water by heating.
Add the agar and dissolve it in the steamer or autoclave. If
necessary, clear by filtration. Adjust pH to 7.5. Add lactose and
neutral red which should be well shaken before use and mix.
Heat in autoclave with free steam (100°C) for 1 hr, then at 115°C
for 15 min. Pour plates. The medium is sterilized by autoclaving.
The medium should be a distinct reddish brown colour. If it
is acid, it assumes rose-pink colour.
Uses
1 For growing enteric bacteria.

FIGURE 91-3 Chocolate agar. 2 To differentiate lactose fermenter (LF) from non-lactose
fermenter (NLF) colonies (Salmonella, Shigella, etc.).
Preparation
LOEFFLER’S SERUM SLOPE
It is prepared by heating 10% sterile blood in sterile nutrient
agar. The agar is melted and cooled it in water bath at 75°C, Loeffler’s serum slope is an enriched medium. This is used
blood is added and the medium continues to remain at 75°C till for culture and isolation of Corynebacterium diphtheriae
the blood becomes chocolate brown in colour. (Fig. 91-5).
Uses
For growing fastidious bacteria such as Neisseria,

Pneumococcus, Haemophilus, etc.
MACCONKEY AGAR
MacConkey agar is a differential or indicator medium. This is a

useful medium for cultivation of enteric bacteria (Fig. 91-4). FIGURE 91-5 Loeffler’s serum slope.
276 Identification of Common Viva Spots
Composition Preparation
Malachite green solution: Prepare 2% solution of malachite
1 Sterile ox, sheep, horse serum : 300 ml.
green in sterile water with sterile precautions by dissolving the
2 Nutrient broth : 100 ml.
dye in incubator for 1–2 hr.
3 Glucose : 1 g.
Preparation of medium
Preparation Mineral salt solution : 600 ml.
Malachite green solution : 20 ml.
Dissolve glucose in nutrient broth and autoclave at 115°C for
Beaten egg (20 to 22 hen’s
20 min. Add glucose broth to the serum with sterile precautions
eggs depending on size) : 1 litre.
and distribute in test tubes or in 2–5 ml amounts in sterile
conditions to fill up nearly one fourth of the bottles. This medium
Mix the complete medium, distribute it in 5 ml volumess in
is sterilized by inspissation.
sterile McCartney bottles and screw the caps tightly on. Lay
the bottles horizontally in the inspissator and heat at 75°–80°C
Uses for 1 hr. This is to solidify the medium.
1 Loeffler’s serum slope is used especially for cultivation of Uses

C. diphtheriae, producing luxuriant growth in 6–12 hr.
2 It is also used to show proteolytic properties, particularly of
1 For growing Mycobacterium tuberculosis (takes 3 to 6 weeks
Clostridium species.
to grow).
2 For antibiotic sensitively testing of M. tuberculosis.
LOWENSTEIN-JENSEN (LJ) MEDIUM

ROBERTSON COOKED MEAT (RCM)
Lowenstein-Jensen (LJ) medium is an enriched medium used
for growing Mycobacterium species. The green colour of the
BROTH
medium is due to the presence of malachite green (Fig. 91-6).
Robertson cooked meat (RCM) broth is a selective medium
used for culture of anaerobic bacteria (Fig. 91-7).
Composition
1 Mineral salt solution Potassium

Composition
dihydrogen phospphate anhydrous KH2 PO4 : 2.4 g.
1 Cooked meat (fresh bullock heart) : 500 g.
Magnesium sulphate, MgSO4 : 0.24 g.
2 Peptone : 2.5 g.
Magnesium citrate : 0.6 g.
3 Sodium chloride : 1.25 g.
2 Asparagine : 3.6 g.
4 Water : 500 ml.
3 Glycerol : 12 ml.
5 Liquid from cooked meat : 500 ml.
4 Water : 600 ml.
5 Egg.
6 Malachite green solution. Preparation
Dissolve mineral salts by heating. Autoclave at 121°C for
25 min to sterilize. Place the meat in each 1 oz bottle to a depth of one inch and
cover with about 10 ml broth. The medium is sterilised by
autoclaving at 121°C for 20 min. After sterilization the pH of
FIGURE 91-6 Lowenstein Jenson’s medium. FIGURE 91-7 Robertson’s cooked meat medium. (RCM ) broth.
broth over meat is 7.5. If test tubes are used the surface of the meat and it is essential that basal medium to which various
medium may be covered with a layer of sterile liquid paraffin carbohydrates added for fermentation tests should be free from
1cm deep but this is not essential. natural sugars (Fig. 91-9).
Uses Composition
1 For growing anaerobic bacteria. Peptone : 10 g.

2 For preservation of stock cultures of anaerobic bacteria. Sodium chloride (NaCl) : 5 g.
Water : 1 L.
SABOURAUD’S DEXTROSE AGAR (SDA)
Sabouraud’s dextrose agar (SDA) is used for culture of fungi.

Low pH and high sugar content of this medium make it
particularly selective for fungi as against bacterial contaminants
(Fig. 91-8).
Composition
Glucose : 40 g.
Peptone : 10 g.
Agar : 20 g. FIGURE 91-9 Peptone water.
Water : 1 L.
Preparation
Dissolve the ingredients in warm water, adjust pH to 7.4–7.5

and filter. Distribute as required and autoclave at 121°C for 15
min. This medium is sterilized by autoclaving.
Uses
1 For growing pathogenic bacteria.

2 For making hanging drop preparation of bacteria.
3 For preparation of sugar media.
FIGURE 91-8 Sabouraud’s dextrose agar (SDA).
4 For growing bacteria for antibiotic susceptibility testing.
5 To test the formation of indole.
Preparation
Dissolve the ingredients in the steamer or autoclave. Filter GLUCOSE BROTH

through cotton gauze and adjust to pH 5.4. Dissolve in stock
bottles or in tubes. Autoclave at 115°C for 15 min. Glucose added to nutrient media promotes luxuriant growth of
many organisms. It also acts as reducing agent and used for
Uses shake cultures of anaerobes (Fig. 91-10).
1 For growing fungi.

2 The agar medium is suitable for the primary isolation of fungi
from clinical material.
3 Fungi can take weeks to grow but yeasts grow in 24 hours to
48 hours on this medium.
PEPTONE WATER
Peptone water is used as the basis for sugar fermentation media

since broth may contain a small amount of sugar derived from FIGURE 91-10 Glucose broth.
Composition STAPHYLOCOCCUS AUREUS ON

1 Nutrient broth.
NUTRIENT AGAR
2 Peptone water.
3 Meat extract 1%. Salient features
4 Glucose (0.5%).
1 Seen as b-hemolytic colonies on blood agar with golden
Preparation yellow pigment.
2 The colonies are opaque convex with a shining surface and
Prepare 20% of glucose solution separately. Add a drop of may be pigmented white (var. albus) yellow, golden yellow
phosphoric acid to endure pH not more than 7.0 and autoclave or golden (var. aureus). Confluent growth appears like oil
at 115°C for 20 mins. This sterile glucose solution is then added paint (Fig. 91-12).
to the sterile basal medium. 3 Gram staining shows as Gram positive cocci in clusters.
4 S. aureus is catalase positive which differentiates it from
streptococci.
Uses
5 It is coagulase positive.
1 Used for growing fastidious organisms such as
Streptococcus pyogenes and Enterococci, and for their
antibiotic susceptibility testing.
CULTURE MEDIA WITH GROWTH
STREPTOCOCCUS PYOGENES ON
BLOOD AGAR
FIGURE 91-12 Staphylococcus aureus on nutrient agar.
Salient features
1 Blood agar colonies 0.5–1 mm in diameter, after 24 hrs PROTEUS SPP. ON BLOOD AGAR
incubation circular, discrete, semi-transparent, low convex
disks, showing a-hemolysis on fresh blood agar plates (Fig. Salient features
91-11).
2 Virulent strains isolated from lesions give a matt type of 1 Group of cells at the edge of developing micro colony migrate
colony whereas avirulent strains produce glossy colonies. to an uninoculated area of the medium and present as
3 A mucoid colony type is also encountered and corresponds swarming. Swarming is seen in blood agar plates and the
in virulence to matt type. growth may eventually appear either as a uniform growth /
4 Str. pyogenes is the commonest organism causing sore film over the whole plate. Continuous swarming or
throat. discontinuous swarming are a series of concentric circles of
5 Seen as b-hemolytic colonies in blood agar plates. growth around point of inoculation (Fig. 91-13).
6 Gram staining shows Gram positive cocci in chains. 2 Proteus spp. are Gram negative, coccobacilli, 1–3 µm long
7 It is catalase negative. This test differentiates it from and 0.6 µm wide. In young culture they may be filamentous
Staphylococcus. as longs as (80 µm).
3 They are motile by peritrichous flagella. They possess more than
one type of fimbriae. Variants are nonflagellate and non motile.
FIGURE 91-11 Streptococcus pyogenes on blood agar. FIGURE 91-13 Proteus species on blood agar.
4 The two common species are Proteus vulgaris and Proteus 4 They are indole positive, urease negative.
mirabilis. 5 They cause urinary tract infections and diarrhoea.
5 PPA and urease tests are positive.
KLEBSIELLA SPP ON
PSEUDOMONAS AERUGINOSA ON MACCONKEY AGAR
NUTRIENT AGAR
Salient features
Salient features
1 Klebsiella spp produces lactose fermenting, mucoid colonies
1 On nutrient agar growth of the organism shows green on MacConkey agar (Fig. 91-16).
diffusible pigment (Fig. 91-14). 2 They are Gram negative, non sporing, non motile bacilli, 1–2
2 Ps. aeruginosa is a strict aerobe, slender, Gram negative µm long and 0.5–0.8 µm wide with parallel or bulging sides
bacillus, 1.5–3.0 µm × 0.5 µm arranged singly, in pairs or and slightly pointed or rounded ends.
short chains. 3 Freshly isolated strains possess a well defined
3 It is non sporing, non capsulate, though mucoid strains may polysaccharide capsule.
sometime occur and usually motile by one or two polar flagella. 4 They are urease positive, and indole negative.
4 It is oxidase positive. 5 They cause urinary tract infections, pneumonia, etc.
5 It is one of the common causes of nosocomial infections.
FIGURE 91-16 Klebsiella species on MacConkey agar.
FIGURE 91-14 Pseudomonas aeruginosa on nutrient agar.
CORYNEBACTERIUM DIPHTHERIAE ON
ESCHERICHIA COLI ON POTASSIUM TELLURITE AGAR
MACCONKEY AGAR
Salient features
Salient features 1 Potassium tellurite agar is used as selective medium for
C. diphtheriae (Fig. 91-17).
1 Escherichia coli shows lactose fermenting, non-mucoid 2 C. diphtheriae produces black coloured colonies in
colonies on MacConkey agar (Fig. 91-15). potassium tellurite agar.
2 It is an aerobe and facultative anaerobe.
3 It is a Gram negative, non capsulated bacillus measuring
1–3 µm × 0.4–0.7 µm. Most of the strains are motile by peritrichate
flagella.
FIGURE 91-15 Escherichia coli on MacConkey agar. FIGURE 91-17 Corynebacterium diphtheriae on potassium tellurite agar.
3 S. aureus is another bacteria which produces black colonies

on this medium BIOCHEMICAL REACTIONS
4 Other media used for C. diphtheriae are Loeffler’s serum
slope, and blood agar containing fresh, lysed or heated blood.
CARBOHYDRATE FERMENTATION
TESTS
MYCOBACTERIUM TUBERCULOSIS ON
LÖWENSTEIN JENSEN (LJ) MEDIUM Salient features
Salient features 1 It is used to determine the ability of an organism to ferment

a particular carbohydrate to produce acid or acid and gas
1 M. tuberculosis takes 3–6 weeks to grow on the LJ medium (Fig. 91-20).
on incubation at 37°C (Fig. 91-18). 2 A large variety of sugars are used as follws:
2 Cultures should not be discarded as negative until they have
been observed for 12 weeks. Pentoses : Arabinose, xylose, rhamnose.
3 The colonies are identified by their rough, tough and buff Hexoses : Glucose, fructose, mannose, sorbose,
coloured eugonic growth. galactose.
Disaccharides : Sucrose, maltose, lactose, trehalose,
cellobiose.
Trisaccharides : Raffinose.
Polysaccharides : Starch, insulin, dextrin, glycogen.
Sugar alcohols : Glycerol, erythritol, adonitol, mannitol,
dulcitol, sorbitol, inositol.
Glycosides : Salicin, aesculin.
3 A suitable indicator that will change colour only as a result

of formation of acids during fermentation of sugar is used in
the test. A small inverted tube (Durham’s fermentation tube)
is placed in each culture tube to detect gas.
FIGURE 91-18 Mycobacterium tuberculosis on Lowenstein- Jensen’s Andrade’s indicator is the indicator used.
medium. Pink colour of the solution is due to acid production by
fermentation of carbohydrates. Air bubble is formed in the
Durham’s tube if gas is produced.
CANDIDA ALBICANS ON 4 The test is done by inoculating a drop or loopful of the
SABOURAUD’S DEXTROSE AGAR (SDA) culture. The inoculated tubes are incubated, aerobically at
37°C for required period, and colour change and gas
formation noticed. Fermentation reactions are observed
Salient features usually after a period of 24 hours of incubation.
5 These tests are used to identify some Gram negative bacilli
1 Candida albicans species grow well on SDA at 25–37°C such as E. coli, Klebsiella species, Salmonella species,
(Fig. 91-19). Proteus species etc.
2 Cream coloured, smooth pasty colonies appear in 1–2 days.
3 Lacto-phenol cotton blue preparation and Gram stained
smears showing budding yeast cells and pseudohyphae.
4 C. albicans can be differentiated from other species by germ
tube test, sugar fermentation and sugar assimilation reactions
+ + + –
FIGURE 91-19 Candida albicans on Sabourauds dextrose agar. FIGURE 91-20 Carbohydrate fermentation test.
4 Examples of indole positive bacteria are E. coli, Proteus

