A HAND BOOK OF

M ICROBIOLOGY

DR SUSHIL SAHU
ASSISTANT PROFESSOR
DEPARTMENT OF MICROBIOLOGY
R.N.T. M EDICAL COLLEGE
UDAIPUR (RAJ.)

VPH
Publisher
Vandana Sahu
Vandana Publishing House

Office: Vandana Publishing House

39, Nepal House
RMV Road
Chhoti Brahmpuri
Udaipur- 313001
Mob. 9571123980, 9460326317

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All rights reserved. No part of the book may be reproduced
or transmitted in any form or by any means, electronic or
mechanical, including photocopying, recording, or any
information storage and retrieval system without permission,
in writing, from the Author.

FIRST EDITION: 2012

Distribution by
Vandana Publishing House
39, Nepal House
RMV Road
Chhoti Brahmpuri
Udaipur- 313001
A HAND BOOK OF
M ICROBIOLOGY

Dr Sushil Kumar Sahu

Professor & Head
[email protected]
[email protected]
Salutations to the supreme Goddess Saraswati...whose face is fair as a jasmine flower, luminescent like the
moon and delicate as a snow flake ; who is dressed in brilliant white (shubhra-) garments (vastraA-). She
holds the musical instrument (vINA-) in her hands to bestow boons (varada.nDa-) to her disciples as she sits
on her white (shveta-) lotus (padma-) throne (aasanA-).

Your right is to work only,

But never to its fruits;
Let not the fruits of action be thy motive,
Nor let thy attachment be to inaction.

This book is dedicated

to my
Mother & Father

“u Roga dke;s jkT;e~ u LoxZ uk iquHkZoe~A

dke;s nq:[krIrkuka izkf.kuke~ vkfrZuk”kue~A”
(Not desire for a kingdom, nor the heaven, nor freedom form rebirth. Let on desire for total
freedom of the ailing creatures from suffering and diseases.)
 Preface of the first Edition
The primary purpose of this book is to serve as a handbook for undergraduate
Medical, Dental and Paramedical students. There is a great need and demand
of the comprehensive book in field of Microbiology. It is examination
oriented, simple and easy to understand book. It is saves several man
hours by covering relevant material. It is ideal as a quick desktop
reference, ready reckoned and last moment revision book. It is increase
the performance and marks in examination. It is focus on the topics
which are frequently asked questions in various university
examinations. Book is covered last years and frequently asked
questions in university examinations. Most important points are
presented in bold and italics to make revision easier. Colored
photographs and diagrams are included to easy understand the topics
of microbiology.

In spite of this, there is still a chance of some changes. I shall be

grateful, if any such is brought to notice for consideration in the next
edition.

I am very grateful to my wife, Son and daughter for their moral support
for this book.

Dr Sushil Kumar Sahu

 CONTENTS
Colour images

1 History of Microbiology 01 Chemical Sterilization 32

Vapour disinfectant 34
Louis Pasteur 01
Pasteurization 02 6 Genetics 35
Robert Koch 02 Transposon 36
Koch’s Postules 03 Gene transfer 36
Koch Phenomenon 03 Transformation 36
Leeuwenhock 04 Transduction 37
Conjugation 37
2 Microscope 06 Lysogenic conversion 38
Fluorescence Microscope 06 Lac-Operon 39
Electron Microscope 07 Mutation 40
Dark-ground Microscope 08 Genetic engireering 41
Phase-Contrast Microscope 08 Restricted endonuclease 42
Interference 09 DNA probe 42
3 Staining 10 Blotting technique 43
Gram’s Staining 10 Microbial Pathogenicity 43
Z.N. Staining 12
Albert’s Staining 13 7 Immunity 45
Innate Immunity 45
4 Morphology of Bacteria 14 Acquired Immunity 47
Cell wall 14 Active Immunity 48
Capsule 16 Passive Immunity 48
Flagella 17 Herd Immunity 49
Fimbriae 19 Adoptive immunity 49
Endospore 20
Sporulation 21 8 Antigen 50
Bacterial growth curve 22 Hapten 50
Heterophil Antigen 51
5 Sterilization 25 Human Leucocyte Antigen 52
Autoclaving 27 Super antigen 53
Hot air oven 29
Pasteurization 30 9 Antibody 54
Inspissation 30 Stucture of Iimmunoglobulin 54
Tyndalization 31 IgG Antibody 55
Disinfectant 31 IgA Antibody 56
IgM Antibody 56

i
IgD Antibody 57
IgE Antibody 58 15 Neisseria 94
Bence Jones Protein 58 N. gonorrhoeae 94
N. meningiditis 95
10 Antigen Antibody Reaction 59
Precipitation Reaction 59 16 Diphtheria 98
Gel Diffusion 61 Diphtheria 98
Immunoelectrophoresis 61 Diphtheria Vaccination 101
Electroimmunodiffusion 62 Schick test 101
Agglutination 63 Virulence Tests 102
Prozone Phenomenon 65 Elek's gel Precipitation test 102
Coomb's test 66 Lysogenic Conversion 103
Immunofluoroscence 67
ELISA 69 17 Bacillus 104
PCR 72 Anthrax 104
Importance of Bacillus 105
11 Hypersensitivity 75 McFadyean’s Reaction 106
Type I Hypersensitivity 76
Type II Hypersensitivity 77 18 Clostridium 107
Type III Hypersensitivity 78 Classification of clostridium 107
Type IV Hypersensitivity 79 Clostridium Perfringens 108
Gas Gangrene 108
12 Immunisation 80 Nagler’s Reaction 109
Immunization 80 Reverse CAMP test 110
Vaccine 80 Tetanus 111
National immunization 81 Clostridium Botulinum 112
Pulse Polio programme 82 Clostridium Difficile 115
Rabies Vaccine 82 Anaerobiosis 115

13 Staphylococci 85 19 Enterobacteriaceae 118

Pathogenesis 85 Escherichia 119
Laboratory diagnosis 87 Traveler’s diarrhea 120
Coagulase test 87 Shigella 121
Salmonella 122
14 Streptococci 89 Enteric Fever 122
Classification of Streptococci 89 Blood Culture 124
Lancefield Classification 90 Widal Test 125
Griffith Typing 90 Typhoid Vaccine 127
Pathogenesis 90
ASO test 92 20 Vibrio 128
CAMP test 92 V. Cholera 128
Quellung Reaction 93 V. Parahaemolyticum 131

ii
21 Plague 132 29 General Virology 165
Plague 132 Laboratory Diagnosis of virus 165
Cultivation of Virus 166
22 Haemophilus 134 Tissue Culture 167
Satallitism 134 Prion 168
Chancroid 135 Inclusion body 169
Viroid 169
23 Bordetella 136
Whooping cough 136 30 Bacteriophage 170
HACEK 137
31 Herpes viruses 173
24 Tuberculosis 138 Herpes Simplex 174
Tuberculosis 138 Varicella- Zoster 175
Tuberculin Test 142 Cytomegalovirus 176
Montoux Test 142 Epstein Barr Virus 177
MOTT 142 Paul Bunnel Test 178
Leprosy 144
Lepromin Test 145 32 Polio Virus 179
Polio 179
25 Spirochaete 146 Polio Vaccine 180
Syphilis 146 Pulse Polio Programme 181
Pathogenesis 147
Laboratory Diagnosis 148 33 Orthomyxovirus 182
VDRL/RPR 150 Swine Flu 182
Non treponemal Disease 151 Bird flu 184
Leptospirosis 152 34 Paramyxovirus 185
Mumps 185
26 Actinomycetes 154 Measles 186
Mycetoma foot 154
Actinomyces 155 35 Rhabdovirus 187
Nocardia 156 Rabies Virus 187
Rabies Vaccine 188
27 Rickettsia 157 Neural Vaccine 189
Typhus Fever 157
Weil-Felix Reaction 158 36 Arbovirus 190
Neil- Mooser reaction 158 Dengue 191
Q-Fever 160 Chikunguniya 192

28 Chlamydia 161 37 Rubella virus 194

Chlamydia Trachomatis 161 Rubella Virus 194
Frei’s Test 163 Rota Virus 195

iii
38 Viral Hepatitis 196 Trypnosoma 237
Hepatitis A Virus 197 Leishmania 240
Hepatitis B Virus 197
Hepatitis C Virus 199 44 Cestode 243
Hepatitis D Virus 199 Taenia Saginata 243
Hepatitis E Virus 199 Taenia Solium 246
Cysticercosis 248
39 Retrovirus 200 Echinococcus 249
HIV/AIDS 200
Viral genes 201 45 Trematode 253
Opportunistic infection 203 Fasciola 253
Laboratory Diagnosis 204 Paragonimus 254
HAART 205 Schistosoma 256

40 General Mycology 207 46 Nematode 260

Classification of Fungus 207 Ascaris 260
Laboratory diagnosis 209 Ankylostoma 262
Larva Migrans 265
41 Medical mycology 210 Wucheria 268
Dermatophytes 210 Guinea Worm 272
Chromomycosis 211
Sporotrichosis 212 47 Normal Flora 275
Rhinosporidiosis 212 48 Nosocomial Infection 277
Histoplasmosis 212 49 Universal Precaution 281
Cryptococcosis 213 50 Biomedical Waste 284
51 P.U.O 288
42 Opportunistic mycosis 214 52 Diarrhoea 294
Candidiasis 215 53 Meningitis 303
Aspergillosis 216 54 STD 306
Penicillosis 217 55 Respiratory infection 311
Pneumocytosis 218 56 Urinary infection 314
57 Differences 318
43 Protozoa 219
Entoamoeba 219
Giardia 223
Trichomanas 225
Plasmodium 227

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 HANDBOOK OF MICROBIOLOGY

t; Jh d`’.kk t; ekW ljLofr

1

Louis Pasteur
Pasteurization
Robert Koch
Koch's postules
Koch's phenomenon
Leeuwenhoek

Louis Pasteur
Q. Write short note on the Louis Pasteur? (B.Sc. Nur.-2007)
Q. Write short note on the contribution of Louis Pasteur in microbiology?
(MBBS-2003)

ANSWER
Louis Pasteur (1822-1895) was a trained chemist of France.
He is known as father of modern microbiology.
He established that fermentation was caused by microbial agents.
He further noted that different types of fermentations were associated with
different kinds of microorganisms.

Important Contributions of Louis Pasteur in Microbiology

1. He established that fermentation was caused by microbial agents.
2. Different types of fermentations were associated with different kinds of
microorganisms.
3. Developed various methods and techniques of bacteriology.
4. Proved conclusively that all forms of life, even microbes, arose only from
their like and not de novo.
5. Introduced sterilization techniques and developed steam sterilizer,

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 HANDBOOK OF MICROBIOLOGY
autoclave and hot-air oven.
6. Studies on anthrax, chicken cholera and hydrophobia.
7. Pasteur developed vaccine for hydrophobia. He obtained the fixed virus of
rabies by serial intracerebral passage in rabbits.
8. Pasteur coined the term ‘Vaccine’.
9. Pasteurization method for sterilization of milk and butter introduced.

Pasteurization
Q. Write short note on the pasteurization?

ANSWER
It is a moist heat at below 100oC temperature sterilization method.
It is first time used by Louis Pasteur for sterilization of milk and butter.
This method was further divided into two types.
(1) Holder Method (630C for 30 minutes)
(2) Flash Method (720C for 20 seconds)
After applying either method do rapid cooling to 130C or lower.
By this method nonsporing organism such as mycobacterium, Brucella and
salmonellae are destroyed.
Coxiella burnetii causative agent of Q-fever may survive in holder method.

Robert Koch
Q. Write short note on the Robert Koch? (B.Sc. Nur.-2011)
Q. Write short note on the contribution of Robert Koch in microbiology?

ANSWER
Robert Koch (1843-1910) was a German general practitioner.
He is known as the father of bacteriology.

Important Contributions of Louis Pasteur in Microbiology

1. Introduced methods for isolation of pure strains of bacteria.
2. Introduced methods of obtaining bacteria in pure cultures using solid
media.
3. Introduced staining techniques.
4. Discovered the anthrax bacillus (1876), tubercle bacillus (1882) and the
Cholera vibrios (1883).
5. Introduced Koch's postulates.
6. Introduced Koch's phenomenon.

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 HANDBOOK OF MICROBIOLOGY

Koch's postules
Q. Write short note on the Koch’s postules? (MBBS-2006)

ANSWER
According to Koch's postulates, a microorganism can be accepted as the
causative agent of an infectious disease only if the following conditions are
fulfilled.

(i) The organism should be constantly associated with the lesions of the
disease.
(ii) It should be possible to isolate the organism in pure culture from the
lesions of the disease.
(iii) The isolated organism (in pure culture) when inoculated in suitable
laboratory animals should produce a similar disease.
(iv) It should be possible to re-isolate the organism in pure culture from
the lesions produced in the experimental animals.
(v) An additional criterion introduced subsequently requires that specific
antibodies to the organisms should be demonstrable in the serum of
patients.
Many infectious disease do not fulfill the 3rd criteria (e.g. gonococci), or 2nd
criteria (e.g. Lepra bacilli, treponema) of Koch’s postulates.

Koch's Phenomenon
Q. Write short note on the Koch’s phenomenon? (MBBS-2003)

ANSWER
Koch’s Phenomenon can demonstrate in Mycobacterium tuberculosis.
It was first explained by Robert Koch.
This phenomenon is a combination of hypersensitivity and immunity.
In this, already infected guineapigs with tubercle bacillus responded with an
exaggerated inflammatory response when injected with the tubercle bacillus
or its protein.
This hypersensitivity reaction is called Koch's phenomenon.
Prior sensitized guinea pig is immunized for tubercle bacilli. This is called
Koch's phenomenon.

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 HANDBOOK OF MICROBIOLOGY
Koch’s Phenomenon can be explained in following manner.

Healthy guinea pig Immunized guinea pig

 Virulent tubercle bacilli inoculate  Virulent tubercle bacilli inoculate
in healthy guinea pig. in prior injection of tubercle
 Puncture site heals quickly and bacilli 4-6 weeks earlier in guinea
there is no immediate visible pig.
reaction.  An indurated lesion appears at
 After 10-14 days, a ulcerated the site of injection in a day or
nodule appears at the site of two which undergoes necrosis in
injection. another day.
 Ulcer persists till the animal dies.  A shallow ulcer developed.
 The regional lymph nodes are  Ulcer heals rapidly.
enlarged and caseous.  No involvement of the regional
lymph nodes or tissues.
 Animal not dies.

Antony Van Leeuwenhoek

Q. Write short note on the contribution of Antony van Leeuwenhoek.

ANSWER
Contribution of Antony Van Leeuwenhoek
He is commonly known as "the Father of Microbiology", and considered to be
the first microbiologist.
Antony van Leeuwenhoek (pronounced Lay-wenhook) (1632-1723), a citizen
of Delft, Holland.
He became expert in the grinding of simple magnifying lenses.
He constructed many of these microscopes each containing a single lens
ground by himself.
The best of lenses magnified about 200 times
He was first time accurately described the different shapes of bacteria (coccal,
bacillary and spiral).
He gave first complete account of the red blood cell.
He demonstrated the capillary connections between arteries and veins and
made other important anatomical observations.
In 1677, he described for the first time the spermatozoa from insects, dogs,
and man.
He studied the structure the optic lens, striations in muscles, the mouth parts
of insects and the fine structure of plants.

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 HANDBOOK OF MICROBIOLOGY

Name of scientist Importance

Antony van leeuwenhoek Father of Microbiology

Louise Pasteur Father of Bacteriology &
Father of Modern Microbiology
Robert Koch Father of Medical Microbiology
Paul Ehrlich Father of Chemotherapy
Joseph Lister Father of Antiseptic surgery
Metchnikoff Discovered Phagocytosis
Ronald Ross Discovered Malaria
Alexander Fleming Discovered Penicillin
Klebs and loeffler Discovered Coryn. Diphtheria
Yersin Discovered Yersinia Pestis
Neisser Discovered Neisseria Gonococci
Hansan Discovered Mycobacterium leprae
Robert Koch Discovered Anthrax, Myco. Tuberculosis and
Vibrio Cholera
Karl Landsteiner Discovered ABO blood groups
Watson and Crick Discovered Double Helix of DNA
Edward Jenner Discovered Small Pox Vaccine
Ruska Discovered Electron Microscope
Stanely B Prusiner Discovered Prion
Kary Mullis Discovered PCR technique
Twort and d’ Herelle Described Bacteriophage

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 HANDBOOK OF MICROBIOLOGY

2
M IC R O SC O P E

Fluorescence Microscope
Electron Microscope
Dark-ground Microscope
Phase-Contrast Microscope

Fluorescence Microscope
Q. Write short note on the Fluorescence Microscope?

ANSWER
When ultraviolet or short-wavelength or invisible light falls on a fluorescent
substance, the wavelength of the invisible light increases so that it becomes
luminous and is said to fluoresce.
If tissues, cells or bacteria are stained with a fluorescent dye and examined
under the microscope with Ultraviolet light, a bright luminous objects seen
against a dark background.
The fluorochrome dyes auramine and rhodamine can be used to demonstrate
acid-fast bacilli.
It is also use for detection of antibody and antigen.
It is viewed by fluorescence microscopy, the bacterial cells appear yellow
against a dark background when potassium permanganate is used as a
counter stain.
Commonly used fluorescent dyes are fluorescein isothiocyanate, lissamine
and rhodamine.
S e e C o l o r Im a g e 6

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 HANDBOOK OF MICROBIOLOGY

Electron Microscope
Q. Write short note on the electron microscope?

Electron Microscope is increase resolving power to observe the detailed

structures of prokaryotic and eukaryotic cells.
A beam of electrons is employed instead of the beam of light used in the
optical microscope.
Electrons have a much shorter wavelength than the photons of white light.
The electron beam is focused by circular electromagnets.
The object which is held in the path of the beam scatters the electrons and
produces an image which is focused on a fluorescent viewing screen.
The wavelength of electrons used in an Electron Microscope is 0.005 nm as
compared to 500 nm with visible light i.e. about 100,000 times shorter than
that of ordinary light.
Important techniques used in electron microsopy
(1) Shadow Casting:
It is deposition of thin layer of metal like platinum on the object.
(2) Negative staining with phosphotungstic acid
(3) Scanning electron microscope:
It is a recent development and provides a three dimensional image of
the surface of the object.
(4) Freeze-etching:
It is use for examine the living cell structure.
In this technique, rapidly cool the specimens by deep freezing in liquid
gas and form replica of carbon-platinum.

Electron Microscope

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 HANDBOOK OF MICROBIOLOGY

Dark-ground (Dark-field) Microscope

Q. Write short note on the dark ground microscope?

ANSWER
Dark ground microscope is used for Treponema pallidum and Flagella
detection.
A special condenser is used by which specimen is illuminated by oblique light
only.
The rays do not enter the tube of the microscope, and in consequence do not
reach the eye of the observer unless they are scattered by objects (e.g.
bacteria).
The organisms appear brightly illuminated against a dark background.
One of the major drawbacks to using a dark field microscope is that it requires
high intensity light, because the light levels tend to be low.
The extremely bright light can damage some specimens

Dark field microscope

Phase-Contrast Microscope
Q. Write short note on the phase contrast microscope?

ANSWER
Light waves passing through transparent objects such as cells.
Emerge different phases depending on the properties of the materials
through which they pass.
Some structures appear darker than others.
It can be used to reveal some details of the internal structures in living cells.

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 HANDBOOK OF MICROBIOLOGY

A light microscope can be converted into phase contrast microscope by using

a special sondenser and objective lens.

Phase contrast Microscope

Interference Microscope
Q. Write short note on the phase contrast microscope?

ANSWER
Interference microscopy (also referred to as quantitative interference
microscopy) uses two separate light beams with much greater lateral
separation than that used in phase contrast microscopy.
Two images are produced of every object (one being the "ghost image").
The main advantage offered by interference microscopy measurements is the
possibility of measuring the projected dry mass of living cells like cell
organelles, lipids, proteins and nucleic acid.

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 HANDBOOK OF MICROBIOLOGY

3
S TA IN IN G

Gram’s Staining
Z.N. Staining
Albert’s Staining

Gram’s Staining
Q. Write short note on the Gram’s staining? (B.Sc. Nur.-2011), (MBBS-2006)

ANSWER
The Gram stain was introduced by Christian Gram a Danish Physician in 1884.
This is the most frequently used differential stain that divides bacteria’s in 2
groups.
1. Gram Positive
2. Gram Negative
Principle:-
 Gram positive bacteria have more acidic protoplasm (PH 2-3)
which binds more strongly with basic primary dye as compare
to gram negative bacteria (PH 4-5).
 The violet basic dye and the iodine form a dye- iodine
complex inside the both Gram positive & Gram negative
organism.
How ever, dye iodine complex is retained in Gram positive
organism by the thick peptidoglycan mesh.
In Gram Positive bacteria:
The cell wall is thicker and 50-90 % of peptidoglcan is present.
The dye iodine complex is not wash out during decolorization
& the organism appear dark Purple.

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 HANDBOOK OF MICROBIOLOGY

In Gram Negative bacteria:

The cell wall is thinner and peptidoglycan is present only 5-10
% of cell wall.
The complex is wash out during decolarization & the organism
take the colour of counter stain ( pink or green ).
Reagents:-
1. Primary stain is Crystal violet (2%)/ Methyl violet 0.5 %/
Gentian violet (1%)
2. Gram’s iodine
3. Acetone/ Alcohol
4. Counter stain dilute carbol fuchsin(1:10)/ Safranin (0.1%)

Procedure:-
1. Place the slide on staining glass rod.
2. Cover the smear with methyl violet and leave for 1minutes.
3. Wash the slide under tap water.
4. Cover the smear with Gram’s iodine and leave it for 1
minutes.
5. Drain off the iodine.
6. Decolourize with alcohol or acetone.
7. Acetone is the stronger decolourizer.
It is applied to smear for only 2-3 seconds while alcohol acts
more slowly than acetone and should be applied and
reapplied for 8-10 seconds.
8. Wash under tap water.
9. Counter stain the smear with dilute carbol fuchsin or safranin
for 30 second.
10. Wash the slide under tap water and allow the stained smear
to dry in air.
11. Put a drop of cedar-wood oil / liquid paraffin on the stained
smear and observe under oil immersion lens (100 X) of
microscope.

S e e C o lo r I m a g e 1 & 2

Result:-
1. Gram-Positive bacteria Dark purple
2. Gram-Negative bacteria Pink
3. Yeast cells Dark purple
4. Other structures Pink

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 HANDBOOK OF MICROBIOLOGY

Ziehl-Neelsen Staining
Q. Write short note on the Z. N. staining? (MBBS-2005)
Q. Write short note on the Acid Fast Staining? (BDS-2009), (BDS-2009 {O})

ANSWER
 Z-N staining method is used for staining the Acid Fast Bacilli.
 It is also known as Acid Fast Staining.
 Acid fast bacilli do not stain readily with dilute solution of dyes.
 Once stained they resist decolourization.
 Original method of AFB staining was in introduced by Ehrlich-1882.
 It was improved by Ziehl-Neelsen in 1885.
Principle:-
Acid fastness has been attributed to the high content of lipid mainly
mycolic acid, fatty acid and higher alcohol.
Basic fuchsin in combination with phenol penetrates the cell wall and
stains the bacilli.
If once stained, they resist decolourization with strong acid.
Procedure:-
1. Prepare smear and pour hot strong carbol fuchsin over the
slide.
2. Heating helps in penetration of dye through mycolic cell wall.
3. Wash the slide with tap water.
4. Decolorize the smear with 1% HCl in 70% alcohol or 20%
sulfuric acid for tubercle bacilli and 5% for lepra bacilli.
Decolourisation is continued with 70% neutral alcohol.
5. Wash the smear under tap water till the film appears faint
pink.
6. Counter stain done with Loeffler’s methylene blue. Wash with
water then allow it to dry.
7. Put a drop of cedar wood oil and observe in microscope.

S e e C o lo r Im a g e 3
Uses
1. Useful in detection of Acid Fast organism.
2. No. of AFB in a smear may be counted and the grading of smear is
done.
Grading of Microscopy smear (Mycobacterium Tuberculosis)
Examination Grading
1 or 2 per 300 oil immersion fields Doubt ful or repeat.
1-9 AFB per 100 oil immersion fields 1+
1-9 AFB per 10 oil immersion fields 2+
1-9 AFB per oil immersion fields 3+
> 9 AFB per oil immersion fields 4+

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 HANDBOOK OF MICROBIOLOGY

E.g. of Acid Fast organism:-

1. Tubercle bacilli. 2. Lepra bacilli.
3. Atypical bacilli. 4. Nocardia sp.
5. Legionella. 6. Oocyst Cryptosparidium
parvam.
7. Isospora belli 8. Actinomyces.
9. Head of sperm

Other method of Acid fast Staining.

1. Kinyoun acid fast stain/ Cold stain
2. Flurochrome stain

Albert Staining
Q. Write short note on the Albert’s staining? (MBBS-2004)

ANSWER
Albert staining method is commonly used for staining of Corynebacterium
diptheriae.
C.diphtheriae contains metachromatic granules which are also known as
Babes- Ernst bodies or Volutin granules & consist of inorganic
polymetaphosphates as a energy storage.
With Albert stain, the granules stain bluish black & cytoplasm green.
Reagents :-
(i). Albert’s stain (ii) Albert’s Iodine
Toludine blue 0.15 gm Iodine 2 g
Malachite green 0.2gm Potassium iodide 3g
Glacial acetic acid 1ml Distilled water 300ml
Alcohol 95% 2ml
Distilled water 100ml
Procedure:-
1. Cover the smear with Albert’s stain allows acting for 3-5 minutes.
2. Drain the staining solution, but do not wash.
3. Cover the smear with Albert’s iodine and allow acting for 1
minute. Wash with water, dry it & observe in 100X.
Result:-
Metachromatic granules- Bluish Black
Bacillary body or Protoplasm- Green
Chinase letters pattern or cuineform C. diphtheria seen.
S e e C o l o r Im a g e 4

Use:-
It is useful in demonstration of metachromatic granules in C.
diptheriae.

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 HANDBOOK OF MICROBIOLOGY

4
MORPHOLOGY OF BACTERIA

C ell W all
C ap su le
F l a g e ll a
F im b ria e
E n d o sp o re

Bacterial Cell Wall

Q. Write short note on the bacterial cell wall? (MBBS-2007), (MBBS-2005)

ANSWER
Cell wall gives bacteria their definite shape.
It is permeable to passage of liquid nutrient material into the cell.
It is about 10-20 nm in thickness and constitutes 20-30% of dry weight at the
cell.
Gram positive bacteria cell walls are generally thicker than those of gram-
negative
The strength of the bacterial] cell wall is due to peptidoglycan, mucopeptide
or murein.
Peptidoglycan consists of three parts:
(1) A backbone, composed of alternating N-acetylglucosamine and
N- acetylmuramic acid.
(2) A set of identical tetrapeptide side chains attached to
N-acetylmuramic acid.
(3) A set of identical pentapeptide cross-linked tetrapeptide side chain.

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 HANDBOOK OF MICROBIOLOGY

Gram positive c ell wall

Gram-positive bacterial cell wall

Thickness 80nm
Peptidoglycan constitutes 50-90% of the dry weight of the wall.
Gram-positive cell wall also contains teichoic acids and polysaccharides.

Gram negative c ell wa ll

Gram-negative bacterial cell wall

The cell wall is thinner than that of gram-positive bacteria
structurally more complex.

Consists of cell wall are

(1) Periplasmic space: Space between cytoplasmic and outer membrane.
Periplasmic space contains degradative enzymes like β-lactamase,
protease etc and specific binding & transport protein for vitamins.
(2) Peptidoglycan: Constitutes 5-10% of the dry weight of the wall.
(3) Lipoprotein
(4) Outer membrane: made by outer membrane protein and porin protein.
(5) Lipopolysaccharide: It is consist
(a) Lipid A It is endotoxin, cause toxicity
(b) Core
(c) O antigen Polysaccharide surface antigen

Acid-fast cell wall

Certain genera (Mycobacterium and Nocardia) have a gram-positive cell wall
structure.

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 HANDBOOK OF MICROBIOLOGY
It is contain a waxy layer of glycolipids and fatty acids (mycolic acid) bound to
the exterior of the cell wall.

Functions of cell wall

 Provides shape to bacteria

 Gives rigidity to organism
 Protects from environment
 Contains receptor sites for phages
 Site of action of antibody
 Provide attachment to complement
 Contains components toxic to host

Capsule
Q. Write short note on the capsule? (B.Sc. Nur.-2009), (MBBS-2010), (MBBS-
2006), (MBBS-2005)

ANSWER
Discrete, thickened and organized gel around cell called capsule.
If capsule is too thin and detected by only quelling reaction know as
microcapsule.
Loose, undemarcated and viscid secretion around cell surface called slime
layer.
In most species, it is made up of a complex polysaccharide.
In some species, it is polypeptide or protein (Bacillus).

Capsule can be visualized by

(1) By suspending the organisms in negative stain (India ink or nigrosin)
(2) By special capsular staining methods such as muir’s stain.
(3) By reaction with specific antibody which causes a characteristic swelling of
the capsule. It is known as Quellung reaction.

Diplococci with Capsule

Capsule containing bacteria Example:

Streptococcus pneumoniae,

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 HANDBOOK OF MICROBIOLOGY

Neisseria meningitidis,
Several groups of streptococci,
Klebsiella,
Haemophilus influenzae
Yersinia and
Bacillus (capsule made up of poly peptide)
Cryptococcus neoformans
Function of capsule:
(1) Inhibit phagocytosis
(2) Increase virulence of bacteria
(3) Protect cell wall from bacteriophage, colicines, complement,
Enzymes
(4) Adherence to host cell and form biofilm to adhere with their other
species.
(5) Use as an antigen to stimulate immune response and in Quelling
Reaction.
It is inhibit phagocytosis thus contributing to the virulence of the bacteria.
Bacteria tend to lose capsules on repeated subcultures in vitro.

Flagella
Q. Write short note on the flagella? (MBBS-2007)

ANSWER
Flagella are long, hollow, helical filaments usually several times the length of
the cell.
There are five types of arrangement of flagella
1. Monotrichous: These organisms have a single polar flagellum. E.g. Vibrio
Cholerae
2. Lophotrichous: They have a tuft of flagella at one pole. E.g. Spirilla
3. Amphitrichous: They have single polar flagellum at both poles.
E.g. Alcaligenes faecalis and Pseudomonas aeroginosa
4. Peritrichous: Flagella are distributed all round the cell. E.g. Salmonella
5. Amphilophotrichous: They have tuft of flagella at both ends.
On microscopic examination of wet films, motile bacteria are seen swimming
in different directions across the field.
Different type of motility are:
(1) Darting (V. cholerae)(200µm/Sec) (2) Very active (Proteus spp.),
(3) Active (Escherichia coli) (4) Sluggish and tumbling
(Listeria monocytogenes)
(5) Gliding (mycoplasma pneumoniae) (6) Corkscrew (campylobacter &
spirochaetes)
(7) Swarming (Proteus)

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Flagella demonstrated by
Direct method
Ordinary light microscope by hanging drop preparation
Dark ground microscope
Electron microscope
Indirect method
Spreading growth on semi solid agar
Cragie`s tube
Swarming on solid medium
Mannitol motility medium
Immobilisation antibody test (Treponema)
The flagellum consists of three parts:
The filament,
The hook and
The basal body
The basal body, anchored in the cytoplasmic membrane, comprises a rod and
two or more sets of encircling rings.
In gram-negative bacteria four types of rings (M,S, P and L) are seen.
Ring M it attaches to the cytoplasmic membrane
Ring S is located just above cytoplasmic membrane
Ring P attached to peptidoglycan
Ring L attached to outer lipopolysaccharide membrane
Rings P and L are absent in gram-positive bacteria.
The motility in spirochaetes is due to one or more pairs of endoflagella which
run between outer membrane and peptidoglycan layer.
Flagella consist mainly of a protein called flagellin.

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Structure of F lagella
Function of flagella
(1) As a locomotive organ
(2) Protect bacteria from unfavourable environment
(3) Increase virulence
(4) Antigenicity
(5) Site of attachment of bacteriophage

Fimbriae or Pili
Q. Write short note on the pili?
Q. Write short note on the sex pili?
Q. Write short note on the fimbriae?

ANSWER
Fimbriae or pili are hair-like microfibrils.
They are straighter, thinner and shorter than flagella.
They are present on many gram-negative cells.
They are composed of protein called pilin.
It is help in adherence to other cells.
They occur in both flagellated and non-flagellated bacteria.
Each bacterium possesses 100-500 peritrichously borne fimbriae.
It can be seen by electron microscopy.
Originate from cytoplasmic membrane.

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 HANDBOOK OF MICROBIOLOGY
Types of fimbriae
(1) Common pili
(2) Sex Or F (fertility) pili
(3) Col (colicin) pili
Sex pili:
It is longer and thinner than the common type.
It is hollow and constitutes conjugation tubes through which DNA is
transferred from one organism to another during conjugation.
They are determined by sex factors.

Function of fimbriae
(1) Adhesion
(2) Transfer of genetic material
(3) For colonization of bacteria
Fimbriae demonstrated by
(1) Electron microscope
(2) Haemagglutination

Spores
Q. Write short note on the spore? (MBBS-2006)
Q. Write short note on the endospore? (B.Sc. Nur.-2007)
Q. Write short note on the sporulation?

ANSWER
Structure of spore

Spores are small, highly resistant, metabolically dormant structures

Spore develops as a response to starvation.
Formation of endospores occurs over a period of 4-8 hours after depletion
carbohydrate supply.

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 HANDBOOK OF MICROBIOLOGY

Type of spore

Free spore

Oval central

Spherical central Non bulging

Oval sub terminal

Oval sub terminal

Oval terminal Bulging

Spherical terminal

Shape & position of bacterial spore

Spores can remain dormant for many years.

They are extremely resistant to chemical and physical agents.
For killing of spore require moist heat at 100-1200C for 10 minutes.
Marked resistance of the spores is due to:
- Impermeability of their cortex and outer coat.
- High content of calcium and dipicolinic acid.
- Low content of water.
- Very low metabolic and enzymatic activity.
Spore can be demonstrated by
In unstained preparation: Within parent cell greater refractivity.
In simple stains preparation:
1 Gram staining: remains unstained and appears as a clear space within
the stained cell protoplasm.
2 Modified Ziehl-Neelsen staining with 0.25 – 0.5% sulphuric acid.
Laboratory use of spore
Some spore use in sterilization control.
(1) Bacillus stearothermophilus: Autoclaving
(2) Bacillus Subtilis: Hot air oven
Sporulation
Sporulation in bacteria, is a method of preservation. Each bacterium forms
one spore which on germination forms a single vegetative cell.

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Sporulation process

Germination
The process of conversion of a spore into a vegetative cell under suitable
environment is known as germination.
Germination may occur in less than two hours in optimal condition.
Germination consist three stages.
(1) Activation (2) Initiation (3) Outgrowth

Bacterial Growth Curve

Q. Write short note on the bacterial growth curve? (B.Sc. Nur.-2008), (MBBS-
2008), (MBBS-2005)

ANSWER
Definition:
When a bacterium is inoculated into a suitable liquid culture medium and
incubated under standard condition, sample is collected at regular interval for
total & viable count. When these data plotted on log paper in relation to time
a characteristic curve is obtained. It is known as Bacteial Growth Curve.

A typical growth curve contains four major phases

1. Lag phase:
It is a period between inoculation and beginning of multiplication.
Organisms adapt themselves
1 to growth in fresh medium
2 increase in size and metabolic activity.
The duration of lag phase varies depend upon
The species
Nature of culture medium
Temperature of incubation

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 HANDBOOK OF MICROBIOLOGY

It is generally shorter in a nutrient-rich than in a minimal medium.

Sep 07 Dr Ekta, M icrobiology, GMCA

B a c t e ri a l g ro w t h c u r v e

2. Log or exponential growth phase:

Bacteria are multiplying at their maximum rate and their number increases
exponentially with time.
The number of organisms present in each generation period is almost twice
than in previous period.

Exponential phase is of limited duration because of:

• Exhaustion of nutrients.
• Accumulation of toxic metabolic end products.
• Rise in cell density.
• Change in pH.
• Decrease in oxygen tension (in case of aerobic organisms).

3. Stationary phase:
The number of viable cells remains constant.
Balance between the bacterial reproduction and bacterial death.

4. Phase of decline:
Stationary phase is followed to phase of decline.
Rate of death exceeds the rate of reproduction
The number of viable cells declines.
After a variable period, all the cells die and culture becomes sterile.

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 HANDBOOK OF MICROBIOLOGY

Morphological and physiological changes of bacterial cell during growth

curve

The end of the lag phase Cell achieve maximum size

In the log phase Smaller and uniformally stain cell
In the stationary phase Gram variable and irregular staining seen
Sporulation occurs
Exotoxin produce
In the phase of decline Involution form seen

Special points
Bacteria can be classified on the basis of nutrition in to following types.
(1) Phototrophs: These derive energy from the sunlight.
(2) Chemotrophs: These derive energy from the chemicals.
(3) Autotrophs: These form energy self from the atmospheric CO2 & N2.
(4) Heterotrophs: These derive energy from preformed compound of
animal and human.
Bacteria can be classified on the basis of temperature in to following types.
(1) Mesophiles: Grown between 250C and 400C.
e.g.majority of pathological bacteria
(2) Psychrophiles: Below 200C. e.g. Soil and water saprophytes.
(3) Thermophilus: Between 550C and 800C.
e.g. Bacillus stearothermophilus
Generation time:
The time required for a bacterium to give rise to two daughter cells.
In E.coli- 20 mintutes,
In tubercle bacilli 20 hours,
In lepra bacilli 20 days.

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5
S T E R IL IZ A T I O N

S te r iliz a tio n
H o t a ir o v e n
A u to c lav in g
P a st e u r iza t io n
T y n d a l iz a t i o n
In sp issa tio n
D is in fe c ta n t

Sterilization

Q. Define sterilization, disinfection and antisepsis. Classified the sterilization

method and describe autoclaving ? (B.Sc. Nur.-2010) , (B.Sc. Nur.-2009),
(BDS-2009), (BDS-2009{O}), (MBBS-2007), (MBBS-2006)

ANSWER
STERILIZATION (MBBS-2005), (MBBS-2004)
The process by which an article, surface or medium is freed of all living
microorganisms either in vegetative or spore state.

DISINFECTION (MBBS-2005)
Destruction of all vegetative forms of organism which might cause disease or
spoilage of food.

ANTISEPSIS
It means prevention of infection, usually by inhibiting the growth of bacteria
in wounds or tissue.

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CLASSIFICATION OF STERILISATION METHODS
A. Physical methods
1. Sunlight
2. Heat
(a) Dry heat (B.Sc. Nur.-2008)
(i) Red heat (ii) Flaming (iii) Incineration (iv) Hot air oven
(b) Moist heat (BDS-2010), (BDS-2006)
(i) Temperature below 1000C
(a) Pasteurisation of milk (b) Inspissation
(c) Vaccine bath
(d) Low temperature steam formaldehyde (LTSF) sterilization
(ii) At a Temperature of 1000C
(a) Boiling
(b) Tyndallisation or intermittent sterlisation
(iii)Temperature above 1000C
(a) Autoclaving
3. Filtration
(i) Candle filters e.g. Berkefeld, Chamberland filter
(ii) Asbestos disc filters e.g. Seitz filter
(iii) Sintered glass filters
(iv) Membrane filters
(v) Air filters HEPA (High Efficiency Particulate Air)
(vi) Syringe filters
4. Radiation
(i) Ionising radiations: Gamma rays, X-rays and cosmic rays
(ii) Non-ionising radiations: Infrared and ultraviolet (UV) radiations
B. Chemical methods
1. Alcohols: Ethyl alcohol and isopropyl alcohol
2. Aldehydes: Formaldehyde, gluteraldehyde
3. Phenols:
Cresol, chlorhexidine, chloroxylenol and hexachlorophene
4. Halogens:
Chlorine: Bleaching powder, Sodium hypochlorite and
Chloramine
Iodine: Betadine
5. Oxidizing agents:
(i) Hydrogen peroxide (ii) Peracetic acid
6. Salts:
(i) Merthiolate (ii) Mercuric chloride (iii) Thiomersal
7. Surface active agents:
(i) Anionic, (ii) Cationic, (iii) Nonionic
(iv) Amphoteric compounds
8. Dyes: (i) Aniline dyes (ii) Acridine dyes
9.Vapour phase disinfectants: (i) Formaldehyde gas (ii) Ethylene oxide

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Autoclaving
Q. Write Short note on the autoclaving? (BDS-2010), (BDS-2008), (BDS-2006),
(BDS-2005) , (MBBS-2006), (MBBS-2005)

ANSWER
It is a moist heat sterilization method under steam pressure.
Saturated steam under pressure is more efficient way of sterilization as
compared to dry heat because
 It provides greater lethal action.
 It is quicker in heating up the exposed articles.
 It penetrates the porous material such as cotton wool, stoppers,
paper, cloth wrapper etc.
 Moist heat coagulates and denatured protein.
Autoclaving is mostly suitable for
 Sterilization of culture media, aqueous solution.
 Decontamination of discarded culture and other laboratory
garbage.
 Gloves, stoppers with rubber liner, glassware with rubber
attachment, glass metal syringes, swabs , dressing material linen
etc.
Procedure
 Arrange the material (pre washed & packed) to be sterilized.
 Ensure that there is sufficient quantity of water in the chamber at
the time of autoclaving by checking water level device provided
with the equipment.
 Place the material to be sterilized in wire basket perforated
container loosely.
 Place the container on perforated rack placed above the level of
the water.
 Fasten the lid tightly with steam discharge valve open.
 Switch on the power and allow the water to boil.
 When water boils, steam will come out of the discharge valve so
that air from chamber may be expelled.
 Wait till total air inside the chamber has been replaced by steam.
This can be checked by connecting one end of the rubber tube to
the discharge valve and immersing other end of tube into the
bucket containing water. The discharge gas will pass through the
water, steam will condense and air will bubble through water.
When bubbles cease, it means that air from chamber has been
expelled.
 Close the steam discharge valve.

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 Adjust the valve to predetermined pressure (Normally autoclaves
are adjusted at 1 5Ibs/inch.
 Allow the pressure to increase to pre adjusted pressure.
 Note the time when pressure gauze indicates the requisite
pressure is achieved.
 Allow to continue the same for required time period as indicated
below.
Temparature Pressure Time
115 0C 10 lbs/ inch 20-30 min.
121 0C 15 lbs/ inch 15-20 min.
132 0C 27 lbs/ inch 2 min.
Holding time increases to 30-45mm at 121°C and 15 lbs pressure if plastic
wares are sterilized.
 At the end of holding time switch off the power supply.
 Allow the autoclave to cool slowly which can be seen by gradual
decrease in pressure till it shows zero reading. Allow the wrapping
paper to be dried.
 Put date on each article and place in dust free area for future use.

Mechanism of sterilization in Autoclave

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Quality Control
Biological control:
Bacillus stearothermophilus is used as a biological control.
An envelope containing a filter paper strip impregnated with Bacillus
stearothermophilus is placed within the load.
After over sterilization strip removed and inoculated into the tryptone soya
broth and incubated aerobically at 550C for five days.
No growth indicates complete and proper sterilization.
Chemical control:
A browne’s tube containing red solution is placed within the load. A change of
colour of the solution to green indicates proper sterilization.
Chemical indicators, autoclave tapes and thermocouples may also be used.

Hot-air oven
Q. Write short note on the hot air oven? (B.Sc. Nur.-2011) , (B.Sc. Nur.-2009),
(B.Sc. Nur.-2008) , (BDS-2007)

ANSWER
It is dry heat method of sterilization.
It is a method of choice for sterilization of
(1) Glassware such as glass syringes, test tubes, petri dishes, pipettes and
flasks,
(2) Metal instruments such as forceps, scissors and scalpels,
(3) Sealed materials such as oils, greases and dry powders which are
impervious to steam and
(4) Swab sticks packed in test tubes.
It is not suitable for materials like fabrics which may be damaged by heat.
Hot-air oven is electrically heated and is fitted with a thermostat that
maintains the chamber air at chosen temperature.
Holding time for sterilization in hot-air oven is
Two hour at 160°C
One hour at 1700C
20 minutes at 180°C.
It is timed as beginning when the thermometer first shows chosen
temperature.

Sterilization controls:
Two types of controls are available.

A. Biological control:
An envelope containing a filter paper strip impregnated with 106 spores of
Bacillus subtilis subsp. niger (NCTC 10075 or ATCC 9372) is placed within the
load.

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After sterilization is over, no growth of B. subtilis subsp. Niger after 5 day in
tryptone soy broth indicates proper sterilization.

B. Chemical control:
A Browne's tube containing red solution is change to green indicates proper
sterilization.

Precautions
(i) It should not be overloaded.
(ii) The material should be arranged in a manner which allows free circulation
of air.
(iii) Material to be sterilised should be perfectly dry.
(iv) Test tubes, flasks etc. should be fitted with cotton plugs.
(v) Petridishes and pipettes should be wrapped in paper.
(vi) Rubber materials (except silicone rubber) or any inflammable material
should not be kept inside the oven.
(vii) The oven must be allowed to cool for two hours before opening the
doors, since the glasswares may crack by sudden cooling.

Pasteurization
Q. Write short note on the pasteurization? (MBBS-2006)

ANSWER
It is a moist heat at below 100oC temperature sterilization method.
It is first time used by Louis Pasteur for sterilization of milk and butter.
This method was further divided into two types.
(1) Holder Method (630C for 30 minutes)
(2) Flash Method (720C for 20 seconds)
After applying either method do rapid cooling to 130C or lower.
By this method nonsporing organism such as mycobacterium, Brucella and
salmonellae are destroyed.
Coxiella burnetii causative agent of Q-fever may survive in holder method.

Inspissation
Q. Write short note on the Inspissation? (MBBS-2003)

ANSWER
It is a moist heat sterilization method below 1000C temperature.
Serum or egg media, such as Lowenstein-Jensen's and Loeffler's serum media
can be sterilized by this method.
In inspissations, media heating at 80-85°C temperature for half an hour daily
on three consecutive days.

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Principal:
First heating at 80-850C for half an hour kill all the vegetative forms and in the
intervals between the heating the remaining spores germinate into vegetative
forms which are killed on subsequent heating.
The instrument used is called inspissator.

Tyndallisation
Q. Write short note on the tyndalization?

ANSWER
Tyndallisation is a moist heat sterilization method at 1000C temperature.
Tyndallisation is also known as intermittent sterilization.
Steam at 100°C for 20 minutes on three successive days is used.

Principal:
First heating at 1000C for half an hour kill all the vegetative forms and in the
intervals between the heating the remaining spores germinate into vegetative
forms which are killed on subsequent heating.
This method is used for sterilization of egg, serum or sugar containing media
which are damaged at higher temperature of autoclave.
The instrument commonly used is Koch's or Arnold's steam steriliser.

Disinfectant
Q. Write short note on the disinfectant?
Q. Write short note on the chemical disinfectant? (MBBS-2007), (MBBS-
2002)

ANSWER
Disinfection
It means the destruction of all pathogens or organisms capable of producing
infections but not necessarily spores.
All organisms may not be killed but the number is reduced to a level that is no
longer harmful to health.
Disinfectants can be divided into three groups:
1. High level disinfectants.
2. Intermediate level disinfectants.
3. Low level disinfectants.
1. High level disinfectants
It is equivalent to sterilization.

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It is used for certain types of endoscopes, cystoscopes and surgical
instruments with plastic components.
Example: Glutaraldehyde, Hydrogen Peroxide, Peracetic acid and Chlorine
compounds.
2. Intermediate level disinfectants
It is not be effective against bacterial spores.
It is used for instruments sterilization. E.g. laryngoscopes, fibroptic
endoscopes.
Examples of intermediate level disinfectants are alcohols, iodophores and
phenolic compounds.
3. Low level disinfectants
Low level disinfectant used for items which come in contact with the patients
but they do not penetrate into tissue.
Stethoscopes, electrocardiogram electrodes etc. are examples of such items.
Examples of low level disinfectants are chlorhexidine and cetrimide.

Chemical disinfectants
1. Alcohols
Ethyl alcohol and isopropyl alcohol are the most frequently used chemical
disinfectant.
It is act by denaturing bacterial proteins.
Human Immunodeficiency Virus is susceptible to 70% ethyl alcohol and 35%
isopropyl alcohol.
It is used mainly as skin antiseptics.

2. Aldehydes (MBBS-2010)
FORMALDEHYDE
It is a bactericidal, sporicidal and virucidal.
A 10% aqueous solution of formalin is routinely used.
Uses
(i) Preservation of tissue for histological examination.
(ii) To sterilize bacterial vaccines.
(iii) To prepare toxoid from toxin.
(iv) For killing of bacterial cultures and suspensions.
(v) For destroying anthrax spores in hair and wool.
(vi) Formalin gas (formaldehyde) also use as ''Vapour phase disinfectants".
GLUTARALDEHYDE
It is effective against bacteria (including M. tuberculosis), fungi and viruses
(including human immunodeficiency viruses and enteroviruses).
It is used as 2% buffered solution.
It is available commercially as 'cidex'.
It is used for delicate instruments having lenses.

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Example cystoscopes, endoscopes and bronchoscope and plastic endotracheal

tubes, face masks, corrugated rubber anaesthetic tubes and metal
instruments.

3. Phenols
Lister, the father of antiseptic surgery, first introduced use of phenol (carbolic
acid) in surgery (1867).
Phenol (1%) has bactericidal action.
Certain phenol derivatives like cresol, chlorhexidine, chloroxylenol and
hexachlorophane are commonly used as antiseptics.
(i) Cresols-
Lysol is a solution of cresols in soap.
It is used for infected glass wares, cleaning floors, disinfection of excreta.
(ii) Chlorhexidine-
Savlon (chlorhexidine and cetrimide) is widely used in wounds, pre-operative
disinfection of skin, as bladder irrigant etc.
(iii) Chloroxylenol-
Ingredient of dettol.
Inactive against Pseudomonas.

4. Halogens
Chlorine and iodine are two commonly used disinfectants.
Chlorine
Chlorine is used in water supplies, swimming pools, food and dairy industries.
Chlorine compounds in three form.
1 Bleaching powder,
2 Sodium hypochlorite and
3 Chloramine are also used.
Bleaching powder or hypochlorite solution are the most widely used for
human immunodeficiency virus (HIV) infected material.
Iodine
Betadine is one example of commonly used iodophores.
5. Oxidising Agents
Hydrogen peroxide
Hydrogen peroxide (H202) is effective at concentration of 3-6%, and for spores
at higher concentration (10-25%).
H202 is used to disinfect contact lenses, surgical prostheses and plastic
implants.
6. Salts
The salts of copper, silver and mercury are used as disinfectant.
They are protein coagulants.
Merthiolate (sodium ethyl mercurithiosalicylateJ is used in a dilution of 1;
10,000 for preservation of sera.
Mercuric chloride,

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Thiomersal and mercurochrome are less toxic and are used as mild
antiseptics.
Copper salts are used as fungicides.

7. Surface Active Disinfectants or Surfactants (MBBS-2002)

It is cause reduction of surface tension.
It is classified into anionic, cationic, nonionic and amphoteric compounds.
(a) Cationic agent:
These include quaternary ammonium compounds which are more active at
alkaline pH and denature proteins. This leads to loss of membrane
permeability. Pseudomonas aeroginosa is particularly resistant.
(b) Anionic Agent:
These include soaps which act better at acid pH and effective against gram
positive. Gram negative resistant due to presence of liposaccharide.
(c) Nonionic agents:
Tween 80 and triton X-100 are example of nonionic agent.
(d) Amphoteric agents:
Tego compounds possess detergent properties of anionic and antimicrobial
activity of cationic compounds.