GLUCOSE WITH DURHAM’S TUBE vulgaris, Edwardsiella species, etc.
5 Kovac’s reagent consists of amyl iosoamyl alcohol, 150ml;
Salient features p-dimethyl – amino benzaldehyde; 10g and conc. HCl; 50ml.
1 It is used to determine the ability of an organism to ferment

a particular carbohydrate to produce acid or acid and gas UREASE TEST
(Fig. 91-21).
2 All members of the family Enterobacteriaceae are glucose
Salient features
fermenters with or without gas production.
3 This test is used to identify some Gram negative bacilli such
as E. coli, Klebsiella species, Salmonella species, Proteus
1 This test determines the ability of an organism to produce
species, etc.
an enzyme urease which splits urea to ammonia (Fig. 91-23).
2 The occurrence of this enzyme, urease can be tested for
alkali (NH3) production by means of suitable pH indicator.
3 Christensen’s urease medium is an example of the medium
which contains phenol red as an indicator. Ammonia
produced from urea makes the medium alkaline and phenol
red changes to pink / red in colour.
4 Development of pink colour indicates positive test while the
persistence of the pale yellow colour indicates the negative
test.
5 Examples of urease producing bacteria are Klebsiella species,
– + Proteus species, Yersinia enterocolitica, Helicobacter
pylori,etc.
FIGURE 91-21 Glucose with Durham’s tube.
INDOLE TEST
Salient features
1 The indole test demonstrates the ability of certain bacteria

to decompose the amino acid tryptophan to indole which NEGATIVE POSITIVE
accumulates in the medium. The production of indole is then
tested for by a colorimetric reaction with p-dimethyl amino FIGURE 91-23 Urease test.
benzaldehyde (Fig. 91-22).
2 The test is performed by inoculating the peptone water
medium with bacterium to be tested and incubated for 24-48 CITRATE UTILIZATION TEST
hours at 37°C. Then 0.5ml Kovac’s reagent is added to the
medium and shaked gently. Salient features
3 Formation of a red coloured ring near the surface of medium
indicates positive test. The presence of yellow coloured ring 1 This is a test for the ability of an organism to utilize citrate as
near surface of medium indicates negative test. the sole carbon and energy source for growth and an
ammonium salt as the sole source of nitrogen (Fig. 91-24).
2 Koser’s liquid citrate medium or Simmon’s citrate agar may
be used. Bromothymol blue is the indicator in Simmon’s
citrate medium.
3 Positive Koser’s citrate medium is indicated by the formation
of a turbidity i.e. growth. No turbidity indicates negative growth.
4 In Simmon’s citrate medium positive test is indicated by
formation of blue colour and streak of growth. Original green
colour and no growth indicates negative growth.
+ 5 Examples of citrate positive bacteria are Klebsiella species,
Salmonella species except Salmonella Typhi, Citrobacter
FIGURE 91 -22 Indole test. species, etc.
NEGATIVE POSITIVE
FIGURE 91-24 Citrate utilization test. FIGURE 91-26 Triple sugar iron agar.
PHENYL PYRUVIC ACID TEST (PPA) 4 Yellow colour formation occurs with fermentation of
carbohydrates, while bubbles in butt show the formation of
Salient features gas during fermentation process.
5 When H2S is produced by the bacteria, the medium shows
1 It is used to determine the ability of an organism to deaminate blackish discolouration.
phenyl pyruvic acid (Fig. 91-25). 6 Combinations of TSI reactions:
2 Positive PPA test is indicated by a green colour and negative
PPA test is indicated by no colour change. Examples of PPA K/A (red/yellow) : Glucose only fermented.
positive bacteria are Proteus species, Providencia species, A/A (yellow/yellow) : Glucose, and lactose or sucrose
and Morganella species. fermented or both fermented.
K/K (red / red) : Neither glucose, lactose nor
sucrose fermented.
Note: K – Alkaline, A- Acidic.
SPECIMENS OF PARASITE
ASCARIS LUMBRICOIDES ADULT WORM
Salient features
NEGATIVE POSITIVE 1 Tail end of male worm is curved ventrally in form of a hook,
while in female worm, it is conical and straight (Fig. 91-27).
FIGURE 91-25 Phenyl pyruvic acid test. 2 Route of infection of the organism is by ingestion of the
food or water contaminated with eggs.
3 Embryonated egg is the infective form of the parasite.
TRIPLE SUGAR IRON (TSI) AGAR
Salient features
1 TSI agar is used to determine the ability of the bacterium to

break down specific carbohydrates incorporated in a growth
medium, with or without the production of gas, along with
the production of hydrogen sulphide (Fig. 91-26).
2 Three carbohydrates i.e., glucose, lactose and sucrose are
present in the TSI agar. It also contains ferric salts for
detection of H2S production.
3 The medium in a test tube has a butt and slant. Phenol red is
the indicator. FIGURE 91 -27 Ascaris lumbricoides adult worm.
HYDATID CYST BIJOU BOTTLE
Salient features Sterilised by hot air oven or autoclave (Fig. 91-30).
1 Caused by Echinococcus granulosus. Uses

2 Cyst wall consists of two layers (i.e.) ectocyst and endocyst
(Fig. 91-28). 1 Specimen collection like CSF, blood, urine, ascitic fluid, etc.
3 Hydatid fluid which is present in cyst is used as antigen in 2 For preparation of media e.g. LJ medium, Loeffers, serum
immunodiagnostic tests. slope, etc.
4 Man is the intermediate host and dog is the definitive host. 3 For preparation of urease medium.
5 Infective agent is the egg, which is present in dog’s faces.
6 Infection is acquired by ingestion of the food or water
contaminated eggs.
FIGURE 91-30 Bijou bottle.
FIGURE 91-28 Hydatid cyst.

GLASS SYRINGE
GLASS WARES Glass syringes (Fig. 91-31) are sterilised by hot air oven.
Uses
UNIVERSAL CONTAINER
1 For collection of blood by venepuncture.
Universal containers (Fig. 91-29) are sterilised by autoclave . 2 To collect body fluids, pus, etc.
3 To collect blood from animals (sheep, rabbit) which may be
used for preparation of blood agar.
4 For injecting medicine to patients.
Uses
1 Specimen collection.
2 For measuring and mixing purposes.
FIGURE 91-31 Glass syringe.
TUBERCULIN SYRINGE
These are of two types: Glass or plastic syringe, and graduated

1ml syringe (Fig. 91-32).
a) Glass syringes are sterilized by hot air oven.
FIGURE 91-29 Universal containers. b) Plastic syringes are sterilized by gamma radiations.
FIGURE 91-32 Tuberculin syringe. FIGURE 91-34 Graduated pipette.
Uses PASTEUR PIPETTE
1 Lepromin test. Pasteir pipettes (Fig. 91-35) are sterilised by hot air oven.
2 Tuberculin test.
3 BCG vaccination. Uses
4 Insulin injection.
5 Tetanus toxoid injection. 1 Used for delivering solutions or reagent in test tubes,
6 To give intradermal and other sub cutaneous injections; and containers etc., during various procedures.
to inject small amount of test material into animals. 2 For mixing the constituents for reactions. Example: RBC s
coated with specific antigen to perform IHA test for detection
of antibodies.
PETRI DISH
Petri dishes (Fig. 91-33) are sterilised by hot air oven.
Uses
1 For preparation of culture media such as nutrient agar, blood

agar, MacConkey agar, etc.
2 For counting colonies as in pour plate method.
FIGURE 91-35 Pasteur pipette.
SWAB TUBE
Swab tubes (Fig. 91-36) are sterilized by hot air oven.

Unplugged swab tubes are contaminated.
Presterilized disposable swabs are commercially prepared.
These are sterilized by gamma radiations.
FIGURE 91-33 Petri dish.
GRADUATED PIPETTE
These are of 2 types: measuring pipette (Fig. 91-34) and delivery

pipette.
Sterilised by hot air oven.
Uses
1 For measuring quantity of fluid used in serological tests or

other tests.
2 For delivering the exact required volume. FIGURE 91-36 Swab tube.
Uses HOT AIR OVEN

1 For collection of specimens from various sites e.g. throat,
cervix, local lesions, etc.
Salient features
2 For streaking in case of lawn culture of bacterial growth as in
antibiotic sensitivity testing 1 Hot air oven (Fig. 91-39) uses the principle of dry heat
sterilization (holding temperature at 160°C for 60 min) method.
2 It is used for sterilization of glass wares such as glass
syringes, Petri dish, flasks, pipettes, test tubes, etc.
NIH SWAB
This is a swab used for collection of perianal scrapings.
Uses
1 Specially used for collection of specimen from perianal region for

demonstration of eggs of Enterobius vermicularis (Fig. 91-37).
FIGURE 91-37 Egg of Enterobius vermicularis, x 400. FIGURE 91-39 Hot air oven.
INSTRUMENTS AUTOCLAVE
Salient features
INCUBATOR
1 Autoclave (Fig. 91-40) uses the principle of moist heat steri-
Salient features lization (121°C at 15 lbs pressure for period of 15 minutes) method.
2 It is used for sterilization of culture media, rubber material,
1 Incubators (Fig. 91-38) are used for incubating culture plates gloves, gowns, dressing, test tubes, etc.
and liquid media at specified temperature for growth of
micoorganisms such as bacteria, fungi, amoebae, etc.
2 Optimum temperature for growing bacteria is 37°C.
3 It contains a thermometer by which periodically temperature
can be monitored.
FIGURE 91-38 Incubator. FIGURE 91-40 Autoclave.

MICROSCOPY SLIDES NEISSERIA GONORRHOEAE
Salient features
GRAM POSITIVE COCCI
1 In Gram stained smear, Neisseria gonorrhoea appear as pink
Salient features coloured Gram negative diplococci with adjacent sides
concave typically kidney shaped (Fig. 91-43).
1 In Gram stained smear, the Gram positive cocci appear violet 2 Found predominantly within the polymorphonuclear cells,
in colour (Fig. 91-41). but some may be seen outside.
2 Gram positive cocci occur either in pairs, chains or clusters.
Examples
S. aureus : Arranged in clusters.

Streptococcus pneumoniae : Arranged in pairs.
Micrococci : Arranged in tetrads.
Sarcina species : Arranged in groups of eight.
Streptococcus pyogenes : Arranged in chains.
FIGURE 91-43 Neisseria gonorrhoeaein a Gram’s stained smear,

x 1000.
GRAM NEGATIVE BACILLI
Salient features
1 In Gram stained smear, the Gram negative bacilli appear red

FIGURE 91-41 Gram positive cocci, x 1000. in colour (Fig. 91-44).
2 They usually do not have any typical arrangement
3 In capsulated organisms, capsule can be seen as unstained
STREPTOCOCCUS PNEUMONIAE structure surrounding the cell.
Salient features Examples
E. coli, Klebsiella species, Proteus species, etc.

1 In Gram stained smear, Str. pneumoniae appear as Gram
positive lanceolate shaped cocci arranged in pairs,
surrounded by capsule (Fig. 91-42).
2 Capsule is seen as a clear halo around the diplococci.
3 Capsule of Str. pneumoniae can be typically demonstrated
by negative staining with India ink.
FIGURE 91-44 Gram negative bacilliin a Gram’s stained smear, x

FIGURE 91-42 Sterptococcus pneumoniae in a Gram’s stained smear, 1000.
x 1000.
HAEMOPHILUS INFLUENZAE
Salient features
1 Gram stained smear shows pink coloured Gram negative

pleomorphic bacilli of Haemophilus influenzae (Fig. 91-45).
2 Coccobacillary forms of the bacteria can also be seen.
FIGURE 91-47 Bacillus anthracis in a Gram’s stained smear, x 1000.
CLOSTRIDIUM PERFRINGENS
Salient features
1 Gram stained films of necrotic muscle tissue shows Gram

positive bacillus of 4-6 µm x 1 µm with straight, parallel sides
FIGURE 91-45 Haemophilus influenzaein a Gram’s stained smear, x and rounded or truncated ends without spores occurring
1000.
singly or in chains (Fig. 91-48).
2 Pus cells are absent or scanty.
VIBRIO CHOLERAE
Salient features
1 Gram stained smear shows pink coloured comma shaped,

curved Gram negative bacilli with rounded or slightly pointed
ends (Fig. 91-46).
2 Vibrios are seen arranged on parallel rows fish in stream
appearance.
FIGURE 91-48 Clostridium perfringens in a Gram’s stained smear, x

1000.
TREPONEMA PALLIDUM
Salient features
1 Treponema pallidum in a smear stained by Levaditi stain

FIGURE 91-46 Vibrio cholerae in a Gram’s stained smear, x 1000. appear as thin delicate spirochaetes of 10 µm long with about
10 regular spirals which are sharp and angular at regular
intervals of about 1 µm (Fig. 91-49).
BACILLUS ANTHRACIS 2 Seen as black spirals against yellowish brown background
Salient features
1 In a Gram stained smear, Bacillus anthracis appears as short

chains of thick purple coloured Gram positive rods arranged
end to end surrounded by capsule (Fig. 91-47).
2 Ends of the bacilli are truncated, often concave and
somewhat swollen giving the chain of bacilli a bamboo stick
appearance. FIGURE 91-49 Triponema pallidum in a Levaditi stained smear, x
1000.
ALBERT STAINING
Salient features
1 It is a special stain for demonstrating metachromatic granules

of C. diphtheriae (Fig. 91-50).
2 The bacteria appears as green coloured bacilli with bluish
black metachromatic granules, arranged in Chinese letter
pattern (V or L pattern). FIGURE 91-51 Ziehl Neelsen staining for Mycobacterium
3 Green colour of the bacilli is due to malachite green and tuberculosis, x 1000.
bluish black granules due to toluidine blue reagents of
Albert’s stain.
4 C. diphtheriae causes diphtheria. ZIEHL - NEELSEN STAINING FOR
5 Potassium tellurite blood agar is the selective medium for C.
diphtheriae. The bacteria forms black colour on this medium
MYCOBACTERIUM LEPRAE
after 36-48 hrs of incubation at 37°C.
6 Loeffler’s serum slope is an egg based medium , on which Salient features
colony of C. diphtheriae can be seen as early as 6-8 hours
after incubation. 1 It is a special stain for demonstrating acid fastness of
Mycobacterium leprae (Fig. 91-52).
2 5% sulphuric acid is used for ZN stain. M. leprae resist
decolourisation with 5% sulphuric acid.
3 M. leprae is only acid fast but not alcohol fast.
4 M. leprae are seen as pink coloured acid fast bacilli arranged
as parallel rows of bacilli giving a ‘cigar bundle’ or ‘globi’
appearance against a blue background.
5 M. leprae causes leprosy.
6 No artificial cell-free media is available for growth of M. leprae,
hence it does not satisfy Koch’s postulates for a pathogenic
bacteria.
7 It can be grown in foot pads of 9 banded armadillo.
FIGURE 91-50 Alberts stained smear of Corynebacterium

diphtheriae, x 1000.
ZIEHL - NEELSEN STAINING FOR

MYCOBACTERIUM TUBERCULOSIS
Salient features
FIGURE 91-52 Ziehl Neelsen staining for Mycobacterium leprae,
x 1000.
1 It is a special stain for demonstrating acid fastness of M.
tuberculosis (Fig. 91-51).
2 20% sulphuric acid is used for ZN stain. M. tuberculosis NEGRI BODIES
resist decolourisation with 20% sulphuric acid.
3 M. tuberculosis is both acid and alcohol fast. Salient features
4 M. tuberculosis appear as approx. 1-3 µm size slender pink
coloured acid fast bacilli with curved ends against a blue 1 Negri bodies are 3 - 27 µm size, intra cytoplasmic inclusion
background. body which appear as round , oval, purplish pink structures
5 M. tuberculosis causes tuberculosis. with characteristic basophilic inner granules (Fig. 91-53).
6 Löwenstein Jensen (LJ) medium is the most frequently used 2 Negri bodies are usually found within the nerve cells of the
medium to grow M. tuberculosis. hippocampus and cerebellar region.
7 It takes 3-6 weeks for M. tuberculosis to grow in this medium. 3 These are usually demonstrated in the impression smears of
the rabid dog brain stained by Seller’s technique (basic

fuschin and methylene blue in methanol).
4 Negri bodies can also be demonstrated by Mann’s and
Giemsa stain.
5 Presence of basophilic inner granules within the Negri bodies
helps to differentiate canine distemper which lacks it.
FIGURE 91- 55 Molluscum bodies, x 1000.
PLASMODIUM VIVAX RING STAGE
Salient features
1 Plasmodium vivax is the causative agent of vivax malaria

(Fig. 91-56).
FIGURE 91- 53 Negri bodies, x 1000. 2 Female Anopheles mosquito transmits the disease and is the
definitive host
3 Man is the intermediate host.
MULTINUCLEATE GIANT CELLS: 4 It is a Giemsa stained thin blood smear, the ring stage appear
MEASLES as a blue stained ring of cytoplasm with a red chromatin dot.
The cytoplasm portion of ring opposite the nucleus is
Salient features thickened.
5 Young erythrocytes are predominantly infected and they
are enlarged.
1 These multinucleate giant cells are produced by measles
virus in Hela cell lines in tissue culture (Fig. 91-54).
2 These giant cells show multinucleate syncytium formation,
with numerous acidophilic nuclear and cytoplasmic
inclusions in Giemsa stained smears.
FIGURE 91- 56 Plasmodium vivax ring stage in a Giemsa stained

blood smear, x (1000.
PLASMODIUM FALCIPARUM RING STAGE
FIGURE 91- 54 Multinucleate giant cells-measles, x 1000. Salient features
1 In a Giemsa stained thin blood smear, ring stage appears as

blue ring of cytoplasm surrounding a central vacuole with
MOLLUSCUM BODIES red chromatin dot at centre.
2 P. falciparum is the causative agent of falciparum malaria
Salient features and causes cerebral malaria (Fig. 91-57).
3 Multiple ring forms are found within a single RBC, along
1 Section of the lesion shows large (20 µm to 30 µm) sized with accole forms.
eosinophilic hyaline inclusion bodies with nuclei displaced 4 All ages of erythrocytes are infected. Ring stages are found
to the margin (Fig. 91-55). within both normal sized young and older RBCs.
2 Molluscum bodies are composed of large nucleus of virus 5 The erythrocyte which is invaded by the parasite is not enlarged
particles, embedded in protein matrix. 6 P. falciparum shows frequent drug resistance to chloroquine.
MALE
FEMALE
FIGURE 91- 57 Plasmodium falciparum ring stage in a Giemsa stained FIGURE 91- 59 Plasmodium falciparum male and female
blood smear, x 1000. gametocytes, in a Giemsa stained blood smear, x 1000.
PLASMODIUM VIVAX
MALE AND FEMALE GAMETOCYTES LD BODIES
Salient features Salient features
1 Male gametocyte (microgametocyte) is spherical and smaller 1 It is a Giemsa stained thin blood smear showing a red
than the female. Cytoplasm stains light blue or pale blue and coloured nucleus and pale blue coloured cytoplasm multiple
nucleus is large (Fig. 91-58). rings in one red blood cell.
2 Female gametocyte (macrogametocyte) is spherical and large 2 Leishmania donovani is the causative agent of visceral
than the male. Cytoplasm is purple and nucleus is small. leishmaniasis or kala-azar (Fig. 91-60).
3 LD bodies, otherwise known as amastigote stage of
L. donovani is found mostly inside the macrophages. Some
LD bodies are found outside macrophages.
4 L. donovani shows frequent drug resistance to pentavalent
antimonials.
FIGURE 91- 58 Plasmodium vivax female gametocyte in a Giemsa

stained blood smear, x 1000.
FIGURE 91- 60 LD bodies in a Giemsa stained blood smear, x 1000.
PLASMODIUM FALCIPARUM
MALE AND FEMALE GAMETOCYTES TOXOPLASMA GONDII
1 Male gametocyte (microgamete) is sickle shaped, broader 1 The typical active multiplying tachyzoites is an important
and shorter. Cytoplasm stains light blue and nucleus is diagnostic form of Toxoplasma gondii (Fig. 91-61).
diffuse (Fig. 91-59). 2 Tachyzoites appear as 3-7 µm, oval to crescent shaped
2 Female gametocyte are typically crescent (banana) shaped structures with pointed anterior end and rounded posterior
with rounded or pointed ends. Cytoplasm stains deep blue end.
and nucleus is compact. 3 Tachyzoites can be stained with periodic acid schiff (PAS),
Gomori methenamine silver, hematoxylin and eosin, and
Wright-Giemsa stain.
4 Tissue cyst is the resting form of T. gondii which appear as

40mm to 50mm spherical structure surrounded by CYST OF ENTAMOEBA HISTOLYTICA/
eosinophilic, weakly PAS positive cyst wall. It contains DISPAR
hundred of strongly PAS positive, slow growing trophozoites
known as bradyzoites. Salient features
1 Iodine wet mount of stool showing quadrinucleate cyst of

Entamoeba histolytica/dispar (Fig. 91-63).
2 Cyst contains 4 nuclei, each nucleus has a central karyosome.
3 The cysts of E. histolytica and E.dispar are morphologically
similar.
4 The quadrinucleate cyst is the infective form of the parasite.
5 The infection is transmitted by ingestion of water and food
contaminated with cysts.
6 It is the causative agent of amoebic dysentery, amoebic liver
abscess, etc.
FIGURE 91- 61 Toxoplasma gondii tachyzoites in Wrisht-Giemsa

stained blood smear, x 1000.
WUCHERERIA BANCROFTI
MICROFILARIA
FIGURE 91- 63 Cyst of Entamoeba histolytica / dispar, x 400.
Salient features
1 It is a Giemsa stained thin blood smear showing the sheathed CYST OF GIARDIA INTESTINALIS
microfilaria.
2 The body of microfilaria shows few nuclei in the body and
more distinct tail is tapering and pointed.
Salient features
3 No nuclei are present in the tail end.
4 Wuchereria bancrofti is the causative agent of lymphatic 1 Iodine wet mount of stool showing iodine stained brown
filariasis (Fig. 91-62). oval cyst of Giardia intestinalis (Fig. 91-64).
5 Anopheles, Culex and Aedes mosquito transmit the disease 2 Cyst contains four nuclei, and an axostyle which lie
and are the intermediate host. diagonally, forming a dividing line within the cyst wall.The
6 Man is the definitive host. cyst is separated from the cyst wall by a clear space.
3 The cyst is the infective form of the parasite.
4 The infection is transmitted by ingestion of water and food
contaminated with cysts.
5 It is the causative agent of diarrhoea, malabsorption , etc.
FIGURE 91- 62 Wuchereria bancrofti microfilaria in a Giemsa stained

blood smear, x 1000. FIGURE 91- 64 Cyst of Giardia intestinalis, x 400.
EGG OF ROUND WORM EGG OF ENTEROBIUS VERMICULARIS
1 Bile stained egg of Ascaris lumbricoides in the saline wet 1 Saline wet mount of stool showing plano-convex and non-
mount of stool (Fig. 91-65). bile stained egg of Enterobius vermicularis (Fig. 91-67).
2 Embryonated egg is the infective form of the parasite. 2 Egg is the infective form of the parasite.
3 A. lumbricoides infection is transmitted by faeco -oral 3 E. vermicularis infection is transmitted by faeco-oral
transmission. transmission.
4 It is the causative agent of intestinal ascariasis. 4 It is the causative agent of pruritus ani in children.
5 Auto infection is characteristic of E. vermicularis infection.
FIGURE 91-67 Egg of Enterobius vermicularis, x 400.
EGG OF HYMENOLEPIS NANA

FIGURE 91-65 Egg of round worm, x 100.
Salient features
1 Saline wet mount of stool showing non-bile stained egg of

EGG OF HOOK WORM Hymenolepis nana (Fig. 91-68).
2 Egg contains oncosphere with three pairs of hooklets.
Salient features 3 Egg is the infective form of the parasite.
3 H. nana infection is transmitted by faeco -oral transmission.
1 Saline wet mount of stool showing non- bile stained egg of 4 It is the causative agent of hymenolepiasis
hook worms: Ancylostoma duodenale and Necator
americanus (Fig. 91-66).
2 The eggs of A. duodenale and N. americanus are
morphologically similar.
3 Filariform larva is the infective form of the parasite.
4 Hook worm infection is transmitted by filariform larva piercing
the intact skin.
5 It is the causative agent of microcytic hypochromic anemia
and tropical pulmonary eosinophilia, etc.
FIGURE 91-68 Egg of Hymenolepis nana, x 400.
EGGS OF TRICHURIS TRICHIURA
Salient features
1 Saline wet mount of stool showing bile stained egg of

Trichuris trichiura (Fig. 91-69).
2 T. trichiura egg is barrel – shaped with mucous plug at each pole.
3 Egg is the infective form of the parasite.
FIGURE 91-66 Egg of Hook worm, x 400.
3 T. trichiura infection is transmitted by faeco-oral 3 They cause opportunistic infection especially in patients
transmission. with HIV.
4 It is the causative agent of gastrointestinal infections. Heavy 4 They cause oral thrush, vaginitis, onychonychia etc., in
infection may complicate as appendicitis. immunocompetent patients.
GERM TUBE TEST
Salient features
1 The test is also called Reynold – Braude phenomenon