8. Dyes
Two groups of dyes, aniline dyes and acridine dyes have been used extensively
as skin and wound antiseptics.

Vapour Phase Disinfectants

Q. Write short note on the Vapour Phase Disinfectant?

ANSWER
FORMALDEHYDE GAS
Employed for fumigation of operation theatres, wards laboratories etc.
Formaldehyde gas is generated by adding 150 gm of KMnO4, to 280 ml
formalin for 1000 cu. feet of room volume.
The doors should be sealed and left unopened for 48 hours.
After completion of disinfection, the effect or irritant vapours should be
nullified by exposure to ammonia vapour.
ETHYLENE OXIDE
Used for sterilising plastic and rubber articles, respirators, heart-lung
machines, sutures, dental equipments and clothing.
It is unsuitable for fumigation of rooms because of its explosive nature.
Bacillus globigi (a red pigmented variant of Bacillus subtilisl has been used as
biological control for testing of ethylene oxide sterilisers.

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6
G e n e t ic s

Transposon
G eneT ransfer
LacO peron
M utation
G eneticE ngireeiring
D NAP robe
Blotting

Transposon
Q. Write short note on the Transposable Genetic Elements?

ANSWER
These are specific sequences of DNA segments that have the ability to move
from one plasmid to another plasmid or from chromosomes to plasmid or
within the chromosomes.
Plasmid
A plasmid is a small, circular molecule of DNA that replicates independently of
the bacterial chromosome.
Plasmids carry information required for their own replication.
Because of this mobile nature they named as “jumping genes” or
“transposons”.
A transposon is a segment of DNA with one or more genes in the centre and
two ends carrying inverted repeat sequences of nucleotides.
Properties of Transposable Genetic Elements
(1) Transposable genetic elements can move from any DNA molecule to any
DNA other molecule or even to another location on the same molecule.
(2) The transposable genetic elements do not exist autonomously.
(3) Transposition requires little or no homology between the current location
and the new site.

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 HANDBOOK OF MICROBIOLOGY
Types of Transposable Genetic Elements
1. Insertion sequences (IS)
Insertion sequences are transposable genetic elements that carry no known
genes except those that are required for transposition.
2. Transposons (Tn)
Transposons are transposable genetic elements that carry one or more other
genes in addition to those which are essential for transposition.
Gene transfer
Q. Describe the methods of gene transfer?
Q. Write short note on the genetic material transfer?

ANSWER
Transfer of genetic material from one bacterium to another can take place in
four ways.
(1)Transformation (2) Transduction (3) Lysogenic conversion (4) Conjugation
Vertical and horizontal gene transfer are two ways in which bacteria can
move around and mix up prokaryotic genetic material.
Vertical Gene Transfer (VGT)
Bacteria reproduce through binary fission producing an exact copy.
When organisms replicate their genomes and provide copies to descendants
(future generations), this is considered vertical gene transfer.
Horizontal Gene Transfer (HGT)
HGT is a process in which a prokaryotic cell can acquire genes from other
microbes. There are three types of horizontal gene transfer.
 Transformation
 Transduction
 Conjugation

Transformation
Q. Write short note on the Transformation?

ANSWER
This is the process in which a recipient cell takes up DNA from the
environment (such as DNA released from a dead organism).
Griffith’s Experiments
In 1928, Frederick Griffith discovered transformation while trying to develop a
vaccine for pneumonia caused by Streptococcus pneumoniae.
Griffith was working with two strains of Strep.
Colonies of the first strain appeared shiny and smooth (strain S) because the
bacteria had a protective capsule and this strain caused deadly pneumonia
when injected into mice.
Colonies of the second strain had a rough appearance (strain R) because they
were mutants that could not make the protective capsule.

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This strain did not cause deadly disease because the white blood cells of the
mice could easily destroy these unprotected bacteria.
Griffith did a number of experiments in which he demonstrated that the live R
strain cells could acquire the smooth, virulent feature when exposed to dead S
strain cells.
His experiments indicated that the R strain was taking up genetic material of
the dead, broken down S strain bacteria.

Transduction
Q. Write short note on the transduction?

ANSWER
This process involves the transfer of DNA from one cell to another via a
replicating virus.
Transduction can occur between prokaryotic cells or between eukaryotic cells.
Transduction occurs in bacterial cells by bacteriophage (phage) virus.
When a phage is being replicated inside a host cell, the new viruses self-
assemble from proteins and viral nucleic acid (genetic material) that the host
cell has produced.
Sometimes some of the DNA of the host gets inside a new virus during viral
self-assembly. When that phage then infects another cell, the new host may
incorporate the donated DNA into its chromosome by recombination.
Transduction are two types
(1) Generalized transduction: When any segment of donor DNA involves
randomly.
(2) Restricted transduction: When a specific bacteriophage transduces only a
particular genetic trait.
Role of transduction:
(1) Penicillin resistance in staphylococci
(2) Genetic mapping of bacteria
(3) Genetic engineering in the treatment of inborn metabolic defects

Conjugation
Q. Write short note on the conjugation?
ANSWER
The third method of gene transfer in prokaryotes is called conjugation.
Unlike the processes of transformation and transduction, the donor cell is not
killed during this type of gene transfer.
This process involves one bacterium making a copy of a portion of DNA, called
a plasmid, and transferring that copy to another bacterium.
Plasmid copies can be transferred during the process of conjugation, during
which the donor bacterium extends a rod-like conjugation pilus that connects
with the recipient bacteria.
The plasmid is transferred via this extension.

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After conjugation, the bacteria separate.
This process is about as close as bacteria can get to having sex.

Gene Transfer Methods

Lysogenic Conversion
Q. Write short note on the lysogenic conversion?
ANSWER
In this the phage DNA becomes integrated with the bacterial chromosomes as
the prophage which multiplies synchronously with the host DNA and is
transferred to the daughter cells.
This process is called lysogeny and bacteria habouring prophages called
lysogenic bacteria. For more detail also see lysogenic conversion in Diphtheria
and Bacteriophage.

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Lac Operon or Operon Model

Q. Write short note on the operan model?

ANSWER
 Operon model is example of phenotypic variation.
 Francois Jacob and Jacques Monad formulated “Operan model” to
describe the regulation of protein synthesis.
 This model is based on the studies of lactose catabolism in E. Coli.
 E.Coli requires Beta-galatosidase, Galatoside permease and
transacetylase enzyme for lactose fermentation.
 These enzymes are coded by the structure genes lac- Z, lac- Y and lac-
A respectively arranged in sequence to form a functional units called
“lac operon”.
 In the control region of lac operon are two relatively short segment of
DNA: Promoter and Operator.
 In the promoter region, RNA polymerase initiates transcription
whereas operator controls transcription of structure genes.
 Near the lac operon is a regulatory gene called lac-I which codes for a
“repressor protein”.
 In the absence of lactose, the repressor protein bind to the operator
site and this prevents the RNA Polymerase from transcribing the
structure genes.
 So no m-RNA is made and no enzymes are synthesized.
 In the presence of lactose, the repressor protein is altered in such a
way that is can not bind to the operator site.
 So in the absence of bound repressor protein, RNA Polymerase
transcribed structure genes to mRNA which is then translated into
enzymes.
 Since enzyme are produce in the presence of lactose, it is said to
induce enzyme synthesis and lac operon called as ‘”inducible operon”.

Operon Model

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Mutation
Q. Define the mutation. Describe the types and effects of mutation?
Q. Write short note on the mutation?

ANSWER
Definition:
It is define as a heritable variation due to any change in the nucleotide
sequence of a gene irrespective of a detectable change in cell phenotype.
Or
It is a random, undirected, heritable variation caused by an alteration in the
nucleotide sequence at some point of the DNA.
Result of mutation:- It result in the production of a protein that has an altered
sequence of amino acids.
Mutagenesis: Induction of mutation is known as mutagenesis.

Type of mutation on the basis of occurrence

(A) Spontaneous Mutation: Those mutations that occur without the
intervention of a mutagen.
Incidence of it is one per 10-2 to10-10 population derived from a single
bacterium.
(B)Induced mutation: When the mutation is induced by some physical or
chemical agents in the DNA.

Mutagens: Agents involved for production of this mutation are known as

mutagens.
(1) Physical agents: 1. Ultraviolet light 2. Ionizing radiation 3. X-ray
(2) Chemical agents: 1. 5-bromouracil and 2-aminopurine
2. Hydroxylamine 3. Nitrous acid
In general four types of alterations that occur in the nucleotide sequence of
DNA hence the mutation may be due to:
(1) Addition: Acquisition of one or more nucleotide.
(2) Deletion: Loss of one or more nucleotide.
(3) Transitions: Purine / Purine or Pyrimidine/ Pyrimidine substitution.
There is substitution of one base pair but the purine and pyrimidine
orientation is preserved.
It is most common type of mutation. E.g :- AT is replaced by GC.
(4) Transversions: The substitution of a purine for a pyrimidinr or vice versa.
It is a less common condition. E.g. GC replaced by CG.
Type of mutation on the basis of gene changes
Mutation is two types.
(a) Point mutation
(b) Multisite mutation

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(a) Point mutation: When mutation affect any one or very few nucleotides. It
is generally reversible.
Point mutation divided into two types.
(1) Base pair substitution:
A single base pair is substituted for another pair and can be subdivided
into two types
(i) Transition
(ii) Transversion.
(2) Frame shift mutation: One or few adjacent base pairs have been inserted
or deleted from the DNA.

(b) Multisite mutation:

It involves extensive chromosomal rearrangement or alteration of DNA by
involving large numbers of base pairs.

Effects of mutation:
(1) Nonsense mutation:
If a nonsense codon (UAA, UGA, UAG) is formed within a gene by
mutation of a sense codon then the protein synthesis is terminated
prematurely and only partial polypeptide is produced during
translation. It is known as nonsense mutation.
(2) Mis-sense mutation:
The effect of both substitution and frame shift mutation gives rise to
an altered codon which specific an aminoacid different from that
indicated by the original codon known as missense mutation.
(3) Suppression mutation:
A Mutation (second) that restores the function of a gene inactivated
by previous mutation (first) is called suppression mutation.
Recognition of mutation:-
It can be recognized by
(a) Gene sequencing
(b) Phenotypic changes.
Genetic Engineering
Q. Write short note on the Genetic Engineering?
Q. Write short note on the Recombination DNA technology?

ANSWER
The technology involved in the isolation of genes coding for any desired
protein from microorganisms or from cells of higher forms of life including
man and inserting them into suitable microorganisms in such a way that the
genes would be functional in directing formation of a specific protein is known
as 'Genetic Engineering' or 'Recombination DNA technology' .
• By using this gene cloning in microorganisms, it is possible to produce large

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quantities of desired protein in pure form.
• DNA fragments containing the desired genes are obtained by using microbial
enzymes, called 'restriction endonucleases'.
• These fragments are inserted into suitable vectors or carriers such as
plasmids or temperate bacteriophages.
• These are then introduced into a bacterial host like E. coli by transformation.
• Large quantities of desired protein are obtained by growing these E. coli in
suitable medium.

Applications of Genetic Engineering

• Production of Proteins-
Genetic engineering or recombinant DNA technology has been used
for the production of proteins of therapeutic value.
Examples: interferon, insulin, human growth hormone.
• Production of antibiotics –
Genetically engineered microbes is used to produce large quantities of
antibiotics.
• Production of vaccines –
Recombinant DNA technology has been used to produce risk free
vaccines that contain specific antigens. Example: hepatitis B, rabies.
• Detection and diagnosis –
Certain bacterial pathogens possess distinctive and unique nucleotide
sequences to infect and cause disease. Recombinant DNA technology
has been used in the detection and diagnosis of such diseases.

Restriction endonucleases
Q. Write short note on the Restriction endonucleases?

ANSWER
• These are microbial enzymes that break DNA into fragments of varying
lengths at specific oligonucleotide sequences.
• Many enzymes that act at different nucleotide sequences have been
recognized.
• These enzymes take part in the destruction of foreign DNA that enters the
bacterial cell.
DNA probes
Q. Write short note on the DNA Probe?
ANSWER
• These are radio-labeled or chromogenically labeled pieces of single stranded
DNA that can be used for the detection of homologous DNA by
hybridization.
• Each microorganism contains a unique nucleic acid sequence that

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differentiates it from other organisms.

• This can be recognized by hybridization using DNA probes.
• Probe containing sequences unique to the microbe to be detected is added
to the culture, body fluid or other specimen suspected to contain the
microbe or its DNA.
• The DNA probe hybridizes with the complementary sequences on the
microbe's DNA.
• DNA probes are extensively used in the diagnosis of various infectious
diseases.
• DNA probes possess high degree of specificity and also sensitivity in
detecting even small quantities of DNA.
• These are highly useful in detecting microorganisms that are either difficult
or not possible to culture.
• DNA probes for diagnostic purposes are commercially available.
Examples: M. tuberculosis, Plasmodium falciparum, HIV, hepatitis B.

Blotting techniques
Q. Write short note on the Blotting technique?
ANSWER
• In this technique, DNA fragments are obtained by digestion with restriction
enzyme and, separated by gel electrophoresis.
• The fragments are transferred from the gel by blotting to nitrocellulose or
nylon membranes that bind the DNA.
• The membrane-bound DNA is denatured and then hybridized with
radioactive single stranded DNA probes.
• This results in formation of radioactive double stranded segments which can
be detected on X-ray film.
• This technique of identifying DNA fragments by DNA: DNA hybridization is
called 'Southern blotting'. This is a highly sensitive technique.
• The same procedure applied for the identification of RNA is known as
'Northern blotting'.
• A similar technique used for the identification of proteins is called
'Immunoblotting' or 'Western blotting'.
Microbial Pathogenicity
Q. Describe the factors predisposing to microbial pathogenicity?
Q. Describe the determinants of virulence of pathogenicity?

ANSWER
Pathogenicity: The ability of a microbial species to cause disease.
Virulence: The degree of pathogenicity within a group or species of
microorganism.
Exaltation: Enhancement of virulence of a strain.

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Attenuation: Reduction of virulence of a strain.

Determinants of virulence
(1) Capsule:
Capsule shield the bacteria from immune and phagocytic response by prevent
interaction between antibodies and C3 and prevent formation of C3b, Bb in
alternative complement pathway.

(2) Adhesins:
Many bacteria adhere to epithelial or endothelial cell by various adhesion
factors like Pili, Lipopolysaccharide, Slime layer, Lipoteichoic acid, Colonization
factor antigen, P1 Protein and M protein.
These factors interact with specific receptors present on the host cells and
tissues for which they have got affinity.
(3) Invasiveness:
It is an ability of an organism to penetrate a tissue after it adheres to cell
surface.
Invasive bacteria either destroy the barrier or penetrate into the cell of the
barrier. Example of invasive type of bacteria are Shigella (by invasion plasmid
antigen), Enteropathogenic Yersinia (by invasion protein), Neisseria
gonorrhoeae (by fimbriae), Salmonella and Enteroinvasive E.Coli.
(4) Enzyme:
Many bacteria release enzymes that can damage host tissue by variety of
mechanisms.
(a) Enzyme that break down collegen and fibrin: Collagenases, Hyaluronidase
and fibrinolysin.
(b) Enzyme that break down cellular material: Proteases and lecithinases.
(c) Enzyme that modify and inactivate antibiotics: β- lactamases
(5) Resistance of phagocytosis: Tubercle bacilli, S. aureus and N. Gonorrhoeae
are resist intracellular killing by preventing fusion of phagosomes with
lysosome.
(6) Toxin: bavterial toxins directly harm tissue or trigger destructive biological
activities. They are classified into two broad categories- Exotoxins and
Endotoxins.
(Difference between exotoxin and endotoxin describe in tabulate form in
chapter Difference.)
Some important exotoxin examples are Tetanospasmin, Choleragen,
Botulinum toxin, Shiga toxin, Verocytotoxin, Diphtheria toxin, pertussis toxin,
toxic shock syndrome toxin-1 and enterotoxin in E. Coli.

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7
IM M U N IT Y

I n n at e I m m u n it y
A c q u ire d I m m u n it y
A c t iv e Im m u n it y
P a s sive I m m u n it y
H e rd Im m u n it y

Innate Immunity
Q. Define and classified the immunity. Describe the innate immunity?
(MBBS-2005), (MBBS-2003)
Q. Write short note on the innate immunity? (MBBS-2010), (MBBS-2004)

ANSWER
Immunity is defined as the state of resistance or insusceptibility exhibited by
the host to toxic molecules, microorganisms and foreign cells.
Classification of immunity
(1) Innate immunity
i. Species immunity
ii. Racial immunity
iii. Individual immunity
(2) Acquired Immunity
i. Active acquired immunity
(a) Natural (e.g. Disease, Infection)
(b) Artificial (e.g. Vaccine)
ii. Passive acquired immunity
(a) Natural (e.g. Breast feeding)
(b) Artificial (e.g. Antibodies)
Innate Immunity
Innate Immunity is also known as native immunity.

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It is a resistance with which a person or animal is born.
It is nonspecific.
This type of immunity is present for life.
Types of innate immunity
1. Species immunity:
e.g., B. anthracis infects human but not chicken, birds are immune to tetanus.
2. Racial immunity:
e.g. (1) Persons with hereditary deficiency of glucose - 6 - phosphate
dehydrogenase are markedly less susceptible to P. falciparum
malaria,
(2) Races or persons with sickle cell anaemia prevalent in the
Mediterranean coast are highly resistant to P. falciparum infection.
3. Individual immunity:
This type of individual immunity is commonly observed in an endemic
outbreak of an infection.

Host factors in innate immunity

1. Age: Fetus and old person carry high susceptibility of infectious diseases.
In the fetus: The immune apparatus is immature
In old age: Gradual waning of their immune response.
2. Hormonal influence:
(i) Diabetes mellitus (elevated level of carbohydrate in tissue due to
decrease insulin) increase susceptibility to pyogenic infection.
(ii) Hypothyroidism and adrenal dysfunction (increased corticosteroid
secretion) influence susceptibility to infection.
(iii) Corticosteroids suppress the inflammatory response and inhibit
antibody formation.
3. Nutrition: Malnutrition predisposes to bacterial infection.

Mechanism of innate immunity

Three-mechanism of innate immunity are the physicochemical, humoral and
cellular.
A . Physiochemical barriers: -
(1) Skin:
The intact skin provides a mechanical barrier to the invading
microorganisms
The sebaceous secretions have bactericidal and fungicidal actions.
(2) Nose, Nasopharynx and Respiratory tract:
The inhaled bacteria are arrested in the tortuous nasal passages on
the moist surfaces of the mucous membrane lining.
The cough reflex plays an important defensive role of respiratory
tract.
Small particulate materials in the alveoli are ingested by alveoli
macrophages.

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Inherited or acquired defects in the function of respiratory cilia or

mucous or both make the lungs vulnerable to infection.
(3) Mouth, Stomach and Intestinal tract:
Saliva possesses mild bactericidal action.
The intestinal anaerobic microflora prevents superinfection by coli
forms during antibiotic therapy.
The acidic pH of gastric acid destroys most of the ingested bacteria.
(4) Conjunctivae: Lysozyme present in tears is bactericidal in action.
(5) Genitourinary tract:
The Slightly acid urine maintains sterility of both male and female
urethra.
The acid reaction of the vaginal secretion in female due to
fermentation of glycogen by lactobacillus (normal flora) is markedly
bactericidal towards most pathogenic organisms.
B. Humoral defence mechanisms:-
(1) Lysozyme: It is function as mucolytic enzyme, splitting sugars of cell wall. It
is present in tears, saliva and nasal secreation.
(2) Gastric secreation: Retard the growth of bacteria in intestine.
(3) Basic polypeptide like spermine, spermidine, arginine, lysine,leukin and
plakin possess antibacterial effect.
(4) Complement: complement possess bacterial activity and causes
destruction of pathogenic bacteria that invade blood and tissue.
(5) Properdin and euglobulin along with complement and mg++ causes lysis
of gram negative nacteria and virus.
(6) Interferon: These are a family of antiviral agents produced by cell
stimulated by live or killed viruses.
C. Tissue factors
Phagocytes like PMN, Macrophages, Kupffer cell, Histiocyte, NK cells,
Monocyte, R.E. cell etc. accumulate at the site of infection along with
outpouring of natural antibacterial substances and deposition of fibrin
entangles or traps the organisms.
Acquired Immunity
Q. Define and classified the immunity. Describe the acquired immunity?
(BDS-2009{O})
Q. Write short note on the acquired immunity? (MBBS-2004)
Q. Define and classified the immunity. Describe the active immunity?
Q. Write short note on the active immunity?
Q. Write short note on the passive immunity? (MBBS-2008),

ANSWER
Resistance acquired by an individual during life is called acquired or specific
immunity.

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Acquired immunity is of two types:
(1) Active acquired immunity.
(2) Passive acquired immunity.
Active immunity
The immune system actively participates by antibody mediated and cell-
mediated immunity.
It is produce by either natural infection or vaccination.
Active immunity develops slowly over a period of days or weeks but persists
for a along time, usually for years.
Mechanism of active immunity
The body's immune apparatus offers protection by synthesizing antibody by B
lymphocytes and plasma cells and/or by sensitized T cells in response to an
antigen.
Types of active immunity
Active immunity may be acquired either naturally or artificially.
(a) Natural active immunity:
It is acquired by natural infection by the organisms.
Such immunity is usually long lasting and plays important roles in preventing
epidemics,
E.g. Poliomyelitis, Tuberculosis.
(b) Artificial active immunity:
It is the resistance produced by vaccination.

Passive immunity
Q. Write short note on the passive immunity?

ANSWER
The resistance is induced in the recipient by transfer of preformed
(readymade form) antibodies against infective agent or toxin in another host,
is called passive immunity.
The immune system plays no active role and the protective mechanism comes
into force immediately after transfer of antibodies (immune serum). Passive
protection is short lasting only for days or weeks.
It is useful when instant immunity is required.

Passive immunity is of two types:

A. Natural passive immunity: (MBBS-2008)
e.g. transfer of maternal antibody to foetus transplacentally and to infant
through milk (colostrum).

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B. Artificial passive immunity:

It is the resistance passively transferred to recipient by the parenteral
administration of antibodies. E.g. Anti Tetanus Sera, Anti Diphtheria sera
(See difference between active and passive immunity in Chapter Difference).
Herd Immunity
Q. Write short note on the herd immunity? (MBBS-2010)

ANSWER
It is also known as immunity of community.
It refers to the collective resistance to the disease displayed by the community
in its environmental setting.
Important factor of herd immunity are
(1) Vaccination,
(2) Quarantine measures,
(3) Effect of carriers and
(4) Environment
When herd immunity is low, there are large number of susceptible persons in
the community and epidemics occur.

Adoptive Immunity
It is a special type of immunisaion by injecting an extract of immunologically
competent lymphocytes known as transfer factor.
This immunity has been attempted in the treatment of lepromatous leprosy.

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8
A N T IG E N

Hapten
HeterophilAntigen
HumanLeucocyteAntigen
Superantigen
Hapten or incomplete antigen
Q. Write short note on the hapten? (BDS-2005), (MBBS-2004)
Q. Write short note on the incomplete antigen?

ANSWER
Hapten is a low molecular weight, usually non-protein substance.
It is unable to induce an immune response by itself but can become
immunogenic only when covalently linked to proteins (called carrier protein).
Haptens can react specifically with its corresponding antibody.
It may be serum protein such as albumin or globulin or synthetic polypeptide.
Haptens are usually low molecular weight lipids and carbohydrates.

Haptens are of two types: complex and simple.

Complex haptens are relatively large molecules and combine with specific
antibodies forming visible precipitate, e.g. capsular polysaccharide of
pneumococci, cardiolipin.
It is polyvalent.
Simple haptens are low molecular weight simple chemical substances when
they combine with specific antibody, no precipitate is produced.
It is univalent.

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Example of hapten
(a) Bacterial: Capsular polysaccharide of pneumococcus,
Polysaccharide "C" of betahaemolytic streptococci.
(b) Drugs and chemicals:
Agents causing allergic contact dermatitis and drug
hypersensitivity.
(c) Blood group (ABO) substance:
Glycoproteins.
(d) Lipids: Forssman, transplantation and cardiolipin antigens.

Heterophile antigen
Q. Write short note on the heterophil antigen? (MBBS-2009)

ANSWER
Certain antigens of similar nature, if not identical, present in different tissues
of more than one species are called heterophile antigens.
Antibody to these closely related antigens produced by one species cross
react with antigens of other species.
Chemically these are Iipoprotein polysaccharide complex.
Examples
1. Forssman antigen :
It is a lipoprotein-polysaccharide complex.
First described by Forssman.
Example of forssman antigen include
Streptococcus pneumoniae,
Some Salmonella serotypes and
Shigella dysenterise.
2. Cross-reacting microbial antigens :
A heterophile antigen present in Rickettsiae causing typhus fever are
share, by certain strains of Proteus (OX 19, OX 2 and OX K).
This forms the basis of Weil-Felix reaction.

3. Human RBC of blood group B shares an antigen with Esch. coli constituting
another example of heterophile antigen.

Common test based on the heterophil antigen are

1. Weil-Felix reaction: Strains of Proteus (OX, 19, 2, K) are agglutinated by
sera of patients suffering from rickettsial infection.
2. Paul-Bunnel test: Sera of infectious mononucleosis patients agglutinate
sheep erythrocytes.
3. Streptococcus MG agglutination test: Positive in primary atypical
pneumonia.

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Major Histocompatibility Complex

Human Leucocyte Antigen
Q. Write short note on the HLA?
Q. Write short note on the Human Leucocyte Antigen?
Q. Write short note on the Major Histocompatibility Complex? (BDS-2008)
(MBBS-2003)

ANSWER
The HLA Complex or Histocompatibility antigens mean cell surface antigens
that evoke immune response to an incompatible host resulting in allograft
rejection.
Alloantigens are present on surface of leucocytes in man and are called
Human Leucocyte Antigens (HLA) and the set of genes coding for them is
named the HLA Complex.
The HLA complex of genes is located on short arm of chromosome 6.
It is grouped in three classes.
Class I - HLA-A, HLA-B and HLA-C
Class II - HLA-DR, HLA-DQ and HLA-DP
Class III - Complement loci encode for C2, C4 and Factor B of
complement system and Tumour Necrosis Factors (TNF) alpha
and beta.

1. Class I MHC Antigens

The MHC class I antigens are present on the surface of all nucleated cells.
They are involved in graft rejection and cell mediated cytolysis.
The cytotoxic T cells (CD8) recognize MHC class I antigens for their action.

2. Class II MHC Antigens

They have found on the surface of macrophages, monocytes, activated T-
lymphocytes (CD4) and B-Iymphocytes.
They are primarily responsible for the graft-versus-host response and the
Mixed Leucocyte Reaction (MLR).

3. Class III MHC Antigens

C2, C4 complement are components of the classical pathway of complement
fixation and Properdin factor B is component of the alternative pathway of
complement fixation.

HLA Typing
HLA typing is done prior to transplantation to lowers the risk of organ
rejection.

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It is also useful in paternity determination and anthropological studies.

HLA A, B, and C are identified by complement dependent cytotoxic reaction.
HLA Class II antigens are identified by mixed leucocyte reactions.
Super antigens
Q. Write short note on the superantigen?
ANSWER
S. aureus secretes two types of toxins with superantigen activity.
(1) Enterotoxins: There are six antigenic types (named SE-A, B, C, D, E and G).
(2) Toxic shock syndrome toxin (TSST-1).
Enterotoxins cause diarrhea and vomiting when ingested and are responsible
for staphylococcal food poisoning.
TSST-1 is expressed systemically and is the cause of toxic shock syndrome
(TSS).
Enterotoxins can also cause toxic shock syndrome.
TSST-1 is weakly related to enterotoxins, but it does not have emetic activity.
Superantigens stimulate T cells non-specifically without normal antigenic
recognition.
Up to one in five T cells may be activated, whereas only 1 in 10,000 are
stimulated during a usual antigen presentation.
Superantigens and the non-specific stimulation of T cells

Cytokines are released in large amounts, causing the symptoms of TSS.

Superantigens bind directly to class II major histocompatibility complexes of
antigen-presenting cells outside the conventional antigen-binding grove.
Superantigens bind directly to class II major histocompatibility complexes
(MHC II) of antigen-presenting cells outside the normal antigen-binding
groove. Up to one in five T cells may be activated. Cytokines are released in
large amounts, causing the symptoms of toxic shock.

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9
A N T I B O D Y

Stuctureof Immunoglobulin
IgG Antibody
IgMAntibody
IgAAntibody
IgEAntibody
IgDAntibody

Structure of Immunoglobulin
Q. Write short note on the structure of immunoglobulin? (MBBS-2006),
(MBBS-2003) (MBBS-2002)

ANSWER
Immunoglobulins are glycoproteins and comprise 20 to 25 per cent of the
total serum proteins.
Antibody molecules are inverted Y-shaped structures.
It is made up of two identical heavy and two identical light polypeptide chains
held together by disulphide (s-s) bonds.
The longer chains are called Heavy (H) and the shorter ones is light (L) chains.
Each L chain is attached to H chain by a disulphide bond.
Based on the structure of constant region of heavy chains, gamma (y), mu (µ),
alpha (α), delta (δ) and epsilon (ε), immunoglobulins are classified into IgG,
IgM, IgA, IgD and IgE, respectively.
The L chains exist in two forms kappa (K) and lambda (A). Immunoglobulin
divided into two fragments Fab (Fragment antigen binding) fragments and one
Fc (fragment crystallisable) fragment with the enzyme papain.
The antigen combining site of the molecule is at its aminoterminus which is
made up of both H and L chains.

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Struc ture of Immunoglobulin

Heavy chain contain 1/5 variable region and 4/5 constant region.
Light chain contain ½ variable and ½ constant region.

Immunoglobulin G
Q. Write short note on the IgG? (B.Sc. Nur.-2011) , (B.Sc. Nur.-2008) , (MBBS-
2007), (MBBS-2005), (MBBS-2002)

ANSWER
IgG is the major serum immunoglobulin making up 85% of the total amount of
Immunoglobulin.
It molecular weight is 150,000 (75).
It is called divalent.
IgM appears early (7 to 8 days after infection) and persists for shorter
duration (4 to 6 weeks) whereas IgG appears later (2 weeks after infection)
but persists for long periods of time.
IgG is the predominant antibody in secondary immune response.
The production of IgG requires T cell help.
IgG participates in precipitation, complement fixation and neutrelisetion of
toxin and viruses.
It is distributed equally between intravascular and extravascular
compartments.
It is the only immunoglobulin that passes through placenta and provides
natural passive immunity to newborn.
IgG is not formed in the foetus in any significant amount.
IgG has a longest half life about 23 days.
When IgG is passively transferred to an individual, it suppresses the
homologous antibody synthesis by feed-back mechanism.
E.g. Isoimmunisation of women is done by anti-RhD IgG immunoglobulin.

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Immunoglobulin A
Q. Write short note on the IgA? (BDS-2008), (MBBS-2007), (MBBS-2003)

ANSWER
IgA is appears in the sero-mucous secretions such as milk, saliva, tears, nasal
fluids, sweat, colostrum and in secretions of respiratory, intestinal and genital
tracts.
It protects the exposed mucous membranes.
It is about 10 to 13 per cent of Immunoglobulin.
It is the second major serum immunoglobulin.
It has half life of 6 days.
IgA is two types
Serum IgA:
It is monomer.
Serum IgA is principally principally a 7 S monomer with molecular weight of
160,000,
Secretion IgA: It is dimer joined by the J chain. (MBBS-2003)
IgA is dimerised within the epithelial cells of glands, intestines and the
respiratory tract.
IgA is largely synthesised locally by plasma cells.
It is forms antibody paste on intestinal and respiratory mucosa.
IgA covers the microorganisms and inhibits their adherence to mucosal cells.

Immunoglobulin M
Q. Write short note on the IgM? (B.Sc. Nur.-2011), (B.Sc. Nur.-2008) (MBBS-
2010), (MBBS-2007)
Q. Write short note on the Millionaire antibody? (MBBS-2004)

ANSWER
IgM constitutes 5-10% of serum immunoglobulins in adults.
It has half life of 5 days.
IgM is a pentamer consisting of 5 immunoglobulin and one molecule of
J chain, which joins the Fc fragments of the basic subunits.
It is often called macroglobulin and the "millionaire molecule.
The valency of IgM is 10.
IgM phylogenetically is the oldest immunoglobulin.
IgM remains largely confined to blood stream (80%).
IgM is the first immunoglobulin synthesized in an antibody response (primary
response) and its presence in serum indicates recent infection.

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IgM appears early (7 to 8 days after infection) and persists for shorter
duration (4 to 6 weeks) whereas IgG appears later (2 weeks after infection)
but persists for long periods of time.
It cannot cross the placenta.

Structure of Imm unoglobulin M (IgM)

The presence of IgM antibody in serum of foetus or newborn indicates
intrauterine infection.
It is the earliest synthesised immunoglobulin in foetus, it appears in blood in
about 20 weeks of age.
It is the most efficient antibody in agglutination, complement fixation,
cytolytic reaction.

Immunoglobulin D
Q. Write short note on the IgD?

ANSWER
The concentration of IgD is less than 1 % serum immunoglobulins.
It is mostly intravascular in distribution.
Nearly all the IgD is present on the membranes (surface) of a proportion of
unstimulated B lymphocytes of blood and serve as recognition receptors for
antigens.
Half life is 3 days.
It does not cross placenta and does not bind to cells via Fc Regoin.
It does not bind complement.

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Immunoglobulin E
Q. Write short note on the IgE?

ANSWER
These antibodies are also referred to as reagins.
The half life of it is 2 days.
It constitutes less than 1 % of the total immunoglobulin.
It is heat labile and does not fix complement or cross placental barrier.
This immunoglobulin is mostly distributed extravascularly bound to Fc
receptors on mast cells.
IgE bound to mast cells serves as a receptor for allergens and parasitic
antigens.
Structurally it resembles IgG.
IgE plays an important role for protection against parasitic infection and is
responsible for anaphylactic hypersensitivity.
IgE mediates Prausnitz- Kustner reaction.
From available information it appears that:
IgG Protects the body fluid
IgA Protect the body surface
IgM Protects the blood stream
IgE Mediate Type I Hypersensitivity reaction
IgD Role yet not known.

Abnormal Immunoglobulins
Apart from antibodies, there are some structurally similar proteins which are
seen in serum of some pathological processes and some time in healthy
person. These are called abnormal immunoglobulins.
Bence-Jones protein
The first abnormal immunoglobulin discovered by Bence-Jones. So these are
called Bence-Jones protein. These are typically present in multiple myoloma.
Their presence in the urine is identified by their characteristic property of
coagulating at 500C and redissolving at 700C.
Bence-Jones protein are present either in Kappa or Lambda form light chain
immunoglobulin but never in both forms.
Abnormal immunoglobulin condition
Multiple myoloma: Unchecked proliferation of one clone of plasma cell.
Waldenstrom’s macroglobilinemia: Over production of IgM and their light
chain.
Heavy chain disease: Over production of the Fc parts of the immunoglobulin
heavy chains.
Cryoglobulinemia: In this condition, gel formation or precipitation occur on
cooling the serum, which redissolves on warming.

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10
ANTIGEN ANTIBODY REACTION

PrecipitationReaction
FlocculationReaction
Agglutination
ImmuneDiffusion
Gel Diffusion
Immunoelectrophoresis
ProzonePhenomenon
Coomb's test
Immunofluoroscence
ELISA
PCR

Precipitation Reaction
Q. Discuss various types of antigen- antibody reaction?
(BDS-2009), (BDS-2005)
Q. Write short note on the precipitation reaction? (MBBS-2005), (MBBS-
2003)
Q. Write short note on the flocculation reaction?
Q. Write short note on the immune diffusion?
Q. Write short note on the gel diffusion?
Q. Write short note on the immunoelectrophoresis?

ANSWER
Various types of antigen antibody reactions
(1) Precipitation: Ring test, Flocculation test, Immunodiffusion test
(2) Agglutination: Slide test, tube test, coombs test, heterophile test, passive

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agglutination test
(3) Complement Fixation test: Direct and Indirect CFT, Immobilisation test,
Immune adherence test, cytocidal test
(4) Conglutination
(5) Neutralization test: Toxigenicity test in Diphtheria, Shick test, ASO test,
nagler rection
(6) Opsonisation
(7) Immunofluorescence: Direct and Indirect
(8) Radia Immunoassay
(9) Enzyme Linked Immuno Sorbent Assay (ELISA): Sandwich, indirect,
competitive and capture ELISA
(10) Chemiluminescence Assay
(11) Immunoelectronmicroscopic tests: Immunoferritin test, Immunoelectron
microscopy, Immunoenzyme test.
(12) Immunoblotting: Southern, Northern and Western.

Precipitation reaction:
When a soluble antigen reacts with its specific antibody in presence of an
electrolyte (NaCI) at an optimal pH (7.4) and temperature (370C), the antigen-
antibody complex forms an insoluble precipitate. This reaction is called
precipitation.
Flocculation reaction:
When the precipitate remains suspended instead of sedimenting, the reaction
is called flocculation.
Precipitation reaction may occur in liquid medium or in semisolid medium like
agar gels, agarose or polyacrylamide.

Mechanism of precipitation:
Antigen and Antibody molecules complex form a large lattice in the zone of
equivalence (precipitate forms). (See prozone phenomenon)

Applications
1. Identification of bacteria, e.g. detection of group specific
polysaccharide of Streptococcus (Lancefield's groupings).
2. Identification of bacterial components in infected tissues.
E.g. B. anthracis (Ascoli's thermoprecipitin test).
3. Detection of unknown antibody, e.g. VDRL and Kahn Test in syphilis.
4. Medico-legal identification of human blood or seminal fluid.
5. Standardisation of toxins and antitoxins.
6. Testing for food adulteration.

Techniques of precipitation
There are three principal types of precipitation technique
 Simple precipitation test--slide and tube techniques.

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 Gel diffusion test

 Immunoelectrophoresis
 Electroimmunodiffusion

Simple precipitation tests

1. Ring test:
After a period of incubation, a white ring of precipitate appears at the junction
of two clear fluids.
E.g. Ascoli's thermoprecipitin test,
Tests for C Reactive Protein and
Lancefield method of grouping β-haemolytic streptococci.
2. Flocculation test
Flocculation test is done in slide (VDRL test for syphilis) and in tube (Kahn
test).
Tube precipitation test is done in standardisation of toxins and toxoids.
Gel Diffusion
Precipitin test done in agar gel is called gel diffusion or immunodiffusion.
The reaction is visible in the forms of distinct band of precipitation.

Different modifications of gel diffusion techniques are

Single diffusion in one dimension (Oudin procedure),
Double diffusion in one dimension (Oakley Fulthorpe procedure),
Single diffusion in two dimensions (Radial immunodiffusion) and
Double diffusion in two dimensions (Ouchterlony procedure)

Immunoelectrophoresis
Q.Write short note on the immunoelectrophoresis?

ANSWER
It is one of type of precipation reaction.
Immunoelectrophoresis combines electrophoresis with immunodiffusion.
Several antigens can be separated from a mixture by electrophoresis in agar
gel.

The test consists of two stages.

In stage I, antigen is placed in a well cut into agar gel on slide or plate and
electric current is passed through the agar (1-2 hours).
The antigen components migrate in the electric field according to their charge
and size and separate from each other.

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In stage 2, a trough is cut in the gel parallel to the direction of migration of
antigen and filled with antibody and allowed to diffuse for an appropriate
period of time.
A series of precipitate arcs are formed where the antibodies encounter the
antigen in optimal proportions.
This technique is useful for determination of the presence or absence of
serum proteins and detection of unusual patterns such as human myeloma
protein.

Immunoelectrophoresis

Electroimmunodiffusion
Q. Write short note on the electroimmunodiffusion?

ANSWER
Immunodiffusion can be speed up if antigen and antibody are driven by
electricity.
It is combination of electrophoresis and diffusion.
Counter-current Immunoelectrophoresis and Rocket Electrophoresis are used
frequently.
Counter-current Immunoelectrophoresis
In this method, during electrophoresis antibodies tend to migrate towards
cathode while most antigenic proteins move towards anode.
As a result antigen and antibody move in opposite directions and form a
precipitation line within few minutes.
This technique is also known as "crossover" technique.
It is ten times more sensitive than the standard double diffusion technique.

Counter-current Immunoelectrophoresis

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Uses
• In the detection of antigens in various body fluids.
• In the sera diagnosis of bacterial, fungal, viral and parasitic infections.
Rocket Electrophoresis (One Dimensional Single Electroimmuno Diffusion)
It is a simple technique described by LaurelJ in 1965.
The antibody is mixed with agarose gel and poured on a slide.
Wells are cut on the cathodic side, charged with antigen and electrophoresed.
Antigens move towards the anode and meet the corresponding antibody in
the gel forming a rocket like precipition peak.
Based on the height of the rocket, concentration of the antigen is estimated.
It is use in the estimation of proteins and other antigens in various body fluids.

Rocket Electrophoresis

Agglutination
Q. Write short note on the agglutination? (B.Sc. Nur.-2009), (B.Sc. Nur.-2007)
(MBBS-2009), (MBBS-2006), (MBBS-2005)

ANSWER
When a particulate antigen reacts with its specific antibody in presence of an
electrolyte (NaCI) at an optimal pH (7.4) and temperature (370C), the antigen-
antibody complex forms a visible clumping (aggregation) of the particles. It is
called agglutination.
Particles like bacteria, erythrocytes, synthetic latex particles etc. coated with
antigen or antibody can be used to detect either soluble antibodies or
antigens, respectively.
Agglutination reaction occurs when antigen and antibody are mixed in optimal
proportions.

Uses of agglutination reaction

1. Identification of unknown culture
2. Blood grouping and cross-matching

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3. Serological diagnosis of enteric fever (Widal test), typhus (Weil-Felix
reaction), brucellosis and infectious mononucleosis (Paul-Bunnel test).
4. Streptococcus MG agglutination test for diagnosis of primary atypical
pneumonia.

Agglutination tests can be done:

In slides or tiles
In tubes
In microtitration plates

Types of agglutination reaction

(1) Slide agglutination reaction
o Blood grouping and cross-matching
o Identification of bacteria
(2) Tube agglutination reaction
o Widal test for enteric fever
o Weil-Felix reaction for typhus
o Paul-Bunnel test for infectious mononucleosis
o Streptococcus MG agglutination test for diagnosis of primary atypical
pneumonia
o Brucellosis
(3) Antiglobulin (Coombs) test
o Direct Coomb’s test
o Indirect Coomb’s test
(4) Heterophile agglutination reaction
o Weil-Felix reaction for typhus
o Paul-Bunnel test for infectious mononucleosis
o Streptococcus MG agglutination test for diagnosis of primary atypical
pneumonia
(5) Passive agglutination reaction
Latex agglutination test:
Use for detection of ASO, CRP, RA, HBV & HCG
Haemagglutination test: Rose-Waaler test
Direct Active Haemagglutination test
Indirect Active Haemagglutination test
Direct Passive Haemagglutination test
Indirect Passive Haemagglutination test
Coagglutination: Protein A of Cowan 1 strain of staph. Aureus used
(6) Reversed passive agglutination reaction:
Antibody absorbed on carrier particles in place of antigen.

The agglutination can be of 2 types

(1) Active agglutination:
In this, direct agglutination of antigen with its corresponding antibody occurs.

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e.g. Slide agglutination of salmonellae, shigellae, or Vibrio cholerae using

specific antibody.
(2) Passive agglutination:
These are tests in which specific antibody or known antigen is attached to
certain inert carrier particles or cells and thereby a precipitation reaction can
be converted to agglutination reaction.
Carriers: Latex particles, bantonite, carbon particles and stabilised
staphylococcal cells.
(a) Antigen-coated latex particles are used in ASO (antistreptolysin 0)
slide test.
(b) Antibody-coated latex particles are used for detection of extracellular
bacterial antigens in CSF.
(c) Carbon particles: used in Rapid Plasma Reagin (RPR) card test in
detection of syphilis where carbon particles are coated with
cardiolipin antigen.

Prozone phenomenon
Q. Write short note on the Prozone phenomenon? (MBBS-2005)
Q. Write short note on the Zone phenomenon?

ANSWER
The amount of precipitate formed depends on the relative proportions of the
antigens and antibodies that take part in the reaction.
Zone of equivalence:
Antigen and antibody are completely precipited and form large three
dimensional lattice structure.
Prozone:
Absence of lattice formation and precipitation due to excess of antibodies. It is
called Prozone Phenomenon.
It may cause precipitation on higher dilutions. E.g. acute brucellosis in which
agglutination occurs with higher dilutions (1 : 40 or 1 : 80).
Post zone:
Absence of lattice formation and precipitation due to excess of antigens. It is
called Post zone Phenomenon.
Ag - Ab aggregates are formed in such a way that a large three dimensional
lattice structure form at zone of equivalence.

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Coomb’s test
Q. Write short note on the Coomb’s test?
Q. Write difference between of indirct and direct coombs test?

ANSWER
Coomb’s test is agglutination test.
It is used for :- (1) Detection of anti Rh antibody.
(2) Detection of incomplete antibodies as in brucellosis &
other disease.

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(A) Direct Coomb’s test:-

Sensitization of erythrocyte with incomplete antibodies takes place in
vivo as for
e.g.- Haemolytic diseases of newborn, due to Rh incompatibility.
(B). Indirect Coomb’s test:-
The sensitization of erythrocytes with incomplete antibodies is
performed in vitro.

Difference between of indirct and direct coombs test

Direct Coombs' test Indirect Coombs' test

Object It detects presence of It detects incomplete
incomplete antibodies antibody present in
adsorbed onto RBC serum.
surface.
Indications of tests Autoimmune haemolytic Detection of anti Rh
anaemia, antibody in serum of Rh
Erythroblastosis foetalis negative pregnant
women of Rh positive
husband
Test procedure Red cells washed in Patient's serum mixed
saline 3 times by with saline washed Rh
centrifugation to remove positive group (or that
serum proteins except of same group of
globulin. patient) red cells and
When washed RBCs incubated at 37 oC for
mixed with Anti Human 30 minutes.
Gamma globulin in Red cells washed in
presence of bovine saline three times.
albumin clumping of Washed cells mixed
RBCs occur. with Coombs' serum.
Agglutination occurs in
positive cases

Immunofluorescence
Q. Write short note on the immunofluorescence? (MBBS-2008), (MBBS-
2005)

ANSWER
Principle:-
Fluorescence dyes absorb invisible UV light between 290-495 nm & emits out
visible longer wave length i.e. 525 nm green light, hence if micro- organisms

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or tissue cells are stained with Fluorescent dye & examined under the
fluorescence microscope with UV instead of visible light then they are seen as
bright objects against a dark background.
Commonly used fluorescent dye:-
(1). Fluorescin isothiocyanate – Blue green fluorescence.
(2). Lissamine Rhodamine - Orange red fluorescence.
Uses of immunofluorescence: - To detect
(1). Tissue Antigen
(2). Antibodies to tissue including autoantibodies.
(3). Antigen of infected organism such as bacteria or viruses in the
body.
(4). Antigen – Antibody complexes.
Types of immunofluorescence:-
(1). Direct immunafluorescence.
(2). Indirect immunafluorescence
(1). Direct immunofluorescence:- (MBBS-2008)
Specific antibodies tagged with fluorescent dye are allowed to
come in contact with antigen on the slide.
After some time wash & examine under fluorescent
microscope.
Uses:-
(1). Diagnosis of Rabies by detection of rabies antigen in brain
smear.
(2). Detection of Gonococci, T.pallidum etc.
(3). Detection of bacteria, viruses etc in blood, CSF, Urine, tissue.
Disadvantage:-
Seperate specific fluorescent labelled antibody has to be prepared
against each antigen be tested.
(2). Indirect immunofluorescence:-
- A known antigen is fixed on a slide.
- Then unknown & unlabelled antibody (pt. serum) is applied & if
antibody is present in serum then it will bind with its specific antigen
& form Antigen- Antibody complex.
-To detect this Ag+ Ab complex fluorescein tagged antibody to
Human globulin is added.
- Wash the slide & examine under UV light.
Uses :- Detection of antibody in serum for ex.- Diagnosis of Syphilis-
Fuorescent treponemal antibody absorption test (FTA-Abs).
Advantage:-
A single antihuman globulin labelled with fluorescent dye can be used
to detect antibody to any antigen because all antibodies are globulin
in nature, therefore antihuman globulin attaches to all antibodies.

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Enzyme Linked Immunosorbent Assay (ELISA)

Q. Write short note on the ELISA? (B.Sc. Nur.-2010), (MBBS-2007), (MBBS-
2006), (MBBS-2004), (MBBS-2003), (MBBS-2002)

ANSWER
ELISA widely use for detection of antibodies and antigens.

Elisa is named as the test technique involves the use of an enzyme system and
an immunosorbent- an absorbing material specific for one of the components
of reaction, antigen or antibody. The absorbing material may be particulate
e.g. cellulose or agarose or a solid matrix, e.g. microwells , membrane.
Chemical conjugation of an enzyme to either antigen or antibody allows
detection of immune complexes formed on a solid phase, once washed
excess reagents and on subsequent substrate interaction colour product
which can be visualised and /or measured by optical density.
Principal Component Involved in ELISA
Solid Phase
Enzyme Conjugate as probe
Enzyme Substrate
Types of ELISA
– Non-competitive

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 Direct ELISA
 Indirect ELISA
 Sandwich ELISA
– Competitive / Inhibition
– Capture Antibody ELISA
Advantages of ELISA
 Versatile
 Simple
 Sensitive
 Robust
 Reagent Stability
 Safe
It is done on a solid phase.
The test can be done in polystyrene tubes (macro-ELISA) or Polyvinyl
microtitre plates (micro-ELISA), Cellulose acetate membrane (CAM) or Nitro
cellulose membrane (NCM).

Enzyme Linked Immunosorbent Assay

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SANDWICH ELISA
It is use for antigen detection in a specimen.
The wells of microtitre plate are coated with specific antibody against the
antigen to be detected.
Specimens to be tested are added in coated wells.
If antigen is present in specimen, it binds to coated antibody.
To detect this antigen-antibody reaction, antiserum (antibody) conjugated
with an enzyme is added.
This conjugated antiserum binds to an antigen already attached to coated
antibody.
A color substrate is added to know the binding of conjugated antiserum to
antigen-antibody complex.
In case of binding (positive result), an enzyme acts on substrate to produce
color.
Intensity of which can be read by spectrophotometer or ELISA reader.
This type of ELISA test is known as sandwich ELISA.

INDIRECT ELISA
It is use for antibody detection.
The wells of microtitre plate are coated with antigen.
Sera to be tested are added in these coated wells.
If antibody is present in specimen, it binds to coated antigen.
To detect this antigen-antibody reaction, a goat antihuman immunoglobulin
antibody conjugated with an enzyme is added.
Enzyme conjugated antihuman immunoglobulin binds to antibody of patient
sera.
To detect this binding, a substrate is added and enzyme acts on substrate to
produce color in a positive reaction.
Intensity of colour can be read by spectrophotometer or ELISA reader.

COMPETITIVE ELISA
It is used for detection of HIV antibodies.
Positive result shows no color whereas appearance of color indicates a
negative test.
There are two specific antibodies, one conjugated with enzyme and other
present in serum (if serum is positive for antibodies).
Competition occurs between two antibodies for same antigen.
The microtitre plate wells are coated with HIV antigen.
Sera to be tested are added to these wells.
If antibodies are present, antigen antibody reaction occurs.
To detect this reaction, enzyme labeled specific HIV antibodies are added.
There is no antigen left for these antibodies to act.
These antibodies remain free and washed off during washing.
Substrate is added but there is no enzyme to act on it.