(Fig. 91-71).
FIGURE 91- 69 Egg of Trichuris trichura, x 400. 2 It is used to identify and differentiate C. albicans from other
Candida species
3 The test is performed by incubating Candida in patients
CANDIDA ALBICANS serum at 37°C for 2hours
4 Germ tube production is due to the formation of
Salient features pseudohyphae by the fungus.
1 It is a yeast-like fungus.
2 In Gram stained smear, C. albicans appear purple in colour
(Fig. 91-70)
FIGURE 91- 70 Candida albicans in a Gram’s stained smear, x 1000. FIGURE 91- 71 Germ tube test, x 400.
FURTHER READINGS
3 Parija SC. Textbook of Medical Parasitology. All India Publishers and Distributors. 3nd Edition. 2006.
294
Index
A Introduction 97
Abbe condenser 4 Principle 97
ABO system 110 Requirements 97
Acetoin 78 Equipments 97
Acetylcholinesterase 140 Reagents and lab wares 97
Acid fast staining method 27 Preparation of stock solutions of antibiotics 97
Acid-alcohol 27 Specimens 97
Acid-fast staining 27 Preparation of suspension of bacteria 97
Learning Objectives 27 Procedure 97
Introduction 27 Test procedure 98
Principle 27 Quality Control 98
Requirements 27 Observations 98
Equipments 27 Results and Interpretation 98
Reagents and glass wares 27 Key Facts 98
Preparation of strong carbol fuchsin 27 Viva 99
Preparation of 20% sulphuric acid 27 Agarose 130
Preparation of 95% alcohol 27 Agglutination 108, 117
Preparation of acid-alcohol decolouriser 28 Albert’s stain 31, 32, 33, 175, 176, 288
Specimen 28 Albert’s staining 31
Procedure 28 Learning Objectives 31
Quality Control 28 Introduction 31
Observation 28 Principle 31
Results and Interpretation 28 Requirements 31
Key Facts 29 Equipments 31
Viva 30 Reagents and glass wares 31
Acid-fast Staining of Stool Smears 198 Preparation of Albert’s stain I 31
Learning Objectives 198 Preparation of Albert’s stain II 31
Introduction 198 Specimen 31
Principle 198 Procedure 31
Requirements 198 Quality Control 32
Equipments 198 Observation 32
Reagents and lab wares 198 Results and Interpretation 32
Specimen 198 Viva 32
Procedure 198 Key Facts 33
Quality Control 198 Alcohol 27, 60
Observations 199 Alkaline peptone water 46, 181
Results and Interpretation 199 Alkaline phosphatase 138, 140
Key Facts 199 Allantoic cavity 243
Viva 200 Allantoic sac 244
Adult hamster 265 Allergic respiratory distress 269
Adult mice 259 Alsever’s solution 121
Adult rabbit 261, 262 Amido black 126, 131
Aedes aegypti 269, 268, 291 Amniotic sac 243, 244
Aerotolerant anaerobes 52 Amoebiasis 269
Aesculin 173 Amoebic antigens 130
Agar 274 Amoebic dysentery 291
Agar dilution method 97, 98 Amoebic liver abscess 291
Learning Objectives 97 Anaerobic bacilli 54
296 Index
Anaerobic bacteria 277 Auramine O 30

Anaerobic cocci 54 Autoclave 285
Ancylostoma duodenale 292 Autolysis 169, 171
Animals and their uses in the laboratory 263 Auxotrophs 144
Learning Objectives 263 Axenic culture medium 208, 209
Introduction 263
Principle 263 B
Requirements 263 Babes Ernst granules 31
Equipments 263 Babesia 204
Reagents and animals 263 Babesia species 203
Specimen 263 Bacilli 6
Procedure 263 Bacillus anthracis 6, 287
Quality Control 264 Bacitracin test 172
Observations 264 Bacterial Agglutination Test 108
Results and Interpretation 264 Learning Objectives 108
Key Facts 266 Introduction 108
Viva 266 Principle 108
Anopheles 268, 289 291 Requirements 108
Anthrax 269 Reagents and glass wares 108
Antibiotic resistance 157 Specimen 108
Antibiotic sensitivity 92 Procedure 108
Antibiotic susceptibility testing 278 Quality Control 109
Antigen 133 Observations 109
Antimicrobial agent 93, 94, 99, 100 Results and Interpretation 109
Antiseptic 59 Positive agglutination 109
Antiseptics and Disinfectants 59 Negative agglutination 109
Learning Objectives 59 Auto agglutination 109
Introduction 59 Key Facts 109
Principle 59 Viva 109
Requirements 59 Bacterial agglutination tests 109
Equipments 59 Bacterial conjugation 155
Reagents and media 59 Learning Objectives 155
Specimen 59 Introduction 155
Procedure 59 Hfr strains 155
Quality Control 59 F’ factors and sexduction 155
Observation 59 Principle 155
Results and Interpretation 59 Requirements 155
Key Facts 60 Equipments 155
Viva 60 Reagents and glass wares 155
antiseptics 59, 60 Specimen 155
Antiseptics 60 Procedure 155
Anti-Streptolysin O (ASLO) Test 121 Quality Control 156
Learning Objectives 121 Observations 156
Introduction 121 Results and Interpretation 156
Principle 121 Key Points to Remember 156
Requirements 121 Viva 157
Equipments 121 Bacterial endospores 38
Reagents and lab wares 121 Bacterial plasmids 145
Specimen 121 Bactericidal drugs 93, 94, 101
Procedure 121 Bacteriostatic drug 93, 94, 101
Serum dilution 121 Balantidium coli 6, 249
Test procedure 121 Bancroftian filariasis 269
Quality Control 121 Barbitone buffer 126
Observations 121 Bartonella quintana 270
Results and Interpretation 122 Basal media 45, 46
Key Facts 122 Basic dye 215
Viva 122 Basic stains 20
Antony Von Leeuwenhoek 2 Bicarbonate buffer 50
Argas persicus 270 Bijou bottle 283
Arginine dihydrolase test 185 Bile aesculin test 173
Armadillo 288 Bile solubility test 169
Asbestos filters 56 Biological false positive (BFP) reactions 123, 125
Ascaris lumbricoides 206, 292 Biotin-avidin ELISA 141
ASLO 122 Bismuth sulfite agar 86
Aspergillus flavus 235 Blastomyces dermatitides 6
Aspergillus fumigatus 234 Blocking solution 139
Aspergillus niger 235 Blood agar 44, 45, 46, 161, 162, 163, 164, 165, 184, 274
Aspergillus species 6 Blood group antigens 110
Blood group antisera 110 Box 90-2 Diseases Transmitted by Hard Ticks 271
Blood grouping 110 Box 7-1 Modifications of Gram’s staining 25
Learning Objectives 110 Box 7-2 Uses of Gram’s Staining 25
Introduction 110 Brain heart infusion agar 46
Principle 110 Bright field microscopy 2
Requirements 110 Brilliant green bile broth 249
Reagents and lab wares 110 Bromothymol blue 81, 281
Specimen 110 Broth dilution agar method 101
Procedure 110 Broth dilution method 100
Quality Control 110 Learning Objectives 100
Observations 110 Introduction 100
Results and Interpretation 110 Principle 100
Key Facts 111 Requirements 100
Viva 111 Equipments 100
Blood parasites 201 Reagents and lab wares 100
Body louse 270 Preparation of stock solutions of antibiotics 100
Boeck and Drbohlav’s medium 208 Specimens 100
Borrelia duttoni 270 Preparation of suspension of bacteria 100
Borrelia recurrentis 270 Procedure 100
Bound coagulase 68, 69 Quality Control 100
Boutonneuse fever 271 Observations 101
Box 1-1 Terminology 5 Results and Interpretation 101
Box 1-2 Size of Different Organisms 6 Key Facts 101
Box 12-1 List of most common capsulated organisms that can be dem- Viva 101
onstrated by negative staining 41 Browne’s tube 56, 57
Box 17-1 List of Anaerobic Bacilli and Cocci 54 Brownian movement 12
Box 18-1 Pasteurisation 58 Brucellosis 253
Box 19-1 Commonly used disinfectants and their mechanism of actions 60 Buffered distilled water 201
Box 2-1 Principle of Dark Ground Microscopy 8 Buffered methylene blue 191
Box 20-1 Uses of catalase Test 63
Box 20-2 Catalase Test for Mycobacteria 63 C
Box 22-1 Free Coagulase 69 CAMP (Christie, Atkins and Munch-Peterson) test 173
Box 29-1 List of media used for detecting production of hydrogen sul- Candida albicans 6, 215, 293
phide 86 Candida albicans on Sabourauds dextrose agar 280
Box 31-1 Glossary of terms 93 Candida species 225
Box 4-1 Demonstrating Motility of Anaerobic Bacteria 12 Candle jar 51
Box 43-1 VDRL-ELISA 125 Candling 244
Box 43-2 Biological False Positive Reactions of VDRL Test 125 Capillary tube method 11, 12
Box 47-1 Reverse Passive Haemagglutination test 133 Capsular antigens 36
Box 49-1 1st, 2nd and 3rd Generation ELISA 140 Capsulated bacteria 36
Box 49-2 Dot ELISA 140 Capsule 36, 286
Box 5-1 Terminology 17 Capsule Staining 3 4
Box 50-1 Role of Plasmids in drug resistance 146 Learning Objectives 34
Box 51-1 Advantages and Disadvantages of Page 150 Introduction 34
Box 52-1 Point Mutations and Large Scale Mutations 153 Principle 34
Box 52-2 Mechanisms of Mutation 154 Requirements 34
Box 52-3 The Importance of Mutation 154 Equipments 34
Box 53-1 Mechanisms of DNA Transfer 156 Reagents and glass wares 34
Box 54-1 Beneficial Effects of Normal Flora 161 Specimen 34
Box 6-1 Simple stains and their uses in microbiology laboratory 21 Procedure 34
Box 61-1 Identification of Escherichia Coli 179 For positive staining of smears 34
Box 61-2 Identification of Klebsiella species 179 For negative staining of smears 35
Box 62-1 Identification of Vibrio Cholerae 182 Quality Control 35
Box 63-1 Identification of Pseudomonasaeruginosa 185 Observation 35
Box 67-1 Acid Fast Parasites and Parasitic Components 199 Observation of positive staining method 35
Box 68-1 The Parasites found in the Peripheral Blood Smear 203 Observation of negative staining method 35
Box 69-1 Advantages and Disadvantages of the Concentration Results and Interpretation 35
Methods 207 Positive staining method 35
Box 71-1 Sabouraud’s Dextrose Agar 214 Negative staining method 35
Box 77-1 Predisposing Factors for Candidiasis 226 Key Facts 35
Box 8-1 Different Modifications of Acid Fast Stain and their uses 29 Viva 36
Box 8-2 Frequently examined specimens for the Detection of Carbohydrate assimilation 229
Mycobacterium Tuberculosis 29 Carbohydrate Assimilation Test 229, 230
Box 87-1 Uses of Mice in Laboratory 259 Learning Objectives 229
Box 88-1 Use of Rabbits in Laboratory 262 Introduction 229
Box 89-1 Use of Laboratory Animals 264 Principle 229
Box 9-1 Rapid staining by direct fluorescent antibody method 32 Requirements 229
Box 90-1 Diseases Transmitted by Sand Fly 271 Equipments 229
298 Index
Reagents and lab wares 229 CIEP test 130, 131

Specimens 229 Citrate negative bacteria 81
Procedure 229 Citrate positive bacteria 81
Quality Control 229 Citrate sulfide agar 86
Observation 229 Citrate utilisation test 80, 81
Results and Interpretation 229 Learning Objectives 80
Key Facts 230 Introduction 80
Viva 230 Principle 80
Carbohydrate Fermentation Test 231, 232, 280 Requirements 80
Learning Objectives 231 Equipments 80
Introduction 231 Reagents and lab wares 80
Principle 231 Specimen 80
Requirements 231 Procedure 80
Equipments 231 Quality Control 81
Reagents 231 Positive control 81
Preparation of indicator broth medium 231 Negative control 81
Specimen 231 Observations 81
Procedure 231 Results and Interpretation 81
Quality Control 231 Key Facts 81
Observations 232 Viva 81
Results and Interpretation 232 Citrate utilization test 185, 281, 282
Key Facts 232 Cladosporium 236
Viva 232 CLED medium 46, 178, 180
Carbohydrate fermentation tests 231 Clostridium perfringens 248, 287
Carbol fuchsin 27, 198 Co-agglutination test 114, 115
Carbonate buffer 139 Learning Objectives 114
Carbonic acid 71 Introduction 114
Cardiolipin antigen 123, 125 Principle 114
Cassette ELISA 141 Requirements 114
Catalase 62, 64 Reagents and lab wares 114
Catalase negative bacteria 63 Specimen 114
Catalase positive bacteria 63 Procedure 114
Catalase test 62, 185 Quality Control 114
Principle 62 Key Facts 115
Reagents and glass wares 62 CO 2 incubator 160
Specimen 62 Coagulase 68
Procedure 62 Coagulase negative bacteria 69
Slide method 62 Coagulase negative staphylococci 168
Tube method 62 Coagulase positive bacteria 69
Quality Control 63 Coagulase reacting factor (CRF) 69
Positive control 63 Coagulase test 68, 166, 167
Negative control 63 Learning Objectives 68
Slide method 63 Principle 68
Tube method 63 Bound coagulase 68
Results and Interpretation 63 Free coagulase 68
Viva 64 Reagents and lab wares 68
Catalyst 53 Specimen 68
Cell culture 240, 242 Procedure 68
Cell lines 240 Slide test 68
Cephalosporium 236 Tube test 68
Cerebral malaria 289 Quality Control 69
Cetrimide agar 46, 185 Positive control 69
Chick cell agglutination 181 Negative control 69
Chick Martin test 60 Observation 69
Chick RBCs agglutination 182 Results and Interpretation 69
Chikungunya fever 269 Key Facts 69
Chlorhexidine 60 Viva 70
Chlorination 248 Coccidian parasites 198
Chocolate agar 44, 161, 163, 275 Coccidioides immitis 6
Cholera 181, 269 Cold agglutination test 120
Chorioallantoic membrane (CAM) 243, 244 Coliforms 248, 251
Christensen’s urea agar 71, 227, 281 Collection of Blood from the Marginal Ear Vein of Rabbit 261
Chromoblastomycosis 6 Learning Objectives 261
Introduction 261 Learning Objectives 130

Principle 261 Introduction 130
Requirements 261 Principle 130
Laboratory wares 261 Requirements 130
Reagents 261 Equipment 130
Specimen 261 Reagents and glass wares 130
Procedure 261 Specimen 130
Quality Control 261 Preparation of Veronal buffer 0.075 M (pH 8.6) 130
Observations 261 Preparation of agarose 130
Results and Interpretation 261 Procedure 130
Key Facts 262 Quality Control 131
Viva 262 Observations 131
Colorado tick fever 271 Results and Interpretation 131
Competitive ELISA 138, 141 Key Facts 131
Compound Microscope 3 Viva 131
Learning Objectives 3 Coxsackie A and B viruses 258
Introduction 3 Craigie’s tube method 11
Parts of the compound microscope 3 Crimean - congo hemorrhagic fever 271
Microscope stand 3 Cryptococcal antigen 130
Main tube 3 Cryptococcus neoformans 6, 221
Body and arm 3 Cryptosporidium parvum 198, 199, 200, 258
Substage 3 Culex 269, 291
Foot 3 Cultivation of Fungi 213
Stage 3 Learning Objectives 213
Microscope optics 4 Introduction 213
Mechanical adjustment of a microscope 4 Principle 213
Coarse and fine focusing adjustments 4 Requirements 213
Condenser adjustment 4 Equipments 213
The light source 4 Reagents and lab wares 213
Principle 4 Specimen 213
Magnification 4 Procedure 213
Principle involved in the magnification of the object 4 Quality Control 213
Importance of numerical aperture 5 Observations 213
Requirements 5 Results and Interpretation 213
Equipment 5 Key Facts 214
Reagents 5 Viva 214
Specimen 5 Cultivation of Viruses in the Cell lines 240
Results and Interpretation 5 Principle 240
Compound light microscope 9 Reagents and lab wares 240
Concentration of stool 205, 207 Specimen 240
Concentration of Stool for Parasites 205 Procedure 240
Learning Objectives 205 Quality Control 241
Introduction 205 Observations 241
Principle 205 Results and Interpretation 241
Requirements 205 Key Facts 242
Equipments 205 Viva 242
Reagents and lab wares 205 Cultivation of Viruses in Embryonated Egg 243
Specimen 205 Learning Objectives 243
Procedure 206 Introduction 243
Saturated salt solution flotation method 206 Principle 243
Formalin-ether sedimentation method 206 Requirements 243
Quality Control 206 Equipments 243
Observations 206 Reagents and lab wares 243
Results and Interpretation 206 Specimen 243
Key Facts 207 Procedure 243
Viva 207 Quality Control 244
Condenser 4 Observations 244
Conjugation 155, 157 Results and Interpretation 244
Conjunctivitis 269 Key Facts 244
Coomassie staining 149 Viva 244
Corynebacterium diphtheriae 31, 175, 275 Culture media 274
Corynebacterium diphtheriae on potassium tellurite agar 279 Culture of anaerobic bacteria 53
Counter-current immunoelectrophoresis (CIEP) 130, 149 Learning Objectives 53
Counter-current Immunoelectrophoresis Test 130 Introduction 53
300 Index
Principle 53 Dobell’s iodine 192

Requirements 53 Dot ELISA 139, 140
Equipments 53 Double diffusion method 126
Reagents and media 53 Dracunculiasis 271
Specimen 53 Dracunculus medinesis 6
Procedure 53 Draughtsman’s colonies 169
Quality Control 54 Drbohlav’s Locke-egg-serum (LES) medium 208
Observations 54 Dry heat sterilization 285
Results and Interpretation 54 Durham’s tube 231, 250
Viva 54 Dysentery 269
Key Facts 55
Culture of Stool for Entamoeba histolytica 208 E
Learning Objectives 208 E-test 103
Introduction 208 Earthenware candles 56
Principle 208 Echinococcus granulosus 283
Requirements 208 EDTA 69
Equipments 208 Egg of Ascaris lumbricoides 190, 193, 196
Reagents and lab wares 208 Egg of Enterobius vermicularis 292
Specimen 208 Egg of hook worm 206, 292
Procedure 209 Egg of Hymenolepis nana 292
Quality Control 209 Egg of round worm 292
Observations 209 Egg of Trichuris trichiura 190, 193, 196, 292
Results and Interpretation 209 Eggs of Taenia saginata 199
Key Facts 209 Ehrlich’s reagent 74, 75
Viva 209 Eijkman test 249, 251
Cutaneous mycoses 212 Electro-immuno transfer blot (EITB) 150
Cyclops 270 Electroimmunodiffusion 127, 149
Cyclospora cayetanensis 195, 198, 199, 200 Electron microscopes 2
Cyst of Entamoeba coli 190193, 196, 206 Electrophoresis 148
Cyst of Entamoeba histolytica/dispar 190, 193, 196, 206, 291 Embryonated eggs 243
Cyst of Giardia intestinalis 190, 193, 196, 206, 291 Endospores 37
Cytopathic effect (CPE) 240, 241 Enriched media 45, 46, 275
Enrichment media 46
D Entamoeba histolytica 8, 208, 248
D’ Antonie’s iodine 192 Enteric fever 116, 118
Dark ground microscopy 2, 7, 11 Enterobius vermicularis 292
Learning Objectives 7 Enzyme-linked immunosorbent assay 138, 141
Equipments 7 The sandwich ELISA for antigen 138
Reagents 7 The indirect ELISA for antibodies 138
Specimen 7 Competitive ELISA for antibodies 138
Procedure 7 Indirect ELISA to Detect Antibodies 139
Observations 7 Requirements 139
Results and Interpretation 7 Equipments 139
Key Facts 8 Reagents and glass wares 139
Viva 8 Carbonate buffer (pH 9.6) 139
Decolourising agent 26, 27, 215 Washing buffer (PBS-Tween 20) 139
Dengue haemorrhagic fever 268 Conjugate 139
Deoxycholate citrate agar (DCA) medium 44, 86, 181 Citric acid phosphate buffer (0.1M) pH 5.0 139
Dettol 60 Substrate 139
Diarrhea 269, 291 Specimen 139
Differential coliform test 249, 250 Procedure 139
Differential media 46 Quality Control 139
Differential stain 21, 26 Observations 139
Diffusion tests 97 Results and Interpretation 139
Dilute carbol fuchsin 215 Sandwich ELISA to Detect Antigen 139
Diphtheroids 163 Requirements 139
Diphyllobothriasis 271 Equipments 139
Diphyllobothrium latum 271 Reagents and glass wares 139
Direct fluorescent antibody test 32, 135, 136 Specimen 139
Direct plate technique 65 Procedure 139
Disc diffusion test 94 Quality Control 140
Disinfectants 59, 60 Observations 140
Disinfection 59, 60 Results and Interpretation 140
DNAase test 167 Key Facts 141
Dobell and O’Connor’s iodine 192 Viva 141
Enzymes 138 Glucose phosphate medium 82

Epidemic relapsing fever 270 Glucose with Durham’s tube 281
Epidemic typhus 270 Glutaraldehyde 60
Epidermophyton floccosum 236 Glycerol 195, 218
Epsilometer test (E-test) 102 Gnathostoma hispidium 271
Learning Objectives 102 Gnathostoma spinigerum 271
Introduction 102 Goat antimouse immunoglobulin 139
Principle 102 Gomori methenamine silver 290
Requirements 102 Gram negative cocci 24
Reagents and lab wares 102 Gram positive bacilli 24
Specimens 102 Gram positive bacteria 24
Procedure 102 Gram positive cell wall 26
Opening an E-test package 102 Gram positive cocci 24, 286
Application of strips 102 Gram variable bacteria 26
Quality Control 103 Gram’s iodine 215
Observations 103 Gram’s iodine 23
Results and Interpretation 103 Gram’s stain 26, 37, 161, 162, 164, 215
Key Facts 103 Gram’s Staining for Fungi 215
Viva 103 Learning Objectives 215
Escherichia coli 178 Introduction 215
Escherichia coli on MacConkey agar 279 Principle 215
Ether 205 Requirements 215
Ethidium bromide solution 145 Equipments 215
Eye pieces 4 Reagents and lab wares 215
Specimen 215
F Procedure 215
F factor 155 Preparation of fungal smear 215
Facultative anaerobes 52, 54 Staining Procedure 215
Faecal Escherichia coli 248 Quality Control 215
Faecal Streptococci 248 Observation 216
Falciparum malaria 289 Results and Interpretation 216
Filariform larva 292 Key Facts 216
Floatation method 207 Grams staining 23
Fluorescence microscopy 2 Learning Objectives 23
Fluorescent microscope 135, 136 Introduction 23
Fluorescent staining 30 Principle 23
Foetal calf serum (FCS) 240 Requirements 23
Forage mite 269 Equipments 23
Formalin 205 Reagents and glass wares 23
Formalin-ether sedimentation method 205, 206, 207 Preparation of methyl violet stain 23
Free coagulase 68 Preparation of Gram’s iodine 23
Fungal elements 219 Preparation of 1% safranine 23
Fusarium 234 Specimen 24
Preparation of bacterial smear 24
G Preparation of bacterial smear 24
Gas gangrene 38 Procedure 24
Quality Control 24
Gaspak system 53
Observation 24
Gel diffusion test 127
Gelatin hydrolysis test 176 Results and Interpretation 24
Key Facts 26
Germ tube test 225, 226, 293
Viva 26
Learning Objectives 225
Introduction 225 Group A streptococci 172
Group B streptococci 172
Principle 225
Group D streptococci 172
Requirements 225
Equipments 225 Guinea pig 263, 264, 265
Reagents and glass wares 225
Specimens 225 H
Procedure 225 H2S producing bacteria 86
Quality Control 225 Haemoparasites 201
Observations 225 Haemophilus influenzae 287
Results and Interpretation 226 Hamsters 263, 265
Key Facts 226 Hanging drop preparation 11, 181
Viva 226 Learning Objectives 11
Germ tubes 225, 226 Introduction 11
Giardia species 248 Principle 11
Giemsa stains 201, 203 Requirements 11
Glucose 6-phosphate dehydrogenase 140 Equipments 11
Glucose broth 277 Reagents and glass wares 11
302 Index
Specimen 11 Fusarium 234