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Therefore, positive results show no color.
If serum to be tested is negative for antibodies, antigen is there to combine
with enzyme conjugated antibodies and enzyme acts on substrate to produce
color.

Uses of ELISA
It has been used for detection of antigens and antibodies of various
microorganisms. Some examples are
(i) Detection of HIV antibodies in serum
(ii) Detection of Dengue, Chikunguniya and Japanese encephalitis
antibody in serum.
(iii) Detection of Herpes virus, Cytomegalo virus antibody in serum.

PCR (POLYMERASE CHAIN REACTION)

Q. Write short note on the PCR? (MBBS-2006), (MBBS-2002)

ANSWER
PCR or Polymerase Chain Reaction is defined as a molecular technique which
allows the production of large quantities of a specific DNA from a DNA
template using a simple enzymatic reaction without a living organism, such as
E. coli or yeast.
It is molecular biology procedure for the in vitro amplification of DNA.
PCR, or Polymerase Chain Reaction was first popularly thought to have been
conceived by Dr. Kerry Mullis in 1983.

Principle of the PCR

The purpose of a PCR (Polymerase Chain Reaction) is to make a huge
number of copies of a gene.
This is necessary to have enough starting template for sequencing.
The cycling Reactions:
There are three major steps in a PCR, which are repeated for 30 or 40
cycles.
This is done on an automated cycler, which can heat and cool the
tubes with the reaction mixture in a very short time.
(1) Denaturation at 94°C :
During the denaturation, the double strand melts open to single
stranded DNA, all enzymatic reactions stop (for example : the
extension from a previous cycle).

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(2) Annealing at 54°C :

The primers are jiggling around and ionic bonds are constantly
formed and broken between the single stranded primer and the
single stranded template.
The polymerase can attach and starts copying the template.
(3) Extension at 72°C :
The bases (complementary to the template) are coupled to the
primer on the 3' side.

Application of PCR
(1) To detect microorganisms that could not be cultured or fastidious and /or
slow growing.
e.g. Bacteria: Mycobaterium Tuberculosis, Helicobacter Pylori,
Chlamydia Trachomatis, Mycoplasma
Virus: Cytomegalovirus Herpes Simplex
Hepatitis B & C HIV
Fungus: Candida Albican Cryptococcus
Pneumocystis carinii
Protozoa: Toxoplasma gondii.
(2) Diagnosis of inherited disorders:
e.g. Sickle Cell Anaemia, β-thalassaemia, Cystic Fibrosis
(3) Diagnosis of cancer: Retinoblastoma
(4) Mediolegal cases

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PCR Thermocycler
Thermocyclers machines which automatically regulate the optimal
temperatures for PCR cycle was first introduced in 1986.

PCR Primers
PCR Primers are short single-stranded, synthesized oligonucleotides
usually shorter than 50 nucleotides (often 18-25 nucleotides).
PCR primers are important as they are complementary to the
beginning and end of the DNA fragment of interest, which one needs
to amplify.
Primers therefore select the boundaries of the region to be amplified
by PCR.
During the PCR annealing cycle, PCR primers anneal to the
complementary region of the DNA.

Taq DNA Polymerase

Taq Polymerase also simply termed "Taq".
It is a thermostable DNA polymerase used to catalyze the DNA
replication reaction in the PCR cycle.
Taq was first isolated from Thermus aquaticus, a bacterium that lives
in hot springs and hydrothermal vents.
Taq was able to withstand the denaturing conditions (over 90°C)
required during PCR cycling.
This allowed a 30-40 cycles of PCR amplification to be performed
without the need to open the PCR reaction tube and add fresh
polymerase.

Real Time PCR

Real Time PCR monitors the amount of fluorescence emitted during
the PCR reaction and this acts as an indicator of the amount of PCR
amplification that occurs during each PCR cycle.
Thus, in newer Real Time PCR machines, one can visually see the
progress of the Amphlification.

Real time RT- PCR

Real time RT- PCR was developed to quantitative differences in mRNA
expression in a easy and quick manner.

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11
H Y P E R S E N S IT IV IT Y

Hypersensitivity Reaction
Type I HypersensitivityReaction
Type II Hypersensitivity Reaction
Type III Hypersensitivity Reaction
Type IVHypersensitivity Reaction

Hypersensitivity
Q. Define and classified hypersensitivity reaction. Describe immediate type
hypersensitivity reaction? (B.Sc. Nur.-2011), (BDS-2010), (BDS-2007),
(MBBS-2006)
Q. Write short note on the antibody mediated hypersensitivity reaction?
Q. Write short note on the type I hypersensitivity reaction? (MBBS-2006)
(MBBS-2004), (MBBS-2002)
Q. Write short note on the anaphylactic reaction? (MBBS-2005)
Q. Write short note on the type II hypersensitivity reaction?
Q. Write short note on the cytotoxic hypersensitivity reaction?
Q. Write short note on the type III hypersensitivity reaction? (MBBS-2010),
(MBBS-2002)
Q. Write short note on the serum sickness? (MBBS-2008)
Q. Write short note on the immunocomplex hypersensitivity reaction?
(MBBS-2008), (MBBS-2006)
Q. Write short note on the type IV hypersensitivity reaction? (BDS-2007)
Q. Write short note on the delayed hypersensitivity reaction? (B.Sc. Nur.-
2011), (BDS-2007)
Q. Write short note on the cell mediated hypersensitivity reaction? (MBBS-
2003)

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ANSWER
Definition: It is an abnormal exaggerated or inappropriate immune response
which produce physiological or histopathological damage in the host.
Hypersensitivity is sometimes also called allergy.

Type of Hypersensitivity

Immediate type:
Immediate type Hypersensitivity reaction develop in less than 24 hours after
exposure to an antigen.
It is an antibody mediated Hypersensitivity reaction.
Type I Anaphylactic Hypersensitivity reaction
Type II Cytotoxic Hypersensitivity reaction
Type III Immune complex Hypersensitivity reaction
Type V Stimulatory or anti receptor

Delayed type:
It is develop more than 24 hours after exposure to antigen.
It is an cell mediated Hypersensitivity reaction.
Type IV Cell mediated Hypersensitivity reaction

Type I (Anaphylactic Hypersensitivity reaction):

It is immediate type Hypersensitivity reaction.

It is an antibody mediated Hypersensitivity reaction.
It is reagin dependent Hypersensitivity reaction.
It is mediated by IgE antibody.
Mast cell sensitized by IgE antibody.
Antibodies (IgE antibodies) are fixed on the surface of tissue cells (mast cells
and basophils) in sensitised individuals.
The antigen combines with the cell-fixed antibody, leading to the release of
active substances from degranulation of mast cell.

Mediator are two types

(1) The primary mediators: Histamine,
Serotonin,
Eosinophil chemotactic factor of anaphylaxis,
Neutrophil chemotactic factor,
Heparin
(2) Secondary mediators: Prostaglandins
Platelet activating factor and
Cytokines.

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MastCellsandtheAllergicResponseinType-I Hypersensitivity

Combined together, the effects of these mediators produce the clinical

symptoms of anaphylaxis.
Allergens that can cause anaphylaxis include nuts, sea food, eggs, insect
venom and some drugs.

Anaphylactic hypersensitivity reaction are two type

(1) Local : e.g. Hay fever, asthma, conjunctivitis, rhinitis and dermatitis.
(2) Systemic : e.g. bee venum, penicillin , horse serum.

Type II (Cytotoxic Hypersensitivity reaction):

It is immediate type Hypersensitivity reaction.

It is an antibody mediated Hypersensitivity reaction.
It is mediated by IgG and IgM antibody.
It is a cell stimulatory hypersensitivity reaction.
Cell or tissue damage occurs in the presence of complement or mononuclear
cells.

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These reactions involve a combination of IgG (or rarely IgM) antibodies with
the antigenic determinants on the surface of cells leading to toxicity
(cytotoxicity) or bursting of cells (cytolysis).

Examples of type II reaction are

(1) Autoimmune anaemias
(2) Haemolytic disease of the newborn.
(3) Transfusion reaction
(4) Rh incompatibility
(5) Erythroblastosis foetalis

Type III (Immune complex Hypersensitivity reaction):

It is immediate type Hypersensitivity reaction.

It is an antibody mediated Hypersensitivity reaction.
It is mediated by IgG and IgM antibody.
It is caused damage of tissue by antigen-antibody complexes.
Immune complex may precipitate in and around small blood vessels,
damaging cells or membranes, and interfering with their function.
These complexes may be deposited at different sites in the body, including the
kidneys, joints, skin and eye.

Immune Complex Mediated Hypersensitivity

Examples of type III reaction are

(1) Arthus reaction: It is a local type III reaction. (MBBS-2007)
(2) Serum sickness: It is a systemic Type III reaction caused by diphtheria
toxin due to use of horse serum.

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Comparison of Hypersensitivity Reactions

Characteristic Type-I Type-II Type-III Type-IV

Antibody IgE IgG, IgM IgG, IgM None
Antigen Exogenous Cell surface Soluble Intracellular
Respons 15-30 min. Min.-hrs 3-8 hours 48-72 hours
e time or longer
Appearance Wheal & Lysis & Erythema Erythema &
flare necrosis & oedema induration

Histology Baso- and Ab and PMN and Monocytes &

eosinophils complement complement lymphocytes
Transfer with Antibody Antibody Antibody T-cells
Examples Hay fever, Pemphigus, Farmers’ TB test,
asthma Goodpasture lung, SLE poison ivy,
granuloma

Type IV (Cell mediated Hypersensitivity reaction):

It is a delayed type Hypersensitivity reaction.

It is a cell mediated Hypersensitivity reaction.
It is mediated by T helper lymphocytes (Th) and T cytotoxic lymphocytes (Tc).
The antigen activates specifically sensitised T helper lymphocytes (Th) and T
cytotoxic lymphocytes (Tc) leading to the secretion of cytokines with fluid and
phagocyte accumulation.
When Th hand Tc lymphocytes come into contact with an antigen with which
they react, they release cytokines that signal to other blood cells, including
macrophages.
The macrophages increase in number in the affected tissues and cause local
damage.
There are two phases to type IV hypersensitivity:
(1) Sensitisation phase: The antigen is presented by antigen-presenting cells
to Th cells.
(2) Effector phase: The antigen reacts with these specific T h lymphocytes,
which release various cytokines.
Two important forms of type IV hypersensitivity are the tuberculin reaction
and contact dermatitis. (MBBS-2005)

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12
IM M U N I S A T IO N

Immunization
Vaccine
National immunizationpregramme
Pulse Polio programme
RabiesVaccine

Q. Write short note on the immunization? (B.Sc. Nur.-2011) ,(BDS-2006)

Q. Write short note on the vaccine? (MBBS-2003)
Q. Write short note on the live attenuated vaccine? (B.Sc. Nur.-2011) (MBBS-
2008), (MBBS-2004)
Q. Write short note on the killed vaccine ? (B.Sc. Nur.-2011)

ANSWER
Immunization is of two types: active and passive.
Active Immunisation
Active immunisation occurs when an immune response is stimulated by an
immunogen.
This can be due to exposure to an infectious agent (natural immunisation) or
to a microbial antigen (vaccines).
Vaccine
Vaccine is an immune-biological substance. It is produce specific protection
against a disease. Vaccines can be broadly divided into three groups:
(1) Live attenuated: (MBBS-2008)
These are prepared from live organisms.
It is lost their capacity to induce full-blown disease.
It is retaining their immunogenicity.
Examples
Bacterial vaccine: BCG, Oral typhoid vaccine (typhoral)
Viral Vaccine: Oral Polio (Sabin), Yellow fever, Measles, MMR (mumps,
measles and rubella) (MBBS-2005), Chikenpox.

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(2) Inactivated or killed vaccines:

These are prepared from organisms killed by heat or chemicals.
When introduced into the body stimulate active immunity.
Examples
Bacterial vaccine: Typhoid, Cholera, Pertussis and Plague.
Viral vaccine: Rabies, Polio (Salk), Hepatitis A, Hepatitis B, Influenza and
Japanese encephalitis.
(3) Toxoids:
These are the exotoxins produced by certain organisms.
Toxoid loose their toxin activity but to retain their antigenicity.
Examples are diphtheria, tetanus

Passive immunization
Passive immunisation is the administration by purified antibodies
(immunoglobulins) or serum.
It is provide rapid, temporary protection.
Newborns receive natural passive immunity from maternal immunoglobulin
that crosses the placenta or is present in the mother's milk.
Passive immunisation may be used to:
1. Prevent disease after a known exposure.
2. Protect immunodeficient individuals who are incapable of making an
immune response.

National Immunization Programme

Q. Write short note on the national immunization programme? (B.Sc. Nur.-
2011)

ANSWER
National Immunisation Schedule in India is as follow
Vaccine name Time of vaccination
FOR INFANT
(1) BCG At the time of birth
(2) OPV 6 wks, 10 wks and 14 wks
(3) DPT 6 wks, 10 wks and 14 wks
(4) Hepatitis B 0, 1 and 6 months
Or
6 wks, 10 wks and 14 wks
(5) Measles 9 Months
FOR BOOSTER
(6) DPT & OPV 16-24 months
(7) DT 5-6 years
(8) Tetanus toxoid 10 & 16 years

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FOR PREGNANT WOMEN
(9) TT-1 or booster: Early in Pregnancy.
(10) TT-2: One month after TT-1.

Diseases covered in immunization are tuberculosis, diphtheria, pertussis,

tetanus, polio, measles and hepatitis B.
• Examples of vaccines stored in the freezer compartment are the polio and
measles vaccines.
• Examples of vaccines stored in a cold refrigerator but never allowed to
freeze are typhoid, DPT, tetanus toxoid, DT, BCG and diluents.

BCG vaccine gives for tuberculosis.

DPT vaccine gives for Diphtheria, Pertussis and Tetanus.
OPV vaccine gives for Polio.
DT vaccine gives for Diphtheria and Tetanus.
TT vaccine gives for Tetanus.

Pulse Polio Immunisation(PPI)

Q. Write short note on the Pulse Polio Immunization? (MBBS-2004)

ANSWER
This is simultaneous, mass administration of OPV on a single day to all
children.
The age group of children is 0-5 years of age.
OPV gives regardless of previous immunizations.
Pulse Polio immunization occur as two rounds, about 4-6 weeks apart.
In India, the peak transmission season is between June and September.
The doses of OPV during Pulse Polio Immunization are extra doses which
supplement and do not replace the routine doses.
This programme is aimed at eliminating polio infection.

Rabies Vaccine
Q. Write short note on the rabies vaccine? (MBBS-2010), (MBBS-2009)
(MBBS-2007), (MBBS-2006), (MBBS-2002)
Q. Write short note on the neural vaccine?

ANSWER
Rabies is caused by rhabdovirus.
In rabies pre prophylaxis and post prophylaxis vaccination can be given.

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In Post-Exposure Prophylaxis includes local treatment, hyper-immune serum

and vaccination.
LOCAL TREATMENT: The wound should be thoroughly washed with soap,
water and disinfectant.
HYPERIMMUNE SERUM: Human antirabies immunoglobulin (20 IV per kg
body weight) is reserved for the high risk cases.

VACCINATION
Vaccines are of two main categories-neural and non-neural
(i) Neural vaccines are (1) Semple vaccine,
(2) Beta propiolactone (BPL) and
(3) Infant brain vaccine:
Brain tissue vaccines are associated with neurological complications due to
the presence of myelin.
(ii) Non-neural vaccines are
(1) Duck egg vaccine,
(2) Cell culture vaccines
(a) The human diploid cell strain (HDCS) vaccine.
(b) The purified chick embryo cell culture (PCEC) vaccine.
(c) Purified vero cell (PVC) vaccine.

VACCINATION SCHEDULES
When the biting animal can be observed for ten days and the animal remains
healthy, rabies may be excluded and vaccine, if already started, may be
discontinued.

Neural vaccines
The vaccine schedule varies according to the degree of risk to which patient
has been exposed.
The vaccine is given subcutaneously on the anterior abdominal wall. It
requires seven to fourteen injections depending on the degree of risk.

Cell culture vaccines

(1) Pre exposure vaccination:
It is recommended for veterinarians, laboratory staff working with the rabies
virus, animal handlers and wildlife officers.
1.0 ml is given by intramuscular route in the deltoid region, on days 0, 7 and
21 day with booster dose after one year. Repeat after every five year.
(2) Post exposure vaccination:
It is recommended to those who were bitten by a suspected rabid animal.
1.0 ml is given by intramuscular route in the deltoid region, on days 0, 3, 7, 14
and 30 with a booster dose (optional) on day 90.

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Special point
Mucosa associated lymphoid tissue (MALT)
Q.Write short note on the MALT?

ANSWER
The lymphoid tissues lining the mucosal layers of alimentary, respiratory,
genitourinary and other surfaces, either as aggregates or as isolated cells, are
collectively known as 'Mucosa-Associated Lymphoid Tissues (MALT)'.
Examples: Payer's patches, adenoids, tonsils
• Lymphoid tissues of the gut are known as Gut Associated Lymphoid Tissue
(GALT).
Those of the bronchus in the respiratory tract are known as
Bronchus Associated Lymphoid Tissue (BALT).
• MALT contains B-cells, T-cells, as well as phagocytic cells.
Functions of MALT is offers local immunity against microbial infection.

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13
S TA P H Y L O C O C C I

Pathogenecity
Coagula se Test
Free Coa gulase
Bound Coagula se

S e e C o l o r Im a g e 7

Staphylococci
Q. Write short note on the Staphylococcus? (BDS-2007)
Q. Write short note on determinant of pathogenecity of Staphylococci.
Q. Write Short note on pathogenesis of staphylococci?
Q. Write short note on the superantigen?

Pathogenesis & Determinants of Pathogenecity

ANSWER
Pathogenesis of S. aureus infections
Staphylococcus aureus causes a variety of suppurative (pus-forming)
infections and toxinoses in humans.
(1) Superficial skin lesions such as boils, styes, impetigo, carbuncles, abscess,
pemphigus and furuncules;
(2) More serious infections such as pneumonia, mastitis, tonsillitis,
pharyngitis, phlebitis, meningitis, and urinary tract infections;
(3) Exfoliative diseases usch as Ritter’s disease , bullous impetigo &

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Staphylococcal scalded skin syndrome;
(4) Deep-seated infections, such as osteomyelitis and endocarditis.

S. aureus is a major cause of hospital acquired (nosocomial) infection of

surgical wounds and infections associated with indwelling medical devices.
S. aureus causes food poisoning by releasing enterotoxins into food, and toxic
shock syndrome by release of superantigens into the blood stream.

Although methicillin-resistant Staph aureus (MRSA) have been entrenched in

hospital settings for several decades, MRSA strains have recently emerged
outside the hospital becoming known as community associated- MRSA( (CA-
MRSA) or superbug strains of the organism.
S. aureus expresses many potential virulence factors:
(1) Surface proteins that promote colonization of host tissues.
(2) Invasins that promote bacterial spread in tissues
(leukocidin, kinases, hyaluronidase).
(3) Surface factors that inhibit phagocytic engulfment
(capsule, Protein A).
(4) Biochemical properties that enhance their survival in
phagocytes (carotenoids, catalase production).
(5) Immunological disguises (Protein A, coagulase).
(6) Membrane-damaging toxins that lyse eukaryotic cell
membranes (hemolysins, leukotoxin, leukocidin).
(7) Exotoxins that damage host tissues or otherwise provoke
symptoms of disease (SEA-G, TSST, ET).
(8) Inherent and acquired resistance to antimicrobial agents.

Pathogenic factor of staphylococci

(A) Antigenic structure
(1) Peptidoglycan
(2) Protein A
(3) Capsule
(B) Toxin
(1) Exotoxin
(i) Pyrogenic exotoxins: Enterotoxin and
Toxic shock syndrome toxin-1
(ii) Leukocidin
(iii) Exfoliatins
(2) Haemolysin
(C) Enzymes
(1) Catalase
(2) Coagulase
(3) Hyaluronidase
(4) Staphylokinase (Fibrinolysin)

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(5) Lipase
(6) Phosphatase
(7) Deoxy ribonuclease
(8) Proteases

Laboratory diagnosis of staphylococci

Microscopy: Gram’s staining
They are gram positive, nonsporing, nonmotile, noncapsulate,
arrange in cluster or bunch of grapes (due to nonseparation of
daughter cell after three planes division).
Culture: Condition: Aerobic and facultative anaerobic condition
Temperature: 370C
PH: 7.4- 7.6
Nutrient Agar: Pin head and deep golden yellow pigmented colonies grown.
Pigmentation best seen in following condition
At temperature 20-250C
In 1% glycerol monoacetate
In milk agar
Blood agar: β- haemolytic, pinhead colonies grown
Liquid medium: Uniform turbidity seen
Mac Conkey’s agar:
Pin head size pink colonies grown due to lactose fermentation
Biochemical reaction:
Positive for:
Catalase, Coagulage, Mannitol fermatation, Hydrolyse urea,
reduce nitrate, liquefy gelatin, MR and VP
Phasphatase test:
For detection of the enzyme, S. aureus is cultured on nutrient
agar containing phenophthalin diphosphate. On exposure to
ammonia vapour, the colonies turn pink due to presence of
free phenophthalin.

Coagulase test
Q. Write short note on the Coagulase?
Q. Write short note on the coagulase test? (MBBS-2010), (MBBS-2007),
(MBBS-2005), (MBBS-2003)
Q. Write short note on the free coagulase? (MBBS-2006)
Q. Write short note on the bound coagulase? (MBBS-2006)
Q. Write short note on the slide coagulase?
Q. Write short note on the tube coagulase?

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ANSWER
Coagulase test is caused by coagulase enzyme.
Coagulase test are two types: free coagulage and bound coagulase.
Coagulase enzyme is produce by staphylococcus aureus.
Coagulase test is performed to differentiate pathogenic (Staph. aureus) strain
from non-pathogenic strains.
There are eight (A-H) antigenic types of coagulase.
Most commonly coagulase produced by type A strain.

Coagulase test are two types.

(i) Slide coagulase test

Slide coagulase test detects bound coagulase.
Bound coagulase also called Clumping factor.
Clumping factor is heat stable constituent of the cell wall.
Clumping factor reacts directly with the fibrinogen and causes clumping of
cocci due to the precipitation of fibrin on the cell surface.
Procedure of slide coagulase test
A few colonies of bacteria are emulsified in a drop of normal saline on a clean
glass slide.
It is mixed with a drop of undiluted rabbit or human plasma.
Prompt clumping of the suspension occurs with coagulase positive strains.

(ii) Tube coagulase test

Tube coagulase test is done for detection of extracellular free coagulase.
Coagulase is secreted free into the culture medium.
Free coagulase requires the cooperation of Coagulase Reacting Factor, CRF.
It is heat labile.
Coagulase converts fibrinogen into fibrin.

Procedure of tube coagulase test

0.1 ml of an overnight broth culture or an agar culture suspension of the
organism is mixed with 0.5 ml of a 1 in 5 dilution of human or rabbit plasma.
The tubes are incubated in a water bath at 37 0C for three to six hours.
In case of a positive test, the plasma clots and does not flow when the tube is
inverted.
Citrated plasma should not be used because some bacteria may utilize the
citrate and produce false positive reaction. E.g. pseudomonas

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14
STR EPTO CO CC I

Classificationof Streptococci
LancefieldClassification
GriffithTyping
Pathogenesisand Itsfactors
ASOtest
CAMPTest
QullungReaction

Streptococci classification
Q. Write short note on the streptococci? (BDS-2006)
Q. Pathogenesis and laboratory diagnosis of streptococci?
(BDS-2008), (BDS-2007)
Q. Write short note on the streptococci classification?
(BDS-2010), (BDS-2008)
Q. Write short note on the Lancefield classification?
Q. Write short note on the Griffith typing?
Q. Write short note on the β- hemolysis?

ANSWER
Streptococci broadly classified in two group on the basis of O2 requirement.

(1) Aerobic and facultative anaerobic

(2) Anaerobic: e.g. Peptostreptococci
Aerobic and facultative anaerobic again divided into three groups on the
basis of haemolysis in Blood agar medium.

(1) α-haemolysis:
(i) They produce a greenish discolouration around the colonies due to

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partial haemolysis.
(ii) Alpha haemolysis is seen with viridans group of streptococci and
pneumococcus.
(iii) These are classified into species by physiological and biochemical
properties.

(2) β-Haemolysis:
(i) These streptococci produce a clear colourless zone of complete
haemolysis around the colonies.
(ii) Erythrocytes are completely lysed due to the production of two types
of streptolysin by the organisms: Streptolysin O and Streptolysin S.
(iii) Most of the pathogenic streptococci fall into this group.
(iv) Streptococcus pyogenes is the most important and is responsible for
many important human infections.

(3) γ-Haemolysis: Non-Haemolytic Streptococci

(i) They produce no haemolysis.
(ii) Streptococci faecalis is a typical example.

Lancefield classification or serological classification

Streptococci further classified by Lancefield classification into 20
groups.
(A to H and K to V) based on the nature of a carbohydrate (C) antigen
on the cell wall.

Griffith typing:
Group A Streptococcus pyogenes further subdivided into
approximately 80 Griffith serotypes (type 1, 2 etc.) based on their
surface proteins (M protein).
S e e C o l o r Im a g e 8

Pathogenesis of streptococci

Q. Describe the pathogenesis and virulence factors of streptococci?

ANSWER
Virulence determinants (factors) of Streptococcus pyogenes
The various virulence factors are produced by group A streptococci.

They have various pathological actions.

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(1) Adherence (colonization) surface macromolecules:

M protein
Lipoteichoic acid (LTA)
Protein F and S fb (fibronectin-binding proteins)
(2) Enhancement of spread in tissues
Hyaluronidase hydrolyses hyaluronic acid, part of the ground
substance in host tissues.
Proteases
Streptokinase lyses fibrin
(3) Evasion of phagocytosis
Capsule: hyaluronic acid is produced.
C5a peptidase: C5a enhances chemotaxis of phagocytes.
M protein is a fibrillar surface protein. It is interferes in phagocytosis.
It also blocks complement deposition on the cell surface
Leukocidins, including streptolysin S and streptolysin O, are proteins
secreted by the streptococci to kill phagocytes.
(4) Defense against host immune responses
Hyaluronic acid capsule
M protein (antigen) is the only effective protective antibody
(5) Production of toxins and other systemic effects
Exotoxin is superantigen that binds directly to MHC II (without being
processed) and binds abnormally to the T cell receptor of many (up to
20% of) T cells.

Diseases caused by Streptococcus pyogenes

Suppurative conditions
Active infections associated with pus occur in the throat, skin, and
systemically.
Throat: Streptococcal pharyngitis.
Skin: Impetigo involves the infection of epidermal layers of skin.
Cellulitis occurs when the infection spreads subcutaneous tissues.
Erysipelas is the infection of the dermis.
Necrotizing fasciitis involves infection of the fascia and may proceed
rapidly to underlying muscle.
Systemic
Scarlet fever is caused by production of erythrogenic toxin.
Toxic shock is caused by toxic shock-like toxin.
Non-suppurative conditions
Some of the antibodies produced during the above infections cross-
react with certain host tissues. These can indirectly damage host
tissues, even after the organisms have been cleared, and cause non
suppurative complications.

Rheumatic fever:

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M proteins cross reacts with sarcolemma. Antibodies of M protein
cross-react with heart tissue, fix complement, and cause damage.
Glomerulonephritis:
Antigen-antibody complexes may be deposited in kidney, fix
complement, and damage glomeruli.

Antistreptolysin O (ASO) TEST

Q. Write short note on the ASO test? (MBBS-2006), (MBBS-2005), (MBBS-
2003)

ANSWER
This test is important in the investigation of post streptococcal disease. In 80-
85 % of the patients with rheumatic fever there is a rise in ASO antibody,
which is highest soon after the onset of the disease. Infection with group A &
also group C & G can also produce a rise in ASO titer.
Principle:-
It is a qualitative slide agglutination test. The reagent contains latex
particle which are sensitized agglutinate in the presence of ASO in
patient serum.
Result:-
Marked agglutination indicates the presence of ASO. The results are
reported of in Todd units/ml or international Units (IU). ASO titers
higher than 200 Todd Units/ml are indicate of prior streptococcal
infection.

Uses:-
(1). Rising titer indicate active & progressive streptococcal infection.
(2). Decreasing titer indicates recovery of the patient.
ASO titer is elevated in Rheumatic fever
- Glomerulonephritis.
- Scarlet fever.
CAMP Test
Q. Write short note on the CAMP test?

ANSWER
CAMP test is performed to detect Group B Streptococci.
Group B Streptococci is produced CAMP factor.
CAMP Factor show synergestic effect with β-lysin of staphylococcus Aureus.
CAMP test was first introduced by Christie, Atkins and Munch-Peterson,
therefore, it is known as CAMP test.

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Procedure of CAMP Test

Inoculate β-lysin producing staphylococcus Aureus on a sheep blood agar
plate.
Group B streptococci inoculate perpendicular to the S. Aureus and incubate
plate at 370C for 24 hours with 10% CO2.
Arrow head shape β- haemolysis appears at the junction due to enhancement
of production β-lysin by CAMP factor.

Quellung reaction
Q. Write short note on the Quellung reaction? (MBBS-2005), (MBBS-2003)
Q. Write short note on the Capsular Swelling Reaction? (MBBS-2010)

ANSWER
Quellung reaction is also known as Capsule Swelling Reaction.
It was described by Neufeld (1902).
It is used to demonstrate the capsule of bacteria.
It is also used to serological typing of pneumococcus.
The Quellung test can be done either in sputum or in culture.
It is used to be a routine bedside procedure in olden days.
In this reaction, a suspension of pneumococci is mixed on a slide with a drop
of specific antiserum and add a drop of methylene blue.
In presence of the homologous antiserum, the capsule around pneumococci
reveals an apparent swelling, sharply delineated and refractile under the
microscope.

Special point
(1) Streptococcus pneumonia or pneumococcus is typical gram
Positive lanceolate shaped cocci arranged in pair with broad ends in
apposition.
(3) On prolonged incubation of colony, autolysis occurs at the centre causing a
central depression (umbonation) of the colony which creates a typical
“draughtsman” appearance.
(4) Bile solubility test: The enzyme, N-acetylmuramyl-L-alanine
amidase(autolysin), produced by pneumococcus solubilises the
peptidoglycan of cell wall. This autolytic process is augmented by surface
active agents such as bile and bile salts.
(5) Pneumococci is soluble in bile and sensitive to optochin.
(6) Antigenic and virulence factors of pneumococci are polysaccharide
capsule, somatic M protein, Cell wall Carbohydrate, IgA proteases, protein
adhesion and teichoic acid.

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15
N e is s e r ia

Morphology
Pathogenesis
Laboratory Diagnosis

Neisseria gonorrhoeae
Q. Describe the pathogenesis and laboratory diagnosis of gonococci?
Q. Write short note on the gonorrhea? (MBBS-2006)
Q. Write short note on the Neisseria gonorrhoeae? (BDS-2009(O))

ANSWER
Gonorrhoea is a sexually transmitted disease involving urethra in both sexes
but in females, the endocervix is the primary site of infection.
It is caused by Neisseria gonorrhoeae.
Neisseria gonorrhoeae is gram negative, oval cocci, arranged in pairs
(diplococci) with adjacent sides concave (pear or bean shaped).
In smear from purulent material, they are intracellular within polymorphs.
The incubation period is 2-8 days.
Gonorrhoea is an acute urethritis characterised by purulent urethral
discharge.
In males, the acute urethritis may extend to the prostate, testes, seminal
vesicles and epididymis.
In females, the primary infection may spread from urethra and cervix to
Bartholin's glands, uterus, fallopian tubes, ovaries and may cause pelvic
inflammatory disease resulting in sterility.

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Antigenic Structures responsible for pathogenesis of gonorrhea are

following:
1. Polyphosphate Capsule: It is resist phagocytosis.
2. Pili: Pili enhance attachment of the organism to host cells and resist
phagocytosis.
3. Lipopolysaccharide (LPS): It is produce endotoxic effects.
4. Proteins (Porin): It forms pores on surface and part in adhesion of
bacterium to host cell.

Laboratory Diagnosis
Gonococci can be diagnosis in laboratory by various methods.
Specimens: Urethral discharge, cervical discharge (in female) and
centrifuged urine.
Transport: Specimens should be collected with charcoal coated swabs
and transported to the laboratory in Stuart's transport
medium.
Direct Microscopy:
Gram negative, oval cocci, arranged in pairs (diplococci) with
adjacent sides concave (pear or bean shaped).They are
intracellular within polymorphs.
S e e C o l o r Im a g e 1 0

Culture: Chocolate agar and Thayer Martin medium are used for
culture of the specimens and incubated at 35°C to 36°C under
5-10% CO2 for 48 hours.
Treatment: Penicillin and / or doxycycline used for gonorrhea.

Neisseria Meningitidis (Meningococcus)

Q. Describe the pathogenesis and laboratory diagnosis of Neisseria
meningitidis?
Q. Write short note on the Neisseria meningitidis?

ANSWER
Determinants of pathogenicity

(1) Capsular polysaccharide: inhibit phagocytosis.

(2) Endotoxin:
It is lipopolysaccharide.
It is cause inflammatory response and vascular necrosis.
It produce shwartzman reaction.

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It is involve in pathogenesis of petechial haemorrhage and
waterhouse Frideichsen syndrome.
(3) IgA1 protease:
It cleaves the heavy chain of monomeric or polymeric human
IgA 1at hinge region and produce intact Fc and Fab Fragment.
(4) Transferring bound iron
(5) Pilli: Adhesion and antiphagocytic action.
(6) Outer membrane proteins

Pathogenicity
The mildest form of disease is characterized by a fever and malaise.
The most serious form is meningitis.
The manifestations of meningococcal meningitis are chills,fever, malaise, and
headache are the usual manifestations of infection. Signs of meningeal
inflammation are also present.
Infants with meningococcal meningitis rarely display signs of meningeal
irritation.
Signs of meningeal irritation such as spinal rigidity, hamstring spasms and
exaggerated reflexes are common.
Petechiae or purpura occurs from the first to the third day of illness in
meningococcal disease, with or without meningitis.
The lesions may be more prominent in areas of the skin subjected to pressure,
such as the axillary folds, the belt line, or the back.

Laboratory diagnosis
Specimen: CSF, Blood, Pus, aspirate and swabs.
Transport medium: Stuart’s transport medium.
Microscopy:
Meningococci is gram negative, diplococci, nonsporing, nonmotile and
capsulated bacteria seen mainly inside polymorphonuclear
leucocytes.
Diplococcic occur in pair with adjacent sides flattened or concave and
long axes parallel.
Culture:
Growth of meningococci occurs in following media
(1) Blood agar
(2) Chocolate agar
(3) Trypticase soy agar
(4) Thayer- martin selective medium
(5) Modified Thayer- martin selective medium
They are strict aerobes and growth is facilitated by 5-10 % CO2 and
high humidity.
The optimum temperature and pH for the growth are 35-360C and
7.0- 7.4 respectively.

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Small, round, gray, non haemolytic and translucent after 24 hours and
large, opaque cremated margin after 48 hours are colony
characteristics of meningococci.
Biochemically meningococci are catalase and oxidase positive.
Serological reaction:
Specific antibodies to capsular polysaccharide may be demonstrated
by haemaggluitnation.

Treatment
Penicillin is the drug of choice to treat meningococcemia and
meningococcal meningitis.
Cefotaxime or ceftriaxone is used in persons allergic to penicillins.
Rifampin is the chemoprophylactic agent of choice.

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16
D IP H T H E R IA

CorynebactriumDiphtheria
Pathogenesis
LaboratoryDiagnosis
Babes-ErnestGranules
DiphtheriaVaccination
VirulenceTests
Elek'sgelPrecipitationtest
SchickTest
LysogenicConversion

Diphtheria
Q. Describe the Pathogenesis and Laboratory Diagnosis of Diphtheria?
(MBBS-2007), (MBBS-2005)
Q. Describe Morphology and Pathogenesis of Corynebactrium Diphtheria?
(MBBS-2006)
Q. Write the Laboratory Diagnostic of Diphtheria? (BDS-2008)
Q. Write short note on the Diphtheria? (BDS-2009(O)), (BDS-2005)
Q. Write short note on the Babes-Ernest Granules?
Q. Write short note on the Volutin granule?

ANSWER
Diphtheria is most commonly seen in children of 2 to 10 years.
It is caused by Corynebacteria Diphtheria.

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Morphology
Corynebacteria diphtheria is thin, slender, gram positive bacilli, nonacid-fast,
non-sporing, non-motile bacilli.
They are club-shaped due to the presence of metachromatic granules at one
or both ends.
These granules are also called Volutin or Babes-Ernst Granules.
These are composed of polymetaphosphates and represent energy storage
depots. (MBBS-2004)

Metachromatic Granules containing

C. Diphtheria in albert’s staining

The bacilli are usually seen in angular fashion resembling the letters V or L.
This has been called Chinese letter or cuneiform arrangement.
This typical arrangement is due to incomplete separation of the daughter cells
after binary fission.

Pathogenesis (MBBS-2006)
Infection is confined to humans only.
The incubation period is 3 to 4 days, but may on occasion be as short as one
day.
Infection occurs by way of droplet spread.
Diphtheria may be of the following clinical types depending upon the site of
infection.
1. Faucial 2. Laryngeal
3. Nasal 4. Conjunctival
5. Otitic 6. Vulvovaginal
7. Cutaneous
Faucial diphtheria is the commonest type.
In diphtheria, pseudomembrane form by necrosis epithelium together with
fibrinous exudate, leucocytes, erythrocytes and bacteria in respiratory tract.

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The diphtheria toxin diffuses into the blood stream and causes toxaemia.
The pathogenicity is due to production of a very powerful exotoxin by virulent
strains of diphtheria bacilli.
Toxin inhibits polypeptide chain elongation in the presence of NAD by
inactivating the elongation factor, EF-2.
Lysogenic or phage conversion process seen in diphtheria.

Laboratory Diagnosis
Laboratory confirmation of diphtheria is necessary for control measures and
epidemiological studies, but not for the treatment of cases.
Specific treatment should be started immediately after clinical diagnosis
without waiting for laboratory reports.
Diagnostic methods
(1) Direct microscopy
(2) Isolation of organism
(3) Demonstration of its toxicity by virulence tests

Specimens:
Two swabs from the lesions (throat, nose, larynx, ear, conjunctiva, vagina, or
skin) are collected.
One swab is used for smear examination.
Second swab is used for culture.
Swabs are collected prior to start of antibiotics and application of antiseptics
in form of gargles.
The swabs are rubbed over the affected area and pseudomembrane.

Direct microscopy
(1) Gram’s staining: Thin, slender, gram positive bacilli seen in smear.
(2) Albert’s staining: Beaded, slender, green rods in typical Chinese letter
pattern in smear. (MBBS-2004)

Culture
The swabs are inoculated on the following culture media:
(a) Loeffler's serum slope: The colonies are small, circular, white or creamy.
(b) Tellurite blood agar: Black or grey coloured colonies grown.
(c) Blood agar
Three different biotypes of C. diphtheriae show different colonies.
Gravis: Daisy- head colony and weakly haemolytic
Intermedius: Frog’s egg colony and non haemolytic
MItis: Poached egg colony and β- haemolytic
(2) Demonstration of its toxicity by virulence tests.
(A) In VIVO test are Subcutaneous test and Intracutaneous test.
(B) In VIRTO test is Elek's gel precipitation test.

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Diphtheria vaccination
Q. Write short note on the Diphtheria vaccination?

ANSWER
Prophylaxis
1. Active Immunisation
Three doses of DPT are given by intramuscular route at an interval of 4--6
weeks.
Booster doses are given at 18 months and at 5 years.
Diphtheria toxoid combined with tetanus toxoid (DT) is used for booster
doses.
2. Passive Immunisation
500-1000 units of antitoxin (antidiphtheric serum, ADS) is administered
subcutaneously in susceptible patient.
3. Combined Immunisation
All persons receiving ADS as prophylactic measure should receive combined
immunisation.
An alum containing preparation like alum precipitated toxoid should be
preferred for combined immunization.
Treatment
C. diphtheriae is sensitive to penicillin, erythromycin and other antibiotics.

Schick Test
Q. Write short note on the Schick test? (MBBS-2003)

ANSWER
This is an intradermal test to demonstrate circulating diphtheria antitoxin.
This antitoxin may be present either due to previous infection (clinical or
subclinical) or immunisation. This test demonstrates immunity or
susceptibility of a person against diphtheria.
This is a neutralisation test (toxin-antitoxin).
Method
A test dose of 0.2 ml diphtheria toxin containing 1/50 minimum lethal dose
(MLD) is injected intradermally on one forearm (test).
Similar amount of inactivated toxin (inactivated at 70'C for 30 minutes) is
injected on another forearm (control).
Results
Results are read after 1, 4 and 7 days. There may be four types of reaction.
(i) Positive reaction:

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o Test forearm show an area of swelling and erythema appears at the
site of injection of toxin after 24-48 hours.
o The control forearm injected with heated toxin will show no reaction.
o A positive test signifies that the person is susceptible to diphtheria due
to either absence or lack of adequate amount of circulating antitoxin.
(ii) Negative reaction:
o There is absence of any reaction in either forearm (control and test).
o It indicates that the toxin has been neutralised by sufficient amount of
antitoxin present in the blood.
o Interpretation that the person is immune to diphtheria.
(iii) Pseudoreaction:
o There is erythema occurring in both forearms within 6-24 hours.
o Erythema disappears completely from both forearms within four days.
o This indicates that the individual is immune to diptheria and
hypersensitive to the components of diphtheria bacilli.
(iv) Combined reaction:
o There is erythema occurring in both forearms within 6-24 hours.
o Erythema disappears in the control forearm within four days but it
progresses in the test forearm to a typical positive reaction.
o It indicates that the individual is susceptible to diphtheria and
hypersensitive to bacillus.

Virulence Tests
Q. Write short note on the Virulence tests of Diphtheria?
Q. Write short note on the Elek’s gel precipitation test? (MBBS-2008)

ANSWER
(A) In VIVO test are Subcutaneous test and Intracutaneous test.
(B) In VIRTO test is Elek's gel precipitation test.

Elek's gel precipitation test

Q. Write short note on the Elek's gel precipitation test?
Elek's gel precipitation test is an immunodiffusion test.
It is a in vitro test used for demonstration of toxicity of virulence strain of
Corynebacteria diphtheria.
The strain most widely used for toxin production is Park-Williams 8 strain.
The toxigenicity of the diphtheria bacillus depends on the presence of a Tox
gene.
Method of test
A rectangular strip of filter paper soaked in diphtheria antitoxin (1000 units
per ml).

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It is placed on the surface of a 20% horse serum agar plate before solidified.
When the agar solidifies, the test strain is streaked at right angle to the filter
paper strip.
The positive and negative controls are also put up.
The plate is incubated at 37°C for 24 to 48 hours.
The toxin produced by the bacterial growth diffuses in the agar and produces
a line of precipitation.
Non-toxigenic stains will not produce any precipitation line.

Elek's gel precipitation test

Lysogenic conversion
Q. Write short note on the Lysogenic conversion?

ANSWER
 Lysogenic conversion also known as phage conversion.
 Lysogenic conversion is seen in Corynebacteria Diphtheria.
 The toxigenicity of the diphtheria bacillus depends on the presence of
a Tox gene.
 Tox gene can be transferred from one bacterium to another by
lysogenic bacteriophages.
 Beta phage is most important lysogenic bacteriophages.
 Non-toxigenic strains may be convert into toxigenic strain by infecting
them with beta phage.
 The toxigenicity remains as long as the bacteria is lysogenic.
 When the bacteria free from phage, it loses the toxigenicity and
becomes non- toxigenic strain.
 This process is known as lysogenic or phage conversion.

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17
B A C IL L U S A N T H R A X

Anthrax
Malignant pustule
McFadyean’sReaction
Importance of Bacillus

Anthrax
Q. Write short note on the Anthrax?
Q. Write short note on the Bacillus anthrax?
Q. Write short note on the Malignant pustule?

ANSWER
Human anthrax caused by Bacillus anthrasis.
It is polypeptide capsulated, spore bearing, gram positive bacilli.

“Bamboo stick appearance”

bacillus in staining
Anthrax is primarily a disease of animals like cattle and sheep, and less often
of horses and swine.
Humans are occasionally secondarily infected from diseased animals.

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Infection occurs in susceptible animals by ingestion of the spores present in

the soil.
There are three clinical type of disease based on route of infection—
(1) Cutaneous,
(2) Pulmonary and
(3) Intestinal anthrax.
All types lead to septicaemia and meningitis.
(1) Cutaneous anthrax
Cutaneous anthrax follows entry of the spores through the abraded skin.
The face, neck, hands and back are commonly affected sites.
This is commonly found in farmers and persons handling carcasses.
The lesion starts as a papule which becomes vesicle containing fluid (pustule).
The acute inflammatory reaction leads to congestion and oedema of the area
with a central necrotic lesion, which is covered by a black eschar.
The name anthrax, meaning coal, comes from the eschar which is black in
coloured.
Black eschar is later surrounded by a ring of vesicles containing serous fluid
and an area of oedema and induration which may become extensive.
This lesion is called malignant pustule.
Cutaneous anthrax also known as hide porter’s disease.
(2) Pulmonary anthrax
Pulmonary anthrax occurs due to inhalation of the dust or filaments of wool
from infected animals.
This is also called wool sorter's disease.
(3) Intestinal anthrax
Intestinal anthrax is found in those who consume improperly cooked infected
meat.

Microbiological importance of bacillus

Q. Write short note on the microbiological importance of bacillus?

ANSWER
Historical importance
(1) Cohn refuted the theory of spontaneous generation with B. subtilis.
(2) Pasteur and Koch established germ theory of disease.
(3) Koch demonstrated the Koch postules on B. anthracis.
(4) Greenfield and Pasteur developed the first bacterial vaccine.
(5) Pollender observed first pathogenic bacteria (B. Anthracis) under
microscope.
(6) First communicable disease was anthrax.
(7) B. anthracis was first to be isolated in the pure culture and shown to

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possess endospore.
Practical importance
(1) Use as a sterilization control:
B. subtilis subsp. niger: Hot air oven and

B. subtilis subsp. Globigi: Ethylene oxide sterilizer

B. stearothermophilus: Autoclave and Low temperature steam
formaldehyde (LTSF) sterilizer
B. pumilis: Ionizing radiation
(2) Use as an antibiotic and drug production:
Bacitracin: B. licheniformis and B. subtilis
Polymyxin: B. polymyxa
Gramicidin: B. Brevis
Folic acid: B. Coagulans
Alfatoxin: B. megaterium
(3) B. subtilis have provided a model for microbial genetics
Mc Fadyean’s Reaction
Q. Write short note on the Mc Fadyean’s reaction?

ANSWER
It is performed for presumptive diagnosis of anthrax.
This is show disintegrated capsule material around the bacillus after staining
with polychrome methylene blue.
In this reaction, anthrax bacilli containing blood film stained with polychrome
methylene blue for 10-12 seconds.
Amorphous purple material seen around the bacillus due to disintegrated
capsule material.
This reaction is known as McFadyean’s reaction.

Special points
(1) In culture, bacillus arranged as “bamboo-stick” appearance.
(2) Under microscope long, interlacing chains of bacillus, resembleing curled
hair-lock. This gives them the “Medusa head” appearance.
(3) In gelatin stab culture, “inverted fir tree” appearance liquefaction seen.

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18
C L O S T R ID IU M

C lostrid ium Pe rfring e ns

G a s Ga ng re ne
Na gle r’s Re a c tion
Re ve rse C AM P te st
Te ta nus
Bo tulinum
C lostrid ium Diffic ile

CLOSTRIDIUM CLASSIFICATION
Q. Write the classification of Clostridium? (MBBS-2006)

ANSWER
Clostridia of medical importance may be classified on the basis of diseases.

Classification of Clostridium.
A Tetanus C. tetani
B Gas gangrene
1 Established pathogens C. perfringens
C. septicum
C. novyi
2 Less pathogenic
C. histolyticum
C. fallax
3 Doubtful pathogens
C.bifermentans
C. sporogenes
C Food poisoning (BDS-2005)
1 Gastroenteritis C. perfringens(type A)

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2 Botulism
Primary agent C.Botulinum
Other agent C. Butyricum
C. Baratii
D Necrotising enteritis C. Perfrigenes
E Antibiotic associated Diarrohoea and colitis
C. Difficile
Clostridium perfringens
Gas Gangrene
Q. Define the gas gangrene. Enumerate the causative agent of gangrene.
Describe the laboratory diagnosis of Clostridium perfringens?
(MBBS-2006)
Q. Write short note on the gas gangrene? (MBBS-2002)

ANSWER
When a wound gets contaminated by faecal matter or soil, it may lead to
simple wound contamination', anaerobic cellulitis or myonecrosis. It is called
gas gangrene.
Causative agents of gangrene are (MBBS-2002)
(A) Established pathogen of gangrene (1) Clostridium Perfringens
(2) Clostridium Septicum
(3) Clostridium Novyi
(B) Less pathogenic of gangrene (1) Clostridium histolyticum
(2) Clostridium fallax
(C) Doubtful pathogens of gangrene (1) Clostridium bifermentans
(2) Clostridium sporoganes
Cl. perfringens is the most important and common aetiological agent of gas
gangrene (60%), followed by Cl. novyi (30--40%) and Cl. septicum (10-20%).
Cl. perfringens also produces food poisoning and necrotising enteritis in man.
Cl. perfringens Type A produce a phospholipidase (lecithinase C) enzyme
which is responsible for profound toxaemia in gas gangrene.
The incubation period varies from six hours to six weeks.
Cl.perfringens is a large, stout, gram positive bacillus with subterminal spore.
It is capsulated and nonmotile.
Cl.perfringens can grow on blood agar, Robertson cooked meat broth and
thioglycollate broth within 24-48 hours.
It is anaerobic and grows over a pH range of 5.5-8.0 and optimum
temperature for growth is 370C.
Cl. perfringens is saccharolytic.

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Laboratory diagnosis
The diagnosis of gas gangrene must be made primarily on clinical grounds and
the laboratory only confirms the clinical diagnosis.
The specimens to be collected are exudates from wound, necrotic tissue and
muscle fragments.
(1) Gram staining: Large number of Gram positive bacilli with spores is
strongly suggestive of Cl. Perfringens.
S e e C o l o r Im a g e 1 3

(2) Culture:
Cl. Perfringens is isolated in fresh and heated blood agar and Robertson's
cooked meat (RCM) broth.
Beta heamolysis, positive nagler’s reaction, stormy clot reaction and positive
reverse CAMP test are culture characteristics.
(3) Biochemical reaction:
Positive reaction:
MR, H2S, gelatin liquefication, meat turns pink and ferment lactose.
Negative reaction:
Indole and VP negative.
Treatment
Gas gangrene organisms are susceptible to metronidazole, penicillin,
sulphonamide, tetracycline and amoxycillin.
All damaged tissue should be removed promptly by surgical procedure.

Nagler’s reaction
Q. Write short note on the Nagler’s reaction? (MBBS-2009), (MBBS-2006),
(MBBS-2005)

ANSWER
Nagler’s reaction can demonstrate in Clostridium Perfringens.
Lecithinase-C enzyme, which also known as alpha toxin is responsible for
Nagler’s reaction.
Principal of nagler’s reaction is that alpha toxin (lecithinase C) splits lecithin
into phosphoryl choline and a diglyceride (lipid).
The lipid deposits around the colonies resulting in opalescence opacity seen
around the colonies.
For demonstration of nagler’s reaction, Cl. perfringens is grown on a medium
containing 6% agar, 5% Fildes' peptic digest of sheep blood and 20% human
serum or 5% egg yolk in a plate.