Procedure 11 Colony morphology 234
Quality Control 11 Microscopy 234
Observations 12 Aspergillus fumigatus 234
Results and Interpretation 12 Aspergillus niger 235
Key Facts 13 Aspergillus flavus 235
Viva 13 Penicillium species 235
Hard ticks 270 Conidiophores 235
Head louse 270 Phialids 235
Heat stable catalase test 63 Cladosporium 236
Hektoen enteric agar 86 Colony 236
HeLa 240 Microscopy 236
Hemolysis on blood agar 167 Cephalosporium 236
Hep 2 240 Colony 236
Hepatitis B antigen 130 Microscopy 236
Heterophile agglutination tests 107 Trichophyton verrucosum 236
Histoplasma capsulatum 6 Colony 236
Holder method 58 Trichophyton violaceum 236
Hook worm egg 190, 193, 196 Colony 236
Horseradish peroxidase 138, 140 Epidermophyton floccosum 236
Hot air oven 58, 285 Colony 236
House dust mite 269 Viva 237
House flies 269 Identification of Common Insects 268
Hungate procedure of anaerobiosis 54 Learning Objectives 268
Hybridization probes 144 Introduction 268
Hydatid antigens 130, 132 Principle 268
Hydatid cyst 283 Requirements 268
Hydatid fluid 283 Equipments and lab wares 268
Hydrogen Sulfide Test 85 Specimen 268
Learning Objectives 85 Mosquitoes 268
Introduction 85 General features 268
Principle 85 Identifying features of Anopheles 268
Requirements 85 Diseases transmitted 268
Equipments 85 Identifying features of Aedes 268
Reagents and lab wares 85 Diseases transmitted 268
Specimen 85 Identifying features of Culex 269
Procedure 85 Diseases transmitted 269
Quality Control 86 Sand Fly 269
Positive control 86 General features 269
Negative control 86 House Flies 269
Observations 86 Identifying features 269
Results and Interpretation 86 Diseases transmitted 269
Viva 86 Itch Mite 269
Key Facts 87 Identifying features 269
Hymenolepis nana 6, 292 Diseases transmitted 269
Trombiculid Mite 269
I Identifying features 269
Identification of Common Fungi 233 Diseases transmitted 269
Learning Objectives 233 Hard Tick 270
Introduction 233 Identifying features 270
Principle 233 Soft Tick 270
Requirements 233 Identifying features 270
Equipments 233 Diseases transmitted 270
Reagents and lab wares 233 Rat Flea 270
Specimen 233 Identifying features 270
Procedure 233 Louse 270
Quality Control 233 Identifying features 270
Observation 233 Diseases transmitted 270
Results and Interpretation 233 Cyclops 270
Rhizopus 233 Identifying features 270
Colony morphology 233 Viva 271
Microscopy 234 Identification of Common Viva Spots 274
Mucor 234 Learning Objectives 274
Colony morphology 234 Culture Media 274
Microscopy 234 Nutrient Agar 274
Alternaria 234 Composition 274
Colony morphology 234 Preparation 274
Microscopy 234 Modifications 274
Uses 274, 275, 276, 277, 278, 283, 284, 285 Universal Container 283
Blood Agar 274 Bijou Bottle 283
Composition 274 Tuberculin Syringe 283
Preparation 275 Graduated Pipette 284
Chocolate Agar 275 Pasteur Pipette 284
Composition 275 NIH Swab 285
Preparation 275 Incubator 285
MacConkey Agar 275 Salient features 285
Composition 275 Hot Air Oven 285
Preparation 275 Salient features 285
Loeffler’s Serum Slope 275 Autoclave 285
Composition 276 Salient features 285
Preparation 276 Microscopy Slides 286
Lowenstein-Jensen (LJ) Medium 276 Gram Positive Cocci 286
Preparation 276 Streptococcus pneumoniae 286
Robertson Cooked Meat (RCM) Broth 276 Salient features 286
Composition 276 Neisseria gonorrhoeae 286
Sabouraud’s Dextrose Agar (SDA) 277 Gram Negative Bacilli 286
Preparation 277 Haemophilus influenzae 287
Peptone water 277 Salient features 287
Composition 277 Vibrio cholerae 287
Glucose Broth 277 Bacillus anthracis 287
Preparation 278 Clostridium perfringens 287
Culture Media with Growth 278 Salient features 287
Streptococcus pyogenes on Blood Agar 278 Treponema pallidum 287
Salient features 278 Salient features 287
Staphylococcus aureus on Nutrient Agar 278 Albert staining 288
Proteus spp. on Blood Agar 278 Ziehl-Neelsen Staining for Mycobacterium tuberculosis 288
Pseudomonas aeruginosa on Nutrient Agar 279 Ziehl-Neelsen Staining for Mycobacterium leprae 288
Escherichia coli on MacConkey Agar 279 Negri Bodies 288
Klebsiella spp. on MacConkey Agar 279 Multinucleate Giant Cells Measles 289
Corynebacterium diphtheriae on Potassium Tellurite Agar 279 Molluscum Bodies 289
Mycobacterium tuberculosis on Lowenstein Jensen (LJ) Medium 280 Plasmodium vivax Ring Stage 289
Candida albicans on Sabouraud’s Dextrose Agar (SDA) 280 Plasmodium falciparum Ring Stage 289
Biochemical Reactions 280 Plasmodium vivax Male and Female Gametocytes 290
Carbohydrate Fermentation Tests 280 Salient features 290
Salient features 280 Plasmodium falciparum Male and Female Gametocytes 290
Glucose with Durham’s Tube 281 Salient features 290
Salient features 281 LD Bodies 290
Indole Test 281 Salient features 290
Salient features 281 Toxoplasma gondii 290
Urease Test 281 Salient features 290
Salient features 281 Wuchereria bancrofti Microfilaria 291
Citrate Utilization Test 281 Salient features 291
Salient features 281 Cyst of Entamoeba histolytica/dispar 291
Phenyl Pyruvic Acid Test (PPA) 282 Salient features 291
Salient features 282 Cyst of Giardia intestinalis 291
Triple Sugar Iron (TSI) Agar 282 Salient features 291
Salient features 282 Egg of Round Worm 292
Specimens of Parasite 282 Salient features 292
Ascaris lumbricoides Adult Worm 282 Egg of Hook Worm 292
Hydatid Cyst 283 Egg of Enterobius vermicularis 292
Glass Wares 283 Egg of Hymenolepis nana 292
304 Index
Salient features 292 Learning Objectives 181

Eggs of Trichuris trichiura 292 Introduction 181
Salient features 292 Specimens 181
Candida albicans 293 Tests for Identification of Vibrio Cholerae 181
Salient features 293 Direct examination 181
Germ Tube Test 293 Hanging drop preparation 181
Salient features 293 Culture 181
Identification of Corynebacterium diphtheriae 175 Biochemical tests 181
Learning Objectives 175 Antibiotic susceptibility testing 181
Introduction 175 Key Facts 182
Specimen 175 Viva 183
Tests for the identification of Corynebacterium diphtheriae 175 Identification of b-haemolytic Streptococci 172
Direct examination 175 Learning Objectives 172
Albert’s stain 175 Introduction 172
Culture 175 Specimen 172
Biochemical tests 175 Tests for Identification of Streptococcus pyogenes 172
Key Facts 176 Direct examination 172
Viva 176 Culture 172
Identification of Lactose Fermenting Enterobacteriaceae 178 Bacitracin sensitivity test 172
Learning Objectives 178 CAMP (Christie, Atkins and Munch-Peterson) test 173
Introduction 178 Bile aesculin test 173
Specimens 178 Viva 173
Tests for Identification of E. Coli and Klebsiella spp. 178 Key Facts 174
Direct examination 178 IHA test 133, 134, 284
Gram’s stain 178 Illuminating source 4
Culture 178 Illumination 5
Biochemical tests 178 Immunodiffusion 127
Antibiotics susceptibility testing 178 Immunoelectrophoresis test 107, 128, 135
Key Facts 180 Learning Objectives 128
Viva 180 Introduction 128
Identification of Pseudomonas aeruginosa 184 Principle 128
Learning Objectives 184 Requirements 128
Introduction 184 Equipments 128
Specimens 184 Reagents and glass wares 128
Tests for Identification of Pseudomonas Aeruginosa 184 Specimen 128
Direct examination 184 Procedure 128
Culture 184 Quality Control 129
Oxidase test 184 Observations 129
Biochemical tests 184 Results and Interpretation 129
Antibiotic susceptibility testing 184 Key Facts 129
Key Facts 185 Viva 129
Viva 185 Immunoelectrophoresis 128, 129
Identification of Staphylococcus aureus 166 Immunofluorescence Test 135
Learning Objectives 166 Learning Objectives 135
Introduction 166 Introduction 135
Specimen 166 Principle 135
Tests for the Identification of Staphylococcus aureus 166 Requirements 135
Direct examination 166 Equipments 135
Culture 166 Reagents and lab wares 135
Coagulase test 166 Specimen 135
Deoxyribonuclease test 166 Procedure 135
Mannitol salt agar 166 Quality Control 136
Novobiocin sensitivity 167 Observations 136
Key Facts 168 Results and Interpretation 136
Viva 168 Key Facts 136
Identification of Streptococcus pneumoniae 169 Viva 136
Learning Objectives 169 In-use test 60
Introduction 169 Incineration 58
Specimen 169 Incomplete antibodies 108
Tests for the Identification of Streptococcus pneumoniae 169 Incubators 285
Direct examination 169 India ink 34, 41, 221, 222, 286
Culture 169 India ink preparation 35, 212, 221, 222
Bile solubility test 169 Learning Objectives 221
Optochin test 169 Introduction 221
Inulin fermentation 170 Principle 221
Identification of Vibrio cholerae 181 Reagents and lab wares 221
Specimen 221 Inulin fermentation 170

Procedure 221 Iodine wet mount 191, 192, 194
Quality Control 221 Iodine Wet Mount of Stool 192
Observations 221 Learning Objectives 192
Results and Interpretation 221 Introduction 192
Key Facts 222 Principle 192
Viva 222 Requirements 192
India-ink method 40 Equipments 192
Indicator broth medium (IBM) 231 Reagents and glass wares 192
Indicator medium 275 Specimen 192
Indirect ELISA 141 Procedure 192
Indirect haemagglutination test 132 Quality Control 192
Principle 132 Key Facts 194
Lab wares 132 Iodine wet mount preparation 189, 192
Reagents 132 Isoantibodies 111
Specimen 133 Isolation of Antibiotic Resistant Mutant 152
Sensitisation of chick RBCs with OSD of the antigen 133 Introduction 152
Performance of the IHA test 133 The importance of mutation 152
Quality Control 133 Principle 152
Observations 133 Requirements 152
Results and Interpretation 133 Equipments 152
Key Facts 133 Reagents and lab wares 152
Viva 134 Specimen 152
Indirect paper strip procedure 65 Procedure 152
Indole negative bacteria 74, 75 Quality Control 153
Indole positive bacteria 74, 75 Observations 153
Indole test 74, 185, 281 Results and Interpretation 153
Learning Objectives 74 Key Facts 154
Introduction 74 Viva 154
Principle 74 Isolation of Plasmids 145
Requirements 74 Learning Objectives 145
Equipments 74 Introduction 145
Reagents and lab wares 74 Principle 145
Specimen 74 Requirements 145
Procedure 74 Equipments 145
Quality Control 74 Reagents and lab wares 145
Positive control 74 Preparation of ethidium bromide stock solution 145
Negative control 74 Preparation of ethidium bromide working solution 145
Observation 74 Preparation of Luria-Bertani medium (LB medium) 145
Viva 75 Extraction of plasmid 145
Infant hamsters 265 Electrophoresis on agarose gel 146
Infant rabbit 262 Quality Control 146
Insects 268 Observations 146
Inspissation 58 Results and Interpretation 146
Intestinal coccidian parasites 199, 200 Key Facts 146
Intravenous Inoculation into Mice Tail Vein 258 Viva 146
Learning Objectives 258 Isolation of Pure Cultures 14
Equipments 258 Requirements 14
Reagents and glass wares 258 Equipment and labwares 14
Specimen 258 Reagents 14
Procedure for loading the syringe for injection 258 Procedure 15
Animal preparation for injection 258 For streak plate method 15, 16
Injection of material 258 For spread plate method 15, 16
Quality Control 259 For pour plate method 15, 16
Observations 259 Quality Control 15
Results and Interpretation 259 Observations 15
Viva 260 Viva 16
306 Index
Key Facts 17 Learning Objectives 112

Isospora belli 198, 199 Introduction 112
Itch mite 269 Principle 112
Requirements 112
J Reagents and lab wares 112
Japanese encephalitis 269 Specimen 112
Jaswant Singh Bhattacharjee stain 203 Procedure 112
Jensen’s Gram method for smears 25 Quality Control 112
Observations 112
K Results and Interpretation 113
Key Facts 113
K antigen 36
Viva 113
Köhler illumination 3, 6
Kala azar 271 LD bodies 290
Lead acetate agar 85, 86
Kinyoun’s modification of acid-fast stain 29
Leishman stains 201, 203
Kirby-Bauer disc diffusion method 92
Kirby-Bauer method 92, 96 Leishman’s 201
Leishman’s stain 201
Leishman’s Staining of Peripheral Blood Smears 201
Introduction 92
Principle 92 Learning Objectives 201
Introduction 201
Requirements 92
Principle 201
Equipments 92
Reagents and lab wares 92 Requirements 201
Equipments 201
Preparation of 0.5 McFarland standard 92
Reagents and lab wares 201
Specimens 92
Preparation of suspension of bacteria 92 Preparation of EDTA anticoagulated blood 201
Preparation of Leishman’s stain 201
Procedure 92
Specimen 202
Quality Control 93
Observations 93 Procedure 202
Preparation of thin blood smear 202
Results and Interpretation 93
Preparation of thick blood smear 202
Key Facts 93
Viva 94 Preparation of combined thick and thin films 202
Leishman’s staining 202
Kirby-Bauer’s chart 93
Quality Control 202
Klebsiella species 178
Klebsiella species on MacConkey agar 279 Observations 202
Kligler’s iron agar median 85, 86, 185
Key Facts 203
Koch’s postulates 288
KOH wet mount preparation 219, 220 Viva 204
Leishmania donovani 290
Kopeloff and Beerman’s Gram method 25
Lepromin test 284
Koser’s citrate 80, 81
Koser’s liquid citrate medium 281 Levaditi stain 287
Light microscopy 2
Kovac’s reagent 74, 75, 281
Listeria monocytogenes 261
Krebs cycle 80
Kyasanur forest disease 271 Locke’s solution 208
Loeffler’s methylene blue 21, 27, 28
Loeffler’s serum slope 175, 275, 276, 280
L Louse 270
Löwenstein-Jensen medium 44 LPCB Wet Mount of Stool 195
Laboratory animals 263
Lactophenol cotton blue (LPCB) stain 195, 217
Introduction 195
Lactophenol Cotton Blue (LPCB) Wet Mount of Fungi 217 Principle 195
Requirements 195
Introduction 217
Equipments 195
Principle 217 Reagents and glass wares 195
Requirements 217
Specimen 195
Equipments 217
Procedure 195
Reagents and lab wares 217 Quality Control 196
Specimen 217
Observations 196
Procedure 217
Scotch tape preparation 217 Key Facts 197
Tease mount preparation 217
Viva 197
Quality Control 218
LPCB wet mount preparation 196
Observations 218 Lugol’s’ iodine 192
Luria-Bertani medium 145
Key Facts 218
Lyme disease 271
Viva 218 Lymphatic filariasis 291
Lactophenol cotton blue (LPCB) wet mount 212, 217
Lysine decarboxylation test 185
Lactose fermenting Enterobacteriaceae 178
Lysine iron agar 86
Lane’s saturated salt solution floatation method 205 Lysol 60
Latex agglutination test 112, 113
M Results and Interpretation 77