Culture plate divided into two half.

(1) One half of the plate, antitoxin is spread on the surface.

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(2) Second half of the plate without the antitoxin.
The inoculated culture plate is incubated at 37°C for 24 hours.

Nagler’s reaction in Cl. Perfringens

Interpretation of result:
Colonies on the one half with antitoxin shows no opacity, due to specific
neutralisation of the alpha toxin.
Colonies on the second half plate without the antitoxin will be surrounded by
opalescence opacity.
This reaction is known as nagler’s reaction.

Reverse CAMP Test

Q. Write short note on the Reverse CAMP test?

ANSWER
Reverse CAMP test performed for detection of Clostridium Perfringens in
presence of group B streptococci.
CAMP test performed for detection of group B streptococci in the presence of
staphylococcus aureus.
CAMP test means Christie, Atkins and Munch-Peterson test.

Principal of reverse CAMP test is that group B streptococci may show some
enhanced haemolysis with other clostridia but only Cl. perfringens exhibits
accentuated zone of haemolysis as butterfly appearance.
Haemolysis is caused by Beta haemolysin toxin.
S e e C o l o r Im a g e 1 1

Procedure of reverse CAMP test

Inoculate the Clostridium Perfringens and group B streptococci perpendicular
in blood agar plate.
Incubate plate at 370C temperature for 18-24 hrs.

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An accentuate zone of haemolysis, as a butterfly appearance seen in blood

agar plate.
This is called Reverse CAMP test.
Tetanus
Q. Write short note on the tetanus? (MBBS-2007)
Q. Write short note on immunization of tetanus?

ANSWER
Clostridium tetani is the causative agent of tetanus.
Clostridium tetani is a gram positive, slender bacillus with spherical, terminal
spores giving the bacillus the characteristic drumstick appearance.
It is non-capsulated and motile (except Cl. tetani type VI) with peritrichous
flagella.
Tetanus develops following the contamination of wound with Cl. tetani
spores.
The source of infection may be soil, dust, faeces etc.
Pathogenesis
Pathogenic effects are mainly due to tetanospasmin (neurotoxin) of Cl. tetani.
Tetanospamin travels along peripheral nerve to anterior horn cells of spinal
cord or brain and interferes the activity of the inhibitory interneurons.
Tetanospasmin is a heat labile, oxygen stable, powerful neurotoxin and rapidly
gets destroyed by proteolytic enzymes. It is protein in nature.
Tetanus classified into four types:
(1) Localised tetanus
(2) Cephalic tetanus: Primary site of infection is head particularly ear.
(3) Generalised tetanus: It is most common form.
Presenting sign of tetanus are
(a) Trismus or lock jaw: Due to spasm of masseter.
(b) Risus sardoricus: Due to sustained contraction of facial muscles.
(c) Opisthotonos: Due to persistant back spasms.
(d) Hyperthermia
(e) Cardiac arhythmias
Laboratory Diagnosis
Specimens generally collected are wound swab, exudate or tissue from the
wound.
Laboratory diagnosis may be made by demonstration of bacilli by microscopy,
culture or by animal inoculation.
1. Microscopy
Gram staining may show Gram positive bacilli with drumstick appearance.
S e e C o l o r Im a g e 1 4

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2. Culture
Specimen is inoculated on freshly prepared blood agar and incubated at 37°C
for 24-48 hours under anaerobic conditions and Cl. tetani produces a
swarming growth.
The specimen is also inoculated in three tubes of Robertson cooked meat
broth.
These cooked meat broths are incubated at 37°C and subcultured on blood
agar plates daily for up to four days.
Gram stained smear from culture shows typical Gram positive bacilli with
drumstick appearance.
3. Toxigenicity Test
Pathogenicity of the isolated organism is established with demonstration of
toxin production.
It is tested in animals.

Immunisation (MBBS-2008)
(a) Active immunisation
Tetanus toxoid is given along with diphtheria toxoid and pertusis vaccine
(DPT) in children.
Three doses are given intramuscularly at 6 weeks, 10 weeks and 14 weeks of
child age.
Booster doses are given at age of 18 months and then at five years.
(b) Passive immunisation
Antitetanus serum (ATS) has been used for preventing tetanus.
It is prepared by immunizing horses with toxoid.
The dose is 1500 IU by intramuscular route immediately after wounding.
(c) Combined prophylaxis
It is given in non-immune person.
First dose of tetanus toxoid inject in first arm and administration of ATS or
HTIG in second arm.
Second and third doses of tetanus toxoid are administrated at monthly
interval in first arm.

Clostridium Botulinum
Q. Write short note on Clostridium Botulinum?
Q. Write short note on Botulism?

ANSWER
Clostridium botulinum is responsible for botulism.
It is a type of food poisoning.
Poisoning occurred after ingestion of poorly cooked sausage

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Morphology
Clostridium Botulinum is large, gram positive, non-capsulated bacillus.
It is motile with peritrichous flagella and bear sub-terminal, oval and
bulging spore.
Pathogenesis
Botulism principally cause by neurotoxin (botulin).
Neurotoxin interferes with the neurotransmission at peripheral
cholinergic synapses and prevents release of acetyl choline which
leads to flaccid paralysis.
Neurotoxin is exotoxin release in the surrounding environment only
after autolysis.
Botulin is heat sensitive but required 20 minutes for boiling.
Toxigenecity is under control of temperate phage.
Pathogenicity is occurs due to preformed toxin in food.

Botulism is three types

Food borne botulism
It is rare life threatening paralytic disease.
Incubation period is 12-36 hrs.
It is caused by contaminated foods like
(1) Home canned vegetables preserved at alkaline PH
(2) Imperfectly treated tinned food
(3) Preserved fish, meat and vegetable
Characteristics of contaminated food
(1) Abnormal appearance and odour,
(2) Bulging of tins
(3) Presence of gas bubbles
Symptom
Vomiting, thirst, constipation, ocular paresis, difficult in swallowing,
speaking and breathing
First sign is constipation,
Diarrhea is not a symptom
Flaccid paralysis
Clinical features due to flaccid paralysis
(1) Diplopia (2) Dysphagia, (3) Flaccid facial
expression
(4) Weakness of peripheral muscles (5) Urinary retention
(6) Respiratory paralysis.

Infant botulism
3 to 20 weeks old infant are affect due to feeding of honey
contaminated by spore.

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Spore germinated and produce toxin.
Clinical features are
(1) Constipation (2) Weakness (3) Lethargy
(4) Cranial palsies (5) Difficulty in sucking and swallowing.
(6) Weak and alter cry (7) Muscle weakness (8) Floppy child
syndrome
(9) Respiratory insufficiency

Wound botulism
It is extremely rare condition.
Type A toxin recovered from most of cases.
Neurological manifestation occurs.
Incubation period is 4-14 days.
No GIT symptoms appears.

Laboratory diagnosis
Specimens
Stool, wound swab, food and vomitus.
Demonstration of the microorganism
Grams staining:
Clostridium Botulinum is large, gram positive, non-capsulated bacillus
and sub-terminal, oval and bulging spore.
S e e C o l o r Im a g e 1 5

Wet mount or hanging drop preparation:

It is motile with peritrichous flagella.
Culture
It can be grown in Blood agar, Robertson cook meat broth and Egg
yolk agar in obligate anaerobic condition at 18-24 hrs.
Colonies characteristics in different media are:
Blood agar:
Colonies are large, irregular, smooth, semitransparent with fimbriate
border.
Haemolysis is seen around colonies.
Egg yolk agar:
Opalescence and pearly effect is seen in colony.
Robertson cook meat broth:
It is predominant proteolytic.
It is turn meat into black color.
Toxigenecity test .
Specimen macerated in sterile isotonic saline and filtrated is divided
into three parts.
One portion heated at 1000C for 10 minutes
Injected into three mice intraperitoneally

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1st mice unheated and polyvalent antitoxin treated

2nd mice unheated and no antitoxin treated
3rd mice heated and no antitoxin treated

2nd mice develop dyspnoea, flaccid paralysis, dies within 24 hrs.

1st and 3rd mice do not show any toxic symptoms.
Prophylaxis and treatment
Gastric lavage with 2-5 % bicarbonate solution used to remove
unabsorbed toxin because toxin is alkaline labile and water soluble.
Saline enema given to remove toxin from colon
Penicillin is drug of choice.

CLOSTRIDIUM DIFFICILE
Q. Write short note on the Clostridium Difficile?

ANSWER
Clostridium difficile is responsible for antibiotic associated colitis.
Lincomycin and Clindamycin antibiotics are prone to cause
pseudomembranous colitis.
Clostridium difficile is a long, slender, gram positive bacillus containing oval
and terminal spores.
It is produce two toxins.
(1) Enterotoxin is primarily responsible for diarrhea.
(2) Cytotoxin is capable of producing cytopathogenic effects.
Laboratory Diagnosis
Toxin can be demonstrated in the faeces by its characteristic effect on human
diploid cells and HEp-2.
ELISA can also be used for the demonstration of toxin.
Treatment
Cl. difficile are susceptible to vancomycin.
Clindamycin and lincomycin should be avoided.
Metronidazole is the drug of choice.

Anaerobiosis
Q. Write short note on the anaerobiosis?
Q. Write short note on the anaerobic culture methods?

ANSWER
Definition
The condition, in which, anaerobic bacteria grow known as an anaerobiosis.

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Methods of anaerobiosis
A. Displacement of oxygen
B. Production of a vacuum.
C. By displacement and combustion of oxygen
D. Absorption of oxygen
E. By reducing method
F. Anaerobic chamber

A. Displacement of oxygen:
(i) By inert gases like H2, N2, CO2, or helium.
(ii) By place a lighted candle in air tight container loaded with
inoculating plates.
B. Production of a vacuum:
Place inoculated plate in vaccum desiccators.
It is outdated method.
C. Displacement and combustion of oxygen:
“McIntosh and filde’s” anaerobic jar is most reliable and widely used
method for displacement and combustion of oxygen.
Catalyst and indicator are used in this jar.
Catalyst / gaspak/ sachet are used to remove O2 from jar.
Indicator (methylene blue strip) is used for verifying the anaerobic
condition. It remains colourless in anaerobic condition and blue in O2.
Alumina pellets coated with palladium containing sachet convert H2
and O2 into water at room temperature.
Gaspak generate H2 and CO2 on the addition of water.

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D. Absorption of oxygen:
Oxygen can remove by chemical and biological methods.
Chemical Method: Following chemical are use for it.
(a) Pyrogallol
(b) Chromium and sulphuric acid
(c) Gas-pak
Biological Method:
This has been attempted by incubating aerobic organisms along
anaerobic bacteria like pseudomonas aeroginosa.
This method is slow and ineffective.
E. By reducing method
Oxygen in culture media can be reduced by various agents such as
glucose, thioglycollate, cooked meat pieces, cysteine and ascorbic
acid.
Two most widely used anaerobic liquid media are
(1) Thioglycollate Broth
(2) Robertson’s Cooked Meat Broth (RCM)
 Unsaturated fatty acids, glutathione and cysteine present in meat
utilized oxygen and liquid paraffin interfere in oxgen exposure.
 Saccharolytic anaerobes turn colour of meat into red.
 Proteolytic anaerobes turn colour of meat into black.
F. Anaerobic chamber:
Anaerobic chamber contain a catalyst, dessicant, hydrogen gas, CO2,
N2, and indicator.

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19
Enterobacteriaceae

ETEC
EPEC
EIEC
EHEC
Tra ve lle r’s Dia rrhoe a

Q. Write short note on the classification of enterobacteriaceae?

ANSWER
Enterobacteriaceae family is classified into various tribes.

Tribe Genus
Escherichiae Escherichia and Shigella
Edwardsielleae Edwardsiella
Salmonelleae Salmonella
Citrobactereae Citrobacter
Klebsielleae Klebsiella, Enterobacter, Hafnia, Serratia
Proteeae Proteus, Morganella, Providencia
Yersinieae Yersinia
Erwinieae Erwinia

Escherichia Coli
Q. Write short note on the ETEC?
Q. Write short note on the EPEC?
Q. Write short note on the EIEC?
Q. Write short note on the EHEC?

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ANSWER
Enterotoxigenic E. coli (ETEC)

ETEC is an important cause of diarrhea in infants and travellers in

underdeveloped countries.
It is also cause traveler’s diarrhea.
ETEC are acquired by ingestion of contaminated food and water.
The disease requires colonization and elaboration of one or more
enterotoxins.
ETEC may produce a heat-labile enterotoxin (LT) that is similar in to the
cholera toxin (Ctx).
It is protein composed of an enzymatically active (A) subunit surrounded by 5
identical binding (B) subunits.
It binds to the ganglioside receptors with B subunit and A subunit convert ATP
into cAMP.
cAMP increase the secretion of water and Cl-, inhibit reabsorption of Na+ and
increase the motility of intestine.
ETEC may also produce a heat stable toxin (ST) that is causes an increase in
cyclic GMP in host cell cytoplasm leading to the same effects as an increase in
cAMP.
This leads to secretion of fluid and electrolytes resulting in diarrhea.
The infective dose of ETEC for adults has been estimated to be at least
108 cells.
ETEC adhesins are fimbriae which are adhere to specific receptors on
enterocytes of the proximal small intestine.

Enteroinvasive E. coli (EIEC)

EIEC closely resemble Shigella in their pathogenic mechanisms.

EIEC penetrate and multiply within epithelial cells of the colon causing
widespread cell destruction.
The clinical syndrome is identical to Shigella dysentery and includes
a dysentery-like diarrhea with fever.
EIEC apparently lack fimbrial adhesins but do possess a specific adhesion.
They do not produce LT or ST toxin.
At least 106 EIEC organisms are required to cause illness in healthy adults.

Enteropathogenic E. coli (EPEC)

EPEC induce a profuse watery, sometimes bloody, diarrhea.

They are a leading cause of infantile diarrhea in developing countries.
EPEC are acquired by ingestion of contaminated meat and water.

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Pathogenesis of EPEC involves a plasmid-encoded protein referred to as EPEC
adherence factor (EAF) that enables localized adherence of bacteria to
intestinal cells.
They do not produce ST or LT toxins.
The infectious dose of EPEC in healthy adults has been estimated to be
106 organisms.

Enteroaggregative E. coli (EAEC)

EAEC strains are associated with persistent diarrhea in young children.

They resemble ETEC strains in that the bacteria adhere to the intestinal
mucosa and cause non-bloody diarrhea without invading or causing
inflammation.
The significance of EAEC strains in human disease is controversial.

Enterohemorrhagic E. coli (EHEC)

EHEC are recognized as the primary cause of hemorrhagic colitis (HC) or

bloody diarrhea, which can progress to the potentially fatal hemolytic uremic
syndrome (HUS).
HUS characterized by (1) Acute Renal Failure
(2) Microangiopathic haemolytic anaemia
(3) Thrombocytopenia
EHEC are characterized by the production of verotoxin or Shiga toxins (Stx).
O157:H7 is the prototypic EHEC and most often implicated in illness of HUS .
The infectious dose for O157:H7 is estimated to be 10 - 100 cells.
EHEC infections are mostly food or water borne and have implicated
undercooked ground beef, raw milk, cold sandwiches, water, unpasteurized
apple juice and vegetables.
The toxin plays a role in the intense inflammatory response produced by EHEC
strains.
Travellers Diarrhoea
Q. Write short note on the Traveller’s Diarrhoea?
Q. Write short note on the ETEC?

ANSWER
The name travellers diarrhoea refers to diarrhoea in persons from the
developed countries within a few days of their visit to one of the developing
countries.
It is caused by Enterotoxigenic Esch. coli (ETEC).

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ETEC form a heat labile enterotoxin (LT) or a heat-stable enterotoxin (ST) or

both.
The identification of ETEC strains only depends on the demonstration of toxins
by biken test.
Biken test is a precipitin test to detect heat labile entero toxin on bacterial
colonies.
This toxin binds to the anti-LT antibodies to form a precipitin line in culture
medium.

S e e C o l o r Im a g e 1 2

Classification of shigella
Q. Write short note on the classification of shigella?
ANSWER
On the basis of biochemical and antigenic structure, shigellae are classified
into four groups
Group A S. dysenteriae
Group B S. flexneri
Group C S. boydii
Group D S. sonnei
Group A (S. dysenteriae)
It is mannitol nonfermaenting shigella.
It consist 10 serotypes.
Serotype 1:- Shiga’s bacillus (it is indole and catalase negative serotpe)
Serotype 2:- Schmidtz bacillus
Serotype 3-7:- Large Sachs groups
Serotype 8-10
Group B (S. flexneri)
This group is most complex antigenically.
They have been classified into eight serotypes.
Serotype -6 is always indole negative and can be divided into 3 biotypes:
Boyd 88, Manchester and Newcastle.
Group C (S. boydii)
It is named after Boyd who first described these strain in India.
19 serotypes have been identified.
Group D (S. sonnei)
It is ferment lactose and sucrose (late).
It is indole negative.
They occur in two forms.
It is causes mildest form of bacillary dysentry.

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Salmonella

Pa thog e n e sis
La b o ra tory Dia g n osis
Typ h oid
Ente ric Fe ve r
Blo o d C ulture
W id a l Te st
Typ h oid Va c c ine

Q. Describe the Pathogenesis and Laboratory diagnosis of salmonella?

(B.Sc. Nur.-2011), (B.Sc. Nur.-2008) , (BDS-2006), (BDS-2005), (MBBS-2007)
Q. Write short note on the enteric fever? (BDS-2007), (MBBS-2006), (MBBS-
2003)
Q. Write short note on the typhoid fever?
Q. Write short note on the blood culture? (B.Sc. Nur.-2009), (B.Sc. Nur.-
2008), (MBBS-2010)
Q. Write short note on the Widal test? (B.Sc. Nur.-2009), (MBBS-2006),
(MBBS-2005)
Q. Write short note on the Typhoid vaccine?

ANSWER
Enteric fever is caused by salmonella typhi and paratyphi (A, B and C).
Salmonellae are Gram negative bacilli, motile, non-sporing and non-
capsulated.
Motility is due to the presence of peritrichous flagella.
Salmonellae cause three types of clinical syndrome in human beings,
Enteric fever,
Septicaemia and
Gastroenteritis or food poisoning
The term enteric fever includes typhoid fever (S. typhi) and paratyphoid fever
(S. paraptyphi A, B, C).
Infections due to S. typhi and S. paratyphi A are prevalent in India.

Pathogenesis of Typhoid fever

The infection is acquired by ingestion through contaminated food and water.
The incubation period is usually 7-14 days.

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Typhoid fever have three stages.

(1) Stage of incubation
(2) Stage of septicaemia
(3) Stage of localization

(1) Stage of incubation

After consuming contaminated food and water, most of bacteria destroyed in
stomach due to gastric acid and lodge into duodenum.
Salmonella are attached with payer’s patches by pili and phagocytosed by
neutrophils and macrophages.
Salmonella multiply within phagocytic cell and enter mesenteric lymphnode.
Salmonella invade the blood stream via thoracic duct leading to a transient
bacteraemia or primary bacteraemia. Blood stream is rapidly cleared by cells
of mononuclear phagocytic system in the liver, Bone marrow, spleen, lung &
lymph node.
(2) Stage of septicaemia
About 10th day, these parasitized cell undergo necrosis and salmonella appear
into the blood stream, leading to secondary and ha=eavier bacteriaemia.
It is caused toxaemia and septicaemia.
(3) Stage of localization
Salmonella localized in the various organs from blood stream. E.g. Gall
baldder, liver, spleen, intestine, bone etc.
In intestine, salmonella produce inflammation in payer’s patches and necrosis
and sloughing in lymphatic follicles, results typhoid ulcer form which lead to
haemorrhage and perforation of intestine.
The illness is usually gradual, with headache, anorexia and congestion of
mucous membranes.
The characteristic features are hepatosplenomegaly, step-ladder pyrexia with
relative bradycardia and leucopaenia.
Skin rashes known as rose spots may appear during the second or third week.
The infecting organisms appear in stool during second to third week and in
urine during third to fourth week.
Salmonellae possess three types of antigens based on which they are
classified by Kauffmann-White Scheme.
(1) Flagellar antigen 'H'
(2) Somatic antigen 'O'
(3) Surface antigen 'Vi'

Laboratory Diagnosis
Bacteriological diagnosis of enteric fever consists of
1. Isolation of bacilli
2. Demonstration of antibodies
3. Demonstration of circulating antigen
4. Other laboratory tests.

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1. Isolation of Bacilli
This may be done by culture of specimens like blood, faeces, urine, aspirated
duodenal fluid etc.
Selection of relevant specimen depends upon duration of illness.

Duration of disease Specimen for examination

1st Week Blood culture
2nd Week Blood culture
Faeces culture
Widal test
3rd Week Widal test
Blood culture
Faeces culture

BLOOD CULTURE
10 ml of blood is collected by venepuncture under aseptic conditions and
transferred into blood culture bottles (glucose broth and taurocholate broth).
Blood contains substances that inhibit the growth of the bacilli and hence it is
essential to dilute out these substances (5 ml blood into the 50 ml culture
media, 1:10 dilution).
Add liquoid (sodium polyanethol sulphonate) to counteracts the bactericidal
action of these substances.
Both blood culture bottles are incubated at 37°C for overnight.
(1) The glucose broth is subcultured on blood agar
(2) The taurocholate (bile) broth on MacConkey's agar and Pale non-lactose
fermenting (NLF) colonies appear on MacConkey's agar.
Subcultures should be done every other day up to ten days before declaring
the culture as negative.

CLOT CULTURE
It is an alternative to blood culture.
Blood clot use in place of whole blood and serum is use in widal test.

FAECES CULTURE
Faecal cultures may be helpful in patients as well as for the detection of
carriers.
Faeces cultures are generally positive after the second week of illness.

URINE CULTURE
Cultures are generally positive only in the second and third weeks.
Culture of bile is usually positive and may be useful in detection of chronic
carriers.

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Culture medium uses for these cultures are

(1) Enrichment medium:
Selenite and
Tetrathionate broth.
(2) Solid medium: MacConkey's agar,
DCA (Deoxycholate citrate agar),
XLD (Xylose lysine deoxycholate) and
Wilson-Blair media.
On Wilson-Blair medium,
S. typhi forms large black colonies with a metallic sheen.
S. paratyphi A produces green colonies due to the absence of H2S production.
On MacConkey's agar or DCA,
Salmonellae grow as pale yellow, non-lactose fermenting (NLF) colonies.
Enrichment media (selenite F and tetrathionate broths) are incubated for 6--8
hours before subculture on to selective media such as MacConkey's agar and
DCA.
Microscopy
Gram`s staining: Gram negative bacilli, non-sporing and non-capsulated.
Hanging drop preparation: Actively motile salmonella seen due to the
presence of peritrichous flagella.

Biochemical Reactions (MBBS-2003)

Salmonellae are Catalase positive,
Oxidase negative,
Nitrate reduction positive and
Ferment glucose, mannitol but not lactose or sucrose.
Slide Agglutination Test
A loopful of the growth from a nutrient agar slope is emulsified in two drops
of saline on a microscopic slide.
Agglutination is first carried out with the polyvalent O and the polyvalent H
antisera.
Positive agglutination indicates that the isolate belongs to genus Salmonella.

2. Demonstration of Antibodies

WIDAL TEST (MBBS-2006)

It is an agglutination test for detection of agglutinins (H and O) in patients with
enteric fever in salmonella infection.
Salmonella antibodies start appearing in the serum at the end of first week
and rise sharply during the third week of enteric fever.
Two specimens of sera at an interval of 7 to 10 days are preferred to
demonstrate a rising antibody titre.

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Procedure
Take two type of tube.
(1) Narrow and conical bottom Drayer’s tube for H agglutination.
(2) Short round bottomed Felix tube for O agglutination.
Take equal volumes (0.4 ml) of serial dilutions of the serum (1:10 to 1:640) in
both type tubes.
The H and 0 antigens are mixed and incubated in a water bath at 37°C for 4
hours and read after overnight refrigeration at 4°C.
H agglutination leads to the formation of loose, cotton wool clumps in
Drayer’s tube.
O agglutination appears as a granular deposit at the bottom of the Felix tube.
Control tubes show a compact deposit (button formation).
Interpretation of widal test
(a) Agglutinins usually start appearing in the serum by the end of the first
week, so first week may give a negative result.
(b) Rising titre: Demonstration of a fourfold or greater rise in titre of both
H and 0 antibodies at an interval of 7-10 days is more meaningful and
diagnostic than a single test.
(c) Single test: O titre of 1:100 or more and H titre of 1:200 or more
signifies presence of active infection.

Special points for Widal test

 In endemic area, low titre of agglutinins is present in the serum of
normal persons.
 In immunisation with TAB vaccine, individuals may show high titres of
antibody to S. typhi, S. paratyphi A and B.
 H-agglutination in serum indicates enteric fever or a latent infection.
 H- agglutinin is more reliable than O agglutinin.
 O- antigen is common in all the salmonellae.
 Anamnestic reaction: Persons who have had past enteric infection or
immunisation may develop anamnestic reaction during unrelated
fever like malaria, influenza etc.
 Anamnestic reaction can be detected by repeating the widal test after
a week.
 Anamnestic reaction show a transient rise in H antibody, whereas, in
enteric fever show a sustained rise in H antibody.
 Cases treated early with chloramphenicol may show a poor antibody
response.
 Test may be positive in many healthy carriers.
3. Demonstration of Circulating Antigen
The antigen can be detected by
(1) Coagglutination test.
(2) Counterimmunoelectrophoresis (ClEP) and
(3) ELISA

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Treatment
Drug of choice for enteric fever: Chloramphenicol
Drug of choice for multiresistant cases:
(1) Fluoroquinolones (Ciprofloxacin, Pefloxacin, Ofloxacin).
(2) Third generation cephalosporins (Ceftazidime, Cefotaxime,
Ceftrioxone, Cefoperazone).

Vaccination
Vaccine is indicated for travellers or who live in endemic areas.
Three type vaccines are use for enteric fever.
(1) TAB Vaccine
(2) Live oral vaccine (typhoral)
(3) Injectable vaccine (typhim-Vi)
Live oral vaccine (typhoral) and Injectable vaccine (typhim-Vi) are
recommended only over five years of age.

TAB vaccine
It is heat-killed whole cell vaccine which contains S.typhi 1,000 millions,
S. paratyphi A and B, 750 millions each per ml and preserved in 0.5 per cent
phenol.
Dose schedule: The vaccine is given subcutaneously in two doses of 0.5 ml at
an interval of 4-6 weeks followed by booster every three years.
Protection: It varies from 3-7 years with an efficacy of 60-80%.

Live oral vaccine (typhoral)

It is a stable mutant of S.typhi strain Ty2 1a, lacking the enzyme UDP-
galactose-4-epimerase.
Dose schedule: One capsule orally an hour before meal with a glass of water
on day 1, 3 and 5.

Injectable vaccine (typhim-Vi)

It is contain purified Vi polysaccharide antigen from S.typhi strain Ty2.
It is given as a single subcutaneous or intramuscular injection.

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20
VIBRIO CHOLERAE

P a th og e ne s is
La b o ra to ry D ia g no s is
C ho le ra
C las s ica l vib rio
E LTo r V ibrio

Classification of Vibrio
Q. Write short note on the classification of vibrio?
ANSWER
The name vibrio is derived from the characteristic vibratory motility (vibrare
meaning is vibrate).
Broadly vibrio classified into two groups
(1) Non- halophilic Vibrio: Capable of growing on media without added salt.
(i) Vibrio cholera O1:
Classical and Eltor (Each further classified into Ogawa, Inaba
and Hikojima serotypes)
(ii) Non O1 Vibrio cholera or Non cholera vibrio (NCV) or
Non agglutinating vibrio (NAG vibrio) or Vibrio cholera O2-O139
(2) Halophilic Vibrio: Unable to grow on media without added salt.
(i) V. Parahaemolyticus
(ii) V. Alginolyticus
(iii) V. Vulnificus
(iv) V. Mimicus

Cholera
Q. Describe the Pathogenesis and Laboratory diagnosis of vibrio cholera?
(B.Sc. Nur.-2008), (MBBS-2010)

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Q. Write short note on the cholera? (MBBS-2009)

Q. Write short note on the cholera toxin?(MBBS-2008)

ANSWER
Cholera is an acute diarrhoeal disease cause by Vibrio Cholerae (both O-1 and
O-139).
The human infection occurs by ingestion of contaminated foods and drink.
Vibrios become adherent to the epithelium.
V. cholerae produces enterotoxin and the disease.

Mechanism of action of enterotoxin

Enterotoxin is a heat labile protein.
It is composed of two type subunits.
(1) A subunit: It has divided into two fragment A1 and A2
Fragment A1: Activates adenyl cyclase in the enterocyte.
Fragment A2: Links the biologically active A1 to the B subunit.
(2) B subunit: Bind to the Gm ganglioside receptor at the brush border of
epithelial cells of small intestine and facilitates the entry of
subunit A1.
The activation of adenyl cyclase converts adenosine triphosphate (ATP) to
cyclic adenosine 5'-monophosphate (cAMP).
The marked increase of cAMP caused following changes.
(1) Intense and prolonged hyper secretion of water and chlorides.
(2) Inhibits the reabsorption of sodium.
(3) The intestinal lumen is distended with fluid.
(4) Hypermotility leads to profuse watery diarrhoea.

The massive loss of water and electrolytes (sodium and bicarbonates) by

action of enterotoxin leads to
1. Dehydration causing haemoconcentration, anuria and hypovolaemic shock.
2. Base-deficit acidosis and
3. Muscle cramps due to hypokalaemia.

LABORATORY DIAGNOSIS
SPECIMENS
(i) Watery stool
(ii) Rectal swab
Collection and Transport:
Specimens should be collected preferably prior to start of antibiotics.
These should not be collected from bedpans due to risk of
contamination.
Specimens should be immediately transported to the laboratory for
processing.
Specimens hold and transport in following media:

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(1) Venkatraman-Ramakrishnan (VR) medium (Holding medium)
(2) Cary-Blair medium (Holding medium)
(3) Alkaline peptone water (Transport medium)
(4) Monsur's medium (Transport medium)
(5) Strips of blotting paper (Transport medium)
Direct Microscopy
Gram’s staining:
It is shows typical Gram negative, comma shaped bacilli, non-sporing,
noncapsulated.
In smear made from mucous flake, vibrio arranged in “fish in stream”
appearance.
Hanging drop preparation:
Vibrio cholera show darting motility by a polar flagellum.
Culture
Stool sample is directly cultured on following media.
(a) Selective media (BSA, TCBS or Monsur's GTTA)
(b) Non-selective media (blood agar and MacConkey's agar).
These plates are incubated at 37'C for overnight.

Vibrio cholera colonies identified in different media.

On Nutrient agar grow colonies which are bluish tinge in transmitted light.
On Blood agar grow Beta heamolytic colonies in Eltor vibrio.
On MacConkey's agar grow pale colonies due to non lactose fermentation.
On Monsur's gelatin taurocholate trypticase tellurite agar medium (GTTA),
grow a black centre with a turbid halo around the colony.
On Thiosulfate citrate bile salt sucrose (TCBS) agar grow yellow colonies.
On Alkaline bile salt agar (BSA) grow translucent colonies.
String test:
A loopful of the vibrio growth is mixed with a drop 0.5% sodium deoxycholate
solution on a slide.
The suspension becomes mucoid. When the loop is lifted fron-m the
suspension, a string is formed.

Cholera red reaction:

V. Cholera produced indole and reduce nitrate into nitrite.
Indole and nitrite produced nitrosoindole.
Addition of a few drops of concentrated sulphuric acid in 24 hours culture
peptone water, produce a red colour due to nitrosoindole.

Biochemical Reaction:
Does not ferment lactose.
Positive reaction for : Catalase (+), Oxidase (+), Indole (+),
Nitrate reduction (+), Urease (+),
Gelatin liquefaction (funnel shape) (+)

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Vibrio cholera further differentiate into ElTor and Classic Vibrio.

(See difference of Classic and ElTor vibrio in chapter Difference)

Agglutination Test
Colonies are picked up with a straight wire and tested with V. cholerae 01
antiserum.
By agglutination test V. Cholera further divided into three types.
Serotype O antigen
Ogawa AB
Inaba AC
Hikojima ABC

Treatment
Oral Rehydration Therapy to correct the severe dehydration and salt
depletion.
Tetracycline is useful in reducing the number of stool and it also shortens the
period of excretion of vibrios.

Vibrio Parahaemolyticus
Q. Write short note on the halophilic vibrio?
Q. Write short note on the Vibrio parahaemolyticus?

ANSWER
It was originally isolated in an outbreak of food poisoning due to sea fish in
japan and India.
It ican be grow in the presence of 7 to 8% Sodium chloride.
It is a gram negative, motile capsulated, curved bipolar stained bacilli.
On TCBS agar, the colonies are green and opaque in colour.
Lactose, sucrose, xylose are not fermented.
Gives positive reaction for oxidase, catalase, nitrate, indole and citrate.
Strains of V. parahaemolyticus associated with gastroenteritis lyse human
erythrocytes in the Wagatsuma’s agar, a special high salt mannitol medium.
This haemolysis is known as Kanagawa Phenomenon and is due to heat
stable haemolysin.
It caused food poisoning associated with abdominal pain, vomiting, diarrhea
and fever.

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21
Y E R S IN IA P E S T IS

P a th og e ne s is
La b o ra to ry D ia g no s is
P lag u e
B u b on ic P lag u e
P ne u m o n ic P la g ue

PLAGUE
Q. Describe the pathogenesis and Laboratory of Yersinia pestis?
Q. Write short note on the plague?
Q. Write short note on the bubonic plague?
Q. Write short note on the pneumonic plague?

ANSWER
Plague is caused by Yersinia pestis.
Plague is a natural disease of rodents and is transmitted to man via the bite of
infected rat flea (X.cheopis).
Y. pestis is a natural pathogen of rodents and causes zoonotic disease called
plague.
The incubation period is 2-6 days.
In man, plague occurs in three forms: bubonic, pneumonic and septicaemic.
1. Bubonic Plague
The inguinal lymph nodes become enlarged and suppurate due to bite of
infected rat flea on the legs. Therefore, it is called bubonic Plague (bubo
means groin).

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2. Pneumonic Plague
It can be transmitted from man to man by droplet infection (airborne route)
and produce haemorrhagic pneumonia in lung.
3. Septicaemic Plague
It is terminal event of bubonic and pneunionic plague.
Massive involvement of blood vessels results in haemorrhages in the skin and
mucosa.
Due to this manifestation, the disease is given the name black death.

Laboratory Diagnosis
1. Specimens
(i) Pus or fluid aspirated from bubo in bubonic plague.
(ii) Sputum and blood in pneumonic plague.
(iii) Blood in septicaemic plague.
(iv) Splenic tissue on postmortem.
(v) CSF in meningeal plague.

Direct Microscopy
Gram’s staining:
Y. pestis is a short, ovoid, gram negative bacillus, with rounded ends
and convex sides, occur singly, in short chains or in small groups.
Methylene blue staining:
It shows bipolar staining (safety pin appearance) with two ends darkly
or densely stained and the central area clear.
S e e C o l o r Im a g e 1 6

Culture
Yersinia pestis is aerobic and facultatively anaerobic.
The optimum temperature for growth (unlike most pathogens) is 27°C but the
capsule develops best at 37°C.
1. Nutrient Agar: Colonies are small and transparent.
2. Blood Agar: Colonies are non-haemolytic and dark brown due to
the absorption of the haemin pigment.
3. MacConkey Agar: Colourless colonies are formed.
4. Nutrient broth: Flocculent growth occurs at the bottom.
5. Ghee Broth: Stalactite growth occurs.
Suspended colonies in ghee broth known as stalactite growth.
Biochemical reaction
Catalase Positive
Indole Negative
Urease Negative
Methyl red (MR) Positive
Voges Proskauer (VP) Negative
Citrate Negative

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22
H A E M O P H IL U S

S a te lliti sm
S o ft C h a n c re
C h a n cr o id

Satellitism
Q. Write short note on the Satellitism?

ANSWER
Satellitism phenomenon is demonstrated in Haemphilus influenza.
Satellitism demonstrates the V factor availability in blood agar media.
Principal
For the growth of Haemphilus influenza, growth factor X and V are essential.
V factor is not freely available in blood agar but remain inside of RBC.
V factor released from RBC by heating agar at 80-900C or by β-haemolyis in
staphylococcus aureus.
Procedure:
Suspected H. influenza inoculates on a blood agar plate.
Staphylococcus auerus is streaked across the same blood agar plate.
It is incubated at 37°C for 18-24 hours.
Interpretation of result
The colonies of H. influenzae will be large near the streak of staphylococci due
to higher concentration of V factor.
The colonies of H. influenzae will be smaller away from the streak of
staphylococci due to lower concentration of V factor.

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Staph. Aureus

H. Influenzae colonies

Satellitism in blood agar

by H. Influenzae

CHANCROID
Q. Write short note on the Soft chancre?
Q. Write short note on the Chancroid?

ANSWER
Chancroid is also known as soft chancre.
Chancroid or soft chancre caused by Haemophilus ducreyi.
It is a highly contagious sexually transmitted disease (STD).
It is characterized by tender, non-indurated, irregular ulcers on the genitalia.
The infection remains localised, spreading only to the inguinal lymph nodes
which are enlarged and painful.
It can leads to inguinal abscess called bubo.
Haemophilus ducreyi is a short, gram negative coccobacillus.
They frequently take bipolar staining.
The microscopic findings have been described as resembling a 'school of fish'
appearance.
It requires X factor but not V factor for its growth.
It can be grown on chocolate agar after adding 1% of iso vitex.

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23
B O R D E T E LL A P E R T U S S IS

Bordetella Pertussis
Whooping Cough
Pertussis Vaccine

WHOOPING COUGH
Q. Write short note on the Whooping cough? (MBBS-2003)
Q. Write short note on the Bordetella pertusis?
Q. Write short note on the Pertusis?
Q. Write short note on the Pertusis vaccine?

ANSWER
Whooping cough is caused by bordetella pertusis.
Whooping cough is a childhood disease.
The incubation period is about one to two weeks.
Infection is transmitted by droplets.
It consists of three stages namely, the catarrhal, paroxysmal and convalescent,
each lasting approximately for two weeks.
Catarrhal stage:
This is maximally infective stage.
Clinical diagnosis is difficult in this stage.
Paroxysmal stage:
The violent spasms of continuous coughing is followed by a long
inrush of air into the almost empty lungs, with a characteristic
'whoop'.

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Convalescent stage:
The frequency and severity of coughing gradually decrease.
Pathogenesis
Lipopolysaccharide, Tracheal Cytotoxin, Pertusis Toxin, Adenylate Cyclase,
Filamentous Haemagglutinin and Haemolysin are responsible for whooping
cough.
Pertusis toxin is also known as lymphocytosis producing factor, histamine
sensitizing factor and islet activating protein.
Subconjuctival haemorrhage, atelactasis, bronchopneumonia , convulsion and
coma are complication of pertusis.
Laboratory diagnosis
Bordetella pertusis is a small, ovoid, gram negative coccobacillus.
It is non-motile, capsulated and non-sporing.
BordetGengou (glycerol-potato--blood agar) is a commonly used medium for
culture.
Modified Stuart’s medium or charcoal agar medium should be used for
transport of the swab.
Cough plate method used to direct inoculation of cough droplet into medium.
In this medium, bisected pearls or mercury drops like colonies grown.
Pertusis vaccine is generally administered intramuscularly in combination with
diphtheria toxoid and tetanus toxoid (DPT) at 6 wks, 10 wks and 14 wks age of
child.
HACEK group bacteria
Q. Write short note on the HACEK?

ANSWER
HACEK refers to a group of fastidious gram negative bacteria, normally
present in the mouth.
Blood cultures from patients with these bacteria take 7 to 30 days to become
positive.
They grow better in blood agar and chocolate agar.
They cause endocarditis, bacteraemia and mixed flora infections.
Drug resistance is common ly present.
H Haemophilus sp. (parainfluenzae, aphrophilus, paraaphrophilus)
A Actinobacillus actinomycetemcomitans
C Cardiobacterium hominis
E Eikenella corrodens
K Kingella kingae

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24
M Y C O B A C T E R IU M

Tuberculosis
Pathogenesis
Laboratory Diagnosis
Tuberculin Test
Montoux Test
MOTT
Leprosy
Lepromin Test

Tuberculosis
Q. Describe the pathogenesis and Laboratory diagnosis of tuberculosis?
(BDS-2010), (MBBS-2006)
Q. Write short note on the tuberculosis?
Q. Write short note on the ZN staining?

ANSWER
Pathogenesis
Tuberculosis may involve lungs (pulmonary) or sites other than lungs
(extrapulmonary).
Approximate all part of body affected by tuberculosis.
Tuberculosis is caused by mycobacterium tuberculosis.
The infection is commonly acquired by inhalation of infected droplets
coughed or sneezed.
Tubercle bacilli are engulfed by alveolar macrophages but they survive and
multiply in macrophages.
The immune mechanism is mainly of cell mediated type.

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The important cell type is the CD4+ helper T-lymphocytes that release
interferon, interleukins and Toxic necrosis factor- α.
An avascular granuloama form in tissue, known as ‘tubercle’

Primary tuberculosis
Tubercle bacilli within the alveolar macrophage multiply to form a subpleural
focus known as Gohn- focus.
Gohn-focus together with the hilar lymph nodes forms the ‘Primary
Complex’.
It heals spontaneously, in most cases, leaving behind a calcified nodule.

Secondary tuberculosis
It is occured due to reactivation of latent infection, lesion undergo necrosis
and tissue destruction leads to cavitation.
Laboratory Diagnosis
Bacteriological diagnosis of mycobacterium tuberculosis is can be established
by
(1) Microscopy,
(2) Culture examination (MBBS-2006)
(3) Serological test
(4) Molecular test
(5) Skin test

Specimen
Specimen collection depends on the site of involvement.
(i) Pulmonary tuberculosis
Sputum is the most common specimen.
Two morning sputum specimen may be collected according RNTCP
(Revised National Tuberculosis Control Programme).
Laryngeal swab or bronchial washings or gastric washings may also be
examined.
(ii) Meningitis Tuberculosis: Cerebrospinal fluid (CSF).
(iii) Renal tuberculosis: Three consecutive days morning samples of urine are
examined.
(iv) Bone and joints tuberculosis: Aspirated fluid.

Specimen concentration methods are in use:

(1) Petroff's method: It is a simple and widely used technique.
(2) Mucolytic agents such as N-acetyl-L-cysteine (NALC) with sodium
hydroxide
(3) Pancreatin
(4) Centrifugation
(5) Homogenisation method

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Petroff’s method
It is a simple and widely used concentration method.
Specimen is mixed with equal volume of sterile 4% NaOH solution and
incubated at 370C for 30 minutes with intermittent shaking.
After centrifugation at 3,000 r.p.m. for 30 minutes, it is neutrilised with 8%
HCl in the presence of a drop of phenol red indicator.
After concentration method, 10-100 bacilliper ml sputum is sufficient for
detection in culture.
Direct Microscopy
M. tuberculosis is a slender, straight or slightly curved bacillus with rounded
ends, occurring singly, in pairs or in small clumps.
These bacilli are acid-fast, non-sporing, non-capsulated and non-motile.
They are Gram positive but are difficult to stain with the Gram stain.
M. tuberculosis is acid fast bacilli.
To be detect microscopically, there must be 50,000-1,00,000 bacilli per ml of
sputum.
Acid fast bacilli can be stain by various staining methods
(1) Ziehl- Neelsen staining (see chapter staining)
(2) Kinyoun acid fast stain/ Cold stain
(3) Flurochrome stain

Fluorescence staining:
Auramine 'O' or auramine rhodamine dyes are used and examined
under ultraviolet light.
The bacilli appear as bright bacilli against dark background.
Culture (MBBS-2006)
Culture is a very sensitive method for detection of tubercle bacilli.
It may detect as few as 10 to 100 bacilli per ml.
Lowenstein Jensen medium is used for culture of tubercle bacilli.
M. tuberculosis is an obligate aerobe.
Optimum temperature for growth is 370C (range 30-40°C).
Optimum pH is 7.0 (range 6.4 to 7.0).
The bacilli grow slowly (generation time 14-15 hours).
It is usually grow in 2 to 8 weeks.
Colonies of M. tuberculosis grow as “ Rough, Tough and Buff” colonies.
Rough due to dry, irregular growth;
Tough due to difficult in lifting the colony from surface;
Buff due to pale yellow colour;
Biochemical reaction
Niacin test, Nitrate reduction test and urease test are positive in M.
tuberculosis.
In radiometric method such as BACTEC, the growth may be detected in about
a week by using middlebrook’s 7H12 liquid medium containin C14 labeled
substrates.

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In this system, multiplication is detected by metabolic activity rather than by

the visible growth.
Serology
Anti mycobacterial antibodies can be detected in patient serum by Enzyme
Linked Immuno Sorbent Assay (ELISA), Radio Immuno Assay (RIA) and Latex
agglutination assay.

Molecular Methods
(1) Polymerase chain reaction (PCR) is a rapid method in diagnosis of
tuberculosis.
(2) Chemiluminescent techniques by using nucleic acid probs.
(3) High performance liquid chromatography (HPLC)
(4) Gas liquid chromatography
(5) Restriction Fragment Length Polymorphism (RFLP)

Treatment
Two type antitubercular drugs are used for treatment of tuberculosis.
Bactericidal antitubercular drugs:
Rifampicin (R),
Isoniazid (H),
Pyrazinamide (Z),
Streptomycin.
Bacteriostatic antitubercular drugs:
Ethambutol (E),
Thiacetazone,
Ethionamide,
Paraaminosalicylic acid (PAS) and
Cycloserine.
Short course regimens of 6-7 months are used.
Combinations of two or more drugs are used due to emerge resistance by
mutation and gene transfer.
The Directly Observed Therapy Short term (DOTS) is being used to prevent
deterioration of resistance problem and ensure compliance.

Vaccination
BCG vaccine is given intradermally in a dose of 0.1 ml.
BCG vaccine should be given soon after birth.
BCG vaccine is live attenuated vaccine.
BCG means Bacille Calmette Guerin.

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Tuberculin Test
Q. Write short note on the tuberculin test? (MBBS-2008)
Q. Write short note on the Montoux test?

ANSWER
Many methods had been describe for tuberculin testing but most widely used
method is Montoux test.
1. Principle
It is delayed or type IV hypersensitivity reaction.
2. Reagents
(i) Old tuberculin (OT)
It was originally described by Robert Koch.
This crude product may lead to serious complications in some patients.
It is now rarely used.
(ii) Purified protein derivative (PPD)
It was prepared by growing M. tuberculosis in a semisynthetic medium.
It is called purified protein derivative (PPD).
The dosage of PPD is expressed in tuberculin unit (TU).
3. Method
Mantoux test
0.1 ml of PPD containing 5 TU is injected intradermally into flexor aspect of
forearm.
A PPD dose of 1 TU is used when extreme hypersensitivity is suspected.
4. Result
In the Mantoux test the site of injection is examined after 48-72 hours and
interpreted as follows:
Positive test
There is induration (local oedema) of 10 mm diameter or more surrounded by
erythema at the site of inoculation.
Positive test only confirms past infection with tubercle bacilli.
It does not indicate presence of active state of the disease.

MOTT (Mycobacteria Other Than Tubercle bacilli)

Q. Write short note on the Non tuberculous mycobacterium?
Q. Write short note on the MOTT?
Q. Write short note on the Atypical Mycobacterium? (MBBS-2007), (MBBS-
2005)
Q. Classified the Non tuberculous mycobacterium?

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ANSWER
Mycobacteria other than tubercle bacilli, cause human disease resembling
tuberculosis, have been called non-tuberculous mycobacteria.
Non-tuberculous mycobacteria also known as
Atypical mycobacteria or
Opportunistic mycobacteria or
MOTT (Mycobacteria Other Than Tubercle bacilli).
Unclassified mycobacteria
Anonymous mycobacteria
They normally exist as saprophytes of soil and water.

Classification
On the basis of pigment production and rate of growth, Runyon (1959)
classified nontuberculous mycobacteria into four groups.

Name of group Species

(I) Photochromogens M. kansasii,
M.simiae,
M.marinum
(II) Scotochromogens M. scrofulaceum,
M. gordonae,
M.szulgai
(III) Non- photochromogens M. avium,
M. intracellulare,
M.xenopi,
M. ulcerans,
M. malmoense
(IV) Rapid growers M. chelonei,
M. fortuitum

Culture
Non-tuberculous mycobacteria grow in Lowenstein-Jensen (LJ) medium.
Colonies appear within two to three weeks.
Rapid growers produce colonies in four or five days.
Photochromogens:
No pigmented colony in the dark, but yellow orange pigment appears
in light.
Scotochromogens:
Only produce pigment in the dark. No pigmented colony in the light.
Non-photochromogens:
Do not form pigment even on exposure to light.
Rapid growers:
Growth occurs within seven days.
It may be photo, scoto or non-photochromogens.

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Leprosy
Q. Write short note on the leprosy?

ANSWER
Pathogenesis
Leprosy is a chronic granulomatous disease of humans.
It is caused by Mycobacterium leprae.
Mycobacterium leprae is a slender, slightly curved or straight and acid-fast
bacillus.
5% sulphuric acid is used for decolourisation after staining with carbol fuchsin
in Z.N. Staining.
The bacilli present as parallel row in the lipid like substance (globi) give
appearance of a cigar bundle.
The only source of infection is patient.
Generation period of leprae bacilli is 20 days.
The bacilli localised primarily in the skin, peripheral nerves and nasal mucosa
but any tissue or organ may be involved.
Incubation period is very long and variable.
Sources of infection are nasal discharge and skin lesions of patients.
Prolonged close contact with patients is necessary for transmission of the
disease.
Lepra bacilli have not yet been grown on artificial culture media or tissue
culture.
The nine-banded armadillo (Dasypus novemcinctus) is highly susceptible to
infection with M. ieprae.
Classification
On the basis of clinical, histopathological and immunological findings, Ridley
and Joplin classified into five groups.
(1) Tuberculoid (TT) Leprosy
(2) Borderline tuberculoid (BT) Leprosy
(3) Borderline (BB) Leprosy
(4) Borderline lepromatous (BL) Leprosy
(5) Lepromatous (LL) Leprosy
According to WHO, leprosy is divided into two groups
(1) Paucibacillary Leprosy
(2) Multibacillary Leprosy
Acid-Fast Staining
Slit-skin smears from skin patches and ear lobes and nasal mucosal scrapings,
is stained by Ziehl-Neelsen method.
5% sulphuric acid is used as decolourising agent.
Acid-fast bacilli (AFB) arranged in parallel bundles within macrophages (Lepra-
cell).

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Grading of Acid-fast bacilli

1-10 bacilli in 100 fields = 1+
1-10 bacilli in 10 fields = 2+
1-10 bacilli per field = 3+
10-100 bacilli per field = 4+
100-1,000 bacilli per field = 5+
More than 1,000 bacilli,
clumps and globi in every field = 6+

Lepromin Test
Q. Write short note on the Lepromin test? (MBBS-2009), (MBBS-2007)

ANSWER
Lepromin test is use for classification, assessment and prognosis of
Mycobacterium laprae.
This reaction was first described by Mitsuda.

Procedure
The lepromin test is carried out by the intradermal injection of 0.1 ml of
lepromin.
It is show two type reaction.
(i) Early reaction of Fernandes:
It consists of erythema and induration developing in 24-48 hours and usually
remains for 3-5 days.
(ii) Late reaction of Mitsuda:
The reaction appears in the form of a nodule that may ulcerate.
It appears 1- 2 weeks after the injection, reaching a peak in four weeks.

Uses of Lepromin test

(i) Classification of leprosy:
Positive in tuberculoid leprosy
Negative in lepromatous leprosy
(ii) Assessment of prognosis:
Positive lepromin test indicates a good prognosis
Negative lepromin test indicates a poor prognosis
(iii) Assessment of immunity:
Positive lepromin test indicates a good immunity
Negative lepromin test indicates a poor immunity

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25
S P IR O C H A E T E

Syphilis
Pathogenesis
Laboratory Diagnosis
Hard Chancre
VDRL/RPR
Non treponemal Disease
Leptospirosis
Weil’s Disease

Syphilis
Q. Describe the types, pathogenesis and laboratory diagnosis of syphilis?
(B.Sc. Nur.-2010), (B.Sc. Nur.-2007), (MBBS-2008)
Q. Write short note on the syphilis? (BDS-2009), (BDS-2006), (BDS-2005),
(MBBS-2005)
Q. Write short note on the hard chancre?
Q. Write short note on the Classification of laboratory diagnosis of syphilis?
(BDS-2010), (BDS-2008), (MBBS-2007), (MBBS-2006)
Q. Write short note on specific test for syphilis? (MBBS-2009)
Q. Write short note on the VDRL? (B.Sc. Nur.-2009), (B.Sc. Nur.-2008),
(MBBS-2010), (MBBS-2005)
Q. Write short note on the RPR?
Q. Write difference between VDRL and RPR (MBBS-2005)

ANSWER
Types
Treponema pallidum is the causative agent of syphilis.
There are four clinical types of the disease.
Primary Syphilis
Secondary Syphilis

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Tertiary Syphilis
Congenital Syphilis (BDS-2006)

Pathogenesis
It is a veneral disease.
Venereal syphilis is acquired by sexual contact.
The treponema enters the body through minute abrasions on the skin or
mucosa.
Incubation period of syphilis is about a month (range 10-90 days).

(i) Primary Syphilis

A papule appears on the genital area that ulcerates forming a classical chancre
of primary syphilis, called hard chancre.
Ulcer is a painless, relatively avascular, indurated and a circumscribed lesion.
The regional lymph nodes are swollen, discrete, non-tender and rubbery.

(ii) Secondary syphilis

The patient is most infectious during the secondary stage.

Following lesion occur in secondary syphilis.

(a) Appearance of widespread macular rash on mucous membranes and skin.
(b) Coalesce rash in intertriginous areas (e.g. the perianal region).
(c) It may cause retinitis, meningitis, periostitis and arthritis.
(d) Multiple, painless, greyish-white plaques 'mucous patches' lesion occur in
oral cavity.
(e) Mucous patches are highly infectious as they contain a large number of
microorganisms.

(iii) Tertiary syphilis

Tertiary lesions contain few spirochaetes.

Following lesion occur in tertiary syphilis.

Cardiovascular lesions (1) Aneurysms,
(2) Chronic granulomata (gummata)
(3) Meningovascular manifestations
Neurological lesions (1) Tabes dorsalis
(2) General paralysis of the insane develop
Oral lesions (1) Intraoral gumma appears as a firm nodular mass
(2) Palate perforation

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(iv) Congenital syphilis (BDS-2006)

Following lesion occur in congenital syphilis.

(i) Frontal bossae, (ii) High palatal arch,
(iii) Saddle nose, (iv) Short maxilla,
(v) Mulberry molars, (vi) irregular thickening of clavicle,
(vii) Hutchinson's triad:
(1) Hyperplasia of the incisor and molar teeth,
(2) Interstitial keratitis
(3) Eighth nerve deafness

Laboratory Diagnosis
Syphilis diagnosis can be done by two ways.
(1) Demonstration of treponemes
(2) Detection of antibodies by serological tests

Classification of laboratory diagnosis for syphilis.

Demonstration of Treponemes
1. Dark ground microscopy
2. Direct fluorescent antibody staining for T. pallidum (DFA-TP)
3. Treponemes in tissue
(i) Silver impregnation method
(ii) Immunofluorescence staining Serological tests
S e e C o l o r Im a g e 1 7

Non-treponemal Tests (Standard test for syphilis)

(1). VDRL (Veneral Disease Research Laboratory)
(2). RPR ( Rapid Plasma Reagin)
(3). Washermann complement fixation test
(4). Kahn test
(5). TRUST (Toluidine Red Unheated Serum test)
Treponemal tests (MBBS-2007)
Specific test of syphilis
These tests may be divided as follows:
1. Tests using Reiter treponeme
Reiter protein complement fixation (RPCF) test
2. Tests using T. pallidum (Nichol's strain)
(i) Using live T. pallidum
Treponema pallidum immobilisation (TPI) test
(ii) Using killed T. pallidum
(a) Treponema pallidum agglutination (TPA) test
(b) Treponema pallidum immune adherence (TPIA) test
(c) Fluorescent treponemal antibody (FTA) test

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(iii) Using an extract of T. pallidum

(a) Treponema pallidum haemagglutination assay (TPHA) test
(b) Enzyme immunoassay (EIA)

DEMONSTRATION OF TREPONEMES
Treponemes can be demonstrated in superficial lesions of Primary, Secondary
and Congenital syphilis.
Treponemes can be demonstrate by
(1) Dark ground microscopy:
Slender, spiral organisms have shown rotational as well as flexion and
extension movements.
A treponemal concentration of 104 per ml is required for the test to
become positive.
(2) Fluorescent microscopy:
It can be done by Direct Fluorescent-Antibody staining for T. pallidum
(DFA-TP)
In this, treponemes appear distinct, sharply outlined and exhibit an
apple green fluorescence.
Treponemes in tissues can be demonstrated by
(1) Silver impregnation method of staining
Fontana’s method for staining smear
Levaditi’s method for staining tissue section
(2) Immuno-fluorescence staining
SEROLOGICAL TESTS
Depending upon the antigen used, serological tests for syphilis are divided
into two types.
(1) Non-treponemal tests (Cardiolipin or lipoidal antigen is used)
(2) Treponemal tests (Treponemes are used as the antigen).

(a) VDRL (Venereal Disease Research Laboratory) test

It is the most widely used simple and rapid serological test.
It is a slide flocculation test.
The VDRL antigen (cardiolipin antigen) must be prepared fresh daily.

(b) RPR (Rapid Plasma Reagin) test

It is almost similar to VDRL test.
Finely divided carbon particles are added to cardiolipin antigen.
Reagin:-
Non specific antibodies that appear in syphilis are known as regain.

Introduction:-
VDRL /RPR test is a non treponemal test most widely employed for
screening of syphilis.

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It is a simple and rapid test which requires only a small quantity of
serum.
Principle:-
RPR test is a modified version of VDRL in which the antigen coated
with carbon particle are allowed to react with the sample and if the
known antibodies for syphilis are present the agglutination will occur
on the slide due to aggression of carbon particle, if the sample does
not contain the antibody sera there will not be any agglutination and
it will give clear background this will indicate negative reaction.

Treponemal Tests:
1. FTA, FTA-ABS (Killed T. pallidum used)
2. TPHA, (T pallidum extract used)
3. TPI test (Live T. pallidum used)

CHOICE OF SEROLOGICAL TESTS

1. VDRL or RPR tests are used for screening or for diagnostic purposes of large
number of sera.
These tests are also used for quantitative measurement of reagin titre for
assessment of clinical activity of syphilis.
2. Treponemal tests (TPHA or FTA-ABS) are used to confirm the diagnosis with
a positive reagin test.

Treatment
1. Early syphilis:
Primary, secondary and latent infection of two years duration or less
are included in early syphilis.
(A) Benzathine benzyl penicillin 24 lacs units intramuscularly in a single
dose after sensitivity test.
(B) Alternatively, doxycycline 100 mg twice a day, orally for 15 days.
2. Late syphilis
Infection more than two years duration is included in late syphilis.
Benzathine benzyl penicillin 24 lacs units, intramuscularly, once
weekly for three weeks.

VDRL
Q. Write short note on the VDRL?
Q. Write short note on the RPR?

ANSWER
VDRL is also known as Venereal Disease Research Laboratory test.
VDRL test is performed for detection of treponema pallidum.

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It is nonspecific treponemal test.

It is the most widely used simple and rapid serological test.
It is a slide flocculation test.
VDRL test is used for screening or for diagnostic purposes of large number of
sera.
This test is also used for quantitative measurement of reagin titre for
assessment of clinical activity of syphilis.
The VDRL antigen (cardiolipin antigen) must be prepared fresh daily.
Heat treated serum used.
Cardiolipin antigen reacts with reagin antibody in syphilitic serum resulting in
formation of visible clumps or floccules.
Results can be read in a few minutes under microscope.
RPR (Rapid Plasma Reagin) test is modification of VDRL test.
It is almost similar to VDRL test.
In RPR, Finely divided carbon particles are added to cardiolipin antigen.

Difference between VDRL and RPR (MBBS-2005)

VDRL RPR
Need heat inactivation of serum No need of heat inactivation of
before use. serum before use.
Antigen freshly prepared daily. No need of preparation of fresh
antigen daily.
Plain antigens are used. Antigens are coated with Carbon
particles.
Microscope required. Microscope not required.
CSF can be test. CSF can not test.
Turn around time of test is more. Turn around time of test is less.

Non-venereal treponemal diseases

Q. Write short note on the non-venereal treponemal diseases?

ANSWER
Non-venereal treponemal diseases occur in communities with poor standards
of hygiene.
Non-venereal treponemal diseases are Yaws, Pinta and Endemic Syphilis.

Yaws
It is caused by T. pallidum subspecies pertenue.
In India, cases have been identified in Orissa, Andhra-Pradesh and Madhya
Pradesh.
The incubation period is 3 to 5 weeks.

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The primary lesion is an extragenital papule with ulcerating granuloma.
Infection is by direct contact with open ulcers.
Flies may act as vectors.

Pinta
Pinta is caused by T. carateum.
Children of 10--15 years are most commonly affected.
The disease is acquired by direct person to person contact.
Incubation period ranges from 7-21 days.
Primary lesion
Non-ulcerating extragenital papule
Lichenoid or psoriaform patch
Secondary lesion
Hyperpigmentation or Hypopigmentation

Endemic Syphilis
Endemic Syphilis is caused by T. pallidum subspecies endemicum.
It is transmitted
by direct person to person contact.
by sharing common contaminated utensils of eating and drinking.
Primary lesion is chancre on nipples of mothers infected by their children.
Secondary lesions are mucous patches and skin eruptions.
Tertiary lesions are gummatous lesions on the skin, bone and nasopharynx.

Leptosporosis
Q. Describe the pathogenesis and laboratory diagnosis of leptospirosis?
Q. Write short note on the Leptospirosis?
Q. Write short note on the weil’s disease?

ANSWER
Pathogenesis
Severe leptospirosis (Weil’s disease) associated with fever, conjunctivitis,
albuminuria, jaundice and haemorrhage .
Leptospirosis (Weil's disease) is usually caused by L. icterohaemorrhagiae
serogroup.
It is a fatal illness with hepatorenal damage.
It is transmitted to humans by direct or indirect contact with water
contaminated by urine of carrier animals.
Leptospires enter the body through cuts or abrasions on the skin, or through
the mucous membranes of the mouth, nose or conjunctiva.
Incubation period is 6-8 days.
The organisms disappear from the blood but enter into the liver, kidney,
spleen and meninges.

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Leptospirosis is an established cause of aseptic meningitis.

L. icterohaemorrhagiae persist in the internal organs especially in kidney.
In later stage, it is appear in urine.

Laboratory Diagnosis
The diagnosis made by
(1) By demonstration of the leptospires microscopically in blood or urine,
(2) By isolating them in culture
(3) By animal inoculation
(4) By serological tests
(i) Microscopy
Leptospires detect in the blood or urine by dark-ground microscopy.
Leptospires present in blood in first week and disappear from blood
after 8 days.
Leptospires present in the urine in the second week.
These are spiral organisms with numerous tightly coiled regular spirals
coil and hooked ends.
(ii) Culture
It is grown aerobic and microaerobic condition.
Optimum temperature and pH are 280C -320C and 7.2-7.5 .
Commonly use medium are
(1) Korthof’s media
(2) Stuart’s media
(3) Fletcher’s media
(4) EMJH (Ellinghausen, McCullough, Johnson & Harris)
(5) Chorioallantoic membrane (CAM) of chick embryo
(iii) Animal inoculation
The blood or urine from the patient is inoculated intraperitoneally
into young guinea pigs.
(iv) Serological tests
It is very useful method of diagnosis.
Antibodies detected by
Complement fixation test,
Haemagglutination test,
Enzyme Linked Immune Sorbent Assay (ELISA) and
Sensitised Erythrocyte Lysis (SEL)

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A C T IN O M Y C E T E S

Mycetoma Foot
Actinomyces
Nocardia

Mycetoma
Q. Write short note on the mycetoma foot? (MBBS-2008), (MBBS-2002)
Q. Enumerate the causative agent of mycetoma foot?
Q. Write short note on the actinomycosis?
Q. Write short note on the nocardia?

ANSWER
Mycetoma is a localized, chronic granulomatous disease of subcutaneous and
deeper tissues affecting commonly the foot.
It is present as a tumour with multiple discharging sinuses.

Etiological agent of Mycetoma are two types.

Bacteriological agent Mycotic (Fungus) agent

(1) Actinomyces (1) Madurella mycetomi
(2) Nocardia (2) Madurella griseus
(3)Actinomadura
(4)Streptomyces

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On the basis of colour granule diagnosis is confirmed.

In Actinomycotic mycetoma White to Yellow granule
In Mycotic mycetoma Black granule

Actinomycosis
Q. Write short note on the actinomycosis?

ANSWER
The actinomycosis is a chronic granulomatous disease characterised by
multiple abscesses, tissue destruction, fibrosis and formation of multiple
sinuses.
Actinomycosis is cause by Actinomycetes israelii.
Actinomycosis is associated with formation of dental plaque, gingivitis and
periodontitis (inflammatory diseases of gums).
A. israelii may occur as commensal in mouth of normal individuals mainly
around the teeth.
The infection is mostly endogenous due to dental extraction.
Actinomycosis occurs in four clinical forms:
1. Cervicofacial: This is the commonest type and it occurs mainly in cheek and
submaxillary regions.
2. Thoracic: It involves lungs.
3. Abdominal: It occurs usually in the ileocaecal region.
4. Pelvic.
Laboratory Diagnosis
1. Specimens
• Pus from lesion or sinuses
• Discharge from fistula
• Sputum in pulmonary disease
• Tissue or biopsy
2. Microscopy
Smear prepare by crushing the ‘sulpher granule’ between two slides.
Smear stained by gram’s stain, by acid fast stain and by
immunofluoroscence staining.
Gram staining:
It is shows “sunray appearance” by a dense network of thin Gram
positive filaments, surrounded by a peripheral radiating Gram
negative 'clubs'.
Actinomyces is gram positive, non-motile, non-sporing, non-acid-fast
organism.
Most of them show true branching.
S e e C o l o r Im a g e 1 8

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Acid fast stain (decolourisation with 1% sulphuric acid):
It is shows central part as non-acid fast surrounded by acid-fast
'clubs'.
Immunofluoroscence staining:
In tissue sections, sulphur granules and mycelia are detected by using
fluorescein conjugated specific antisera.
3. Culture
Medium used for culture are:
Thioglycollate broth: White fluffy ball colonies.
Brain heart infusion agar: White smooth colonies resemble to molar
teeth in 8-10 days.
Blood agar
4. Biopsy
Haematoxylin-Eosin stained section shows mycelial mass surrounded
by pus cells and chronic inflammatory cells.
Treatment
Surgical removal of affected tissue along with penicillin therapy is
effective.

Nocardia
Q. Write short note on the nocardia?

ANSWER
Nocardiae is Gram positive bacteria and form a mycelium.
Nocardia resembles Actinomyces.
Some species are acid-fast and few are non-acid-fast.
They are strict aerobes.
They can be grown on nutrient agar, Sabouraud dextrose agar (SDA) and brain
heart infusion agar (BHI agar).
Nocardiae form dry, granular, wrinkled colonies with pigmentation (white,
yellow, pink or red).
Nocardiae is produce nocardiosis in immunocompromised.
Man acquires infection by inhalation of the bacteria from environmental
sources.
It causes pulmonary disease, pneumonia, lung abscess or other lesions
resembling tuberculosis. It may also cause mycetoma.
It presents as a tumour with multiple sinuses.
It came to be known as Madura foot or Madura disease.
Gram positive filamentous bacteria and Acid-fast bacilli are detected in
smears by staining with Gram staining and Ziehl-Neelsen (ZN) technique using
decolourisation with 1% sulphuric acid respectively.
Nocardia can also be isolated by paraffin bait technique.
Cotrimoxazole is given for 3 months or more as a treatment.

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R IC K E T T S IA
Typhus Fever
Epidemic Typhus
Endemic Typhus
Recrudescence Typhus
Weil-Felix Reaction
Coxiella
Q-Fever
Typhus Fever
Q. Write short note on the Typhus fever?
Q. Write short note on the Epidemic typhus?
Q. Write short note on the Endemic typhus?
Q. Write short note on the Recrudescent typhus?
Q. Write short note on the Brill-Zinsaar disease?

ANSWER
Typhus fever is caused by rickettsial bacterial infections.
Rickettsiae are generally transmitted to humans by the bite or by the faeces of
an infected arthropod vector.
Rickettsial are obligate intracellular organism.
Rickettsiae are unable to grow in cell free media.
They are cultivated in yolk sac of chick embryo.
They multiply locally and enter the blood stream.

Epidemic typhus ( Classical typhus)

Causative organism is R.Prowazekii.
Vector is Human body louse.
Incubation period is 5-12 days.
Symptom and sign
Severe headache, chills, myalgia high fever and vomiting

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A macular rash appear after 4-7 day of illness
Rashes appear on trunk and then on limb
Types of Typhus fever caused by Rickettsial infection and Diseases.

Disease Species Vector

Epidemic typhus R.Prowazekii Human body louse
( Classical typhus)
Brill-Zinsser disease R.Prowazekii Human body louse
(Recrudescent typhus)
Endemic typhus R.typhi (R. Mooseri) Rat Flea
(Murine typhus)
Scrub typhus Orientia tsutsugamushi Tromniculid mites

Brill-Zinsser disease (Recrudescent typhus)

Causative organism is R.Prowazekii.
Vector is Human body louse.
Reactivation of epidemic typhus is called recrudescent typhus.
It is milder and shorter disease as compared epidemic typhus.

Endemic typhus (Murine typhus)

Causative organism is R.typhi (R. Mooseri).
Vector is Rat flea (Xenopsylla cheopis).
Rat is reservoir.
Human infection is dead end of it.
It is gives immunity to both endemic and epidemic typhus.
Typhus fever rickettsiae can be detected by
(1) Stain with giemsa and Castaneda stain.
(2) Isolation in yolk cell wall of chick embryo, Hela, HEp2 cell line.
(3) Neil mooser or tunica reaction.
Neil-mooser or tunica reaction is positive in endemic typhus.
Neil-mooser or tunica reaction is negative in epidemic typhus.
(4) Weil-felix serological reaction.
Treatment
Tetracyclines or chloramphenicol are drug of choice.
Weil - Felix Reaction
Q. Write short note on the Weil-Felix reaction? (MBBS-2007)

ANSWER
It is a heterophile agglutination test.
Weil -Felix reaction detects anti-rickettsial antibodies.

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It is a heterophile antibody that cross react with certain non-motile proteus

strains OX 19, OX 2 and OX K.

Principal of test:
An alkali stable carbohydrate antigen of some rickettsiae sharing with non-
motile strains of proteus sp. E.g. P. vulgaris OX 19, and OX 2 and P. mirabilis
OX K.

Weil-Felix Reaction interpretation in different Rickettsial Diseases.

Disease Agglutination with proteus strain

OX 19 OX 2 OX K
Epidemic typhus ++++ ± -
Brill-Zinsser ± - -
disease
Endemic typhus +++ ± -
Spotted fever ++ ++ -
group
Scrub typhus - - +++
Rickettsial pox - - -
In Epidemic and Endemic typhus, strongly agglutinates OX 19.
In spotted fevers, both OX19 and OX2 are agglutinated.
In Scrub typhus, only OX-K agglutinated.
In Rickettsial pox, agglutination is absent.

Neil-Mooser or tunica reaction

Q. Write short note on the Neil-Mooser or tunica reaction?

ANSWER
A male guinea-pig is inoculated intraperitoneally with blood of a case of
endemic typhus (R. Typhi).
The scrotum becomes enlarged and can not be pushed back into the
abdomen.
Inflammatory adhesions developed between the layers of tunica vaginalis.
Neil-mooser or tunica reaction is positive in endemic typhus (R. Typhi).
Neil-mooser or tunica reaction is negative in epidemic typhus (R. Prowazekii).

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Q-Fever
Q. Write short note on the Q-fever?
Q. Write short note on the Coxeilla?

ANSWER
Q-fever is caused by Coxiela burnetii.
C. burnetii is an gram negative & obligate intracellular pathogen.
In milk it may survive pasteurisation by the holder method, but the flash
method is effective.
It can be transmitted by
(i) Consumption of infected milk
(ii) Handling of infected wool
(iii) Contaminated soil
(iv) Contaminated clothing
In Q-fever, patient develops headache, chills, myalgia, pneumonia,
endocarditis, hepatitis and meningoencephalitis.
No skin rash occurs in Q fever.
The incubation period is 2-4 weeks.
Weil-Felix test is negative in Q fever.

Special points
Rickettsiae possess properties in between those of bacteria and viruses.
They are obligate intracellular gram negative bacilli reside in the cytoplasm
and nucleos of the cell.
They do not grow on artificial media.
They contain muramic acid in the cell.
All member of genus rickettsia are transmitted to human by bites of infected
arthropods.
Coxiella burnetti survives in holder method of pasteurization of milk and
borderline inactivation in flash method.

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C H L A M Y D IA

Chlamydia Trachomatis
Trachoma
OphthalmiaNeonatorum
LGV
Frei’s Test

Trachoma /Ophthalmia Neonatorum/

Lympho Granulum Venereum(LGV)
Q. Classified the chlamydial species? (MBBS-2006)
Q. Write short note on the Trachoma?
Q. Write short note on the Chlamydia trachomatis? (MBBS-2005)
Q. Write short note on the laboratory diagnosis of Chlamydia? (MBBS-2008)
Q. Write short note on the Ophthalmia neonatorum?
Q. Write short note on the LGV?
Q. Write short note on the Lymphogranulum Venereum?

ANSWER
These are sexually transmitted diseases.
It is cause by Chlamydiae trachomatis.

Subtype of Chlamydia Disease

(1) C. trachomatis A, B, Ba, C Hyperendemic trachoma
(2) C. trachomatis D, E, F, G, H, I, J, K Inclusion conjunctivitis (neonatal and
adult),
Ophthalmia neonatorum
(3) C. trachomatis L1, L2, L3 Lymphogranuloma venereum

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Chlamydiae trachomatis transmitted by sexual intercourse.

Chlamydia is obligate intracellular parasites which are small, non-motile and
Gram negative.
Chlamydiae exist in two forms.
The elementary body The reticulate (initial) body.
Extracellular form Intracellular form
Infective form Non-infective form and
replicating form
The duration of development cycle is about 24-28 hours.

Trachoma:
Causative agent of trachoma is C. trachomatis A, B, Ba, C.
Trachoma is a chronic kerato-conjuctivitis and one of the major cause of
blindness in India.
It is characterised by follicles, papillary hyperplasia, pannus formation and
cicatrization.
Incubation period is 7-14 days.
It is more prevalent in children below 9 years of age.

Inclusion conjunctivitis or Ophthalmia neonatorum

Causative agent of Inclusion conjunctivitis is C. trachomatis D to K.
Inclusion conjunctivitis is occurs by genital secreation and bathing in
swimming pool .
Ophthalmia neonatorum is occurs while passing through the birth canal of
infected mother.

Lympho-Granuloma Venereum (LGV)

Causative agent of LGV is C. trachomatis L1, L2, L3.
It is a sexually transmitted disease.
Incubation period is 3-5 days
Clinical features
 Small painless papule or vesicle on the external genitalia
 Enlarged and tender lymphnode
 Bacteria multiply in the lymph node and form “bubos”.
 Bubos break down to form pus discharging “sinuses”.
Laboratory diagnosis
Chlamydiae trachomatis is Gram negative nature but they stain better with
other staining methods.
Staining method use for this are
Giemsa
Castaneda,
Machiavello or
Gimenez stains.

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The basophilic inclusion bodies are present in cytoplasm.

The inclusion bodies (Halberstaedter Prowazek or HP bodies) can be stained
with Lugol's iodine because of the presence of glycogen matrix.

Inclusion body
Chlamydiae can be isolated by
1. Animal inoculation:
Mice are inoculated by intranasal, intraperitoneal or
intracerebral inoculation.
2. Yolk sac inoculation: Yolk sac of chick embryo is used.
3. Tissue culture: McCoy cells treated with cycloheximide are the
most commonly used cell line.
Isolated bacteria from above methods can be demonstrated by staining
methods and serological methods.
Antibody of Chlamydia can be detected by serological method.
(1) Complement fixation test (CFT)
(2) Microimmunofluorescence test
(3) ELISA
Treatment
Tetracycline or erythromycin is drug of choice.
Frei's Test
Q. Write short note on the Frei’s test?

ANSWER
Frei’s test is used to detect lymphogranulum venerum (LGV).
It is an intra-dermal skin test.
It is an example of delayed type cell mediated hypersensitivity reaction.
A heat inactivated LGV (0.1 ml) grown in yolk sac of embryonated egg.
It is injected intradermally on the forearm.
Non-infected yolk sac inject into the other forearm (control).
The test is read 48-72 hours after injection.
An inflammatory nodule (7 mm in diameter) appears at the test site in two
days and reaching a maximum in 4-5 days.
This skin test becomes positive 2-6 weeks after infection and remains positive
for several years.
The test is now not outdated due to false positive reactions.

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29
G E N E R A L V I R O L O G Y

Laboratory Diagnosis
Cultivation of Virus
Tissue Culture
Prion
Inclusion Body

Laboratory diagnosis of Viral infection

Q. Discuss the laboratory diagnosis of viral infections? (B.Sc. Nur.-2007) ,
(MBBS-2004)
Q. Discuss the cultivation of virus in the laboratory? (MBBS-2010), (MBBS-
2004)
Q. Write short note on the tissue culture? (MBBS-2009), (MBBS-2005)
Q. Write short note on the method of detection of virus growth in the cell
Cultures? (MBBS-2008)

ANSWER
Viral infection can be detected in laboratory by following methods.
A. Direct demonstration of virus and its components
B. Isolation of virus
C. Detection of the specific antibodies
A. Direct Demonstration of Virus and its Components
Following methods are used for direct detection of virus and it components.

1. Electron Microscopy
Electron microscopy (EM) is useful for viruses that are difficult to
culture.

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2. Immuno-electron Microscopy
It is increase the sensitivity to aggregate virus particles by adding
specific antibody.
3. Fluorescent Microscopy
In this, virus can be detected by direct or indirect fluorescent antibody
technique and detected under fluorescence microscope.
E.g. Paramyxoviruses, Orthomyxoviruses, Adenoviruses and
Herpesviruses.
4. Light Microscopy
Negri bodies and other inclusion bodies of virus can be
demonstration in the specimens.
5. Viral Antigens
Viral antigen may be detected by Enzyme Linked Immune Sorbent
Assay (ELISA), Radioimmunoassay, Chemilumiscene, Complement
Fixation Test and Latex Agglutination.
6. Nucleic Acid Probes
Cytomegalovirus, Papillomavirus and Epstein-Barr virus have been
identified by use of nucleic acid probes.
7. Polymerase Chain Reaction (PCR).
A target DNA sequence can be amplified to the point and identified by
using labelled probes in a hybridisation assay.
PCR can be used for the diagnosis of infections caused by HIV-l, HIV-2,
human Papillomaviruses, Herpes Simplex Virus, Hepatitis B Virus,
Rubella Virus and Epstein-Barr virus.

Cultivation of virus
B. Isolation of Virus
Virus cannot be grown on any of the inanimate culture medium.
Viruses multiply only in living cells.
For the cultivation of viruses three methods are employed:
A. Animal inoculation
B. Embryonated egg inoculation (MBBS-2004)
C. Tissue culture

a. Animal Inoculation
Infant (suckling) mice, guinea pigs, rabbits and ferrets are used for animal
inoculation.
Animal inoculation is used for
1. Primary isolation of certain viruses
2. To study pathogenesis of viral diseases
3. To study viral oncogenesis

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b. Embryonated Egg Inoculation:

Embryonated hen's eggs (7 to 12 days old) are inoculated by one of the
several routes such as chorioallantoic membrane (CAM), allantoic cavity,
amniotic sac and yolk sac.

Structure and Utility of Fertilized Egg

1. Chorioallantoic Membrane (CAM): For growing poxviruses.

2. Allantoic Cavity: For growing influenza virus for vaccine production.
3. Amniotic Sac: For the primary isolation of the influenza virus.
4. Yolk sac Inoculation: For viruses and certain bacteria (chlamydia and
rickettsiae).

Tissue Culture
c. Tissue Culture
Three types of tissue cultures are available:
1. Organ culture
2. Explant culture
3. Cell culture
Cell Culture:
This is the type of culture routinely employed for diagnostic virology.
Cell cultures are classified into three different types on the basis of their
origin, chromosomal characters and the number of generations through which
they can be maintained.
(i) Primary cell culture:
Chromosomal number: As the parent cells and are diploid.
Number of generations: 5-10 subcultures
Cell origin from: Rhesus monkey kidney cell culture,

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Human amnion cell culture,
Chick embryo fibroblast cell culture
(ii) Diploid cell line (MBBS-2005)
Chromosomal number: As the parent cells and are diploid.
Number of generations: 50 subcultures
Cell origin from: WI-38 (Human embryonic lung cell),
HL--8 (Rhesus embryo cell strain).
(iii) Continuous cell lines
Chromosomal number: Half of the parent cells and haploid.
Number of generations: Serially indefinite subculture
Cell origin from: BeLa (Human carcinoma of cervix cell)
HEP-2 (Human epithelioma of larynx cell line),
Vera (Vervet monkey kidney cell line),
McCoy (Human synovial carcinoma cell line),
BHK-21 (Baby Hamster kidney cell line) &
KB (Human carcinoma of nasopharynx cell
line).

Detection of virus growth in cell cultures (MBBS-2008)

Virus growth in cell cultures can be detected by the following methods:
1. Cytopathic effect seen like (i) Syncytium formation,
(ii) Cell necrosis and lysis,
(iii) Cellular clumping,
(iv) Rounding of cells,
(v) Discrete focal degeneration
2. Haemadsorption 3. Interference
4. Transformation 5. Immunofluorescence
6. Electron microscopy 7. Detection of enzymes

C. Detection of Specific Antibodies

The rise in titre of antiviral antibodies (IgM, IgG) demonstrated by
Neutralisation, ELISA, Haemagglutination Inhibition, Complement Fixation
Test,radio immune assay, Immunofluorescence and Latex Agglutination tests.

Prions
Q. Write short note on Prions? (MBBS-2007), (MBBS-2004)

ANSWER
Prions are infectious proteins without any detectable nucleic acid.
Properties of prion
 Highly resistant to physical and chemical agents

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 Long incubation period (in years)

 Proteinaceous, filterable
 Multiply in cell culture but no CPE
 Do not elicit inflammatory reaction and antibody in host
 Do not exhibit virus morphology in electron microscope
Diseases caused by them are
(1) Scrapie of sheep and goats (2) Mink encephalopathy
(3) Bovine spongiform encephalopathy (4) Kuru
(5) Creutzfeld Jakob disease

Inclusion bodies
Q. Write short note on the inclusion Bodies? (MBBS-2008)

ANSWER
These are intranuclear or cytoplasmic aggregates of products of viral
replication.
They can be seen in stained preparation under a light microscope.
They may be acidophilic or basophilic, single or multiple, large or small and
round or irregular.
Intracytoplasmic bodies:
Small Pox (Guarnieri bodies), Rabies (Negri bdies), Yellow Fever
(Councilman bodies) and Fowl pox (Bollinger bodies)
Intracytoplasmic bodies:
Herpes virus (Cowdry A bodies), Adenoviru and poliovirus (Cowdry B
bodies)
In measles virus both type intracytoplasmic and intranuclear inclusions bodies
seen.
Viroids
Q. Write short note on the viroids?

ANSWER
Viroids exist inside cells as particles of RNA without capsules.
Viroids particles are not apparent in infected tissues without using special
techniques to identify nucleotide sequences in RNA.
Compared to viral nucleic acids, the RNA of viroids is a low molecular weight
material.

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30
Bacteriophage
Bacteriophage
Q. Write short note on the bacteriophage? (MBBS-2008)

ANSWER
Bacteriophage (phage) are obligate intracellular parasites that multiply inside
bacteria by making use of some or all of the host biosynthetic machinery (i.e.,
viruses that infect bacteria.).
Bacteriophage are used in the diagnostic laboratory for the identification of
pathogenic bacteria (phage typing).
It is used in reference laboratories for epidemiological purposes.
A. Composition
Bacteriophages may contain nucleic acid and protein.
Depending upon the phage, the nucleic acid can be either DNA or RNA
but not both.
The proteins function in infection and to protect the nucleic acid from
nucleases in the environment.
B. Structure
The basic structural features of bacteriophages are
1. Size –
T4 is among the largest phages.
Most phages range in size from 24-200 nm in length.
2. Head or Capsid –
All phages contain a head structure which can vary in size and shape.
Some are icosahedral (20 sides) others are filamentous.
The head or capsid is composed different proteins.
Inside the head is found the nucleic acid.
The head acts as the protective covering for the nucleic acid.
3. Tail –
Many but not all phages have tails attached to the phage head.
The tail is a hollow tube through which the nucleic acid passes during
infection.
Bacteriophage T4 tail is surrounded by a contractile sheath which
contracts during infection of the bacterium.

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T4 have a base plate and one or more tail fibers attached to it.

Structure of Bacteriophage

INFECTION OF HOST CELLS

Infection of host cells having four steps.
A. Adsorption:
The tail fibers of bacteriophage attach to specific receptors on the
bacterial cell.
B. Irreversible attachment:
Irreversible binding of phage to a bacterium is mediated by one or
more of the components of the base plate.
C. Sheath Contraction:
The contraction of the sheath pushed through the bacterial envelope.
D. Nucleic Acid Injection:
When the phage has gotten through the bacterial envelope the
nucleic acid from the head passes through the hollow tail and enters
the bacterial cell. Usually, the only the nucleic acid enters in to the
cell.
The remainder of the phage remains on the outside of the bacterium.

PHAGE MULTIPLICATION CYCLE

Phage multiplication cycle are two type.
A. Lytic or Virulent Phages
B. Lysogenic or Temperate Phage

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A. Lytic or Virulent Phages
Lytic or virulent phages are phages which can only multiply on
bacteria and kill the cell by lysis at the end of the life cycle.
Lytic phage contain three phases
a. Eclipse period:
During the eclipse phase, no infectious phage particles can be found
either inside or outside the bacterial cell.
The phage nucleic acid takes over the host biosynthetic machinery
and phage specified m-RNA's and proteins are made.
b. Intracellular Accumulation Phase
In this phase the nucleic acid and structural proteins that have been
made are assembled and infectious phage particles accumulate within
the cell.
c. Lysis and Release Phase
The bacteria begin to lyse due to the accumulation of the phage and
intracellular phage are released into the medium.
3. Assay for Lytic Phage: Lytic phage is enumerated by a plaque assay.

B. Lysogenic or Temperate Phage

Lysogenic or temperate phages are those that can either multiply via
the lytic cycle or enter a quiescent state in the cell.
In this quiescent state most of the phage genes are not transcribed;
the phage genome exists in a repressed state.
The phage DNA in this repressed state is called a prophage.
The phage DNA actually integrates into the host chromosome and is
replicated along with the host chromosome and passed on to the
daughter cells.
The cell harboring a prophage is termed a lysogen.

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H E R P E S V IR U S E S

Classification
Herpes Simplex
Varicella- Zoster
Cytomegalovirus
Epstein Barr Virus
Paul Bunnel Test

Herpes Virus
Q. Classify the human Herpes viruses? Discuss the laboratory diagnosis of
herpes simplex virus? (MBBS-2009), (MBBS-2003), (MBBS-2002)

ANSWER
Herpesvirus is an icosahedral capsid composed of 162 capsomers, containing
linear double stranded DNA genome and is surrounded by a lipid envelope
containing peplomers. Between capsid and envelope is 'tegument'.

Classification of Human Herpesviruses

Sub family Scientific name Common name
Alpha Human herpesvirus 1, Herpes simplex virus-1
Herpesvirinae Human herpesvirus 2 Herpes simplex virus-2
Human herpesvirus 3 Varicella-Zoster virus
Beta Human herpesvirus 5 Cytomegalovirus
Herpesvirinae Human herpesvirus 6 Human B-cell lymphotropic virus
Human herpesvirus 7 R.K. virus
Gamma Human herpesvirus 4 Epstein-Barr (EB) Virus
Herpesvirinae Human herpesvirus 8

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Structure of Herpes virus

Herpes Simplex Virus (HSV)

Man is the only natural host.
There are two types of the Herpes Simplex Virus.
Herpes simplex virus type 1 is usually associated with oral and ocular lesions
like acute gingivostomatitis, herpes labialis, keratoconjunctivitis, eczema
herpeticum, encephalitis and dendritic keratitis.
Herpes simplex virus type 2 is responsible for the majority of genital infections
like genital herpes (penis, urethra, cervix, vulva, vagina), neonatal herpes and
aseptic meningitis.
Pathogenesis
After entering through the skin or mucous membrane the virus multiply
locally and spread to the neighbouring cells.
HSV-2 travels to nerve roots near the spinal cord and migrates to skin and
mucosa via ganglia and produced lesions.
Laboratory Diagnosis

1. Specimens
Specimens include vesicle fluid, skin swab, saliva, corneal scrapings, brain
biopsy and CSF, according to the site of involvement.

2. Direct Examination
Smears prepared from scrapings from the base of vesicles are stained with
toludine blue.
Multinucleated giant cells with faceted nuclei and homogeneously stained
'ground glass' chromatin (Tzanck cells) are present in a positive smear.
Cowdry type A intranuclear inclusion bodies may be seen in Giemsa stained
smears.
Herpes virions may be demonstrated in specimens by electron microscopy.

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Viral antigens can also be demonstrated by immunofluorescent staining.

S e e C o l o r Im a g e 1 9

3. Tissue Culture
Virus can be isolated on human fibroblasts, HEp-2 cells, Vero cells and
chorioallantoic membrane.

4. Polymerase Chain Reaction (PCR)

PCR can be used for detection of HSV DNA in CSF.
5. Serological reaction: ELISA , RIA, CFT etc.
Treatment
HSV infection can be treated with acyclovir (acycloguanosine).
Valaciclovir and famciclovir are more effective oral agents.
Varicella-Zoster
Q. Write short note on the Varicella –Zoster?
Q. Write short note on the Chickenpox?
Q. Write short note on the Shingles?

Varicella (chickenpox) and herpes zoster (shingles) are caused by a single virus
Varicella-zoster virus (VZV).
Chickenpox follows primary infection in a non-immune individual.
Herpes zoster is a reactivation of the latent virus when immunity falls to
ineffective level.

A. Varicella/ Chickenpox
Chickenpox is one of the commonest childhood exanthemata.
The virus enters through respiratory route.
The source of infection is a chickenpox or herpes-zoster patient.
It is characterized by vesicular rash mostly on the trunk.
The rash progresses through macule, papule, vesicle, pustule and scab.
The rash is centripetal in distribution.
Small blister-like lesions are present and involve buccal mucosa, gingiva,
tongue, palate and mucosa of pharynx.

B. Herpes zoster/ Shingles

Herpes zoster is a disease of old age and occurs usually in persons who had
chickenpox several years earlier.
The virus remains latent in the sensory ganglia.

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When the immunity has fallen to ineffective levels, the virus may be
reactivated and triggered by some precipitating stimulus.
The vesicles are usually confined to the area supplied by a single sensory
ganglion. The vesicles are like those of varicella.
Herpes zoster may involve the face.
There is usually unilateral involvement of skin areas supplied by ophthalmic,
mandibular or maxillary nerves.
Oral lesions appear as extremely painful vesicles.
One attack of chickenpox confers life long immunity.

Laboratory Diagnosis
1. Direct Microscopy
Smears stained by Toludine blue or Giemsa stains and Flourescent
monoclonal antibody technique.
Specimen collects from the base of early vesicles.
Smear show multinucleated giant cells and type A intranuclear
inclusions bodies under light microscope, fluorescent microscope and
electron microscope.
2. Virus Isolation
Virus can be isolated in human fibroblast cells, human amnion, HeLa
or Vero cells.
Cytopathic effect is focal with refracticle ballooned cells.
3. Serology
Varicella-zoster specific IgM antibody in patient's serum can be
detected by ELISA.
Treatment
Acyclovir and Vidarabine are effective in the treatment of severe
varicella and zoster.

Cytomegalovirus
Q. Write short note on the Cytomegalovirus?
Q. Write short note on the Infectious Mononucleosis?
Q. Write short note on the Glandular Fever?

ANSWER
Cytomegalovirus was originally known as “salivary gland virus”.
CMV is the largest virus in herpes family.
Cytomegalovirus is shed in urine saliva, semen, cervical secretions, tears and
breast milk.
Cytomegalovirus can be transmitted transplacentally from a mother with
latent infection to the foetus.

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This may be acquired by sexual intercourse, blood transfusion and organ

transplantation.
Congenital infection may lead to cytomegalic inclusion disease which is often
fatal.
Postnatal infections are usually
Clinical disease in adults associated with hepatospleenomagaly, jaundice,
thrombocytopenia, haemolytic anaemia, microcephaly and chorioretinitis.
CMV is an important pathogen in AIDS.

Laboratory Diagnosis
1. Specimens
CMV can be isolated from urine, saliva, breast milk, semen, cervical
secretions and blood leucocytes.
2. Demonstration of Cytomegalic Cells
Enlarged cells with large intranuclear "owl's eye" appearance
inclusions (cytomegalic cells) can be demonstrated in the centrifuged
deposits from urine or saliva.
S e e C o l o r Im a g e 2 0

3. Isolation of Virus
Virus can be grown in human fibroblast cultures.
Cytopathic effects may take 2-3 weeks to appear.
These cultures may be stained by immunofluorescence technique
using monoclonal antibody.
4. Serology
CMV -specific IgM can be detected in the serum by ELISA.

Treatment
Ganciclovir is the drug of choice.

Epstein Barr Virus

Q. Write short note on the Epstein Barr Virus?
Q. Write short note on the Paul Bunnel test? (MBBS-2003)

ANSWER
The B lymphocytes of human beings have receptors (CD21 molecules) for EBV,
therefore, EBV has affinity for lymphoid tissue.
The source of infection is usually the saliva of infected persons.
The main mode of transmission is kissing.
The following clinical manifestations may result from EBV infection:
1. Infectious mononucleosis or Glandular fever

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2. Infections in immunocompromised hosts
3. EBV-associated malignancies
(i) Burkitt's lymphoma
(ii) Nasopharyngeal carcinoma
(iii) B-cell lymphoma

Laboratory Diagnosis
1. White Blood Cell Count
During the initial phase, patient develops leucopenia and later there
is leucocytosis with a predominance of abnormal or atypical
lymphocytes.
2. Paul-Bunnell Test
During infectious mononucleosis, heterophile antibodies (IgM) appear
in the serum of the patient.
It is heterophile reaction.
Certain antigens of similar nature, if not identical, present in different
tissues of more than one species are called heterophile antigens.
Antibody to these closely related antigens produced by one species
cross react with antigens of other species.
Chemically these are Iipoprotein polysaccharide complex.
Procedure
Inactivated serum (56oC for 30 minutes) in doubling dilutions is mixed
with equal volumes of 1% sheep erythrocytes suspension.
These tubes are incubated at 37oC for four hours and examined for
agglutination.
A titre of 100 or above is suggestive of infectious mononucleosis.
3. EBV-Specific Antibodies
These are specific antibodies against EBV viral capsid antigen (VCA).
These can be demonstrated by indirect immunofluorescence or ELISA.
4. Nucelic Acid Hybridisation:
It is the most sensitive method for detection of EB virus in patient
material.

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32
P O L I O V I R U S

Polio
Pathogenesis
Laboratory Diagnosis
Polio Vaccine
Salk Vaccine
Sabin Vaccine
Pulse Polio Programme

Classification of Picornavirus
Q. Write down classification of Picornavirus?

ANSWER
Genus Species
Enterovirus Polio virus
Coxsackie A & B
Echovirus
Enterovirus
Perechovirus Perechovirus
Rhinovirus Rhinovirus
Hepatovirus Hepatitis A Virus
Polio Virus
Q. Describe the pathogenesis and laboratory diagnosis of Polio Virus?
(MBBS-2006)
Q. Write short notes on the Vaccination for polio virus? (MBBS-2007) (MBBS-
2006)

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ANSWER
Poliovirus is the causative agent of poliomyelitis.
It is belong to genus picornavirus and family picornaviridae.
Poliovirus is RNA genomic, icosahedral in symmetry virus.
Children are the most susceptible.
The virus enters the body by ingestion of contaminated water and food.
The mode of transmission is oral- faeco route.

Pathogenesis
(1) The virus enters the body by ingestion of contaminated water and food.
(2) Multiplies in lymphatic tissues of oropharynx and intestine.
(3) Virus comes in Bloodstream.
(4) Virus destroys the Spinal cord and anterior horn cells of brain.

Clinical Features
There are four types of poliovirus infection:
(A) Inapparent infection (90-95%): No Clinical feature
Virus present in stool or throat
(B) Minor illness (4-8%): Mild influenza like illness
(C) Non-paralytic poliomyelitis (1-2%): Stiffness of the neck, headache
(D) Paralytic poliomyelitis (0.1-2%): Flaccid paralysis
Spinal, bulbar or bulbospinal paralysis

Laboratory Diagnosis
1. Virus can be isolated from the faeces, the pharyngeal washings and
autopsy specimen (spinal cord and brain).
Virus usually cannot be recovered from CSF.
2. Virus can be demonstrated in stool by direct electron microscopy or
by immune electron microscopy.
S e e C o l o r Im a g e 2 1

3. Isolation of virus
Primary monkey kidney cells, HeLa and HEp-2 are usually use for
isolation of virus.

Prophylaxis
Polio Vaccine
Two types of vaccines are available.
Killed polio vaccine (Salk)
Live attenuated oral polio vaccine (Sabin).
1. Salk's killed polio vaccine
The vaccine is given by deep subcutaneous or intramuscular injection.
Three doses are given 4-6 weeks apart followed by a booster to be given 6
months later.

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The first dose should be given after the age of six months.
This vaccine produces long-lasting immunity to all three poliovirus types.
It does not induce secretory IgA in the intestine.
It is safe to administer to immunocompromised individuals.

2. Live attenuated oral polio vaccine (OPV) (Sabin vaccine)

It contains live attenuated strains of polio virus types, 1, 2 and 3 grown either
in monkey kidney cells or human diploid cell cultures.
It is administered orally.
It stimulates both local secretory IgA antibodies in the intestine as well as
humoral antibodies (IgM and IgG).
It protects the individual and the community.
It is not safe to administer to immunocompromised individuals.
Three doses are given.
First dose of OPV is given along with DPT at the age of 6 weeks.
Second dose of OPV is given along with DPT at the age of 10 weeks.
Third dose of OPV is given along with DPT at the age of 14 weeks.
Zero dose is also given at birth in institutional deliveries.

Pulse Polio Immunization(PPI)

Q. Write short note on the Pulse Polio Immunization?

ANSWER
This is simultaneous, mass administration of OPV on a single day to all
children.
The age group of children is 0-5 years of age.
OPV gives regardless of previous immunizations.
Pulse Polio immunization occur as two rounds, about 4-6 weeks apart.
In India, the peak transmission season is between June and September.
The doses of OPV during Pulse Polio Immunization are extra doses which
supplement and do not replace the routine doses.
This programme is aimed at eliminating polio infection.

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33
O R T H O M Y X O V IR U S

Influenza
Swine Flu
Antigenic Drift
Antigenic Shift

Swine flu / Influenza virus

Q. Describe antigenic variation and laboratory diagnosis of influenza virus?
(MBBS-2008), (MBBS-2002)
Q. Write short note on the Swine flu? (MBBS-2010)
Q. Write short note on the Influenza virus? (MBBS-2003)
Q. Write short note on the Antigenic Drift? (MBBS-2007)
Q. Write short note on the Antigenic shift? (MBBS-2007)

ANSWER
It is Orthomyxo influenza A virus.
In swine flu, haemagglutinin subtype H1 and the neuraminidase subtype
N1antigen present. Therefore, it is also known as H1N1.
It is a spherical or filamentous, pleomorphic enveloped viruses with a single
stranded RNA genome which is segmented and exists as eight pieces.
The nucleocapsid has a helical symmetry.
The nucleocapsid is enclosed by an inner protein layer called the matrix or 'M
protein' and outer lipid layer.
S e e C o l o r Im a g e 2 2

Attached to the lipid layer of the envelope are two types of glycoprotein
peplomers or spikes, the haemagglutinin (HA) and the neuraminidase (NA).

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Haemagglutinin spikes are triangular in cross section while neuraminidase

spikes are mushroom shaped which are less numerous.

Structure of Influenza Virus

Depending on the degree of antigenic change of HA and NA, two distinct

forms of antigenic variation are known.
1. Antigenic Drift:
Antigenic drift refers to the minor antigenic changes in either the
haemagglutinin or neuraminidase or both due mutations in genes.
Periodic epidemics of influenza are associated with antigenic drift.
2. Antigenic Shift:
Major antigenic changes in HA or NA are called antigenic shift due to
mutation and gene reassortment (recombination) in doubly infected
cells.
It is leading to major epidemics and pandemics.
Pathogenesis
Influenza virus enters the body by respiratory tract.
The ciliated epithelial cells of respiratory tract are the main sites of
infection.
Incubation period varies from 1--4 days.
Clinical features vary from mild coryza to fulminating pneumonia.

Laboratory Diagnosis
Laboratory diagnosis depends on demonstration of virus antigens, isolation of
the virus and serology.

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1. Demonstration of Virus Antigens:
Viral antigens in clinical specimens can be detected by fluorescence
microscope after stain smears with fluorescent-tagged influenza
antiserum.
2. Isolation of the Virus:
Throat gargling is inoculated into
(i) Amniotic cavity of 11 to 13 days chick embryo
(ii) Monolayers of monkey kidney cell cultures
(iii) Human embryo kidney cells
It is can be detected.
by haemagglutination or
by haemadsorption or
by immunofluorescence.
3. Serology:
The routinely serological tests for the diagnosis of influenza include
(i) Complement Fixation Test (CFT)
(ii) Haemagglutination Inhibition test (Hl test)
4. RNA genome detection by RT-PCR amplification method.

Treatment
Oseltamivir is the recommended drug both for prophylaxis and treatment.