MacConkey agar 45, 46, 178, 184, 275 Key Facts 77
MacConkey broth 249 Viva 77
Macleod’s potassium tellurite agar media 175 Methyl violet stain 23, 215
Macrocapsule 34 Methylene blue (counter stain) 198
Macrogametocyte 290 Methylene blue reduction test 252
Magnesium sulphate floatation method 206 Methylene blue test 252
Magnification 6 Mice 263
Malabsorption 291 Microbiology of Air 254
Malachite green solution 276 Learning Objectives 254
Malachite green stain 37 Introduction 254
Malaria 268 Principle 254
Male gametocyte 290 Requirements 254
Mannitol salt agar 166, 167 Equipments 254
Mc Fadyean reaction 21 Reagents and lab wares 254
McFadyean reaction 35 Procedure 254
McFarland standard 92, 95 Quality Control 254
McIntosh and Fildes jar 53 Observations 254
Measurement of Microorganisms 9 Results and Interpretation 254
Principle 9 Microbiology of Water 248
Requirements 9 Learning Objectives 248
Equipments 9 Introduction 248
Reagents 9 Coliforms 248
Specimen 9 Faecal or thermotolerant coliforms 248
Procedure 9 Faecal Escherichia coli 248
Quality Control 10 Faecal streptococci 248
Observations 10 Clostridium perfringens 248
Results and Interpretation 10 Collection of water samples 249
Key Facts 10 Principle 249
Viva 10 Plate count 249
Media for Routine Cultivation of Bacteria 44 Detection of coliform bacteria 249
Learning Objectives 44 Presumptive coliform test – Multiple tube technique 249
Introduction 44 Differential coliform test 249
Principle 44 Membrane filtration method 249
Basal media 44 Detection of faecal streptococci 249
Enriched medium 44 Examination for Cl. perfringens 249
Enrichment media 44 Requirements 249
Selective media 44 Equipments 249
Differential media 44 Reagents 249
Requirements 45 Preparation of MacConkey broth 249
Equipments 45 Preparation of double strength medium 249
Reagents and media 45 Preparation of single strength medium 249
Specimen 45 Preparation of brilliant green bile broth 250
Quality Control 45 Procedure 250
Observations 45 Quality Control 250
Results and Interpretation 46 Observations 250
Viva 46 Differential coliform count 250
Membrane filtration method 249 Key Facts 251
Mesocyclops 271 Viva 251
Metachromatic granules 31, 32, 176, 288 Microbiology of Milk 252
Methyl alcohol 201 Learning Objectives 252
Methyl red test 76 Introduction 252
Learning Objectives 76 Principle 252
Introduction 76 Requirements 252
Principle 76 Equipments 252
Requirements 76 Reagents and glass wares 252
Equipments 76 Specimen 252
Reagents and lab wares 76 Procedure 252
Specimen 76 Quality Control 253
Procedure 76 Observations 253
Quality Control 77 Results and Interpretation 253
Positive control 77 Key Facts 253
Negative control 77 Viva 253
Observation 77 Microcapsule 34
308 Index
Microfilaria of Brugia malayi 203 Positive control 88

Microfilaria of Loa loa 203 Negative control 88
Microfilaria of Mansonella perstans 203 Observation 88
Microfilaria of Wuchereria bancrofti 203 Results and Interpretation 88
Miliary tuberculosis 29 Key Facts 89
Minimum bactericidal concentration (MBC) 100, 101 Viva 89
Minimum inhibitory concentration (MIC) 97, 98, 100, 101, 102 Nitrates 89
Missense mutations 153 Non agglutinable vibrios 183
Modified acid fast staining 199 Non motile bacteria 12
Modified acid fast staining of faeces 198 Non-bile stained eggs 193
Modified Ziehl-Neelsen stain 37 Non-fermenters 184
Moist heat sterilization 285 Nonsense mutations 153
Molluscum bodies 289 Normal flora 160
Monkeys 263, 265 Normal Microbial Flora of the Mouth 160
Monoclonal antibodies 139 Learning Objectives 160
Mordant 24, 215 Introduction 160
Mosquitoes 268 Principle 160
Motile bacteria 12 Requirements 160
Mouse 258, 260, 264 Equipments 160
Mouse virulence 170 Reagents and lab wares 160
MR negative bacteria 77 Specimen 160
MR positive bacteria 77 Procedure 160
MR test 76, 77 Quality Control 160
Mucor 234 Observations 160
Multinucleate giant cells 289 Results and Interpretation 161
Mutations 152, 154 Key Facts 161
Mycobacterium leprae 258, 288 Viva 161
Mycobacterium tuberculosis 28, 276 Normal Microbial Flora of the Skin 164
Mycobacterium tuberculosis on Lowenstein- Jensen’s medium 280 Learning Objectives 164
Introduction 164
N Principle 164
N-N tetramethyl para-phenylene diamine hydrochloride 65 Requirements 164
NCCLS QC ranges 103 Equipments 164
NCCLS table 98 Reagents 164
Necator americanus 292 Specimen 164
Negative staining 40, 41 Procedure 164
Principle 40 Viva 164
Requirements 40 Key Facts 165
Equipments 40 Normal Microbial Flora of the Throat 162
Reagents and glass wares 40 Learning Objectives 162
Specimen 40 Introduction 162
Procedure 40 Principle 162
Quality Control 40 Requirements 162
Observations 41 Equipments 162
Results and Interpretation 41 Reagents and lab wares 162
Key Facts 41 Specimen 162
Viva 41 Procedure 162
Negri bodies 288 Observations 162
Neisser’s stain 31, 33, 176 Results and Interpretations 162
Neisseria gonorrhoea 286 Key Facts 163
Neutral red 180 Viva 163
Neutralization test 107 Normal flora in the throat 162
Nigrosin staining 34, 40 Normal flora on the skin 164
NIH Swab 285 Normal microbial flora 163
Nitrate agar slant 88 Northern blotting 149
Nitrate broth 89 Norwegian itch of man 269
Nitrate reduction test 88, 89, 185 Nosocomial fungal infections 212
Learning Objectives 88 Novobiocin sensitivity 167
Introduction 88 Novobiocin-sensitive staphylococci 168
Principle 88 Numerical aperture 5, 6
Requirements 88 Nutrient agar 45, 46, 184, 274
Equipments 88 Nutrient broth 274
Reagents and lab wares 88
Specimen 88 O
Procedure 88 O/129 reagent 183
Quality Control 88 Obligate aerobes 52
Obligate anaerobes 52, 53 Penicillium species 235

Ocular micrometer 9 Peptone water 46, 74, 274, 277
Onychonychia 293 Periodic acid schiff (PAS) 290
Optimum sensitizing dose (OSD) of the antigen 132 Permanent staining of blood smear 201
Optochin (ethyl hydrocupreine hydrochloride) 169 PH requirement of bacteria 49
Optochin sensitivity 170 pH Requirement for Growth of Bacteria 49
Optochin test 169 Learning Objectives 49
Oral thrush 293 Introduction 49
Oriental sore 271 Principle 49
Orientia tsutsugamushi 269 Requirements 49
Ornithodorus lahorensis 270 Equipments 49
Ornithodorus species 270 Reagents 49
Ornithodorus tholozani 270 Specimen 49
Oroya fever 271 Procedure 49
Osmk hemorraghic fever 271 Quality Control 49
Oxidase negative bacteria 66 Observations 49
Oxidase positive bacteria 66 Results and Interpretation 49
Oxidase reagent 65 Key Facts 50
Oxidase test 65, 66, 185 Viva 50
Learning Objectives 65 Phase-contrast microscope 2, 38
Introduction 65 Phenol 60, 218
Principle 65 Phenol red 82, 281, 282
Requirements 65 Phenyl pyruvic acid test 282
Reagents and glass wares 65 Phosphatase test 252
Specimen 65 Phosphate buffer 50
Procedure 65 Phosphate buffer saline 132
Direct plate technique 65 Phthirus pubis 270
Indirect filter paper strip procedure 65 Physiological saline 189
Quality Control 66 Pikes medium 46
Positive control 66 Plasmids 145, 146, 157
Negative control 66 Plasmodium 204
Observations 66 Plasmodium falciparum 203
Direct plate technique 66 Plasmodium Falciparum male and female Gametocytes 290
Indirect filter paper strip procedure 66 Plasmodium Falciparum Ring stage 289
Results and Interpretation 66 Plasmodium malariae 203
Viva 66 Plasmodium ovale 203
Key Facts 67 Plasmodium vivax 203
Oxygen Requirement for Growth of Bacteria 51 Plasmodium Vivax male and female Gametocytes 290
Learning Objectives 51 Plasmodium vivax ring stage 289
Introduction 51 Pneumococcal antigen 114
Principle 51 Point mutations 152, 153, 154
Aerobes 51 Polar bodies 31
Microaerophiles 51 Polar flagellum 11
Obligate anaerobes 51 Poliomyelitis 269
Aerotolerant anaerobes 51 Polyacrylamide Gel Electrophoresis 148
Facultative anaerobes 51 Learning Objectives 148
Requirements 51 Introduction 148
Equipments 51 Principle 148
Reagents and media 51 Requirements 148
Specimen 51 Equipments 148
Procedure 52 Reagents and lab wares 148
Quality Control 52 Stock solutions 148
Observations 52 Working solutions 148
Results and Interpretation 52 Separating gel buffer (4x) 148
Key Facts 52 Stacking gel buffer (4x) 148
Viva 52 10% Ammonium persulfate (APS) 148
Electrophoresis/Running Buffer (1x) 149
P Sample buffer 149
P-dimethyl amino benzaldehyde 75, 281 Staining solution: 1000ml. 149
P-phenylene diamine dihydrochloride 67 Destaining solution: 1000ml. 149
Paracoccidioides brasiliensis 6 Specimen 149
Parvo virus 6 Procedure 149
Passive agglutination tests 106 Quality Control 149
Pasteur pipette 284 Observations 149
Pasteurisation 56, 58 Results and Interpretation 149
Paul-Bunnel test 120 Viva 150
Pediculus capitus 270 Key Facts 151
Pediculus corporis 270 Polychrome methylene blue 20, 21
310 Index
Polymetaphosphate 175 Testing unknown serum samples 127

Polymyxin-B (50U) sensitivity 181, 182 Quality Control 127
Polysaccharide capsule 36, 279 Observations 127
Polyxenic culture medium 208, 209 Results and Interpretation 127
Ponder’s stains 176 Key Facts 127
Positive staining technique 35 Viva 127
Potable water 248 Rat Flea 270
Potassium hydroxide (KOH) wet mount 212, 219 Reaginic antibodies 123
Potassium Hydroxide Wet Mount of Fungi 219 Recombinant DNA technology 144
Learning Objectives 219 Relapsing fever 270
Introduction 219 Resolution 5, 6
Principle 219 Resolving power 5
Requirements 219 Restriction endonucleases 144
Equipments 219 Reverse passive haemagglutination test 132, 133
Reagents and lab wares 219 Reynold – Braude phenomenon 293
Specimen 219 Rheumatoid factor (RF) 112
Procedure 219 Rhinosporidium seeberi 6
Quality Control 219 Rhizopus 217, 233
Observations 219 Rhodamine 135
Results and Interpretations 219 Rickettesia orientails 269
Key Facts 220 Rickettsia prowazaki 270
Viva 220 Rickettsial infection 119
Potassium nitrate broth (KNO3 ) 88 Rideal Walker test 60
Potassium tellurite blood agar 275, 279, 288 Rift valley fever 269
Pour plate inoculation procedure 14 Robertson cooked meat (RCM) 53, 276
Pour plate method 14 Robertson’s cooked meat (RCM) medium 52
Pox virus 6 Robinson’s medium 208
PPA test 282 Rocky mountain Spotted fever 271
Precipitation tests 107 Romanowsky’s stains 201, 204
Preston and Morrell’s Gram method 25 Russian spring summer encephalitis 271
Presumptive coliform count 249, 250
Protein-A ELISA 141 S
Proteus mirabilis 279 Sabouraud’s dextrose agar (SDA) 164, 213, 214, 215, 225, 229, 277
Proteus OX 19, OX 2 and OX K antigens 119, 120 Safranine 23
Proteus species on blood agar 278 Safranine stain 37
Proteus vulgaris 279 Saline Wet Mount of Stool 189
Prototrophs 144 Learning Objectives 189
Protozoal cysts 195, 205 Introduction 189
Protozoal infections 201, 204 Principle 189
Prozone reaction 108 Requirements 189
Pseudomonas aeruginosa 184 Equipments 189
Pseudomonas aeruginosa on nutrient agar 279 Reagents and glass wares 189
Pubic louse 270 Specimen 189
Puch’s stain 31, 33 Procedure 189
Pulmonary tuberculosis 29 Quality Control 189
Pure culture 14, 16, 17 Observations 189
Pyocyanin 184 Results and Interpretation 190
Pyruvic acid 78 Key Facts 191
Viva 191
Q Saline wet mount preparation of faeces 189
Q fever 253 Salmonella-Shigella agar 86
Quellung reaction 170 Sand Fly 269
Sand fly fever 271
R Sandwich ELISA 141
Rabbit 261, 263, 264 Sarcocystis hominis 199
Rabbit plasma 68, 69 Sarcoptes scabies var hominis 269
Radial immuno diffusion 126 Saturated salt solution flotation method 205, 206, 207
Radial Immunodiffusion Test 126 Scabies 269
Learning Objectives 126 Schaeffer Fulton method 37, 38
Introduction 126 Scolices of Echinococcus granulosus 199
Principle 126 Scotch tape preparation 217
Requirements 126 Scrub typhus mite 269
Equipments 126 SDS-PAGE 150
Reagents and glass wares 126 Sedimentation method 207
Specimen 126 Selenite F broth 45, 46
Procedure 126 Seller’s technique 289
Preparation of antibody containing gels 126 Semi quantitative catalase test 63
Calibration of reference graph 126 Sereny’s test 262
Settle plate method 254, 255 Spores of B. stearothermophilus 57