Avian influenza (Bird Flu)

Q. Write short note on the avian influenza or bird flu?
ANSWER
Avian influenza or “Bird flu” is a contagious disease of animals caused by
H5N1 or H7 serotype of Influenza A virus that normally infect only bird or less
commonly pigs.
Direct contact with infected poultry or surface and objects contaminated by
theirfaeces is main route of human infection.
Infected bird shed large quantities of virus in their faeces.
Normal temperatures used for cooking will kill the virus.
Some migratory birds directly spread the H5N1 virus in highly pathogenic
form.
Laboratory diagnosis:
(1) Direct antigen detection by immunofluorescence and radio immune assay
(2) Isolation in cell culture
(3) Detection of influenza RNA by Reverse Transcriptase PCR.

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34
P A R A M Y X O V IR U S

Mumps
Measles
Koplic’s Spot

Mumps
Q. Write short note on the Mumps? (BDS-2010), (BDS-2008)

ANSWER
Mumps is caused by mumps virus.
Mumps virus is a paramyxovirus.
Mumps is also known as “Epidemic Parotitis”.
It has spherical enveloped with two types of HN (haemagglutinin,
neuraminidase) and F (fusion protein) peplomers.
Nucleocapsid is helical symmetry and contains a single stranded RNA genome
as a single piece.
Mumps or epidemic parotitis is predominantly a disease of childhood.
It is acquired from direct contact with infected saliva or aerosols from infected
patients.
Respiratory tract is the portal of entry.
Virus multiplies in upper respiratory tract and in local lymph node and spread
to other part of body.
Incubation period is 15-18 days.
The most characteristic feature is nonsuppurative inflammation of the parotid
glands (parotidis).

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Meningitis, deafness, myocarditis, pancreatitis, orchitis and oophoritis is a
common complication.
It is provide life long immunity due to only one serotype.

Measles Virus
Q. Write short note on the Measles Virus?
Q. Write short note on the Koplic’s spots?

ANSWER
Measles virus is paramyxovirus.
It has spherical enveloped with two types of H (haemagglutinin ) and F (fusion
protein) peplomers.
Nucleocapsid is helical symmetry and contains a single stranded RNA genome
as a single piece.
Measles is highly infectious childhood disease spread by respiratory
secretions.
Measles virus is acquired by inhalation.
Measles is characterised by high fever, cough and conjunctivitis.
Koplik's spots can be seen on the buccal mucosa and are pathognomic of
measles.
Widespread maculopapular rash appears first on the neck and then spreads to
the rest of the body.
The rash fades in about a week and the patient recovers uneventfully by 10-14
days.
Complications
(i) Patient may develop secondary bacterial infections such as otitis
media, bronchopneumonia and croup.
(ii) Post-measles encephalitis and subacute sclerosing panencephalitis
(SSPE).
It is provide life long immunity due to only one serotype.
Laboratory diagnosis:
Multinucleated giant cells can demonstrated into smear by staining with
giemsa stain.
IgM measles specific antibody can be demonstrated in the serum by ELISA.
Measles can be isolated in tissue cell culture.

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35
R H A B D O V IR U S

Rabies Virus
Pathogenesis
Laboratory Diagnosis
Hydrophobia
Rabies Vaccine
Negri’s Bodies
Neural Vaccine

Rabies Virus
Q. Describe the pathogenesis and Laboratory diagnosis of Rabies virus?
(MBBS-2003)
Q. Write short note on the Rhabdovirus?
Q. Write short note on the Rabies virus?
Q. Write short note on the Hydrophobia? (MBBS-2003)
Q. Write short note on the Negri’s bodies?

ANSWER
Rhabdoviruses are bullet or rod shaped (rhabdos, meaning rod) enveloped
viruses with single stranded RNA genome. They are included in the family
Rhabdoviridae.
Morphology
It is bullet shaped and outer lipoprotein envelope contains haemagglutinating
peplomer spikes.
Nucleocapsid shows helical symmetry containing a single stranded RNA
genome and a RNA-dependent RNA polymerase.
The genome is unsegmented.
Pathogenesis
Rabies is a natural infection of dogs, foxes, cats, wolves and bats.
Man is infected by the bite of rabid dog or other animals.

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Saliva containing viruses is deposited in the wound.
The rabies virus multiplies in muscle or connective tissue at or near the site of
introduction before it attaches to nerves and travel towards the brain and
spinal cord.
Rabies virus multiplies in the brain and spreads along the peripheral nerve to
various parts of the body including salivary gland.
Incubation period depending upon the site of bite, which related to the
distance the virus has travel to reach the brain.
The disease undergoes four stages:
(a) Prodromal phase: malaise, headache, fatigue anxiety are symptom seen.
(b) Encephalitic phase: patient develops hydrophobia, hyperactivity, difficulty
in drinking.
(c) Coma
(d) Death: due to respiratory arrest during convulsion.
Laboratory Diagnosis
(i) Immunofluorescence test
Viral antigens and negri’s bodies can be detected in corneal impression
smears and facial skin biopsies or saliva by direct immunofluorescence using
antirabies serum tagged with fluorescein isothiocyanate. Diagnosis may be
made postmortem using brain as a specimen.
(ii) Demonstration of Negri bodies
This is demonstrated in brain. Negri bodies appear as intracytoplasmic, oval or
round, purplish pink (3-27 mm) bodies with characteristic basophilic inner
granules. Seller's technique is used to demonstrate these bodies.
S e e C o l o r Im a g e 2 3

(iii) Isolation of the virus

The virus can be isolated from specimens like CSF, saliva and urine, by
intracerebral inoculation in mice and examined by immunofluorescence
technique.
(iv) Antibody detection
High titre antibodies in the CSF can be used for diagnosis.
(v) PCR
PCR can be used for detection of rabies virus RNA.

Rabies vaccine
Q. Write short note on the Rabies vaccine? (MBBS-2010), (MBBS-2003)
Q. Write short note on the post exposure prophylaxis for rabies? (MBBS-
2003)
Q. Write short note on the Neural vaccine?

ANSWER
In rabies pre prophylaxis and post prophylaxis vaccination can be given.

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In Post-Exposure Prophylaxis includes local treatment, hyper-immune serum

and vaccination.
LOCAL TREATMENT: The wound should be thoroughly washed with soap,
water and disinfectant.
HYPERIMMUNE SERUM: Human antirabies immunoglobulin (20 IV per kg
body weight) is reserved for the high risk cases.
VACCINATION
Vaccines are of two main categories-neural and non-neural
(i) Neural vaccines are
(1) Semple vaccine (2) Beta propiolactone (BPL) and
(3) Infant brain vaccine:
Neural vaccines are not satisfactory because
(1) Brain tissue vaccines are associated with neurological complications
due to the presence of myelin.
(2) They are poor immunogens
(3) Many vaccine contain infectious particles.
(ii) Non-neural vaccines are
(1) Duck egg vaccine,
(2) Cell culture vaccines
(a) The human diploid cell strain (HDCS) vaccine.
(b) The purified chick embryo cell culture (PCEC) vaccine.
(c) Purified Vero cell (PVC) vaccine.
VACCINATION SCHEDULES
When the biting animal can be observed for ten days and the animal remains
healthy, rabies may be excluded and vaccine, if already started, may be
discontinued.
Neural vaccines
The vaccine schedule varies according to the degree of risk to which patient
has been exposed.
The vaccine is given subcutaneously on the anterior abdominal wall.
It requires seven to fourteen injections depending on the degree of risk.
Cell culture vaccines
(1) Pre exposure vaccination:
It is recommended for veterinarians, laboratory staff working with the
rabies virus, animal handlers and wildlife officers.
1.0 ml is given by intramuscular route in the deltoid region, on days 0,
7 and 21 day with booster dose after one year.
sRepeat after every five year.
(2) Post exposure vaccination:
It is recommended to those who were bitten by a suspected rabid
animal.
1.0 ml is given by intramuscular route in the deltoid region, on days 0,
3, 7, 14 and 30 with a booster dose (optional) on day 90.

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36
A R B O V IR U S

Arbovirus
Encephalitis
Hemorrhagic Fever
Dengue
Chikunguniya

ArboVirus
Q. Classified the Arbo viruses? (MBBS-2010),(MBBS-2007),(MBBS-2005),
(MBBS-2003)
Q. Classified the Orthropods borne viruses?
Q. Classified the Encepahlitis viruses?
Q. Classified the Hemorrhagic fever?

ANSWER
Viruses from various families which are transmitted via arthropods from one
vertebrates to another.
Diseases caused by arboviruses
– Encephalitis
– Febrile diseases
– Hemorrhagic fevers
Classification of the arbovirus is basis on the clinical features.
Arbo virus is classified into three groups.
(1) Encephalitis:
Western equine encephalitis, Eastern equine encephalitis,
Venezuelan equine encephalitis, St. Louis encephalitis,
Ilheus, West Nile,

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Murray Valley encephalitis, Japanese B encephalitis,

Russian spring summer encephalitis, Powassan
California encephalitis

(2) Febrile illness:

Chikungunya, O'nyong-nyong,
Semliki Forest, Sinduis,
Ross river virus, Dengue, types 1-4,
Sandfly fever, Chandipura virus
Rift-valley fever, Nairobi sheep disease,
Ganjam virus, Colorado tick borne
Vesicular stomatitis virus Chandipura virus

(3) Hemorrhagic fever:

Chikungunya , Dengue ,
Yellow fever, Kyasanur Forest disease,
Omsk Haemorrhagic fever, Chittor virus

Dengue
Q. Describe the Pathogenesis and Laboratory Diagnosis of Dengue
Virus? (MBBS-2010), (MBBS-2007), (MBBS-2005)
Q. Write short note on the Dengue fever? (B.Sc. Nur.-2009) (MBBS-2009)
Q. Write short note on the Dengue hemorrhagic fever?
Q. Write short note on the Dengue shock Syndrome?

ANSWER
Dengue virus belong to family flavi viridae and genus flavi virus.
It is icosahedral in symmetry, enveloped and single strand RNA genomic virus.
Dengue virus has four serotypes:
Dengue 1 (DEN 1), Dengue 2 (DEN 2), Dengue 3 (DEN 3) and Dengue 4 (DEN 4)
It is transmitted by bite of Aedes aegypti.
Clinical Features
The disease may occur in three forms;
(1) Classical Dengue fever,
(2) Dengue Haemorrhagic fever (DHF) and
(3) Dengue Shock Syndrome (DSS).
(1) Classical Dengue Fever:
Dengue is characterised by fever of sudden onset, headache, retrobulbar pain,
conjunctival infection, pain in the back and joints (also called “break-bone
fever”) lymphadenopathy and maculopapular rash.
This usually affects older children and adults.
The fever is typically biphasic (saddle back).

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Incubation period is 5-8 days.
The febrile illness lasts for about 10 days after which recovery is generally
complete.

(2) Dengue Haemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS).
DHF/DSS remains mostly confined among children of 5-10 years age group in
area where multiple dengue viruses cause disease.
It is seen in patients previously infected with dengue virus.
On reinfection with a different serotype of dengue virus, antibody formed
against the first virus reacts with the second serotype virus forming immune
complexes (virus-antibody complex).
Initial symptoms are like those of dengue fever but associated with
haemorrhagic rash, thrombocytopenia and shock.
The mortality rate is 5-10%.
All four types of dengue virus are present in India.

Laboratory Diagnosis
Serum used as a specimen for detection of antibody of dengue.
Demonstration of IgM antibody in serum provides early diagnosis.
IgM antibody appears within 2 to 5 days of the onset of illness and persists for
one to three months.
ELISA is used for detection of IgM antibody.
A strip immunochromatographic test for IgM is also available for rapid
diagnosis.

Treatment:
Classical dengue fever treated by symptomatically.
DHF and DHS treated by replacement of fluid, blood transfusion and
transfusion of platelet.

Prophylaxis
Control measures include elimination of mosquitoes. No effective vaccine is
available.

Chikungunya
Q. Write short note on the Chikungunya?

ANSWER
Chikunguniya name is derived from Swahili word.
Chikungunya meaning is that which bends up.
Chikungunya is a virus that is transmitted from human to human mainly by
infected Aedes albopictus and Aedes aegypti mosquitoes.

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Chikungunya causes sudden onset of high fever, severe joint pain, muscle pain
and headache
It is belong to family Togaviridae and genus Alpha virus.
It is a enveloped virions spherical in shape, icosahedral symmetry, positive-
sense, single-stranded RNA genome.
Out breaks occur during rainy season with increasing densities of Aedes
aegypti mosquito.
Mosquitos bites and infect the Humans in day time.
Aedes mosquitoes are feed in day time, breed in stagnant water and small
puddle.
Incubation period is 3 – 12 days

Clinical feature
Fever (1030C to 1040C) with rigors Crippling Joint pain
Lymphadenopathy Conjunctivitis
Maculopapular rash
Some time may lead to hemorrhagic manifestations
Fever is biphasic with remission after 1 - 6 days of fever
Fever, rash, nausea, fatigue, arthralgia lasting days to weeks
Arthritis may be long-term sequellae.

LABORATORY DIAGNOSIS
Routine Diagnosis with serology
Detection of IgM antibody provides a specific and reliable means for
early diagnosis.
ELISA and Dot blotting methods are used.
Isolation of Virus
Amplification of Nucleic acid

TREATMENT
Treatment is symptomatic.
Aspirin should be avoided.
There is no vaccine or specific antiviral treatment currently available for
Chikungunya fever.
PROPHYLAXIS
When staying in affected areas:
o Wear long-sleeved shirts and long trousers.
o Use mosquito repellents, coils or other devices that will help fend off
mosquitoes.
o If possible, sleep under bed nets pre-treated with insecticides.

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37
R U B E L L A V IR U S

Rubella Virus
German Measles

Rubella Virus
Q. Write short note on the Rubella Virus?
Q. Write short note on the German measles?

ANSWER
Rubella or German measles is primarily a mild childhood disease.
It may be acquired congenitally or postnatally.
It is a member of togaviridae family.
Rubella virus is a pleomorphic, spherical, single stranded RNA genome and
enveloped carries haemagglutinin peplomers.

(i) Postnatal rubella

Infection is acquired by inhalation.
Virus multiplies locally and in the cervical lymph nodes.
Incubation period is 2 to 3 weeks.
Patient develops fever, and fine, pink, macular rash, which first
appears on the face and then spreads to the trunk and legs.

(ii) Congenital rubella

Rubella virus can cross the placental barrier and can cause congenital
defect, stillbirth and abortion.

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Laboratory Diagnosis
(i) Virus isolation
Rubella virus can be isolated from adult throat swab by tissue culture.
(ii) Serology
Serological diagnosis is the method of choice in rubella.
Antibodies in blood are detected by ELISA.
Demonstration of rubella IgM antibody in a single specimen of blood
has a diagnostic value.
In a newborn baby, rubella specific IgM antibody is diagnostic of
congenital rubella as IgM antibodies do not cross the-placenta.
In case of rubella IgG antibody, four-fold rise in titre must be
demonstrated between two serum samples collected at 10 days
interval.

Prophylaxis
A combined measles-mumps-rubella (MMR) vaccine is recommended
for all infants at the age of 15 months, followed by a booster at the
age of 4-6 years.

Rota Virus
Q. Write short note on the rotavirus?

ANSWER
Rota (rota,latin wheel) virus is double walled icosahedral capsid resembling a
wheel under electron microscope.
The genome consists of double stranded segmented RNA.
Rotavirus is common cause of infantile diarrhea.
Incubation period is 2-4 days.
After infection, rotavirus multiplies in the columnar epithelial cells of small
intestine leading to shorting of the microvilli and mononuclear cell infiltration
in lamina propria.
Watery diarrhea occurs due to
(1) Infection prevents absorption of water and electrolyte.
(2) Crypt hyperplasia causes hypersecretion of water.
Virus can be demonstrated into stool by immunoelectron microscopy, ELISA or
immunodiffusion.
Rotavirus polyvalent “cocktail” vaccine containing attenuated strains of two or
more serotypes are now in use.

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38
V IR A L H E P A T IT IS

Hepatitis A Virus
Hepatitis B Virus
Hepatitis C Virus
Hepatitis D Virus
Hepatitis E Virus

Viral Hepatitis
Q. Describe different Hepatitis Viruses? (BDS-2005), (MBBS-2007), (MBBS-
2004), (MBBS-2003)
Q. Write short note on the Hepatitis A Virus?
Q. Write short note on the Hepatitis B Virus? (BDS-2009), (BDS-2009 {O})
(MBBS-2009), (MBBS-2007), (MBBS-2004), (MBBS-2002)
Q. Write short note on the hepatitis B virus markers?
Q. Write short note on the Hepatitis C Virus? (MBBS-2007), (MBBS-2002)
Q. Write short note on the Hepatitis D Virus?
Q. Write short note on the Hepatitis E Virus?
Q. Write short note on the Australia Antigen?
Q. Write short note on the Serum Hepatitis?
Q. Write short note on the Fulminating Hepatitis?

ANSWER
Viral hepatitis is a systemic disease with primary inflammation in the liver.
There are six hepatitis viruses types A, B, C, D, E, and G.
Hepatitis B is a DNA virus while others (A, C, D, E, G) contain RNA genome.

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HEPATITIS A VIRUS (HAV) (INFECTIOUS HEPATITIS)

Infectious hepatitis is occurring mainly in children and young adults.

The hepatitis A virus enters the body by oral route.
The clinical disease has two stages:
The prodromal or preicteric stage
The icteric stage
Symptoms include fever, malaise, anorexia, nausea, vomiting, liver tenderness
and jaundice.
It is non-enveloped single stranded RNA virus with an icosahedral symmetry.
It belongs to the picornavirus family.

Laboratory Diagnosis
HA V can be detected in faeces by ELISA and Immunoelectron microscopy.
The virus has been grown in human and simian cell cultures.
Liver function tests such as Alanine aminotransferase (ALT) and bilirubin
supplement the diagnosis.

HEPATITIS B VIRUS (HBV) (SERUM HEPATITIS)

Hepatitis B virus (HBV) belongs to the family Hepadnaviridae.

It is enveloped, double shelled particle, circular double stranded DNA virus
with an icosahedral symmetry.

HBV antigenic structures (markers) are

1. HBsAg: Surface antigens (envelope protein).
This antigen is also named as Australia antigen.
It is the first marker to appear in blood after infection.
It persists for years in carriers.
Peak levels are seen in preicteric phase of disease.
Antibody to HBsAg appears within weeks after the disappearance of
HBsAg and is persists for very long periods.
Anti-HBs is the protective antibody.
2. HBcAg:
It is the core (nucleocapsid) antigen of the virus.
It is not detectable in the serum but can be demonstrated in liver
cells.
It remains life long and thus serves as a useful indicator of prior
infection with HBV.
3. HBeAg:
It is the hidden antigenic component of core.
Sera containing HBeAg are believed to be highly infectious and those
with anti-HBe of little infectivity.
4. Viral Genes: Two linear strands of DNA.

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Modes of Transmission
There are three important modes transmission of HBV infection:
1. Parenteral 2. Perinatal 3. Sexual
Clinical Features
The incubation period varies from 6 weeks to 6 months.
HBV infection can be divided into three phases: preicteric, icteric and
convalescent.
1. Preicteric Phase
Patient develops malaise, anorexia, weakness, myalgia, nausea and
vomiting.
2. Icteric Phase
Patient develops jaundice, pale stools and dark urine (bilirubinuria).
3. Convalescent Phase
This phase is long and drawn out with malaise and fatigue.
HBV convert into chronic carrier stage, cirrhosis and hepatocellular
carcinoma.
Two types of carriers super carriers and simple carriers present in
hepatitis B virus.

Laboratory Diagnosis
HBsAg detection is specific and gold standard antigen of HBV
infection. It is the first marker to appear in blood after infection.
HBsAg disappears with recovery from clinical disease.
It persists for years in carriers.
Antigenic marker can be detected by ELISA, Immunochromatography,
immunofluoroscence microscope and PCR.

Prophylaxis
1. General Preventive Measures
Use unsterile needles, syringes and other material must be avoided to prevent
hepatitis B infection.
2. Immunisation
(i) Passive immunisation
Hepatitis B immunoglobulin can be given in doses of 300-500 IU
intramuscularly.
(ii) Active immunisation
Two type of killed type vaccine use for active immunization.
(a) Plasma derived vaccine
(b) Recombinant yeast hepatitis B vaccine
Both vaccines are adsorbed with aluminium hydroxide as adjuvant, stored in
cold but not frozen.
Three doses at 0, 1 and 6 months are administered intramuscularly into
deltoid muscle.

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HEPATITIS C VIRUS (HCV)

Hepatitis C virus (HCV) belongs to the family Flaviviridae.

It is a linear single stranded RNA.
It is transmitted by parenteral, perinatal and sexual route.
Clinical Features
Clinical infection with hepatitis C is generally less severe, with milder
symptoms, absent or less marked jaundice.
Laboratory Diagnosis
It can be established by detection of anti-HCV (antibody) by ELISA.
Viral genome (RCV RNA) can be detected by polymerase chain
reaction (PCR) and by immunofluorescence.
Prophylaxis
Only general prophylaxis, such as blood or blood products screening, is
possible.

HEPATITIS D VIRUS (HDV) (DELTA ANTIGEN)

The HDV is a defective virus as it is dependent on the helper function of HBV

for its replication and expression.
It belongs to genus Deltavirus.
It is spherical, single stranded small circular molecule of RNA particle
surrounded by HBsAg envelope.
It is co-infect with HBV.
HDV infection can occur in presence of HBV under two situations:
(i) Simultaneous infection with both HDV and HBV (coinfection).
(ii) Superinfection of an HBsAg carrier by HDV.
Transmission occurs parenterally.

Laboratory Diagnosis
Diagnosis can be made by detecting the IgM anti-delta antibody in
serum.
ELISA and RIA kit are commercially available for detection of
antibodies to HDV.

HEPATITIS E VIRUS (HEV)

Hepatitis E virus belongs to family Calciviridae and genus Calcivirus.
They are spherical, nonenveloped, possess single stranded RNA genome,
which is surrounded by icosahedral capsid.
It is primarily associated with ingestion of faecally contaminated drinking
water.
HEV is mostly seen in pregnant lady and cause of fulminating hepatitis.
Hepatitis E can be prevented by improved standards of sanitation and
chlorination of water.

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39
R E T R O V IR U S

HIV/AIDS
Pathogenesis
Opportunis tic infection
Laboratory Diagnosis
HAART
ART

Human Immunodeficiency Virus(HIV)

Q. Describe the pathogenesis and laboratory diagnosis of HIV? (B.Sc. Nur.-
2010), (B.Sc. Nur.-2008), (MBBS-2007) (MBBS-2006)
Q. Write short note on the opportunistic infection in HIV? (MBBS-2007),
(MBBS-2006), (MBBS-2004)
Q. Write short note on the viral genes?
Q. Write short note on the HIV markers?
Q. Write short note on the HAART?
Q. Write short note on the ART?
Q. Write short note on the HIV/AIDS? (BDS-2007)

ANSWER
Morphology (MBBS-2010)
HIV is RNA viruses that belong to family Retroviridae.
HIV is a spherical enveloped virus.
It contains two identical copies of single stranded RNA genome.
It is possess reverse transcriptase (RNA directed DNA polymerase) enzyme
which prepares a DNA copy of the RNA genome in host cell.
The virus core is surrounded by a nucleocapsid composed of protein, which
has an outer icosahedral shell and an inner cone shaped core.

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72 glycoprotein peplomers are projected from the bilayered lipoprotein

envelop surface.

Viral genes and antigens

Q. Write short note on the viral genes?
Q. Write short note on the HIV markers?

ANSWER
HIV genome is made up of three structural and seven non-structural genes.
Structure genes
Structure genes are three types:
(1) Gag gene:
 It is encode for the core and shell of virus.
 Precursor protein P55 protein is cleaved into three proteins P15, P18
and P24.
 P24 is the major core antigen and is detected in the serum during the
early stages of the HIV infection till the antibodies appear.
 In the later stage, decline anti P24 antibodies and reappearance of
P24 antigen indicates progression of illness.
(2) Env gene
 Envelope glycoprotein gp 160 is cleaved into two components gp120
and gp41.
 Gp 120 form the surface spike and gp 41 form transmembrane
pedicle.
 Anti gp 120 antibodies are first to appear in circulation and remain till
the terminal stage of the infection.
(3) Pol gene
 It encodes for the polymerase reverse transcriptase and other viral
enzymes.
 Precursor protein P100 is cleaved into P32, P51 and P66.

Non- structural gene

These are regulatory genes and are specific to HIV only.
(1) tat gene (Transactivating gene): Enhances the expression of all genes
(2) vif gene(Viral infectivity factor): Influence the infectivity
(3) rev gene(Regulator of virus): Enhances the expression of structure
gene
(4) nef gene (Negative factor gene): Regulate the latent state of the virus
(5) vpr gene (Promoter gene): Stimulates the promoter region
(6) vpu gene: Unknown functions in only HIV-1
(7) vpx gene: Unknown functions in only HIV-2
(8) LTR sequence (Long terminal repeat): Gives promoter, enhancer and
integration signals

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S e e C o l o r Im a g e 2 4

Mode of transmission of HIV: (MBBS-2005)

There are three modes of transmission: sexual contact, parenteral and
perinatal.
1. Sexual contact:
This is the most important mode of transmission.
Sexual transmission occurs among both homosexual as well as
heterosexual individuals.
2. Parenteral transmission:
It may occur through blood after receiving infected blood
transfusions, blood products, sharing contaminated syringes and
needles as in intravenous drug abusers or accidental inoculation.
3. Perinatal transmission:
Infection may be transmitted from an infected mother to her child
either transplacentally or perinatally.

Pathogenesis
Q. Describe the pathogenesis HIV? (B.Sc. Nur.- 2010), (B.Sc. Nur.-2008),
(MBBS-2007) (MBBS-2006)

ANSWER
On entering HIV into the blood stream of a person, the virus infects the CD4+
T lymphocytes.
It also affects macrophages, monocytes, alveolar macrophages, microglial cell
of brain and dendrite cell of skin.

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The virus binds specifically to CD4 receptor with the envelop glycoprotein
gp120 by participation of a coreceptor molecule CXCR4 for T cell and CCR5 for
macrophages.
After entering viral core, viral RNA is transcribed by ‘reverse transcriptase’
into double stranded DNA which is integrated into the genome of infected cell
by ‘integrase’ enzyme.
Viral DNA is replicate with host DNA at low level.
HIV infection causes damage to CD4+ T lymphocytes resulting in a decrease in
CD4 cell count.
Window period:
This is a period between the HIV infections to the appearance of antibodies
upto detectable level by available diagnostic tool.
This is also called “seronegative infective stage”.
Usually window period is 3 weeks to 3 months.
In this period viras can be detected by P24 antigen detection and viral DNA
sequence (DNA hybridization) in CD4 lymphocytes.
Clinical Features
Clinical courses of HIV infection.
Group I Acute HIV infection
Group II Asymptomatic infection
Group III Persistent generalized lymphadenopathy
Group IV
Subgroup A Constitutional disease -ARC
Subgroup B Neurological diseases
Subgroup C Secondary infectious diseases
Subgroup D Secondary cancers
Subgroup E Other conditions

Opportunistic Infections and Malignancies Commonly Associated with HIV

Infection. (MBBS-2007), (MBBS-2003)
Bacterial:
1. Mycobacterial infections
2. M. avium complex
3. Salmonellosis
Viral
1. CMV
2. Herpes simplex
3. Varicella-zoster
4. Epstein-Barr (EB) virus
Mycotic
1. Pneumocystis carinii pneumonia
2. Candidiasis
3. Cryptococcosis
4. Aspergillosis

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5. Histoplasmosis
6. Coccidioidomycosis
Parasitic
1. Toxoplasmosis
2. Cryptosporidiosis
3. Isosporiasis
4. Generalised strongyloidiasis
Malignancies
1. Kaposi's sarcoma
2. B-cell lymphoma or non-Hodgkin's lymphoma

Laboratory diagnosis
Q. Describe thelaboratory diagnosis of HIV? (B.Sc. Nur.- 2010), (B.Sc. Nur.-
2008), (MBBS-2007) (MBBS-2006)

ANSWER
HIV can be diagnosed by various methods
(i) Antigen detection
The p24 antigen is the earliest (2 weeks) virus marker to appear in the blood.
ELISA can be used for detection of this antigen.
It is highly useful in diagnosis in early infections, screening of blood and tissue
for transfusion & for transplantation and in new born babies or infant.
(ii) Virus isolation:
HIV can be isolated from CD4 cell of Blood, Bone marrow, serum & body fluid.
For diagnosis, virus is not routinely isolated.
(iii) Viral nucleic acid can be detected by polymerase chain reaction (PCR).
Two types of PCR have been developed: DNA PCR and RNA PCR.
DNA PCR:
In this method peripheral lymphocytes are lysed and proviral DNA is
amplified.
The Method is highly sensitive and specific.
RNA PCR:
This method is used for the diagnosis and monitoring of the level of
viremia.
(iv) Antibody detection:
Demonstration of antibodies is the simplest and most commonly employed
technique for diagnosis.
IgM antibodies appear first usually in about 3-4 weeks after infection, to be
followed by IgG antibodies.
Diagnosis of HIV is made by detecting serum antibodies to P24, gp 120 and gp
41.
HIV infected persons remain negative for antibodies during window period.

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There are two types of serological tests for HIV diagnosis.

Screening and supplemental

1. Screening (E/R/S) tests are (a) ELISA

(b) Rapid tests - Dot blot assay
-Particle agglutination
-HIV spot and comb tests
(c) Simple tests
2. Supplemental tests are -Western blot test
-Indirect immunofluorescence test
-Radio Immuno Precipitation assay

Confirmatory test of HIV diagnosis

(1) Detection of Viral nucleic acid
- In situ hybridization
- Polymerase Chain Reaction (PCR)
(i) DNA- PCR
(ii) RNA- PCR
(2) Detection of P24 antigen
(3) Virus isolation
Non-specific Tests
(i) In AIDS, there is leucopenia with a lymphocyte count less than 400 per
mm3.
The count of CD4 lymphocytes falls below 200 per mm3.
(ii) The normal CD4 : CD8 T-cell ratio of 2 : 1, is reversed to 0.5 : 1 in cases of
AIDS.
(iii) Skin test for cell mediated immunity is diminished.
Prevention
The following preventive measures are recommended.
1. The use of condoms can prevent transmission of the virus.
2. Contaminated syringes or needles should not be shared.
3. All blood, blood products and tissue are to be screened for HIV.

Treatment
Highly active antiretroviral therapy (HAART) is effective in inhibition of HIV
replication in most of HIV-infected individuals.
Antiretroviral Drugs used in HAART are
(1) Nucleoside reverse transcriptase inhibitors: Lamivudine and Zidovudine
(2) Non-nucleoside reverse transcriptase inhibitors: Nevirapine
(3) Nucleotide inhibitor: Tenofovir
(4) Protease inhibitor: Indinavir and Nelfinavir
(5) Fusion inhibitor: Enfluvirtide

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40
G E N E R A L M YC O LO G Y

Classification of Fungus
Laboratory Diagnosis

Classification of fungus
Q. Write down the Classification of the fungus? (MBBS-2006)
Q. Describe the pathological fungus?
Q. Enumerate the medical importance fungus?
Q. Describe the laboratory diagnosis of fungus? (MBBS-2003)

ANSWER
Fungus classified by three types of classification.
(A) Taxonomical Classification
(B) Morphological Classification
(C) Clinical Classification

(A) Taxonomical classification

On the basis of Hyphae, asexual spore and sexual spore fungus, classified into
four classes.
(1) Zygomycetes: Non-septate hyphae
Asexual spore (sporangiospores)
Sexual spores (Zygospores and oospores)

(2) Ascomycetes: Septate hyphae

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Sexual spore (Ascospores)
(3) Basidiomycetes: Septate hyphae
Sexual spore (Basidiospore)
(4) Deuteromycetes: Septate hyphae
(Fungi imperfecti) Lack of sexual stage

(B) Morphological Classification

On the basis of morphology, it is classified into four classes.
(1) Yeast Round unicellular fungi
e.g. Cryptococcus neoformans
(2) Yeast like fungi Partially yeast and partially budding cells
Form pseudohyphae
e.g. Candida
(3) Mould Septate or non septate branching hyphae or
mycelium present
e.g. Dermatophytes, Aspergillus, penicillium and
Rhizopus.
(4) Dimorphic fungi Both forms present.
(MBBS-2006) Yeast form (In host and in culture at 370 C).
(MBBS-2004) Hyphae form (In soil and in culture at 22-250C).
e.g. Blastomyces, Histoplasmosis, Paracoccidioides,
Coccidioides, Sporothrix.

D i ffe re n t st ru c t u re s o f fu n g u s

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(C) Clinical classification

On the basis of involvement of tissue or organ classified into three groups.
(1) Superficial mycoses: Dermatophyte, Tinea nigra , Piedria,
Pityriasis.
(2) Subcutaneous Mycoses: Mycetoma, sporotrichosis, Rhinosporidiosis.
(3)Systemic Mycoses: Blastomycosis, Histoplasmosis, Candidiasis,
(MBBS-2006), (MBBS-2003) Cryptococcosis.

Laboratory diagnosis
Fungus can be detected in laboratory by three ways.
(1) By Microscopy
(2) BY culture
(3) By tissue section
(1) BY microscopy:
Prepare smear from specimen and stain with different stains.
Potassium Hydroxide (KOH) Preparation:
Yeast cell, septate or non-septate hyphae, mycelium may be
observed.
KOH with calcofluor white: Fluoresce fungus cell element observed.
Gram’s staining: Gram’s positive yeast seen e.g. Candida.
Indian Ink preparation: Used for detection of capsulated
Cryptococcus neoformans.
(2) By Culture:
Fungal specimen inoculated in Sabouraud's Dextrose Agar (SDA) with
or without adding Chloramphenicol and Cycloheximide at 25°C and
37°C for three weeks.
Chloramphenicol is added in the culture to suppress growth of
contaminating bacteria.
Cycloheximide is added in the culture to suppress growth of
contaminating fungi.
Fungal colonies identified by
(1) Rapidity of growth
(2) Colour of the colony
(3) Morphology of the colony
(4) Pigmentation on the reverse of SDA.
Arrangement of hyphae and spore detected by stained with Lacto phenol
cotton blue (LCB).

(3) Tissue Sections:

Fungal elements in tissue can be identified by methenamine silver stain and
Periodic Acid Schiff (PAS) stain.

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41
M E D IC A L M Y C O L O G Y

Dermatophytes
Chromomycosis
Sporotrichosis
Rhinosporidiosis
Histoplasmosis
Cryptococcosis

Dermatophytes
Q. Write short note on the dermatophyte? (MBBS-2010), (MBBS-2009),
(MBBS-2008), (MBBS-2007), (MBBS-2006), (MBBS-2004), (MBBS-2003)
(MBBS-2002)
Q. Write short note on the ring worm?

ANSWER
Dermatophytes are infecting only superficial keratinised I tissue (skin, hair and
nails).
Dermatophytes are also known as tinea or ringworm.
According site involvement, it is also named as
Name of disease Part of body
Tinea capitis Scalp
Tinea corporis Non hairy skin of the body
Tinea cruris Groin
Tinea pedis Foot or athlete's foot
Tinea barbae Bearded areas of the face and neck.
Fevus Hair follicles
Dermatophytes antigen caused hypersensitivity reaction. It is known as
dermatophytids or 'id' reaction.

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Dermatophytes are classified into three genera as follows

Genus Infected part of body Conidia amount

Trichophyton Hair, skin, nail Microconidia- Plenty
Macroconidia- Few
Microsporum Hair, skin Microconidia- Few
Macroconidia- Plenty
Epidermophyton Skin, nail Microconidia- Absent
Macroconidia- Plenty

Laboratory diagnosis
(1) 10% KOH used to show hyphae in direct microscope.
(2) Culture in SDA with or without Chloramphenicol / Cycloheximide and
revealed Microconidia and Macroconidia by Lactophenol cotton blue stain in
microscope.
Dermatophyte diffentiate on the basis of
Microconidia and Macroconidia
Pigment production in colony
Morphology of colony

Treatment of choice is oral griseofulvin and topical antifungal like fluconazole.

Chromomycosis
Q. Write short note on the chromomycosis?

ANSWER
It is a chronic, localised infection of skin and subcutaneous tissue.
It is caused by several darkly pigmented fungi of the family dematiaceae.
In H and E staining they appear as yeast like bodies with septae, called
sclerotic bodies.
These sclerotic bodies can be demonstrated in KOH mounts and by culture on
SDA.
Amphotericin Band 5-fluorocytosine are used in treatment.

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Sporotrichosis
Q. Write short note on the sporotrichosis? (MBBS-2002)

ANSWER
Sporotrichosis is a nodular, ulcerating disease of skin and subcutaneous tissue.
The fungus gains access through thorn pricks or some injuries.
Causative agent is Sporothrix schenckii.
It is dimorphic fungus.
Yeast form: Appears as cigar-shaped cells
Mould form: Flower like clusters of small conidia borne on delicate
sterigmata.
S e e C o l o r Im a g e 2 6

Rhinosporidiosis
Q. Write short note on the rhinosporidiosis? (MBBS-2005), (MBBS-2002)

ANSWER
Rhinosporidiosis is a chronic granulomatous disease characterised by
formation of friable polyps in mucous membrane.
It is confined to the nose, mouth or eye.
Causative agent is Rhinosporidium seeberi.
The fungus has not been cultivated.
Tissue sections stained with H &E stain show large number of endospore
within the sporangia embedded in a stroma of tissue and capillaries.
S e e C o l o r Im a g e 2 5

Histoplasmosis
Q. Write short note on the histoplasmosis? (MBBS-2003)

ANSWER
Causative fungus of Histoplasmosis is Histoplasma capsulatum.
It is a dimorphic fungus & intracellular fungus.
Source of infection is inhalation of spores containing excreta of birds or bats.
Reticulo-endothelial system is mainly affected in Histoplasmosis.
On microscopy with Giemsa or Wright stains, H. capsulatum show yeast and
mycelia form.

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Yeast form: Small oval yeast cell packed within the cytoplasm of
macrophages or monocytes at 37°C.
Mycelial form: Spherical spores with tubercles or finger like projections25°C.
Amphotericin B is drug of choice.

S e e C o l o r Im a g e 2 7

Cryptococcosis
Q. Write short note on the cryptococcosis? (MBBS-2010), (MBBS-2009).
(MBBS-2007), (MBBS-2005), (MBBS-2004), (MBBS-2002)

ANSWER
Causative fungus of Cryptococcosis is Cryptococcus neoformans.
It is polysaccharide capsulated true yeast and found in the faeces of pigeons.
Cryptococcosis may lead to a mild pneumonitis and meningitis in
immunocompromised individual (AIDS).
Cryptococcus can be demonstrated in specimen by various staining
methods.
Gram’s staining : Gram positive yeast cell seen
Indian ink or nigrosin stain: Capsule appear as clear halo around yeast.
Haemotoxin & Eosin stain: For Histopathological examination of tissue.
Pariodic Acid Schiff stain For Histopathological examination of tissue.
Mucicarmine stain For Histopathological examination of tissue.
Lactophenol cotton blue To demonstrate yeast from colony.
S e e C o l o r Im a g e 2 8

Cryptococcus can be culture in following medium.

Sabouraud’s Dextrose Agar: Cream color colony form
Niger seed agar: Brown color colony form
Bird seed agar: Brown color colony form
Cryptococcal capsular polysaccharide antigen can be detected in CSF, serum
or urine by latex agglutination test.
C. neoformans has ability to
• Grow at 37°C.
• Hydrolyse urea.
• Produce brown colonies on niger seed agar.
• Produce disease in mice (animal inoculation test positive).

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42
O P PO R T U N IST IC M YCO S ES

Candidiasis
Aspergillosis
Penicillosis
Pneumocytosis

Opportunistic Mycosis
Q. Write short note on the opportunistic mycoses? (B.Sc. Nur.-2011) ,(MBBS-
2003)
Q. Write short note on the candidiasis?
Q. Write short note on the aspergillosis?
Q. Write short note on the penicillosis?
Q. Write short note on the pneumocytosis?

ANSWER
Some saprophytic fungi usually do not produce disease but may cause
infection in immunocompromised individuals.
These fungi are called opportunistic mycoses.
Example of opportunistic mycoses
(1) Candidiasis
(2) Aspergillosis
(3)Zygomycosis
(4) Penicillosis
(5)Pneumocytosis carinii
(6) Cryptococcisis

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Candidiasis
Q. Write short note on the candidiasis?
(B.Sc. Nur.-2010) , (B.Sc. Nur.-2009) , (B.Sc. Nur.-2008) , (B.Sc. Nur.-2007),
(BDS-2010), (BDS-2009), (BDS-2009 {O}),(MBBS-2007), (MBBS-2006),
(MBBS-2004), (MBBS-2003, (MBBS-2002))

ANSWER
Causative fungus of Candidiasis is Candida albicans.
Candida albicans is the normal inhabitant of skin, gastrointestinal tract, oral
and vaginal cavities.
Candidiasis is an opportunistic endogenous infection.
Predisposing factors for candidiasis are
Diabetes
Immunodeficiency
Malignancy
Prolonged administration of antibiotics
Patients on immunosuppressive drugs
Intravenous catheters
Candidiasis is cause infection of skin, mucosa and internal organs.
(i) Skin and nail infection:
Paronychia
Onychia
Napkin dermatitis
(ii) Mucosal infection:
Oral thrush
Vulvovaginitis
Balanitis
Conjunctivitis
Keratitis
(iii) Internal organ infection
Urinary tract infection
Intestinal candidiasis
Pulmonary candidiasis
Endocarditis
Meningitis
Septicaemia
(iv) Oro-dental infection
Angular stomatitis
Circumoral candidal dermatitis
Oral thrush
Chronic oral mucocutaneous candidiasis

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Laboratory diagnosis
Gram stained smears and KOH mounts from lesions of skin, nail or
mucous membranes show budding gram positive yeast cells.
Cream coloured, smooth, pasty colonies appear in SDA at 25-37°C
within 24 hours.
Gram stained smear from colonies shows Gram positive budding yeast
cells.
C. albicans can be diffentiate from other species by the following
tests.
S e e C o l o r Im a g e 2 9 & 3 0

(a) Germ tube test:

C. albicans has ability to form germ tubes within two hours when
incubated in human serum at 37°C. This is known as a Reynolds-
Braude phenomenon.
(b) Chlamydospores:
Chlamydospores develop in a nutritionally deficient medium such as
corn meal agar at 20°C.
They can be seen at the end of pseudohyphae
(c) Carbohydrate fermentation and carbohydrate assimilation tests

Treatment
Topical treatment: Polyene (nystatin)
Imidazole (miconazole, clotrimazole)
Systemic infection treatment: Amphotericin B with 5-fluorocytosine.
Aspergillosis
Q. Write short note on the aspergillosis? (MBBS-2005), (MBBS-2002)

ANSWER
Aspergillosis is caused by inhalation of Aspergillus conidia or mycelial
fragments which are present on the decaying matter, soil or air.
Aspergillosis is caused by infection of Aspergillus fumigates, Aspergillus niger
or Aspergillus flavus.
Following clinical manifestation occur by aspergillosis.
Aspergillus asthma
Bronchopulmonary aspergillosis
Aspergilloma
Invasive or disseminated aspergillosis
Sinusitis
Mycotic keratitis
Otomycosis

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Laboratory Diagnosis

(i) Specimens:
Sputum, Bronchoalveolar lavage and tissue biopsy used as a
specimens.

(ii) Direct microscopy

Specimens stained by KOH preparation and tissue biopsy section with
H & E and PAS staining.
Non-pigmented septate hyphae with characteristic dichotomous
branching at an angle of 45°seen.

S e e C o l o r Im a g e 3 1

(iii) Culture
Velvety to powdery coloured colonies appear in 1-2 day on SDA
without cycloheximide at 25°C.
Species are diffentiate, on the basis of colour of colonies and
arrangement of conidia.
A. fumigates -Green colonies
A. niger -Black colonies
A. flavus -Golden yellow colonies

Penicillosis
Q. Write short note on the penicillosis?

ANSWER
Penicillium may cause opportunistic human infections.
It causes penicillosis, keratitis,otomycosis and deep infections.
This saprophytic fungus contains septate hyphae and bears flask -shaped
Phialides.
Phialide contain chains of round conidia.
Brush or brooms like arrangement of conidia are seen in this species.
S e e C o l o r Im a g e 3 2

The colonies are initially velvety and white but later on become powdery and
blue green on SDA.
P. marneffei has been reported as an important opportunist pathogen in the
HIV infected individuals.

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Pneumocytis Carinii
Q. Write short note on the pneumocytosis? (MBBS-2002)

ANSWER
Pneumocystis carinii causes pneumonia in immunocompromised patients.
Previously, it is known as protozoan but now it is known as fungi due to
molecular study.
P. carinii has two stages.
Thin walled trophozoites
Thick walled spherical cysts.
Cyst contains 4 to 8 nuclei.

Life Cycle

Life cycle of pneumocystis Carinii

Bronchoalveolar lavage, lung biopsy and induced sputum are the specimens
used for diagnosis.
Trophozoites and cysts can be demonstration by Giemsa, toludine blue,
methenamine silver and calcofluor white stains.

Methenamine silver staining: Cyst wall stains black.

Fluorescent staining: 'Honeycomb' appearance of the cyst.
Serological tests used for diagnosis are
Complement fixation test.
Latex agglutination test.
Trimethoprim-sulphamethoxazole or pentamidine are used in treatment of
acute cases of pneumonia.

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43
P R O T O Z O A

Entoamoeba
Giardia
Trichomanas
Malaria
Trypnosoma
Leishmania
Entamoeba histolytica
Q. Describe the life cycle, pathogenesis and laboratory diagnosis of
Entmoeba Histolitica? (B.Sc. Nur.-2011) , (MBBS-2005)
Q. Write short note on the E.histolytica? (BDS-2009 {O})
Q. Write short note on the amoebic ulcer?
Q. Write short note on the amoebic dysentery? (BDS-2005), (MBBS-2010)
Q. Write short note on the amoebic liver abscess? (MBBS-2009), (MBBS-
2003) (MBBS-2002)
Q. Write the difference between of amoebic and bacillary dysentery?

ANSWER
Habitat
Trophozoites of E. Histolytica live in the mucous and sub mucous layers of
large intestine of man
Morphology
Three form
A Trophozoite
It is vegetative feeding stage.
Actively motile in fresh passed dysentery stool.
It is move with the help of pseudopodia, Crawling or gliding
movement.

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Nucleus: stained by Iron haematoxylin or Gomorri`s
trichrome stains
Dot like central Karyosome surrounded by clear halo.
Nuclear membrane lined by a single layer of chromatin.
Spoke like radial arrangement b/w karyosome and nuclear
membrane.
Trophozoite divided every 8 hours by binary fission.
Trophozoite do not survive for any length of time in stool
outside the body
B. Pre cyst
Round or oval with blunt pseudopodium contain 1-2 nucleus.
Nucleus are same as trophozoite.
C. Cyst (MBBS-2003)
It is spherical, surrounded by highly refractile membrane.
Nucleus initially one with chromatoid bar and glycogen mass
but mature cyst containing four nucleus without chromatoid
bar and glycogen mass.

Life cycle
Man only host
Mature quadrinucleate cyst is infective form in faeces of convalescent
and carriers
Faeco-oral route is mode of transmission.
Excystation
Cyst wall is damaged by trypsin in the alkaline medium of caecum or lower
part of the ileum.
Amoeboid movement causing a tear in the cyst wall and quadrinucleate
amoeba(metacyst) emerge out.
Metacyst contain 8 actively motile amoebulae.
Amoebulae lodge in the glandular crypts of submocous tissue of colon and
multiply by binary fission.

Encystation
This is a process of transformation of trophozoites into cysts and occurs in the
intestine lumen of infected person.

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Life cycle of E. histolytica

Pathogenesis
Mature quadrinucleate cyst is infective form in faeces ingested by human
being through contaminate water and vegetable.
Noninvasive infection
It is localized in large intestine with minimal lesion and normal immune
response.
Invasive infection
With weaker immune response.
(a) Intestinal wall:
Trophozoite penetrate the epithelium and produce ulcer due to
histolysin and motility of trophozoite.

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Ulcer is pinhead to one inch, transverse, ragged margin and base is
formed by muscular coat.
Ulcer are multiple, most common in caecum and next in the sigmoido-
rectal region.
(b) Amoebic liver abscess:
Single abscess in superior posterior surface of right lobe of liver form
due to supply by right branch of portal vein.
Reddish brown in colour with a semifluid or grumous consistency or
anchovy sauce pus (mixture of sloughed liver tissue and blood)
formed in abscess.
Pulmonary amoebiasis
Cerabral amoebiasis
Splenic abscess
Cutaneous amoebiasis

Clinical syndromes
Incubation period is 4 days to a year or longer (average 1 to 4 months).
A Asymptomatic infection most frequent
B Symptomatic infection
1 Uncomplicated intestinal infection: Diarrhea, Cramp and
flatulence
2 Amoebic dysentery
Moderate to profuse diarrhea containing blood and mucous
Severe abdomen pain
Resemble to bacillary dysentery
Chronic amoebic dysentery (amoebic colitis)
Intermittent diarrhea over a long period of time often misdiagnosed
as ulcerative colitis or irritable bowel syndrome.
Laboratory diagnosis
Specimen
Stool, Blood stained mucous, colonic scraping from the edge of
ulcerated area.
Specimen is examination within 15 minutes because amoeboid
movement of trophozoite rapidly lost in cold surrounding.
Microscopic stool examination
Saline preparation: For studying the trophozoite.
Observations are
1 Motile trophozoite throwing pseudopodia containing RBC
Endoplasm bluish or ground glass appearance
Nucleus faint line visible due to motility
2 Cyst Smooth and thin cyst wall
Round, refractive chromatoid bars
Glycogen mass not visible
3 RBC and pus cell found

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4 Charchot Leyden Crystal: Diamond shape, Refractive structure

Iodine preparation: Show morphological details of cysts.

1 Trophozoite: Yellow to light brown
2 Nucleus: Clearly visible with a central karyosome
3 Cyst: Hyaline cytoplasm, 1 to 4 nucleus, brown glycogen
mass,
Not stained chromatoid bar

Serological test
Positive only in invasive amoebiasis
Test are I Indirect haemagglutination assay (IHA) :
II ELISA
III Latex agglutination
IV Gel diffusion precipitation
V Counter current immunoelectrophororesis
IV Cellulose acetate precipitin test (CEPT)
Treatment

Luminal amoebiasis: Diloxanide furoate, iodoquinol paromomycin or

tetracycline

Tissue amoebiasis: Dehydroemetine or chloroquine

Metronidazole use in both condition.
Metronidazole: 400-800mg thrice daily for 5-7 days

Giardia lamblia
Q. Write short note on the Giardria lamblia?

ANSWER
Giardia was first seen by leuwenhoek (1681) in self stool.
It was named by professor Giard of paris and studied by professor lambol of
prague.
Habitat
Lives attached to mucosa of duodenum and upper part of ileum of man.
Morphology:
Two form:
(1) Trophozoite
(2) Cyst
Trophozoite:
Shaped is tear drop or racket or heart or pyriform.

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Possesses two bilateral symmetrical nuclei one on each side and four
pairs of flagella.
Four pair Flagella originate from basal body between the nuclei.
Movement of trophozoite like “falling leaf”.
It is also contain two sausage shaped parabasal or median bodies
posterior to the suckling disc.
Suckling disc used for attachment to the surface of intestinal
epithelium.
Trophozoite is convex dorsally and concave ventrally.
Cyst
It is surrounded by a tough hyaline cyst wall.
Young cyst possesses two and mature possesses four nuclei.
Flagella remain of margin of the sucking disc within cytoplasm.
Cysts are passed from the faeces.

Different stages of Giardia

Life cycle
Infective stage is cyst.
Mode of infection is faecal-oral route.
Cyst wall is weaken by acidic PH in stomach and excystation complete
in duodenum.
Multiply by binary longitudinal fission.
Residing in the crypt of liberculum in duodenum.
Attached with ventral disc (suckling disc).
Feed by pinocytosis.
Encystment occur in large intestine in adverse environment.
Cysts are passed through stools and remain viable outside in soil &
water.
Pathogenesis
Drinking contaminated water is the most common mode of infection.

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It is causes
Disturbance of intestine function
Malabsorption of lipid and lipid soluble vitamins
Mucous diarrhea
Clinical disease:
Malabsorption, diarrhea, biliary colic, jaundice,
Hypogammaglobulinaemia, lactose intolerance and achlorhydria are
common menifestation.
Laboratory diagnosis:
Microscopy
Stained by iodine or Giemsa or trichome.
Seen trophozoite and cyst in stool sample.
Trophozoite may be recovered from bile.
Serology
Antigen and antibody detection by ELISA and FAT (Fluoroscent
antibody test).
Treatment:
Metronidazole & tinidazole

Trichomoniasis
Q. Write short note on the trichomoniasis?

ANSWER
Trichomonas vaginalis (a flagellate) is caused trichomoniasis.
Morphology
The trophozoite form is half pear shaped with a single nucleus, four anterior
flagella and a lateral flagellum attached by an undulating membrane.
Two axostyles are arranged asymmetrically.
The organism does not encyst.
Life cycle
T. vaginalis colonizes the vagina of women and the urethra (sometimes
prostate) of men.
Infection occurs primarily via sexual contact.
The organism does not encyst and divides by binary fission which is favored by
low acidity (pH > 5.9; the normal pH is 3.5 to 4.5).
Symptoms
T. vaginalis infection may cause mild urethritis or occasionally prostatitis.
In women, it is often asymptomatic, but heavy infections in a high pH
environment may cause mild to severe vaginitis with copious foul-smelling
yellowish, sometimes frothy discharge

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Pathology
The organism causes contact-dependent damage to the epithelium of the
infected organ.
Diagnosis
Clinical suspicion may be confirmed by finding the organism in Giemsa-stained
smears and wet mount preparation of vaginal discharge.
It can be cultivate in Diamond's medium.
Trophozoites must be distinguished from the non-pathogenic
flagellate Trichomona hominis.
Treatment
Metronidazole is effective in both males and females.
Vinegar douche may be useful.
Personal hygiene and the use of condoms are helpful.

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Malaria
Q. Enumerate the type of plasmodium. Describe the life cycle, pathogenesis
and laboratory diagnosis of malaria? (B.Sc. Nur.-2010) ,(BDS-2010)
Q. Write short note on the laboratory diagnosis of malaria? (MBBS-2006)
Q. Write short note on the malaria? (BDS-2009)
Q. Write short note on the Schizogony?
Q. Write short note on the life cycle malaria?

ANSWER
Malaria parasite first time discovered by Alphonse laveran(1880).
Ronald Ross (1897) has established mode of transmission and found oocyst in
stomach wall.
Type of species of plasmodium and their diseases
(1) P. Vivax Benign tertian malaria
(2) P. Falciparum Malignant tertian malaria
(3) P. Malariae Benign quartan malaria
(4) P. Ovale Benign tertian malaria

Life cycle of plasmodium

Plasmodium completes their life cycle in two hosts.
1 Man Intermediate host
2 Female anapheles Definitive host

Schizogony: Asexual reproduction in hepatocyte and RBC.

Sporogony: Sexual reproduction, maturation and fertilization in mosquito.
(I) Human life cycles are
A. Pre-erythrocytic (tissue) Schizogony : Asexual reproduction in
hepatocytes.
B. Erythrocyctic Schizogony: Asexual reproduction in RBC.
C. Gametogony: Develop in red cells of
capillaries of internal organs
such as spleen and bone
marrow
(II) Mosquito cycle is sporogony: Sexual reproduction,
maturation and fertilization.
Human cycle:
Human cycle is started by infective stage sporozoite.
The sporozoite stage is form in salivary gland of female anopheles and enters
into blood stream of man by bite of female anopheles.
After enter into blood stream, sporozoites convert into various stages in
different cycle in following sequence.

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A Pre-erythrocytic (tissue) Schizogony stages are
(1) Sporozoite
(2) Pre-erythrocytic schizont ( and Hypnozoite in P. Vivax).
(3) Merozoite
B. Erythrocyctic Schizogony stages are
(1) Young trophozoite (Signet ring)
(2) Late trophozoite (Amoeboid form)
(3) Schizont
(4) Merozont
C. Gametogony stages are
(1) Female gametocyte (Macrogametocyte)
(2) Male gametocyte (Microgametocyte)

Mosquito cycle:
Female anopheles sucked both asexual and sexual stages with blood
of human.
All the asexual stages are digested and sexual stages further
developed into following stages into stomach of female anopheles.
Sporogony stages are
(1) Male gamete (2) Female gamete
(3) Zygote (4) Ookinete
(5) Oocyst (6) Sporozoite
Sporozoites are deposit into the salivary gland of female anopheles
and released sporozoite into blood stream by biting.

Duration of different cycle.

Species type Pre-Erythrocytic cycle Erythrocytic cycle Gamatagony

P. Falciparum 7-8 days 48 hrs 9-10 days
P. Vivax 5.5 -7 days 48 hrs 8-10 days
P. Malariae 14-16 days 72 hrs 14-16 days
P. Ovale 9 days 48 hrs 12-14 days

Pathogenesis and clinical feature of plasmodium

Incubation period of different species of plasmodium are
P.Vivax 13-17days
P. Falcifarum 12 days
P.Malariae 28-30 days
P.Ovale 13-17 days

Plasmodium broadly caused two type of malaria.

(A) Benign malaria
(B) Malignant malaria

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(A) Benign malaria:

This type of malaria seen in P. Ovale, P. Vivax and P. Malariae.

LifeCycleof MalariaParasiteinHumanandMosquito
Clinical feature of benign malaria
(1) Series of febrile paroxysms –
Fever is caused by the release of merozoites & toxins from ruptured
erythrocytic schizont which in turn causes the release of cytokines
(interleukin-1 and tumour necrosis factor alfa).
Each febrile paroxysm has 3 stages –
Cold stage (rigors),
Hot stage (high temperature, body & joint pains, vomiting &
diarrhoea) and

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Perspiration stage (fall in temperature)
On the basis of periodicity, malaria further divided into two types.
Quartan malaria - every 72 hrs
Tertian malaria - every 48 hrs
(2) Anaemia
Malaria caused anaemia due to destroy of RBC by parasite.
Malarial pigments release from rupture cells.
(3) Splenomegaly
Marked hyperplasia of mononuclear phagocytic system throught the
body and massive destruction of RBC in spleen leading to
splenomegaly .
(B) Malignant malaria
It is caused by P. falciparum.
It is also known as pernicious malaria or cerebral malaria or malignant
tertian malaria or black water fever.
This occurs when more than 5 % RBC are infected.
In falciparum malaria, Erythrocytic schizogony occurs in the deeper
capillaries of internal organ.
Deformed parasite red cells become sticky and adhere to one another
and also on the capillaries endothelium.
Late stage schizont secretes protein of surface of red cells and
promotes aggregation to other noninfected RBC and endothelium
cells.
This causes obstruction of blood flow to organs and lead to necrosis of
affected organ.
Complication of falciparum
Due to obstruction of capillaries, following complications developed.
(1) Cerebral malaria
(2) Algid malaria
(3) Black water fever
Laboratory diagnosis of malaria
Q. Write short note on the Laboratory diagnosis? (B.Sc. Nur.-2007), (MBBS-
2008), (MBBS-2003)
Q. Write short note on the newer approaches of diagnosis of malaria?
(MBBS-2009)
Q. Write short note on the QBC?
Q. Write difference between different erythrocytic stages of P. vivax and P.
Falciparum? (MBBS-2004), (MBBS-2003)

ANSWER
In laboratory malaria parasite can be detected by following methods.

(I) Demonstration of parasites by

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(a) Thick and thin blood film

(b) Quantitative Buffy Coat (QBC) Method
(c) Micro concentration method

(II) Immunodiagnosis of parasite by

(a) Indirect fluorescent Antibody assay
(b) Indirect haemagglutination Assay
(c) ELISA
(d) Radio immune assay

(III) Newer Approaches of diagnosis

(a) Rapid dipstick test
(b) Parasite antigen and DNA detection method
(c)Fluorescent dye test

(I) Demonstration of parasites by

(a) Thick and thin blood film:

It is definitive and gold standard method for plasmodium detection.
Two types blood smear prepared for detection of parasite.
(1) Thick film smear:
It is preferred due to-
- 30 to 40 times more sensitive than thin films.
- More suitable for detection of malarial parasite when they are few in
number.
- Blood is not fixed, RBCs are lysed during staining (only parasitic
forms will be seen).
(2) Thin film smear:
It is preferred due to-
- to confirm the Plasmodium species.
- assists in the identification of mixed infections
- blood is fixed, parasites are seen within the RBCs
- also helps in assessing the response to treatment especially in areas
where drug resistance is suspected (by counting the number of
parasitised RBCs before & after the treatment).
Staining:
 Thick and thin smear prepared on same slide or separated slide.
 Place 2-3 drops of blood separately on the slide for each smear.
 Thin smear prepared by spreading blood drops with another slide.
 Thick smear prepared by stirring blood drops to prevent formation of
fibrin.
 Thin smear is fixed with absolute alcohol for 1 to 2 mins and stained
with Romanowsky stain or giemsa stain or field’s stain or wright’s
stain.

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 Stained smear examined for different morphological forms of parasite

in blood smear under microscope.
S e e C o l o r Im a g e 3 3 & 3 4

(MBBS-2004), (MBBS-2003)
Following findings are seen in microscope for different stags in P. Vivax and
P. Falciparum
Trophozoite/ Signet Ring form

Character P. Vivax P. Falciparum

Size 1/3rd of RBC 1/5th of RBC
Cytoplasm Thick opposite to Uniform thickness
nucleus
Nucleus One per ring More than one per ring
Number of ring in RBC One More than one
Location in RBC Always inside Inside as well as on the
surface (Accole’s form)
Type of RBC infected Mainly young RBC & All types
Reticulocytes

Ring form of P. Vivax and P. Falcipaum

Schizont
Character P. Vivax P. Falciparum
Size of RBC Increase in size Does not change
No. of merozoites 16 8 to 32
Arrangement of In rosette Asymmetrical
merozoites
Presence in peripheral Present Absent
blood

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Gamatocyte form of P. Vivax and P. Falcipaum

Character P. Vivax P. Falciparum

Shape- Male Spherical Cresentic
Female Spherical Sausage shaped
Nucleus- Male Central, Diffuse Central, Diffuse
Female Peripheral, Small Central, Compact
Infected RBC Enlarged Deformed with
membrane stretched

P. Vivax P. Fa lc ipa ru m

Malarial pigment produce in various species.

P.Vivax: Numerous fine golden brown dusts like particle
P. Falcifarum Scanty (1-3) solid blocks of black pigment
P.Malariae Numerous Coarse dark brown particle
P.Ovale Blackish brown particle

(b)Quantitative Buffy Coat (QBC) Method

 It is one another method for detection of malaria parasites.
 It is use to concentrate malaria parasite.
 A specially prepared and precoated with acridine orange stain &
potassium oxate enzyme in haemotocrit QBC tube used.
 56-65 µl of blood collected from the finger or ear lobe into QBC tube
and centrifuged at 12000 rpm for 5 min.
 Buffy coat layer is formed between the RBCs & the plasma.

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 Parasitic RBC detected by acridine orange stain and appears as bright
specs of light among non-fluorescing RBC.
(c) Micro concentration method
Blood collected in QBC tube and centrifuged it.
Break the tube & transfer buffy coat & RBCs to a slide, make a thin smear, fix
with ethanol and stain with Giemsa.

(II) Immunodiagnosis
Immunodiagnosis can not match the sensitivity of microscopic detection.
It may only assist diagnosis.
It may not differentiate between active and past infection.
Following Immunodiagnostic test are used.
(a) Indirect fluorescent Antibody assay
(b) Indirect haemagglutination Assay
(c) ELISA
(d) Radio immune assay

(III) Newer Approaches of diagnosis

(a) Rapid dipstick test

It is developed to diagnose falciparum malaria rapidly & without a
microscope.
It can also detect vivax malaria.
Two types of tests are available commercially which detects either
HRP2 Ag (Histidine rich protein-2) or specific pLDH (parasite lactate
dehydrogenase).
Both HRP2 & pLDH are produced by the parasites during their growth
& differentiation in RBCs.

HRP-2 test is based on the detection of a monoclonal antibody against

P.Falciparum histidine Rich Protein 2 Antigen.
It is very useful in the diagnosis of drug resistant P. falciparum.
pLDH test is used to detection of P.falciparum & P.vivax.
pLDH is produced by all human malarial parasites.

(b) PCR (Polymerase Chain Reaction)

Parasite antigen and DNA can be detected by PCR (Polymerase Chain
Reaction).

(c) Fluorescent dye test:

Benzothiocarboxypurine dye stain nucleic acid of malaria parasite but
dye does not staion Nuclei of WBC.
It is useful for mass screening because of rapid staining and
evaluation.

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Life cycle, pathogenesis and laboratory diagnosis are same in all plasmodium
species.

Apart of these, some special characteristic also present in various species.

P .vivax / benign tertian malaria/ Relapse

Q. Write short note on the Vivax?
Q. Write short note on the Benign Malaria?

ANSWER
Specific characteristics of P. Vivax.
 P. vivax invades on reticulocyte and young RBC.
 All stage take place in peripheral blood.
 Degree of parasitism more than 1-2% of total RBC.
 RBC size enlarged and show stippling called schuffner`s dots.
 1/3rd of RBC size single signet ring is present in RBC.
 It is caused relapse.
Relapse
 Relapse is reactivation of hypnozoite lead to initiation of fresh
erythrocytic schizogony and new attacks of malarial fever.
 It will take 24 wks to 5 yrs after primary attack.
 Hypnozoite is a dormant stage in the hepatocyte.
 Relapse is seen in P. vivax and P. Ovale.

P.falciparum or Malignant tertian or

Pernicious malaria or Celebral malaria
Q. Write short note on the Falciparum?
Q. Write short note on the Black water fever?
Q. Write short note on the Malignant malaria?
Q. Write short note on the Celebral malaria?

ANSWER
Specific characteristics of P. falciparum

 Falciparum mean sickle shape gametocyte.

 Single cycle of Pre erythrocytic schizogony is occur in P. falciparum.
 No hypnozoite stage and relapse seen in in P. falciparum.

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 Erythrocytic schizogony is occur in capillaries of internal organs like
spleen, liver, bone marrow
 Ring and gametocyte stages are seen in PBF.
 More than one parasite( 2-6) invade on all stages of RBC.
 RBC consist more than 1 signet ring and bi-lobed nuclei in RBC.
 Size of signet ring is 1/6th or 1/5th of RBC.
 Accole or appliqué form is present in P.falciparum.
 In Accole or appliqué form, Signet ring form are often seen attached
along the margin or the edge of the RBC.
 Size of RBC is normal.
 Maurer`s dots or cleft (stain brick red) found.
 Basophilic stippling seen.
 Pernicious manifestation anticipated when >5% RBC are parasitized.
 P. falciparum is responsible for recrudescence.

Recrudescence
Q. Write short note on the Recrudescence?

ANSWER
 The occurrence of clinical malaria caused by persisting erythrocytic
form in the circulation at a subclinical level following a previous attack
is called recrudescdence.
 Erythrocytic schizont is reactived and start erythrocytic schizogony.
 All species may cause but mostly in P.Falciparum and malariae
Causes of recrudescence are
1 Waning immunity of the host e.g. pregnancy
2 Antigenic variation in the parasite
3 In adequate drug therapy
4 Drug resistance
It occur upto 25-30 years.
Black water fever malaria
Q. Write short note on the Black Watre Fever?

ANSWER
It is caused by Plasmodium falciparum.
It is associated with repeated malaria infection and inadequate quinine
therapy.
Attributed to intravenous haemolysis with rapid destruction of RBC caused
haemoglobulinuria and haemoglobulinaemia by antierythrocytic
autoantibody.

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G6PD deficient may also develop this condition.

Clinical manifestations in Black water malaria.
(1) Bilious vomiting and prostration
(2) Dark red or blackish urine
(3) Urine acidic
(4) High methaemoglobulin content

Trypanosomes
Q. Write short note on trypanosomes?
Q. Write short note on the sleeping sickness?
Q. Write short note on the chagas disease?(MBBS-2002)

ANSWER
Trypanosomes are haemoflagellate protozoan.
They are move with help of flagella in blood therefore they known as
haemoflagellate.

Two species of Trypanosomes are pathogenic.

Species Disease Vector
(1) Trypnosoma brucei Sleeping sickness Tse Tse fly
(2) Trypanosoma cruzi Chagas disease Reduviid bug

Trypnosoma brucei
It is habitat and multiplies in connective tissues.
It invades regional lymph node & blood stream and localized in the brain.
Morphology
It is elongated, flattened spindle shaped organism.
It consist nucleus, kinetoplast and flagella.
It consist two forms trypomastigote and epimastigote.
Life cycle:
It passes life in two types of hosts.
Definitive host: Man
Intermediate host: Tse Tse fly (glossina).
 Infective stage is metacyclic stage of trypomastigote.
 Mode of transmission of infection is inoculation of infective stage into
the skin due to biting by tse tse fly during blood meal.
 Infective stage transform into the trypomastigote stage in blood.
 Trypomastigote stage is taken by fly during blood meal and converts
into the epimastigotes which further developed into metacyclic stage
of trypomastigote.

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Clinical feature
It is develop a hard, painful, nodular trypomastigote chancre.
Chancre fluid contains active dividing tryponosomal chancre.
Fever, headache, loss of nocturnal sleep and feeling of oppression and
other clinical features present in trypanosomasis.
Laboratory diagnosis
Specimen
(1) Peripheral blood
(2) Bone marrow by sternum puncture
(3) Juice aspirated from the swollen lymph node
(4) CSF by lumber puncture
Microscopy:
Parasite can be demonstrated in the thick and thin film after staining
with field’s or giemsa stain.
Parasite also demonstrates in wet mount preparation due to their
active motility.
Serological reaction:
Various serological methods like IFA, ELISA, IHA and Card
agglutination test are useful for detection of parasite.
Treatment:
Suramin and pentamidine are drug of choice.

Trypanosoma cruzi
It is habitat and multiplies in muscular, nervous tissue and Reticulo-
endothelium system.
Morphology
It consist two forms trypomastigote and amastigote.
Trypomastigote form:
It is elongated, flattened and spindle shaped organism.
It is consist nucleus, kinetoplast and flagella.
S e e C o l o r Im a g e 3 7

Amastigote form:
It is round or oval bodies.
It consist nucleus and kinetoplast.
No flagella present.
Life cycle:
It passes life in two types of hosts.
Definitive host: Man
Intermediate host: Reduviid bug
Development in reduviid bug:
Trypanomastigote is taken by bug which transform into amastigote
form by binary fission.

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Amastigote transform into epimastigote which further developed into

metacyclic form of trypanomastigote.

Metacyclic form of trypanomastigote

Development in human:
Metacyclic form of trypanomastigote is infective stage.
Man is infected by faecal matter of bug through break skin or mucosa.
Trypanomastigote invade tissue and transform into amastigote form
by binary fission.
Amastigote further transform into promastigote, epimastogote and
trypanomastigote.
Clinical feature
 Portal of entry produce swelling.
 In skin, it is known as Chagoma.
 In conjunctivae, it is known as Roman’s sign.
 Incubation period is 7-14 days.
 It is caused chagas disease.
Chagas disease characterized by
 Fever
 Conjunctivitis
 Unilateral oedema of face
 Spleenomagaly and lymphadenopathy
 Anaemia
 Meningoencephalitis
 Heart block
 Spastic paralysis
 Psychological change
 Magacolon and maga oesophagus
 Cardiomagaly.

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Laboratory diagnosis
Specimen
(1) Peripheral blood
(2) Bone marrow by sternum puncture
(3) Juice aspirated from the swollen lymph node
(4) CSF by lumber puncture
Microscopy:
Parasite can be demonstrated in the thick and thin film after staining
with field’s or giemsa stain.
Parasite also demonstrates in wet mount preparation due to their
active motility.
Serological reaction:
Various serological method like IFA, ELISA , IHA and Card agglutination
test are useful for detection of parasite.
Treatment
Nifurtimox is the drug of choice.

Leishmaniasis
Q. Write short note on the leishmaniasis? (B.Sc. Nur.-2008), (MBBS-2004),
(MBBS-2003)
Q. Write short note on the Kala azar? (MBBS-2010)

ANSWER
Leishmaniasis is a group of protozoal diseases caused by parasite of the genus
leishmania and transmitted to man by the bite of female phlebotomine
sandfly.
They are responsible for various type of syndrome in human.
(1) Visceral leishmanisis or Kala azar: Leishmania donovani
(2) Post kala azar dermal leishmanisis: Leishmania donovani
(3) Old world cuteneous leishmanisis: L. tropica , L. Major, L.aethipica
or oriental sore
(4) New world cuteneous leishmaniasis: L. maxicana, L. branzelensis

Leishmania is a intracellular parasite.

They infect and divide within macrophages.

Life cycle:
It is complete their life cycle in two hosts in two different stages.
Definitive host: Man (Amastigote stage)
Intermediate host: Sand fly (Promastigote stage)
Development in the sand fly
(1) Female sand fly ingests intracellular and free amastigote from

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infected person.
(2) Amastigote convert into promastigote within 72 hrs and
promastigote migrate and deposit into salivary gland.
Development in the human
(1) Promastigote (infective stage) enters into blood circulation after
bite of sand fly and converts into amastigote stage.
(2) Amastigote engulf by macrophages. They are multiple by binary
fission.
(3) Amastigote again taken by sand fly.

Clinical feature:
 The most common symptoms of visceral leishmaniasis are a
prolonged undulant fever, weight loss, decreased appetite, signs of
anemia, and abdominal distension with splenomegaly and
hepatomegaly.
 Thrombocytopenia may cause bleeding tendencies, including
petechiae or hemorrhages on the mucous membranes, and
leukopenia can result in increased susceptibility to other infections.
 Other symptoms may include coughing, chronic diarrhea, darkening of
the skin, lymphadenopathy, and in many cases, signs of chronic kidney
disease.
 Post-kala azar dermal leishmaniasis (PKDL) occurs after recovery in
some cases of visceral leishmaniasis caused by L. donovani.
 PKDL is characterized by hypopigmented patches, erythrmatous
patches and yellowish pink nodule around the mouth.

Laboratory diagnosis (MBBS-2003)

Leishmaniasis can be diagnosed by
(1) Direct observation of the parasites in peripheral blood smear, bone
marrow, spleen aspirate, skin scrapings, impression smears or skin
biopsies stained with Giemsa, Leishman’s, Wright’s or other stains.
Amastigotes are easiest to find in recent or active lesions.
S e e C o l o r Im a g e 3 5 & 3 6

Amastigote stage of leishmania seen in within macrophages

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(2) Leishmania spp. can also be cultured in Novy-MacNeil-Nicole (NNN)
medium, brain–heart infusion (BHI) medium.
Promastigote stage is grown in culture.

Promastigote stage of leishmania

(3) Serology can also be helpful in this form of leishmaniasis.

Common serological tests used are
(a) Immunofluorescent antibody test (IFA),
(b) Direct agglutination,
(c) Enzyme-linked immunosorbent assay (ELISA),
(d) Fast agglutination-screening test (FAST), and
(e) Rapid immunochromatographic assay (K39 dipstick or strip-test).
(4) The species, subspecies and/or strain can be identified by PCR, DNA
hybridization techniques.
(5) Animal inoculation into hamsters may also be valuable.
(6) A delayed hypersensitivity test, the leishmanin skin test (Mon-tenegro
skin test), is useful in the diagnosis of cutaneous and mucocutaneous
leishmaniasis.
Treatment
The standard treatment for kala azar is Pentavalent antimonial sodium
stibogluconate.

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44
C E S T O D E S

Taenia Solium
Taenia Saginata
Echinococcus

Taenia Saginata
Q. Write the short note on the tapeworm?
Q. Write the short note on the taenia solium? (B.Sc. Nur.-2008)
Q. Write the short note on the taenia saginata? (B.Sc. Nur.-2008)

ANSWER
Taenia saginata is also known as a Beef tapeworm or Asian taenia or the
unarmed tapeworm of man.
Man is the definitive host.
The ingestion of cysticercus cyst (larvae) in raw or undercooked meat of cattle
is the source of infection.
Adult parasite lives in the small intestine (upper jejunum) of man attached to
the mucosa by scolex.
Life span is above 10 years.

MORPHOLOGY
Adult worm consist Head (scolex), Neck, Strobila (trunk) and Proglottids
(segments).
Eggs: Each gravid segment contains about 80,000 eggs.

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Infected person may be discharging more than 500,000 eggs per day in the
environment.
The characteristic features of the egg are as follows:
(i) Spherical and brown in colour (bilestained).
(ii) A thick brown radially striated wall (embryophore) surrounds the embryo.
(iii) Within the egg there is an embryo with 3 pairs of hooklets, the
oncosphere.
S e e C o l o r Im a g e 3 8

Eggof
Taeniasaginata

LIFE CYCLE
T. saginata passes its life in two hosts:
Definitive host: Man, harbours adult worm.
Intermediate host: Cattle (cow or buffalo), harbours larval stage.
Life cycle of T. saginata and T. solium
Developmental stages of T. saginata / T. solium are as follow:
(1) Cysticercus reach in the small intestine and attaches to the mucosal
surface by its four suckers when eat raw or undercooked beef / pork
containing larval stage.
(2) Sexually mature male and female cross fertilize and starts producing egg.
(3) Egg passed in faeces along gravid segment.
(4) Cow or buffalo or pig infected by ingesting the eggs with segments.
(5) The oncospheres are liberated from eggs in their alimentary canal, pass
into a blood vessel and muscles.
(6) Common sites affected are the skeletal muscles of tongue, neck, shoulder
and hand.
(7) In muscle tissues, the embryos grow and develop into infective cystic
larvae called cysticerci.

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Clinical feature
Adult tapeworms may cause nausea, abdominal discomfort, hunger pain,
chronic indigestion and diarrhoea alternating with constipation.
The larva of T. saginata (cysticercus) does not occur in man.

DIAGNOSIS
Direct evidences
Laboratory confirmation of diagnosis of T. saginata infection is made
by demonstration of gravid segments and eggs passed in faeces by
microscopic examination of stool.

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Serology
Serodiagnosis by IHA, ELISA and IFA have been used for detection of T.
seginata.

TREATMENT
Praziquantel in a single dose is the drug of choice for treating taenia
infections.
Niclosamide is also highly effective.
PREVENTION
1. Proper hygienic disposal of human faeces.
2. Education regarding proper cooking of beef.
3. Adequate inspection of beef for cysticerci in slaughter house.

Taenia Solium
Q. Describe the life cycle, pathogenesis and laboratory diagmosis of taenia
solium?
Q. Write the short note on the taenia solium? (B.Sc. Nur.-2008)
Q. Write the short note on the cysticercosis? (MBBS-2009), (MBBS-2004),
(MBBS-2003)
Q. Write the short note on the Neurocysticercosis? (MBBS-2002)

ANSWER
Taenia solium is also known as a Pork tapeworm or armed tapeworm of man.
Humans are definitive host for T. solium.
Human infection occurs following ingestion of undercooked pork containing
cysticercus (called cysticercus cellulosae).
Modes of infection are oral-faeco route and auto inoculation.

MORPHOLOGY
Adult worm:
Adult worm lives in the small intestine (upper jejunum) of man.
Commonly a single worm is present.
Adult worm consist Head (scolex), Neck, Strobila (trunk) and Proglottids
(segments).
Eggs:
Each gravid segment contains about 80,000 eggs.
Infected person may be discharging more than 500,000 eggs per day in the
environment.
The characteristic features of the egg are as follows:
(i) Spherical and brown in colour (bilestained).
(ii) A thick brown radially striated wall (embryophore) surrounds the embryo.
(iii) Within the egg there is a embryo with 3 pairs of hooklets, the oncosphere.

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Eggof
Taeniasolium

Human infection occurs following ingestion of undercooked pork containing

cysticercus (called cysticercus cellulosae)
CLINICAL SYNDROMES
A. lntestinal taeniasis:
Tapeworm infection cause abdominal pain with intestinal
disturbances.
B. Cysticercosis:
Common sites affected by cysticercus cellulosae include subcutaneous
tissue, skeletal muscle and brain.
It may also affect eyes, brain, heart, liver, lungs, spinal cord and
abdominal cavity.
This is called human cysticercosis.
C. Neurocysticercosis:
The presence of cysticerci in brain represents one of the most
frequent parasitic infections of human nervous system.
It is the most common cause of adult onset epilepsy.

DIAGNOSIS

1. Intestinal taeniasis:
Laboratory confirmation of diagnosis is made by
(a) Identifying gravid segments passed in faeces and
(b) Detection of eggs in faeces.
2. Human cysticercosis:
(a) Calcified cyst in muscles can be recognised radiologically and by
biopsy.
(b) CT scans or MRI techniques may reveal the presence of cysticerci
in the brain.

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(c) Fine needle aspiration (FNA) cytology is useful and cost-effective
since it eliminates the biopsy.
(d) Serologic tests: detect antibody in blood.

TREATMENT
Adult worm
Praziquantel in a single dose is the drug of choice for treating taenia
infections.
Niclosamide is also highly effective.
Cysticercosis
1. Remove by surgical procedure.
2. Two drugs, praziquantel and alberdazole are effective against the
Cysticercosis.

PREVENTION

1. Proper hygienic disposal of human faeces.

2. Education regarding proper cooking of pork.
3. Adequate inspection of pork for cysticerci in slaughter house.
4. Vaccination of pigs against cysticercosis.

Cysticercosis
Q. Write short note on the cysticercosis?

ANSWER
It is cause by taenia solium by ingestion of undercooked pork containing
cysticercus (called cysticercus cellulosae).

Cysticercus of Taenia Solium

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Modes of infection are oral-faeco route and auto inoculation.

Common sites affected by cysticercus cellulosae include subcutaneous tissue,
skeletal muscle and brain.
It may also affect eyes, brain, heart, liver, lungs, spinal cord and abdominal
cavity.
This is called human cysticercosis.

Neurocysticercosis :
The presence of cysticerci in brain represents one of the most frequent
parasitic infections of human nervous system and is the most common cause
of adult onset epilepsy.
Most common symptoms of cysticercosis are epileptiform seizures abnormal
behaviour, transient paresis, meningo encephalitis and visual problems.

Echinococcus
Q. Write short note on the Echinococcus? (MBBS-2006)
Q. Write short note on the Hydatid cyst? (MBBS-2004)
Q. Write short note on the casoni test? (MBBS-2007), (MBBS-2002)

ANSWER
Echinococcosis, which is often referred to as hydatid disease or echinococcal
disease, is a parasitic disease that affects both humans and other mammals,
such as sheep, dogs, rodents and horses

Morphology

Adult worm
Echinococcus adult worms develop from protoscolices and have a scolex, neck
and typically three proglottids, one of which is immature, another of which is
mature and the third of which is gravid (or containing eggs).
The scolex of the adult worm contains four suckers and a rostellum that has
about 25-50 hooks.

Egg
Echinococcus eggs contain an embryo that is called an oncosphere or
hexcanth.
The name of this embryo stems from the fact that these embryos have six
hooklets.
The eggs are passed through the faeces of the definitive host and it is the
ingestion of these eggs that lead to infection in the intermediate host.

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Larval/hydatid cyst stage
The embryo released from an egg develops a hydatid cyst.
Cysts can contain several liters of fluid.
The cyst is lined by a multilayer parasite tissue with the innermost layer being
the germinal layer.
This layer is an undifferentiated “stem cell” layer that can spawn the
formation of “brood capsules” which are themselves lined by Germinal Layer.
The daughter cysts (the encircled body) "bud" into the center of the fluid-filled
cyst.
This is a very small portion of the cyst which may become quite large.
Each of the smaller bodies will develop into diminutive tapeworms should this
be eaten by a definitive or final host such as a canine.
Thousands of protoscolices can fill the hydatid (hydatide sand).
Protoscolices are the infective stage for dogs.
Hydatides usually grow slowly but steadily (1-5 cm per year).
They are usually well tolerated until their size becomes a problem or they
rupture.
Cyst rupture or leakage can result in allergic reactions and metastasis.

Incubation period
Incubation period may be month to year or even decades.

Transmission
Echinococcus is transmitted to intermediate hosts via the ingestion of eggs
and are transmitted to definitive hosts by means of eating infected, cyst-
containing organs.

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Humans are accidental intermediate hosts that become infected by handling

soil, dirt or animal hair that contains eggs.

Host
Definitive hosts are normally carnivores such as dogs.
Intermediate hosts are usually herbivores such as sheep and cattle.
Humans function as accidental intermediate hosts, because they are usually a
'dead end' for the parasitic infection cycle.

Life Cycle
(1) An adult worm resides in the small intestine of a definitive host.
(2) Afterwards, gravid proglottids release eggs that are passed in the feces of
the definitive host. The egg is then ingested by an intermediate host.

(3) The egg then hatches in the small intestine of the intermediate host and
releases an oncosphere that penetrates the intestinal wall and moves

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through the circulatory system into different organs, in particular the liver
and lungs. Once it has invaded these organs, the oncosphere develops
into cyst.
(4) The cyst then slowly enlarges, creating protoscolices and daughter cysts
within the cyst.
(5) The definitive host then becomes infected after ingesting the cyst-
containing organs of the infected intermediate host.
(6) After ingestion, the protoscolices attach to the intestine.
(7) They then develop into adult worms and the cycle starts all over again.

Symptoms
The symptoms depend upon the location of the cyst.
Large abdominal cysts produce increasing discomfort.
Liver cysts cause obstructive jaundice.
Peribronchial cysts may produce pulmonary abscesses.
Brain cysts produce intracranial pressure and Jacksonian epilepsy.
Kidney cysts cause renal dysfunction.
The contents of a cyst may produce anaphylactic responses.

Diagnosis
Clinical symptoms of a slow-growing tumor accompanied by eosinophilia are
suggestive.
Casoni test: (MBBS-2007)
It is intradermal test.
It is preformed to detect echinococcus.
It is an immediate hypersensitivity test.
Hydatid fluid inoculated intradermally.
Intradermal (Casoni) test with hydatid fluid is useful.
Pulmonary cysts and calcified cysts can be visualized using x-rays, CT
Scan & MRI.
Antibodies against hydatid fluid antigens have been detected in a
sizable population of infected individuals by ELISA or indirect
hemagglutination test.

Treatment and control

Treatment involves surgical removal of cyst or inactivation of hydatid sand by
injecting the cyst with 10% formalin and its removal within few (4-5) minutes.
Prazequantel has been shown to be effective in many cases.
Albendazole, in high doses, is an alternative.
Preventive measures involve avoiding contact with infected dogs and cats and
elimination of their infection.

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45
T R E M A T O D E

Fasciola
Schistosoma
Paragonimus

Fasciola Heaptica
Q. Write the short note on the fasciola Hepatica? (MBBS-2004)
Q. Describe the life cycle of the fasciola Hepatica?

ANSWER
Fasciola hepatica known as sheep liver fluke.
The parasite produces severe parenchymal disease of liver and the disease is
known as "liver rot".
The adult fasciola hepatica live for 9 years in the bile ducts of the liver of
sheep, goats, man and cattle, produce eggs that are carried by bile into the
duodenum and then comes out with the faeces.
Adult lays approximately 20,000 eggs/day.
Egg is operculated and unembryonated (contains a big unsegmented ovum).
Transmission occurs by the ingestion of metacerceriae encysted on aquatic
vegetation, such as watercress in salads.

F. hepatica requires two hosts to complete its life cycle.

Definitive hosts: Herbivores like sheep, goat, cattle and man. The adult worm
lives in the biliary passage.
Intermediate hosts: Snails.

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Developmental stages in F.haptica are as follow:

(1) The egg developed into a miracidium in water & penetrates the tissues of
intermediate host(snails).
(2) In the snail,the miracidium metamorphoses into sporecyst, the 1st and
2nd generation redia and become the cercariae in about 1-2 months.
(3) Metacercariae encyst on aquatic vegetation, ingestion of which leads to
infection in herbivorous hosts.
(4) The metacercariae excyst in the duodenum, enter into the peritoneal
cavity and reach the biliary passages.
Humans usually acquire infection by eating contaminated wild watercress.

CLINICAL SYNDROME
Acute infection is characterised by nausea, vomiting, pain, chills and fever
and marked eosinophilia are commonly observed.
It leads to inflammatory, oedematous and fibrotic changes in the bile ducts.

Laboratory diagnosis
Operculated eggs seen in faeces or aspirates of duodenal fluid in stool
samples by microscope in wet mount preparation.

Treatment of choice is praziquantel.

Paragonimus Westermani
Q. Write the short note on the paragonimus?
Q. Describe the life cycle of the paragonimus?

ANSWER
It is also called oriental lung fluke.
Human infections occur from ingestion of flesh or juice of insufficiently
cooked, or pickled crab or cray fish that contains Paragonimus metacercariae.
Paragonimus spp. requires three hosts to complete their life cycle.
Definitive host: Man and carnivorous animals (pigs, dog, cat, tiger, leopard).
First intermediate host: Fresh water snail.
Second intermediate host: Fresh water crab or cray fish.
Eggs is oval in shape and possesses a flat operculum (lid).
Eggs has unsegmented ovum with mass of yolk cells.
Developmental steps of paragonimus are as follow:
(1) Adult flukes live in the lung parenchyma of the definitive hosts.
(2) The eggs escape & reach the bronchioles.
(3) Sputum swallowed and egg passed in faeces.

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(4) The eggs hatch in water and releasing ciliated miracidium.

(5) Miracidia undergo successive developments - sporocysts, two generation
of rediae, finally to cercariae in the snail.

L ife c y c le o f p a ra g o n im u s

(6) The mature free-swimming cercariae penetrate in the gills, muscles and
viscera of second intermediate host, crabs or cray fish, and encyst and
become metacercariae.
(7) Metacercariae hatch in the duodenum of man after ingestion of infected
crab or cray fish raw or undercooked.
(8) Young worms penetrate the gut wall, and reach the peritoneal cavity, the
thoracic cavity and into the lung and bronchi.

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(9) The eggs are then discharged into a bronchiole to be expectorated with
sputum.

PATHOGENESIS
1. Larval effect:
During migratory phase produce localised haemorrhage and
leucocytic infiltration.
2. Effects due to adult flukes:
The flukes cause an inflammatory reaction in the lung that results in
cough, fever and increased sputum.
3. Effects due to eggs:
The eggs may evoke a foreign body granulomatous reaction.
Incubation period is 2 to 30 days.

LABORATORY DIAGNOSIS
Specimens:
Paragonimus eggs can be detected in sputum, faeces and occasionally
found in aspirated pleural fluid.
Direct microscopy:
Oval shape, unsegmented ovum with mass of yolk cells egg seen in
sputum.
The eggs possess prominent opercula.
Egg can also detect in faeces.
Serological tests
Parasitic-specific IgG and IgE anti-paragonimus antibodies in patient's
serum can be detected by ELISA using adult excretory secretory
antigens.

TREATMENT AND PREVENTION

1. Praziquantel (25 mg TDS for 1-2 days) is the drug of choice.
2. Education on adverse effects of consumption of undercooked crabs and
cray fish.
3. Proper sanitation and control of disposal of human faeces.

Schistosomasis or Birharziasis
Q. Write the short note on the schistosomiasis? (MBBS-2002)
Q. Describe the life cycle of the Schistosoma haematobium?

ANSWER
The three schistosomes most frequently associated with human disease.
They are S. haematobium, S. mansoni and S. Japonicum.

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Schistosoma Haematobium
ANSWER
It is also known as vesical blood fluke.
Schistosoma haematobium is a causative agent of urinary schistosomiasis.
Adult worms live in the pelvic venous plexus (veins surrounding the bladder),
Adult gravid female contains 20 to 30 eggs in its uterus at anyone time and
may pass upto 300 eggs per day.
The body is covered by fine tuberculated cuticles.
The eggs are elongate with a brownish-yellow transparent shell carrying a
terminal spine at one pole.
It contains a fully developed miracidium.
Human infection is acquired by contact with fresh water (bathing, washing
clothes, agriculture purposes, fishing or recreation) contaminated by urine of
infected patients that contain egg of S.haematobium.

Elongated Schistosoma haematobium eggs s

with a prominent terminal spine

LIFE CYCLE
Definitive host: Man.
Intermediate host: Fresh water snails.
Reservoir hosts: Monkeys, baboons, chimpanzees.
Adult S. haematobium residing in vesical and pelvic venous plexus of definitive
host and eggs are discharged in urine.
Many of the eggs penetrate through the mucosa of bladder by the piercing
action of spine and passed into the lumen of bladder and then to urine.
S e e C o l o r Im a g e 3 9

Life cycle of Schistosoma

Development steps are as follow:
(1) Embryonated egg, when passed in the urine and readily hatch when
deposited in fresh water.
(2) The miracidium enters its intermediate host (snail).
(3) The miracidium loses its cilia and undergoes development and
multiplication stages of the first and second generation sporocysts.

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(4) Fork-tailed cercariae are produced by asexual reproduction within each
second generation sporocyst.

(5) Infective stage cercariae attached to the skin and enter the peripheral
venules through the subcutaneous tissues into blood vessels.
(6) The young flukes are carried through the vena cava into the right side of
the heart, the pulmonary circulation, the systemic circulation, ultimately
reached the liver.
(7) After mating of male and female fluke leave liver and reach into vesical
plexus.

PATHOGENICITY AND CLINICAL FEATURES

Pathogenic effect depends on the stages of schistosomes.
1. By cerceriae at the site of entrance: Skin rash (called swimmer's itch) and
Cercarial dermatitis is seen at the site of cercarial penetration.

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2. By toxic metabolites liberated by schistosomulae: Anaphylactic or toxic

symptoms appear.
3. By egg deposition and extrusion: (a) Haematuria and proteinuria
(b) Formation of granulomas
4. Long term complications: (i) Hydronephrosis
(ii) Bladder cancer.

LABORATORY DIAGNOSIS
(a) Microscopy:
Definitive diagnosis is made by detection of eggs in urine and seminal
fluid.
Centrifuged deposit of urine shows eggs with characteristic terminal
spine by wet mount.
(b) Biopsy:
Eggs may also be found in the wall of the rectum as well as in the
bladder wall.
(c)Serological tests:
ELISA, latex agglutination, complement fixation test perform to detect
antibodies in serum.
Plasma card test is now available for mass screening.
(d) Blood cell count:
Eosinophilia is a common finding in Blood cell count.

TREATMENT
Praziquantel is the drug of choice.
Praziquantel, metrifonate and neridazole are equally effective in a
single dose in all schistosomiasis.

CONTROL
Long term control can be achieved by improvement of sanitation and
patient education.

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46
N E M A T O D E

Ascaris
Ankylostoma
Larva Migrans
Wucheria
Guinea Worm

Ascaris Lumbricoides
Q. Write short note on the life cycle of Ascaris lumbricoides?
Q. Write short note on the on the life cycle of round worm?
Q. Describe the life cycle and laboratory diagnosis of ascaris?
Q. Write down difference between Fertilised egg & Unfertilised egg of
ascaris? (BDS-2007)

ANSWER
The common name of ascaris lumbricoides is Roundworm.
It is the largest nematode parasite of human intestine.
The adult worms live in the lumen of small intestine (about 85% in jejunum,
15% in ileum).

MORPHOLOGY
A. Adult worm :
Females are larger than the male.
The adult is rounded in shape.
The body is filled with an irritating fluid called ascarion or ascarase,
can cause allergic manifestation.
The life span of adult is 1-2 year.

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The tail (posterior) end of male is curved ventrally in the form of hook
having a conical tip, a property that distinguishes males from females,
which have straight tails.
One gravid of female round worm may contain about 27 million ova
and liberates approximately 2,00,000 eggs daily.

B. Eggs:
There are two types of eggs liberated by the female:
Fertilised and unfertilized eggs.
S e e C o l o r Im a g e 4 1

(See difference between fertilized and unfertilized egg in chapter Difference)

LIFE CYCLE
Man is the only definitive host of the worm.
Infection occurs by ingestion of infective embryonated eggs in contaminated
food, drink or raw vegetables or from faecally contaminated hands.

The various stages of the parasite in its life cycle are as follows:
Fertilized eggs are passed with faeces. A rhabditiform larva is developed from
the unsegmented ovum within the egg shell in 10-14 days time in soil.
Four moulding occur in human body.

Moulding Site Days

1st moulding Within egg shell ---
2nd moulding Lung On the 5th or 6th day,
3rd moulding Lung After the 10th day
4th moulding Intestine Between 25th to 29th day of
infection
Migration of ascaris
(1) Fertilized egg comes in faeces
(2) Moult in soil
(3) Embryonated egg comes in stomach & duodenum
(4) Rhabtidiform larva reaches in liver & lung
(5) 2nd and 3rd moult in lung
(6) Again come in stomach by cough sputum
(7) Adult development in intestine.

CLINICAL DISEASE
In mild infection, there is intestinal disorders, malnutrition and/or colicky
pain.
Effects due to migrating larvae
1. Larvae in the lungs cause transient pneumonitis & Loeffler's syndrome.
2. Hypersensitivity reaction.

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3. Larvae occluding a small blood vessel in heart, brain and kidney.
Effects due to adult worms
Incubation period is 60-70 days.
(1). Nausea and vomiting
(2). The worm derives nutrition (protein and vitamin content of the worm is
very high) by robbing the host of its nutrition causing malnutrition.
(3). Obstruction or perforation of the intestine and occasionally obstruction
of bile duct and pancreatic duct.
(4). Migrating worms may cause liver abscess and appendicitis

LABORATORY DIAGNOSIS
A. Direct evidences
1. Demonstration of adult worm in stool or vomitus.
2. Ascaris egg can be detected in stool by direct wet mount and also by
concentration method.
Usually both fertilized and unfertilized eggs are found.
Duodenal aspirate also shows eggs.
Ascaris larvae may be detected in sputum in loeffler's syndrome along with
eosinophils and charcot leyden crystals.
B. Indirect evidences.
1. Eosinophil count may be increased.
2. Skin test with A. lumbricoides antigen gives a positive result.
3. Serological test include ELISA, IHA and microprecipitation for antigen.

TREATMENT
1. Pyrantel pamoate and mebendazole are drugs of choice.
Other effective drugs include piperazine and albendazole.
2. Surgery may be necessary for intestinal obstruction.

CONTROL
1. Improvement of personal hygiene.
2. Proper treatment and disposal of sewage.
3. Filtering of drinking water.
4. Vegetables and hands should be washes before meals.

ANCYLOSTOMA DUODENALE
Q. Write the short note on the ankylostoma?
Q. Write the short note on life cycle of ankylostoma?
Q. Write the short note on the laboratory diagnosis?

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ANSWER
Morphology
It is also called hookworm because anterior end bent slightly dorsally.
It is possesses four teeth on its ventral surface and a pair of smaller teeth on
its dorsal surface.
Male have two copulatory spicules projecting from the bursa.
Female hind end is conoid with a subterminal anus situated ventrally.
Vulva opens ventrally at the junction of middle and posterior thirds of body.
The life span of adult is 2-7 years.

Hook worm larva

Egg
Each female hookworm can lay 15,000 to 20,000 eggs per day in faeces.
The egg is oval or elliptical in shape, Colourless (not bile stained), surrounded
by a thin translucent hyaline shell membrane, clear space between the egg
shell and segmented ovum, contains a segmented ovum usually with four
blastomeres and floats in the saturated solution of common salt.

S e e C o l o r Im a g e 4 2

LIFE CYCLE
Man is the definite host.
Third-stage, filariform larvae is infective stage.
Mode of infection is direct penetration of the skin, usually through feet.

Life cycle of Hook Worm

Development of ankylostoma
(1) Segmented ovum with four blastomeres passed in faeces.
(2) First-stage rhabditiform larva hatches out in about 48 hours.
(3) The rhabdtiform larva moults twice on 3rd day & 5th day and develop into
an infective third stage filariform larva.
(4) Penetrate the human skin exposed to contaminated soil.
(5) The larvae migrate into the subcutaneous tissue, lymphatics or small
Venules.
(6) Enter into pulmonary capillaries via the right heart.

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(7) The larvae migrate to the bronchi, trachea, larynx, epiglottis, pharynx and
then pass down the digestive tract.
(8) Third moulting leading to development of a fourth-stage larva.
(9) Fourth stage larva converts into adult stage.
Life spans of adult worms of both species range from 1-3 years.

CLINICAL SYNDROMES
Asymptomatic in individuals less than 5 eggs/mg of faeces.
Significant anaemia is seen in patients passing more than 20 eggs/mg of
faeces.
Effects due to migrating larva
1. An erythematous papular rash develops that becomes vesicular due to
severe itching at the site of penetration.
This condition is called "ground" itch or ancylostoma dermatitis.
2. Loeffler's syndrome

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Effects due to adult worms

1. Abdominal pain, nausea, vomiting, and diarrhoea with blood loss.
2. Chronic infection leads to iron deficiency anaemia, listlessness, pallor and
general retardation of development in affected children.
A. duodenale ingests about 0.15 ml to 0.25 ml blood per day.

LABORATORY DIAGNOSIS
The diagnosis of hookworm infection is made by demonstration of eggs in the
faeces by direct Microscopy or by concentration method.

I. Direct evidence
1. Demonstration of characteristic eggs in direct wet mount of faeces.
The rhabtidiform larva of hookworm has a long, narrow buccal
chamber and an inconspicuous genital operculum.
It is seen in prolonged stool sample.
2. Aspirate obtained from duodenum by duodenal intubation (Ryle's
tube) may sometimes contain eggs or adult hookworms.
3. Stool culture may be done using Harada-Mori filter paper strip
technique or charcoal culture method.

II. Indirect evidences

1. Occult blood (benzidine) test of stool is positive.
2. Anaemia - microcytic hypochromic.
3. Blood picture - Hb, PCV, MCV, MCHC all decreased.
4. Eosinophilia in blood.
5. Charcot Leyden crystals are found in stool.
TREATMENT
1. The drug of choice is mebendazole, pyrantel pamoate or albendazole.
2. Iron therapy & blood transfusion is indicated in anaemia.
CONTROL AND PREVENTION
1. Proper disposal and treatment of sewage.
2. Wearing shoes in endemic areas.
3. Education on personal hygiene.
LARVA MIGRANS
Q. Write the short note on the Larva migrans?
Q. Write the short note on the Cutaneous larva Migrans? (MBBS-2010)
(MBBS-2009), (MBBS-2007)
Q. Write the short note on the Visceral Larva Migrans? (MBBS-2002)

ANSWER
Animal nematodes larvae wander around, aimlessly in the body. This is known
as larva migrans.

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(a) Cutaneous larva migrans (CLM), also known as creeping eruption, when
larval migration occurs in the skin.
(b) Visceral larva migrans (VLM) when larval migration takes place in deep
tissues.

CUTANEOUS LARVA MIGRANS (CLM)

DEFINITION
CLM or Creeping eruption (also called ground itch) refers to the production of
serpigenous inflamed trails in the skin, resulting in intense pruritus, may be
caused by larvae or various nonhuman species of Ancylostoma, strongyloid,
Uncinaria stenocephata, Gnathostoma spinigerum & Bunostomum
phlebotomum.