Sheather’s sucrose floatation method 200, 206 Spores of Bacillus stearothermophilus 56
Significant bacteriuria 25 Spores of Microsporidia 199
Simmon’s citrate agar 280, 281 Sporothrix schenckeii 6
Simmon’s citrate medium 80, 81 Spread plate inoculation procedure 14
Simple stain 21 Standard test of syphilis 125
Simple staining 20 Staphylococcus aureus 6, 166, 278
Learning Objectives 20 Staphylococcus aureus on nutrient agar 278
Introduction 20 Sterile Mueller Hinton broth 97
Principle 20 Sterilisation 56, 60
Requirements 20 Sterilization of Commonly Used Culture Media 56
Equipments 20 Learning Objectives 56
Reagents and glass wares 20 Introduction 56
Preparation of Loeffler’s methylene blue stain 20 Principle 56
Preparation of polychrome methylene blue 20 Sterilization by heat 56
Specimen 20 Sterilization by filtration 56
Procedure 20 Requirements 56
Observations 20 Reagents 56
Sintered glass filter 56 Autoclave 57
Slide agglutination tests 106, 108 Hot air oven 57
Slide coagulase test 70 Filters 57
Slide culture 223, 224 Observations 57
Slide Culture for Fungi 223 Results and Interpretation 57
Principle 223 Sterilization of culture media 56
Requirements 223 Stokes Method 95, 96
Reagents 223 Introduction 95
Specimen 223 Principle 95
Procedure 223 Requirements 95
Observations 224 Reagents and lab wares 95
Slide culture preparation 217 Observations 96
Slide culture set 223 Results and Interpretation 96
Slide flocculation test 123 Key Facts 96
Slide test 68 Viva 96
Slit sampler method 254, 255 Streak plate inoculation procedure 14
Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS- Streptococcus MG agglutination 120
PAGE) 148, 150 Streptococcus pneumoniae 169, 170, 261
Sodium hypochlorite 60 Streptococcus pyogenes 172, 278
Soft Tick 270 Streptococcus pyogenes on blood agar 278
Southern blotting 149 Streptolysin O 121
Specific soluble substance 36 Streptolysin S 121, 122
Spore of Clostridium tetani 56 Streptomycin-resistant mutants 152
Spore Staining 37 String test 183
Learning Objectives 37 Stuart’s broth 72
Introduction 37 Subcutaneous mycoses 212
Principle 37 Suckling mouse 259, 258
Requirements 37 Sulphur indole motility medium 86
Equipments 37 Superficial mycoses 212
Reagents and lab wares 37 Swarming 11
Preparation of malachite green stain 37 Systemic mycoses 212
Preparation of safranine stain 37
Specimen 38 T
Procedure 38 TAB vaccine 117
Quality Control 38 Table 1-1 Types of condensers 4
Observations 38 Table 13-1 List of different type of media commonly used forisolation
Results and Interpretation 38 of bacteria 46
Viva 38 Table 14-1 Examples of bacteria showing different temperaturesfor their
Key Facts 38 growth 48
312 Index
Table 16-1 Examples of bacteria grouped depending on theirrequirement Specimen 47

of oxygen 52 Procedure 47
Table 18-1 Different methods of sterilization of varioussubstances 58 Quality Control 47
Table 19-1 List of commonly used disinfectants and antiseptics 60 Observations 48
Table 20-1 Catalase positive and negative bacteria 63 Results and Interpretation 48
Table 21-1 List of oxidase positive and negative bacteria 66 Key Facts 48
Table 22-1 List of coagulase positive bacteria 69 Viva 48
Table 23-1 Urease producing bacteria and fungi 72 Test for coliform bacilli 252
Table 24-1 List of indole positive and negative bacteria 75 Test for pathogenic bacteria 249
Table 25-1 List of MR positive and negative bacteria 77 Tetramethyl paraphenylene diamine dihydrochloride 160
Table 26-1 VP positive and negative bacteria 79 Thick blood smear 201, 202, 204
Table 27-1 Differences between Simmon’s citrate and Koser’scitrate 80 Thin blood films 201, 202, 204
Table 27-2 List of citrate positive and negative bacteria 81 Thioglycollate broth 52
Table 29-1 List of H2S positive bacteria 86 Thioglycollate broth culture 53
Table 32-1 Comparison of Kirby-Bauer and Stokes methods 96 Tick borne encephalitis 271
Table 34-1 Preparation of stock dilutions of the antibiotic Tick paralysis 271
stocksolutions 101 Ticks 270
Table 4-1 Motile and non-motile bacteria 12 Toluidine blue 31
Table 43-1 Advantages and disadvantages of VDRL test 124 Total coliform count 248
Table 44-1 Uses of gel diffusion tests 127 Toxoplasma gondii 199, 258, 290
Table 48-1 Advantages and disadvantages of the Trachoma 269
immunofluorescencetest 136 Transcription 144, 156
Table 49-1 Enzymes and substrates used in the ELISA 140 Transformation 156
Table 49-2 Advantages and disadvantages of ELISA test 140 Transversions 154
Table 51-1 List of other types of electrophoresis in gels 149 Trench fever 270
Table 51-2 Calculation for X% separating or stacking gel 150 Treponema pallidum 287
Table 52-1 Different types of mutations and their role inmicrobial in- Trichinella spiralis 6
fections 153 Trichomonas vaginalis 8
Table 53-1 Differences between F+ strain and Hfr strain 156 Trichophyton verrucosum 236
Table 57-1 Laboratory tests for differentiation of staphylococcalspecies 167 Trichophyton violaceum 236
Table 58-1 Differences between Streptococcus pneumoniaeand Triple sugar iron (TSI) agar 82
Streptococcus mitis 170 Triple sugar iron agar (TSI) test 82
Table 62-1 Differences between V. cholerae biotypeclassical and V. Learning Objectives 82
cholerae biotype El Tor 182 Introduction 82
Table 65-1 List of bile-stained and non-bile stained eggs 193 Principle 82
Table 69-1 Other floatation methods of concentration stool 206 Requirements 82
Table 7-1 List of Gram positive and Gram negative bacteria 24 Equipments 82
Table 71-1 List of media used for fungal culture 214 Reagents and lab wares 82
Table 77-1 Differences between germ tubes and pseudohyphae 226 Specimen 82
Table 79-1 Preparation of Yeast Nitrogen Base 230 Procedure 83
Table 8-1 List of acid-fast structures 29 Quality Control 83
Table 82-1 The cell lines and indications for the viruses andcytopathic Observations 83
effects they produce 241 Results and Interpretation 83
Table 83-1 The routes of inoculation of the egg and the Viva 83
virusesisolated 244 Key Facts 84
Table 84-1 Grades of the Quality of Drinking Water Supplies Deter- Trombicula akamush 269
mined by results of Periodic Escherichia coli andColiform Trombicula dilensis 269
counts 250 Trombiculid Mite 269
Table 85-1 List of bacteria that can be found in contaminatedmilk 253 Tryptone broth 74
Table 86-1 List of bacteria commonly found in air 254 TSI agar 82, 84, 85, 86, 282
Table 89-1 Laboratory animals and their usage 265 Tube agglutination tests 106
Table Applications of various tests used in a microbiologylaboratory 106 Tube coagulase test 69, 70
Tachyzoites 290 Tube dilution method 100
Taenia saginata 6 Tuberculin test 284
Taenia soluim 6 Tuberculous meningitis 29
Tautomerization 154 Turbidity test 252
TCBS 181 Tyndallization 58
Tease mount preparation 217
Temperature Requirement for Growth of Bacteria 47 U
Learning Objectives 47 Universal Container 283
Introduction 47 Universal donors 110
Principle 47 Urea 71, 227
Psychrophilic bacteria 47 Urea hydrolysis test 176
Mesophilic bacteria 47 Urease 71, 72, 227, 228
Thermophilic bacteria 47 Urease negative bacteria 72
Requirements 47 Urease negative fungi 227
Equipments 47 Urease positive bacteria 72
Reagents and media 47 Urease positive fungi 227
Urease producing bacteria 72 Equipments 78

Urease producing fungi 72 Reagents and lab wares 78
Urease test 71, 72, 185, 227, 228, 281 Specimen 78
Learning Objectives 71 Procedure 78
Introduction 71 Quality Control 79
Principle 71 Positive control 79
Requirements 71 Negative control 79
Equipments 71 Observations 79
Reagents and glass wares 71 Results and Interpretation 79
Specimen 71 Key Facts 79
Procedure 71 Viva 79
Quality Control 72 Volutin granules 31
Positive control 72 VP broth 78
Negative control 72 VP negative bacteria 79
Observation 72 VP positive bacteria 79
Results and Interpretation 72 VP test 79
Key Facts 72
Viva 72 W
Urease Test for fungi 227, 279 Water fleas 271
Learning Objectives 227 Weil-Felix test 119
Equipments 227 Requirements 119
Reagents and lab wares 227 Equipments 119
Specimen 227 Reagents and glass wares 119
Quality Control 227 Procedure 119
Observations 227 Preparation of master dilution of the serum 119
Results and Interpretation 227 Performance of the test 119
Key Facts 228 Quality Control 120
Viva 228 Observations 120
V Key Facts 120
VDRL test 123, 125 Viva 120
Learning Objectives 123 West Nile fever 269
Introduction 123 Western blot 150
Principle 123 Wet mount preparation 191, 206
Requirements 123 Widal test 116, 117
Reagents and glass wares 123 Introduction 116
Preparation of buffered saline solution 123 Principle 116
Preparation of un buffered saline solution 123 Requirements 116
Specimen 123 Equipments 116
Procedure 123 Reagents and glass wares 116
Preparation of serum 123 Specimen 116
Preparation of antigen emulsion 124 Procedure 116
Maturation of the antigen 124 Preparation of master dilution of the serum 116
Qualitative serum test 124 Performance of the test 116
Quantitative serum test 124 Quality Control 117
Quality control 124 Observations 117
Observations 124 Results and Interpretation 117
Results and Interpretation 124 Viva 117
Qualitative serum test 124 Key Facts 118
Quantitative serum test 124 Wright’s stain 201
Key Facts 125 Wuchereria bancrofti 291
Viva 125 Wuchereria bancrofti microfilaria 291
VDRL-ELISA 123, 125
Veronal buffer 130 X
Viable count 252 Xylose lysine deoxycholate agar 86
Vibrio cholerae 181, 287
Vibriostatic (O/129) agent 182 Y
Voges-Proskauer (VP) test 182
Yaws 269
Voges-Proskauer Test 78
Yeast-like fungi 229
Learning Objectives 78 Yellow fever 269
Introduction 78
Yolk sac 243
Principle 78
Yolk sac 244
Requirements 78
314 Index
Z Zinc dusts 89
Zephiran 60 Zinc sulphate floatation method 206
Ziehl Neelsen staining 288 Zone reactions 124, 125
Ziehl-Neelsen acid-fast staining 27 Zygomycetes 6
Ziehl-Neelsen stain 29, 37
1 / Running Head 13
TEXTBOOK OF
PRACTICAL MICROBIOLOGY
The intent of the book is to provide recent information and explain in

detail the routine diagnostic methods performed in a Microbiology
laboratory. Every effort has been made to incorporate all aspects of
practical microbiology. A sincere effort is made to provide the
essential underlying principles of practical microbiology, to help
students to perform various practicals, and to learn and apply the
knowledge of practical microbiology in clinical medicine

is Director-Professor and Head of Microbiology
at JIPMER, Pondicherry.
Rs
Ahuja Publishers New Delhi

Textbook of Practical Microbiology

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1 / Running Head 1

Subhash Chandra Parija

Dr. Subhash Chandra Parija