PATHOGENESIS
CLM results from skin contact with filariform larvae present in animals
defecate.
They wander in the superficial layers of the skin, especially of the feet, legs,
thighs, buttocks and back causing serpigenous ulcers and allergic reactions.

CLINICAL FEATURES
The larva develops a serpigenous tunnel in the epidermis.

DIAGNOSIS
No larva is found in the biopsy. There is no eosinophilia and immunological
test available.

TREATMENT
1. Oral and topical treatment of thiobendazole is effective.
2. Freezing the advancing edge of creel eruption with ethyl chloride is
effective.

VISCERAL LARVA MIGRANS (VLM)

DEFINITION
Visceral larva migrans (VLM) is a syndrome caused by nonhuman parasitic
larva in the viscera of the host for months and year.

AETIOLOGY
1. Commonest causative agents:
(i) Toxocara canis (dog ascarid),
(ii) Toxocara catis (cat ascarid)

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2. Other worms include:

(i) Angiostrongylus cantonensis.
(ii) A. costericensis,
(iii) Anisakis spp.,
(iv) Gnathostoma spinigerum.
3. Human nematodes :
(i) Ascaris lumbricoides
(ii) Strongyloides stercoralis may produce VLM in auto and heavy
infections.
The children are exposed more readily to egg contaminated soil who has a
tendency to put objects in their mouths.

PATHOGENESIS
Human infection occurs by ingestion of infective eggs.
Larvae hatch in the small intestine and penetrate the gut wall, and are carried
in the blood to lungs, eyes and/or other parts of the body.
This is called visceral larva migrans.

CLINICAL FEATURES
It is most commonly seen in children of 1 to 4 years of age.
The most outstanding feature of the disease is a high peripheral eosinophilia.
Symptoms of VLM manifest as hepatomegaly, eosinophilia and serious retinal
lesions.

DIAGNOSIS
 Diagnosis of VLM is made on the basis of clinical findings and
immunodiagnostic procedures.
 Serology: ELISA employing excretory and secretory (ES) antigens of
2nd stage larvae of T. canis is highly sensitive and specific for diagnosis
of VLM of T. canis origin.
 Other findings include an outstanding blood eosinophilia and
increased gamaglobulin level.

TREATMENT
1. Symptomatic therapy.
2. Specific : Diethylcarbamazine 100 mg TDs for 3 weeks in an adult kills the
larva and arrest the disease.

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Wucheria Bancrofti
Q. Write the short note on the Wucheria Bancrofti? (MBBS-2003)
Q. Write the short note on the life cycle of Wucheria Bancrofti?
Q. Write the short note on the laboratory diagnosis of Wucheria Bancrofti?
(MBBS-2003)
Q. Write the short note on the filariasis? (MBBS-2009), (MBBS-2006), (MBBS-
2005)
Q. Write the short note on the elephantiasis? (MBBS-2003)

ANSWER
W bancrofti causes bancroftian filariasis, elephantiasis.
The adult worm lives in the lymph nodes and lymphatic vessels of human only.

MORPHOLOGY

Adult
They are long, hair-like, transparent and filariform in shape.
Both ends of the worm are tapered and the head end is slightly swollen with
two rows of ten papillae.
Males and females usually remain coiled together in abdominal and inguinal
lymphatics and in testicular tissues.

Embryo (microfilaria)

Passing through lymph nodes, the actively motile embryos find their way by
the main lymphatic trunks into circulatory blood.
The microfilaria does not undergo any further development in human body.
Further developments of the larvae occur in their appropriate intermediate
hosts. The life span of the microfilaria could be up to 70 days.

Life Cycle

Wucheria bancrofti completes its life cycle in two hosts.

The definitive host is man.
The intermediate host is female mosquito Culex fatigans.
Vector is culex fatigans.
Mode of infection is bites of mosquito.
Infective stage is 3rd stage filariform larvae.
The larva develops into lymphatic vessels and lymph nodes.

Definitive host
In man adult worms live in the lymphatic vessels and glands.

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Males and females remain coiled together & difficult to separate them.
The females lay embryos (microfilariae) which travel via the lymphatics to
blood stream without undergoing any developmental metamorphosis.

Nocturnal periodicity:
The microfilariae of W bencrotti, (periodic variant) take rest in the capillaries
of lungs, kidneys (glomerular tufts), heart and the big arteries (such as carotid
artery) during day time and invade peripheral circulation at night.
Man-biting mosquitoes are more active at night from 10 PM to 4 AM
(nocturnal periodicity).

S e e C o l o r Im a g e 4 0

Life cycle of Wuchereria

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Intermediate host
1. The sheathed microfilaria is ingested by mosquito during its blood meal.
2. In thoracic muscles, it moult and undergo development to 1st, 2nd and
then to 3rd stage (filariform) larvae.
This activeIy motile 3rd stage larva is infective form.
Microfilaria does not multiply in mosquito and only one microfilaria
develops into one infective larva only.
The infective larva is injected into man by mosquito during its blood meal
and the life cycle is repeated.
3. Larval development in the mosquito takes 11-14 days.

CLINICAL DISEASE
Lymphatic filariasis
(a) Early infection:
Patients experience fever, lymphangitis, headache, nausea and
urticaria.
(b) Chronic infection:
It cause lymphadenitis, lymphoedema and other complications.
Mechanical irritiation and absorption of toxic metabolites cause
lymphangitis and lymphadenitis.
Dilatation of lymphatics leads to Iymphangiovarix.
Other complications
(a) Lymphorrhagia (b) Chylorrhagia (c) Hydrocele.

Elephantiasis
It is a delayed sequel to repeated and progressive W bancrofti infection.
It is caused by:
(i) Mechanical blocking of the lumen or lymph vessels,
(ii) Obliterative endolymphangitis
(iii) Excessive fibrosis of lymphatic vessels,
(iv) Fibrosis of afferent lymph nodes.
Elephantiasis most commonly affects limbs, genital organs (especially of
spermatic cord) and breasts.
Elephantiasis in the leg

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LABORATORY DIAGNOSIS
Specimens for diagnosis are blood, chylous urine or hydrocele fluid, Iymp
node.
Collection time of blood is night between 22.00-04.00 hrs.

Method of diagnosis
(1) Detection of microfilaria in peripheral blood, Chylous urine &
Hydrocele fluid by microscopy.
(2) Demonstration of adult worm in biopsy specimen of lymph node by
microscopy.
(3) Detection of antigen & antibody by Immunodiagnosis.
(4) Indirect evidence by skin test & blood count (eosinophilia).

Microscopy:
Definitive diagnosis is made by detection of microfilariae in a thick
blood smear and chylous fluid stained by Leishman/Giemsa stain and
examined microscopically.

Microfilariae in Blood film

Wet-slide preparation:
Two drops of deposit is mixed with equal volume of water (to lyse the
red cells) on a slide.
The preparation is covered with a cover-slip and examined
microscopically using low power (10 X).
DEC provocation test: This is useful in mass screening.
Serological reaction:
Circulating Filarial Antigen can be detected with monoclonal
antibodies by serological reaction.
Specific antibodies against filarial antigen can be detected by
serological tests like ELISA & FAT.
Skin test:
Intradermal skin tests using filarial antigen show immediate type
hypersensitivity.
Blood count:
Increase eosinophil in Differential Leukocyte count is indirect evidence
for diagnosis.

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TREATMENT
1. Diethylcarbamazine (DEC) is the drug of choice.
2. Antibiotic soaps and topical creams are helpful in reducing elephantiasis.

PREVENTION AND CONTROL

1. Eradication of vector mosquito is the major measures of prevention.
2. Protection from bites of insect by mosquito net, insect repellant etc.

Dracunculus medinensis or Guinea worm

Q. Write the short note on the guinea worm?
Q. Describe the Life cycle and laboratory diagnosis of guinea worm?

ANSWER
The name Dracunculus medinensis means little Dragon of Medina.
Adult females reside in the subcutaneous tissue, especially of the legs, arms
and back.

Morphology:
Female worm is a slender, posterior end is blunt and bent to form a hook.
It is viviparous.
Life span of female is 1 year.
Male worm is rarely seen.
Embryos (larvae):
Embryo (larvae) is large, unsheathed, anterior end is rounded, tail is pointed
and long.
The cuticle shows prominent striations.
The larva swims about with a coiling and uncoiling motion.

Life cycle:
Transmission: Human infection occurs by drinking of infected Cyclops water.
Definitive host: Man is the definitive host.
Intermediate host: Cyclops.

Developmental steps of guinea worm:

(1) The gravid female discharges larva from lesions on the arms, legs, feet and
ankles in water.
(2) The larvae infect Cyclops.
(3) Larva undergoes metamorphosis in the body cavity of the Cyclops.
(4) Cyclops containing infective larvae is swallowed by man with raw
unfiltered drinking water.
(5) Cyclops is killed by the gastric acidity and the larvae are liberated.

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(6) The larvae penetrate the duodenal wall and enter the retroperitoneal
connective tissues and live as adult.
(7) The gravid female migrates to subcutaneous tissue of those parts of the
body liable to come in contact with water.
(8) At the site of localisation, the worm produces a papule and blister.
(9) The blister rupture and discharge milky fluid containing numerous larvae
by stimulation of water.
(10) The larvae are back again in water and the cycle is repeated.

Life cycle of Dracunculus Medinensis

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CLINICAL SYNDROMES
Incubation period is about a year.
1. Systemic symptoms: Nausea, vomiting, diarrhoea, urticarial rash and
intense pruritus.
2. Blister formation:
After one year, head of the gravid worm produce papule and blister in lower
limbs (98%), umbilicus, groin, palm, wrist and upper arm.
Due to itching sensation, blister ruptured and head of female worm
protrude through small hole whenever the part of the body comes in
contact with water and discharge milky fluid containing numerous larvae.

LABORATORY DIAGNOSIS
1. Detection of adult worm:
Diagnosis is made by observing the typical ulcer and the female worm
at the surface of the skin.
2. Detection of embryo:
The affected (ulcer) is flooded with water that induces the release of
embryos which can be examined under the microscope.
3. Intradermal test:
Dracunculus antigen leads to a wheal type reaction after 24 hrs.
4. X-Ray examination:
It may show coiled structure or linear density of calcified worm in
deeper tissues.

TREATMENT
(1) Niridazole is the drug of choice.
(2) Removal of the worm:
Worm can be removed from wound around a stick slowly.
It may take several days for complete removal of the worm.
It can be removed by surgical procedure.

PREVENTION
(1) Drink safe filtered water only.
(2) Prohibition of bathing or washing of clothing in well.
(3) Increase general awareness regarding the life cycle of guinea worn.

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47
N o rm al F lo ra
NORMAL FLORA
Q. Write short note on the Normal flora?

ANSWER
Normal flora is also known as bacterial flora.
They are bacterial flora residing in different organ without damage host.
They make symbiotic equilibrium with host.
Viruses and parasites are always considered as pathogen.
Bacterial flora is two types
(1) Resident flora or commensals: It is a constant population and when
disturbed, it reestablished itself.
(2)Transient flora: They can eliminated by mechanical mean and not
reestablished itself.

Role of normal flora

(1) Beneficial role:
They prevent or suppress the colonization or invasion of the body by
pathogen by means of bacterial interference.
They form a mucous blanket and prevents attachment of organism.
They are responsible for production of vitamins (K and B Vitamins).
Antibodies production increase due to response of cross reaction of
commensals.
(2)Disease production
They act as opportunistic pathogens when resistance of the host is
lowered.
They act pathogen out side of the habitat.
(3) Role of antibiotics
Use of broad spectrum antibiotics, disrupt the composition of normal
flora by inhibiting sensitive bacteria and allowing over growth of
resistant bacteria.

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Normal flora in different organs

Organ Normal flora

(1) Skin (i) Staph. Epidermidis (ii) Diphtheroid (iii) Peptostreptococcus

(iv)Strepto. Viridens (v) Enterococcus (vi) Micrococcus
(vii) Clostridia (viii) E. coli (ix) Candida albicans
(x) Propionibacterium
(2) Conjunctiva (i) Corynebecterium xerosis
(ii) S. Epidermidis
(3) Teeth (i) Strepto. Mutans (ii) Bacteroides (iii) Fusobacterium
(iv)Streptococci (v) Actinomyces

(4) Nose (i) Staph. Aureus (ii) Staph. Epidermidis

(iii) Diphtheroids (iv)Streptococci
(5) Mouth (i) Strepto. Mitis (ii) Trichomonas tenax (iii) Candida
(iv)Pepto streptococcus (v) Veillonella (vi) Bacteroides
(vii)Treponema
(6) Colon (i) Bacteroides (ii) Bifidabacteria (iii) Cl. Perfringens
(iv) Lactobacillus (v)E.coli (vi) Strepto. Faecalis
(vii)Klebsiella (viii) Candida (ix) Enterobacter
(x) Mycoplasma
(7) Urethra (i)Coagulase Negative Staphylococci (ii) Strepto. Viridens
(iii) Propionibacterium
(8) Vagina (i) Lactobacillus (ii) Enterococcus

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48
N o socom ia l In fe ctio n
Hospital Acquired Infection
Q.Write short note of Nosocomial infection? (B.Sc. Nur.-2010), (B.Sc. Nur.-
2008) , (B.Sc. Nur.-2007), (MBBS-2005)
Q.Write short note on the Infection control committee?
Q.Write short note on the role of microbiologist for control of Hospital
acquired infection?
Q.Write short note on the role of nursing staff for control of Hospital
acquired infection? (B.Sc. Nur.-2011), (B.Sc. Nur.-2008)

ANSWER
Nosocomial infection also called Hospital Associated Infection or Hospital
Acquired Infection.
An infection occurring in hospital or other health care facility in person the
infection was not present or incubating at the time of admission.
This includes infections acquired in the hospital even appearing after
discharge and also occupational infections among staff of the facility.
HAI increased economic and human impact because of:
- Increasing numbers and crowding of people in hospital.
- More frequent impaired immunity (age, illness, and treatments).
- New microorganisms.
- Increasing bacterial resistance to antibiotics
Factors influencing HAI :-
(1). Heavily laden with wide variety of pathogen in hospital.
(2). Micro organism laden in air, dust, antiseptic, lotions, water & food.
(3). Hospital microbial flora is multi drug resistant due to injudicious use of
antibiotic.
(4). Diabetic, immuno-compromised are more susceptible.
(5). Use Blood & blood product .
Source of infection
(1). Exogenous:-
(a) From patient
(b) Member staff.

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(c) Inanimate object – e.g. Endoscope, cystoscope, catheters,
needles, pad pans, food , water, secretion,
blood.
(2). Endogenous:-
(a) Patient’s own flora- Spontaneous invade,
- Surgical operation,
- Instrumentation,
- Nursing procedure.
Common Micro organism in HAI:-

(1). Aerobic gram negative rod – 60%

(2). Gram positive cocci - 30%
(3). Virus & fungi - 10%

(1) UTI E.coli, Klebsiella, Proteus, Serratia, Pseudomonas,

Providencia, Coagulage Negative Staphylococci, Candida,
Enterococci.
(2) RTI H.influanza, Strepto.pneumoniae, Staph.aureus, Klebseilla
Serratia, Proteus, E.coli, Pseudomonas.
(3) Wound Staph.aureus, E.coli, Proteus, Bacteroides,
Coagulage Negative Staphylococci.
(4) Burn Staph. Aureus, Pseudomonas, Acinetobactor.
(5) G.I. Salmonella, Shigella, Virus.
(6) Eye Staphylococcus aureus, Pseudomonas.

Transmission of infection:
Bacteria that cause nosocomial infections can be transmitted by several ways:
(1) Gram-negative bacteria in the digestive tract frequently cause surgical site
infections after abdominal surgery.
(2) Urinary tract infection in catheterized patients.
(3) Through direct contact between patients or health worker (hands, saliva
droplets or other body fluids),
(4) Through air (droplets or dust contaminated by a patient’s bacteria),
(5) Through objects contaminated by the patient (including equipment), the
staff’s hands,
(6) Visitors or other environmental sources (e.g. water, other fluids, food).

Infection control committee:- (ICC)

-Every hospital must have ICC.
-ICC consists- (1). Chairman: Medical Superindent
(2). Secretary: Microbiologist.
(3). Member:
Physician, surgeon, Nurse teacher,
Nurse representatives, gynecologist

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Sterile service manager,

Purchasing officer etc.
Function of Infection control committee -
(1).Surveillance.
(2).Control of infection.
(3).Monitoring of hygiene practice.
(4).Education of all staff.
(5).Close working link between Microbiology laboratory & different
department.
(6).Supportive service Sterile service
Laundry
Pharmacy
Engineering
(7).Maintain policy
(8). Ensure rational based
(9). Recommended procedures are practical

Role of the microbiologist in Infection control committee

The microbiologist is responsible for:
1 Handling patient and staff specimens to maximize the likelihood of a
microbiological diagnosis.
2 Developing guidelines for appropriate collection, transport, and
handling of specimens.
3 Ensuring laboratory practices meet appropriate standards.
4 Ensuring safe laboratory practice to prevent infections in staff.
5 Performing antimicrobial susceptibility testing and providing summary
reports of prevalence of resistance.
6 Monitoring sterilization, disinfection and the environment where
necessary.
7 Timely communication of results to the Infection Control Committee
or the hygiene officer.
8 Epidemiological typing of hospital microorganisms where necessary.

Role of the nursing staff

Implementation of patient care practices for infection control is the role of the
nursing staff.
Nurses should be familiar with practices to prevent the occurrence and spread
of infection, and maintain appropriate practices for all patients throughout
the duration of their hospital stay.
The senior nursing administrator is responsible for:
 Participating in the Infection Control Committee.
 Promoting the development and improvement of nursing techniques,
and ongoing review of aseptic nursing policies, with approval by the
Infection Control Committee.

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 Developing training programmes for members of the nursing staff.
 Supervising the implementation of techniques for the prevention of
infections in specialized areas such as the operating suite, the
intensive care unit, the maternity unit and newborns.
 Monitoring of nursing adherence to policies.

The nurse in charge of a ward is responsible for:

 Maintaining hygiene, consistent with hospital policies and good
nursing practice on the ward.
 Monitoring aseptic techniques, including handwashing and use of
isolation
 Reporting promptly to the attending physician any evidence of
infection in patients under the nurse’s care.
 Initiating patient isolation and ordering culture specimens from any
patient showing signs of a communicable disease, when the physician
is not immediately available.
 Limiting patient exposure to infections from visitors, hospital staff,
other patients, or equipment used for diagnosis or treatment
 Maintaining a safe and adequate supply of ward equipment, drugs
and patient care supplies.

The nurse in charge of infection control is a member of the infection control

team and responsible for :
 Identifying nosocomial infections
 Investigation of the type of infection and infecting organism
 Participating in training of personnel
 Surveillance of hospital infections
 Participating in outbreak investigation
 Development of infection control policy and review and approval of
patient care policies relevant to infection control
 Ensuring compliance with local and national regulations
 Liaison with public health and with other facilities where appropriate
 Providing expert consultative advice to staff health and other
appropriate hospital programmes in matters relating to transmission
of infections.

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49
U nive rsal P recau tio n
Universal Safety Precaution
Q. Write short note on the universal safety precaution? (B.Sc. Nur.-2010),
(B.Sc. Nur.-2008)

Definition:
The practice of Universal Safety Precautions (USP) is the most effective
method of preventing the transmission of HIV and other hospital acquired
infection in health care settings.
This terminology states that all patient blood and body fluids are to be
considered as potentially infectious.

Universal precautions include

1. Hand-washing with soap and water before and after all procedures.
2. Use of protective barriers such as gloves, gowns, aprons, masks and
goggles, to avoid direct contact with blood and other body fluids and
airborne organisms.
3. Safe disposal of waste contaminated with blood or body fluids.
4. Safe handling and disposal of needles and sharp instruments. DO NOT recap
or manipulate needle in any way.
5. Proper disinfection of instruments and other contaminated equipment.
6. Proper handling of soiled linen.
7. Adherence to correct hospital sterilization and disinfection protocols.
8. Processing of all laboratory specimens as potentially infectious.
9. Immunization against HBV.

Body fluids to which universal precautions apply

• Blood
• Vaginal secretions
• Semen
• Cerebrospinal fluid
• Synovial fluid
• Pleural fluid

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• Peritoneal fluid
• Amniotic fluid
• Pericardial fluid
• Other body fluids containing blood
Body fluids to which universal precautions DO NOT apply
• Tears
• Sputum
• Sweat
• Urine
• Vomitus
• Nasal secretions
The risk of transmission is extremely low or negligible unless these contain
visible blood.

Good Biosafety Practices

Q. Write short note on good biosafety practices?

ANSWER
Good Biosafety Practices are as following.
 The working area should be kept neat, clean and free of
materials that are not pertinent to the work.
 All blood samples are to be treated as potentially infectious
samples.
 Exposure may occur during blood collection, handling,
processing, testing, disposal of waste and transport.
 Aprons, gowns or uniforms must be worn at all times for the
work in the working area.
 It is prohibited to wear protective working area clothing
outside the working area, e.g, in canteens, coffee rooms,
offices, libraries, staff rooms and toilets.
 Appropriate gloves must be worn for all procedures that may
involve direct or accidental contact with blood, body fluids
and other potentially infectious materials.
 After use, gloves should be removed and hands must be
washed.
 Personnel must wash their hands thoroughly after handling
infectious materials and before they leave the working area.
 Open toed foot wear must not be worn in laboratories.
 Eating, drinking, smoking, applying cosmetics and handling of
contact lenses is prohibited in the working areas.
 Storing foods or drinks anywhere in the laboratory working
areas is prohibited.

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 Pipetting by mouth must be strictly forbidden.

 All spills, accidents and overt or potential exposures to
infectious materials must be reported to the supervisor.
 A written record of such accidents and incidents should be
maintained
 Written procedures for the clean -up of all spills must be
maintained.
 Contaminated liquids must be decontaminated before
discharge to the sanitary sewer.
 Work surfaces must be decontaminated after any spill of
potentially dangerous material and at the end of the working
day.
 Safe handling of sharp items and prevention of accidents with
sharps.
 Safe handling of specimens (blood etc) during collection,
processing and transport.
 Whenever possible avoid the use of sharps.
 Never recap a used needle.
 Ensure sharps container is nearby, well placed, and not over-
filled.
 Keep one bottle with 70% ethanol, one with 10% hypochlorite
solution ready.
 Replace container with hypochlorite solution for discarding
contaminated pipette & tips everyday.
 Work surface to be wiped with 70% alcohol.

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50
B iom edical W aste
Biomedical Waste Management
Q. Write short note on the biomedical waste disposal? (B.Sc. Nur.-2010),
(B.Sc. Nur.-2009) , (B.Sc. Nur.-2008) , (B.Sc. Nur.-2007) , (MBBS-2007)

ANSWER
Biomedical waste, (BMW), consists of solids, liquids, sharps, and laboratory
waste that are potentially infectious or dangerous and are
considered biowaste.
It must be properly managed to protect the general public, specifically
healthcare and sanitation workers who are regularly exposed to biomedical
waste as an occupational hazard.
Biomedical waste differs from other types of hazardous waste, such
as industrial waste, in that it comes from biological sources or is used in the
diagnosis, prevention, or treatment of diseases. Common producers of
biomedical waste include hospitals, health clinics, nursing homes, medical
research laboratories, offices of physicians, dentists, and veterinarians, home
health care, and funeral homes.

Categories of biomedical waste

Categories No. Biomedical waste Example

1 Human anatomical waste Tissue, organ
2 Animal waste Tissue, organ
3 Microbiological waste Culture, stocks, vaccine
4 Sharp Waste Needle, blade, scalpels,
syringes
5 Medicinal waste Medicines and cytotoxic drug
6 Soiled waste Cotton, dressing bedding with
blood & body fluid
7 Solid waste Tubings, catheter, i.v sets
8 Liquid waste Waste of washing, cleaning
and disinfecting
9 Incineration ash Incineration ash

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10 Chemical waste Chemical of disinfection and

insecticides

Colour coding and treatment options of biomedical waste

Waste categories Colour coding Treatment option

1,2,3 and 6 Yellow Incineration/ Deep burial
3,6 and 7 Red Autoclaving/microwaving/
Chemical treatment
4 and 7 Blue/ white translucent Autoclaving/microwaving/
Chemical treatment/
Shredding
5, 9 and 10 Black Disposal in secure landfill

Biomedical Waste Disposal management

o
Before disposal, the objects or materials should be effectively
decontaminated or disinfected by an approved procedure.
o Materials for decontamination and disposal should be placed
in containers, e.g, autoclavable plastic bags, that are color
coded according to whether the contents are to be
autoclaved and or incinerated.
o After use, hypodermic needles should not be recapped,
clipped or removed from disposable syringes.
o The complete assembly should be placed in a sharp disposal
container and incinerated with prior autoclaving.
o Sharps disposal containers must be puncture proof and must
not be filled to capacity. When they are three-quarters full,
they should be replaced with new containers.
o Discard containers, pans or jars preferably unbreakable (e.g.
plastic) should be placed at every work station.
o When disinfectants are used, waste materials should remain
in intimate contact with the disinfectant for the appropriate
time according to the disinfectant used.
o Discard containers should be decontaminated and washed
before reuse
Management of spills of blood or body fluid:
 Wear gloves throughout.
 Cover spill with absorbent material and pour disinfectant
around the spill and over the absorbent material for 30 min.
 Remove the absorbent material and place in the biohazard
bag for infectious waste.
 Wipe the surface again with disinfectant.

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 Sweep broken glass etc with a brush and discard into the
waste container.
 Report the spills to the laboratory in-charge.
 Keep a written record of all such accidents.

Management of Needle stick injury:

 Needle stick, puncture wounds, cuts, open skin contaminated
by spills or splashes should be washed thoroughly with soap
and water.
 Do not put your injured finger reflexively in mouth.
 Report the injury to the in-charge.
 Keep a written record of all such accidents.
 Appropriate medical evaluation, surveillance, treatment and

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 counseling should be provided.

Preparation of Sodium Hypochlorite solution (B.Sc. Nur.-2008)

 Bleach is a good disinfectant.
 Concentration prescribed by WHO is 10 g bleach in 1 L water (1%
Available chlorine).
 Household bleach contains 4%–5% available chlorine, should be
used after diluting.
 Minimum contact time recommended is 30 minutes.
 Always use freshly prepared diluted hypochlorite solution every
day.
Recommended strength of the sodium hypochlorite for various purposes
Purpose Strength
Spills 1%
Surface decontamination 1% (smooth surface)
10% (porous surface)
Discard bins 2.5%
Liquid infectious waste 10%
(with large amount of organic matter)

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51
P . U . O .

Pyrexia Unknown Origin

Q. Write short note on the Lab. Diagnosis of P.U.O?

ANSWER
Defination:- Any febrile illness i.e.
(1).Body temp. more than 38 oC or more than 100 o F.
(2).Lasting more than few days i.e. fever of 3 weeks or longer.
(3).Without any obvious cause or remain uncertain inspite of
investigations is known as Pyrexia of unknown origin.
Types of fever:-
(1).Sustained or continuous fever:-
When the temperature elevation is persistent with minimal
variations, i.e. temp. remains high throughout the day with a
difference between maximum & minimum daily temp. of less
than 2 0F.
E.g. Typhoid fever.
(2).Remittent fever:-
When the temp. falls each day but not to normal, i.e.
difference between maximum & minimum daily temperature
more than 2 0F.
E.g. Kala- azar.
(3).Intermittent fever:-
When the temperature in between touches to normal.
E.g. Malaria.
(4).Relapsing fever:-
When febrile episode are separated by interval of normal
temperature.
E.g. Caused by Borrelia recurrentis.
(5).Pel-Ebstein fever:-
Fever lasting 3-10 days followed by aferbile period of 3-10
days.
E.g. Classic of Hodgkin’s disease & other lymphomas.

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Causes:-
(I). Infections:- 35%
(A).Bacterial:-
(1).Enteric fever (2).Brucellosis.
(3).Septicemia associated with pneumonia, infective
endocarditis etc.
(4).Tuberculosis (5).Rheumatic fever.
(6).Relapsing fever (7).Leptospirosis
(8).Typhus fever (9).Q.fever
(10).U.T.I. (11).Hepatobiliary sepsis
(12).Lung abscess (13).Intra-abdominal abscess
(14).Dental infections (15).Pneumonia
(16).S A B E
(B).Parasitic:-
(1).Filariasis (2).Kala-azar
(3).Toxoplasmosis (4).Trypanosomiasis
(5).Amoebic hepatitis or Liver abscess.
(6).Malaria
(C).Viral:-
(1).Infectious mononucleosis-Epstein-Barr virus
(2).Cytomegalovirus infection
(3).Hepatitis A & B
(4).HIV
(5).Rubella & other viral infection fever
(6).Influenza
(D).Fungal infections:-
(1).Histoplasmosis
(2).Coccidioidomycosis.
(II).Neoplasms:- 20%
(1).Hodgkin’s & Nonhodgkin’s lymphoma
(2).Leukaemia
(3).Hypernephroma
(4).Hepatoma
(5).Desseminated maliganancy.
(III).Connective tissue Disorders:- 20%
(1).Systemic lupus erythematosus
(2). Polyartertis nodosa
(IV).Granulomatous diseases:- 15%
(1).Sarcoidosis
(2).Crohn’s disease
(3).Granulomatous hepatitis
(V).Miscellaneous:- 10%
(1).Drug induced fever
(2).Psychogenic fever.

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Laboratory Diagnosis:-
Sample Collection:- Depends on
- Clinical history
- Clinical examination
Important samples may be:-
- Blood - Urine
- Sputum - Pus
- Faecal matter - Body fluid
- Biopsy etc.
(I).Haematology:-
(i).T.L.C. & D.L.C. –
(a). High count with polymorphonuclear leucocytosis often with Toxic
granulations suggest Acute bacterial infection, such as -
- Pneumonia
- Dental abscess.
- Intra abdominal abscess.
(b). Low count with relative lymphocytosis –
- Intra abdominal abscess
- Typhoid
- Brucellosis
- Viral infections.
(c). Neutropenia:- ↓ Neutrophils.
- Viral infections.
- Systemic lupus erythematosis
- Histoplasmosis.
(d). Atypical lymphocytes:- with lymphocytosis
- Infections mononucleosis.
(e). Immature cells:- Different types of leukaemia.
(ii). P.B.F. Examination:-
(a). All stages of P.vivax – Signet ring, Trophozoite, Schizont,
Gametocyte.
(b). Signet ring & Gametocytes of P.falciparum.
(c). L.D. Bodies in cases of Kala-azar in circulating monocytes.
(d). Trypanomastigote form – African trypanosomiasis
- Chagas disease.
(e). Microfilariae – Filariaris.
(f). Borrelia can also be demonstrated in cases of Relapsing fever in
blood.
(iii). Dark ground Microscopy:-
- During the pyrexial period the Borrelia which is the causative agent of
Relapsing fever can be demonstrated in a wet film of blood.
– They show active movements.
- During the first week of illness- Leptospira can be observed by Dark
ground microscopy in Blood & Urine.

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(iv) Quantitative buffy coat (QBC) test: - For diagnosis of malaria.

(v). Blood Culture:- Indications
- Enteric fever – During 1st week.
- Brucellosis – Castoneda method & incubation in 10% CO2.
- Bacterial endocarditis – Single sample is adequate as bacteraemia is
constant.
- S A B E :- Series of culture over a period of several hours as
bacteraemia is intermittent.
- Leptospirosis
- Septicaemia & Bacteraemia
Time of collecting the blood:-
- Before any antibiotic therapy.
- Ideally shortly before the expected rise of temperature.

Quantity of Blood & Medium :-

- 5 -10 ml in 50 -100 ml of medium.
Medium used:-
- 0.5% Glucose broth / BHI broth/ Thioglycollate broth – for pyogenic infection
or
- 0.5% Bile broth – for enteric fever.
- Modified korthof ’s medium – Leptospira.
Sub culture on various solid media like Blood Agar, McConkey etc.
Observe colony character
Perform Biochemical test accordingly
Final confirmation by serological agglutination.

(II). Serological Tests:-

(1). Enteric fever:-
(i).Coagglutination test: – Antigen detection during early
period of infection.
(ii). Widal test – IInd week of infection.
(iii). IgM Antibody Detection e.g.- Typhidot test.

(2).Infectious mononucleosis :-
Paul-Bunnel test- Heterophile Agglutination test.

(3). Typhus fever:-

Weil- Felix test-
Heterophile Agglutination test by using OX-19, OX-2 & OX-K
Antigen from proteus strain

(4). Syphilis:-
Different serological tests-
V.D.R.L.

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R.P.R.
TPHA
FT Ab-Absorption test.
(5). Kala-azar:- Aldehyde test.
(6). Malaria:-
(i). Malaria Antigen can be detected:-
HRP II (Histidine- Rich Protein II ):
- Water soluble protein produced by trophozoite & young
gametocytes of P.falciparum .
pLDH parasite lactate dehydrogenase:
- Produced by asexual & sexual stages of malaria parasite.
E.g. Optimal test.
(ii). QBC - Quantitative Buffy Coat test.
(7). Viral & other infections:-
Various types of ELISA test to detect specific IgG or IgM Ab.
(8). Various serological tests for HIV.
(9). Indirect haemagglutination test for hepatic amoebiasis.

(III). Urine Examination :- To exclude U.T.I.

(a). Microscopy:-
(1). Pus cells in pyogenic infections.
(2). Leptospira can be demonstrated by dark ground
microscopy.
(b). Culture:-
(1). Pyogenic infection:- mid stream urine sample inoculated
on solid media such as Bloo agar (BA), Mac Conkey agar
(MC).
(2). Genito Urinary tuberculosis :- early morning sample for
three consecutive days.
- Z.N. staining – AFB.
- Culture on Lowenstein-Janson (L.J.) Medium.

(IV). Sputum Examination:- For pulmonary conditions.

(a). Gram’s staining:- GPC, GNB,Candida, Fungal elements.
(b). Z.N. staining:- AFB
(c). Giemsa stain:- H.capsulatum appear as small oval yeast cells
2-4 µm in diameter in the cytoplasm of
macrophages or monocytes.
(d). Pyogenic culture:- Inoculate on Blood agar & McConkey agar
(e). A.F.B. culture:- L.J. media
(f). Fungus culture:- Sabouraud dextrose agar (SDA) or Brain
Heart Infusion(BHI) agar.

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(V). Pus examination:-

(a). Gram’s staining :- GNB, GPC, GNC etc.
(b). Z.N. staining:- A.F.B.
(c). Pyogenic culture :- Inoculate on Blood agar & McConkey agar
(d). A.F.B. culture:- L.J. media
(VI). Faecal Examination: -
(a). Wet mount :- Pus cells, Ova
(b). Iodine preparation:-Cyst
(c). Culture:- For organism of enteric or Dysentry group

(VII). Body fluid:-

(a). Routine microscopic.
(b). Culture –
Aspirated Bile :- Suspected cases of bladder, liver, billiary
passage infection.
CSF:- Meningitis & Encephalitis.
Bone marrow:- Enteric fever.

(VIII). Immunological tests:-

(a). L.E. cell:- S.L.E.
(b). Antinuclear antibody test:- S.L.E.

(IX). Skin Test:-

In a number of sub acute & chronic infections hypersensitivity to the
constituents of the causative organism develops, hence the
inoculation of small quantities of suitable preparations of infective
agent in to skin (intradermal or subcutaneous).
A localized Delayed type Hypersensitivity reaction at the site of
inoculation developed in patients with present or past infections.
E.g:- (1).Tuberculin or Mantaux test - For T.B.
(2). Kveim test - For sarcoidosis.
(3). Histoplasmin skin test - For Histoplasmosis.
(4). Coccidioidin Ag test - For Coccidioidomyconis.

(X).Biopsy:- Lymphnode or other tissue.

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52
D ia rr h o e a
Diarrhoeal Diseases
Q. Define the diarrhoeal. Classified the etiological agent & describe
laboratory diagnosis of diarrhoeal ?

ANSWER
Defination:- It is an inflammation of mucous membrane of Stomach &
Intestine resulting in:-
a) Frequent loose motion
b) With or without blood & mucous
c) Pain abdomen
d) With or without temprature rise

For all practical purpose the term

Diarrhoea
Dysentry are placed under one heading as
Food poisoning “ Diarrhoeal Diseases”
Gastroenteritis

Diarrhoea:-
It is defined as increase in the frequency, fluidity or volume of
bowel movement relative to the usual habit of each
individual.
Dysentry :-
Presence of blood & mucous in the stool with abdominal pain,
tenesmus & fever known as dysentry.
Food poisoning:-
Any type of illness acquired through consumption of food or
drink contaminated with micro-organisms, their toxins or
chemical poisons.

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Causes of Diarrhoeal diseases:-

According to mechanism
(I) Non inflammatory:-
Characterized by
- Inovlement of proximal small bowel
- Watery diarrhoea
- No faecal leucocytes
V. cholera ETEC
Clostridium perfringens Bacillus cereus
Staph aureus Rotavirus
Norwalk like viruses Enteric adenoviruses
Giardia lamblia Cryptosporidium
Isospora belli Cyclospora cayetanensis
II Inflammatory ;-
Characterized by
- Colon or distal small bowel involvement
- Dysetry or inflammatory diarrhoea
- Presence of faecal polymorphonuclear leucocytes.
1. Shigella sp 2. Salmonella enteritidis
3. Campylobacter jejuni 4. EHEC
5. EIEC 6. Vibrio para haemolyticus
7. Clostridium difficile 8. Entamoeba histolytica
III Penetrating:-
Characterized by
- Distal small bowel involvement
- Systemic manifestations
- Faecal mononuclear leucocytes
1 Salmonella typhi
2 Yersinia erterocolitica

(B) Causes of infective diarrhoea according to the causative agents:-

I- Viruses:-
1 Rotavirus 2 Calicivirus
3 Adenovirus 4 Astrovirus
II- Bacteria:-
1. V. cholerae 2. V. parahaemolyticus
3. Esch. coli 4. Sal. enteritidis
5. Sal. typhimurium 6. Campylobacter sp.
7. Yersinia enterocolitica 8. Shigella sp
9. Clostridium perfringens 10. Clostridium difficle
11. Staph. aureus 12. Bacillus cereus

III Prorozoa:-
1. Entamoeba histolytica 2. Giardia lamblia

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3. Cryptosporidium parvum

IV Fungi:-
1. C. albicans

Clinical manifestations:-
Depends on how the enteric pathogen causes the disesase.

(a) Patient infected with an enteric pathogen that upsets fluid &
electrolyte balance have.
1) No faecal leucocytes
2) Watery diarrhoea
3) Fever is usually absent or mild
4) Nausea, vomiting, abdominal pain may be present
5) Dominant feature is intestinal fluid loss.
E.g. :- V. cholerae, ETEC, Rotavirus , B. cereus.

(b) Patient infected with enteric pathogen that causes significant

cell destruction & inflammation have.
1. Faecal leucocytes in the stool
2. Mucous & Blood in the stool.
3. Fever is a predominent complaint
4. Abdominal pain, tenesmus.
E.g.:- Shigella sp, EIEC, Sal. enteritidis, E. histolytica

(c) Pt. infected with a pathogen that is able to penetrate the

intestinal mucosa of small intestine without producing
enteroclitis & then subsequently spread and multiply at other
sites & produces symptoms of systemic illness such as
- Headache, malaise, Sorethroat, fever
- Diarrhoea is not a predominant features
E.g.:- -Salmonella typhi
-Yersinia enterocolitica

3. Bacterial food poisoning:-

Q. Write short note on the food poisoning?
ANSWER

It is conventionally restricted to acute gastroenteritis that results from

presence of bacteria or their products (toxins) in food.

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Classification of causative agent of food poisoning:-

(A) Infective type:-

Multiplication of the organisms occurs in vivo
E.g.:-
- Salmonella typhimurium
- Sal. enteritidis
- Campylobacter jejuni
- Vibrio parahaemolyticus
- Vibrio mimicus
Incubation period :-
8-24 hrs (usually more than 16hrs )
Common food sources:-
Shell fish, meat, Egg, Salad, Raw vegetables, Cheese, Dairy products
and Water

(B) Toxin type:-

Disease occurs following ingestion of food with preformed toxin,
resulting in nausea, vomiting and diarrhoea.
Incubation period:- 1- 6 hrs.
Common food sources:-
Potato, Salad, Milk & milk products, Cream pastries, Rice and egg
E.g.:- 1) Staphylococcus aureus
2) Bacillus cereus- due to contaminated fried rice

(C) Intermediate type:-

In this bacteria ingested with food & produces toxin in the gut which
induces abdominal pain, diarrhoea & rarely vomiting.
Incubation period:- 8- to 16 hrs
E.g.:- 1 Clostridium perfringens
2 B. cereus
Common food:-
Beef, poultry, cereals, Dried beans.

History of Patient:-
1 Recent travel
2 Exposure to potentially contaminated food or drink
3 Presence of similar disease in friends & family
Association of food poisoning infection with specific food:-

1. Back packing & Drinking from mountain streams:- G. lamblia.

2. Dairy products:- Salmonella Sp, Campylobacter Sp, Yersinia sp
3. Egg & Potato, Salads, Pastries:- Staph. aureus
4. Egg :- Salmonella

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5. Fresh fruits :- Cryptosporidium Sp , Cyclospora sp.
6. Fried rice :- Becillus cereus
7. Hamburger :- E. coli 0157 :H7
8. Shell fish :- Vibrio sp, Norvo viruses, Hepatitis A virus

LABORATORY DIAGNOSIS
Specimen collection & transport:-
Specimen:-
- Faeces - Rectal swab
- Vomit - Food material
- Duodenal aspirate

General comments:-

1. Specimen that can be delivered to the Lab. within 1 hr. may be

collected in a clean, sterile waxed cardboard or plastic container.
2. Volume of Liquid stool- 5 ml or 1 teaspoon
Or
Formed stool- Pea size piece
3. Stool sample for bacterial culture – If a delay of longer than 2 hrs
is anticipated then the specimen should be placed in a transport
medium.
4. If stool is not available then rectal swab may be collected &
immediately inoculated on culture media or transported to
Laboratory in suitable transport medium.
Swab is not acceptable for
i) Detection of parasites
ii) Toxins or viral antigens.
5. Stool for virus culture must be refrigerated if they are not
inoculated within 2 hrs
- A rectal swab transported in modified Stuart’s transport
medium or other viral transport medium is adequate for
most viruses.
6. For detection of ova & parasites – Specimen preservation with a
fixative is recommended.
7. Duodenal aspirate for detection of Giardia or Strongyloides.
8. String test:- Useful for diagnosis of duodenal parasites such as
Giardia & for isolation of Sal. typhi from carriers & from patient
with Acute typhoid fever.

Transport media:-
1. Cary-Blair transport medium:- Best preserves the viability of
intestinal pathogen.

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2. Glycerol & 0.033 M phosphate buffer (PH 7.0) – For shigella,

which is a delicate organism & supports the viability of shigella
better than Cary- Blair medium.
Special for V. cholerae:-

Sample collection:-

By introducing rubber catheter No. 26 or 28 into the rectum & stool

flow is directly collected in sterile screw caped container.

Transport media:-
1) V.R. Transport medium:- 10- 15 ml of medium + 1-3 ml of stool
Vibrio do not multiply but remain viable for several weeks.
2) Cary-Blair medium
3) Bile peptone transport medium
4) Autoclaved sea water
5) Alkaline peptone water:- PH 8.6 (Transport & Enrichment)
6) If transport medium is not available then a 5 cm X 1.5 cm strip of
thick blotting paper can be soaked in the faecal matter, then
placed is a sealed plastic beg & sent to laboratory.

Examination:-
(1) Wet mount:-
- For detection of motile trophzoites such as Entamoeba/Giardia.
- Occasionally the larvae or adult worm may be seen.
- Refractile form of Cryptospordium & many cyst can be seen.
- Ova of intestinal parasites can be seen.
- Direct wet mount of faecal material with blood/ mucus + equal
volume of loeffler’s methylene blue for detection of leucocytes.
(2) Hanging drop preparation:-
Specially for vibrio cholerae-Darting motility/ Swarm of gnats
appearance in cases of Acute cholera stool.
(3) Immoblisation test:-
Add one drop of V. cholerae Poly ‘o’ antisera to hanging drop
preparation and again observe motility V. cholerae →becomes
immobilized.
(4) Gram’s Stain:-
- Thin Comma shped GNB- Campylobacter/ V. cholerae
- Polymorphs can also be seen.
- Sheaths of Gram +ve cocci in cluster- Staph. aureus in
Pseudomembranous enterocolitis
- Smear from suspected food, intestinal content & faeces show Gram
+ve sporing bacilli in Cl. botulinum food poisoning, but it is to be
confirmed only by demonstrating toxin production.

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- Smear from mucous flakes in cholera shows Fish in stream
appearance.
(5) Z.N. Staining:-
- Cryptosporidium sp.
- Isospora sp.
(6) Iodine preparation:-
For ova & cyst.
(7) Antigen detection:-
Direct immunofluorescence by using monoclonal
antibody fluorescent stain.
- Giardia
- Cryptosporidium
- V. Cholerae
(8) Enzyme immunoassay or Latex agglutination:-
- Rotavirus
- Cryptosporidium
- Giardia
- Certain bacterial pathogens
(9) Molecular biological techniques:-
- PCR: For detection of Enteric pathogen.
(10) Culture:-
(a) Fresh specimen should be immediately inoculated on routine culture
media.
(b) If specimen is received in transport medium then first inoculate into
one of the enrichment medium & incubate at 37 0c for 6-8 hrs before
plating on a selective medium
(c) If Specimen has been collected in Enrichment medium, then it should
be incubated as such for 6-8 hrs including transit time before
subculture on solid media.
Enrichment media are:- E.g.:-
(1) Selenite-F broth: For Salmonella & Shigella
(2) Tetrathinate broth : For Salmonella & Shigella
(3) Alkaline peptone water: For Vibrio cholerae
Culture Media used are:-
1) Blood agar:-
It is an excellent general supportive medium it allows the growth of
- Yeast - Staphylococcus
- Entero cocci - Gram Negative Bacilli
- Clostridium.
2) MacConkey agar:-
- A moderate selective medium for growth of most
enterobacteriaceae, vibrio & other possible pathogens.

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- Other moderate selective media especially for:-

(a) Salmonella & Shigella are:-
- Hektone enteric agars
- Xylose- lysine deoxycholate (XLD)
- Salmonella & Shigella agar
- Deoxycholate Citrate Agar
These culture media inhibit growth of most enterobacteriaceae &
allow salmonella & shigella to grow.
(b) For vibrio:-
- T.C.B.S. (Thiosulphate citrate bile salt sucrose agar)
- Monsur’s medium
- Alkaline Bile salt agar
(c) For campylobacter:-
Thayer- martin medium containing vancomycin & polymyxin.
Incubate at 42-43 0C under 5-10 % Co2
(d) For clostridium perfringens typeA:-

(Gram’s stain show Gram Positive Bacilli with subterminal

spore).
 Inoculated blood agar plates are incubated at 370C for 18-24
hrs.
 Next day morning observe & note down the colony characters
Gram’s stain – Gram’s stain show Gram Positive Bacilli with
Subterminal spore
Motility test–
Salmonella: Highly motile
Vibrio: Darting motility
Shigella: Non motile
 NLF colony :- Oxidase test
Salmonella /Shigell: - Negative
Vibrio , campylobacter: - Positive
 Biochemical test :-
IMViC
Urease
Triple sugar iron
Sugar Fermentation
Decarboxylation of amino acid
 Agglutination with specific antisera –
Polyvalent & Monovalent:-
Emulsify the colony in a drop of normal saline on clean glass
side at two different site- one control & other as test site.
To the test side aid specific polyvalent antisera→
If Positive repeat with monovalent antisera

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 Special test for individual Organisms:-

(a). Staphylococcal enterotoxin of food, vomit , culture filtrate of

isolated strain can be detected by :-
- E.L.I.S.A.
- Reverse Passive Agglutination test.
(b). Test for production of Enterotoxin by Eschi. Coli
-Radio –Immunoassay.
- E.L.I.S.A.
(c). Sereny test :- To detect EIEC & Shigell.
(d). For production of verocytotoxin
-cultivate the strain on vero cells in which toxin causes
cytopathic effect.
(e). Nagler’s reaction for Cl. perfringeng.
(f). Demonstration of botulinum toxin by animal inoculation test.

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53
M e n in g it is
Meningitis
Q. Classified the etiological agent & describes laboratory diagnosis of
Meningitis?

ANSWER
Infection of the membranes surrounding the brain and cord is called
meningitis.
Etiology:-
Common pathogens:-
80 % infection of Pyogenic meningitis are caused by:
(i) Streptococcus pneumoniae
(ii) Neisseria meningitidis, and
(iii) Haemophilus influenzae
Infrequent pathogens:-
20% cases of pyogenic meningitis are
(i) Listeria monocytogenes
(ii) Gram-negative bacilli
(e.g., Klebsiella, Psuedomonas, E.coli)
(iii) Staphylococcus aureus
(iv) Streptococcus pyogenes
(v) Staphylococcus epidermidis
LABORATORY DIAGNOSIS:-

Specimen:-
CSF is obtained by lumber puncture.
The fluid is collected in 3 separate sterile vials, one for cell count, one
for chemical examination and one for culture.
1. Microscopy
Gram film of centrifuged deposit shows plenty of pus cells and few
Gram positive or Gram-negative organisms.
India Ink stain:-

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The large polysaccharide capsule of Cryptococcus neoformans allows
these organisms to be visualized by the India ink stain.

2. Chemical analysis
It shows diminished glucose levels and increased protein.

Features Normal CSF Pyogenic Tuberculosis Virus

Meningitis Meningitis Meningitis
Pressure Highly Increase Moderately Slightly
Increase Increase
Chemical
Total 30-45 mg% Highly Slightly Slightly
protein increased increased increased
(100-600 mg%) (80-120mg%) (60-80 mg%)
Sugar 40-80 mg % Dimished (10- Dimished Normal
20 mg%) or (30-50 mg%)
absent
Cell count
Total cell 1-3 500-10,000 50-500 4-10
/cu.mm
Type of cell Lymphocyte Neutrophil Lymphocyte Lymphocyte
Lymphocyte Neutrophil
Bacteriologi
cal
Gram stain Nil GPC or GNC or
GPB or GNB Nil Nil
found
Acid fast Nil Nil AFB found Nil
stain

3. Culture
(i). CSF:- Centrifuged deposit is inoculated onto blood agar, chocolate agar
and plates are incubated up to 24 hours in an atmosphere of 5-10
% CO2 .
MacConkey agar is also inoculated & incubated at 37oC for 24 hrs
and follows according to gram stain morphology.
(ii). Blood:- Blood culture is positive in about 50% of cases of meningitis due
to N. meningitides, H. influenzae and S. pneumoniae.
4. Demonstration of bacterial products
I. Bacterial antigen:- Bacterial antigen can be detected by latex
agglutination of N. meningitides, H. influenzae, S. pneumoniae.
II. Bacterial endotoxin:- Limulus Lysate test can be done.

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ASEPTIC MENINGITIS

Aseptic meningitis is labelled when clinical features of meningitis, with

leucocytes but no bacteria in the cerebrospinal fluid.
Aseptic meningitis is most commonly caused by a viral agent.
Aseptic meningitis may also result from tumour, cysts, chemicals,
sarcoidosis or other noninfectious causes.
Viral etiology is established by IgM anti bodies or IgG antibodies.
T. Pallidum is diagnosed by VDRL test is CSF.

Tuberculous meningitis
The CSF in tuberculous meningitis shows moderate rise in cell count
(50-100/cm. mm.).
For Tubercular meningitis CSF is allowed to stand a fibrin web is
formed (cob-web).

Acid-fast Bacilli :-
May be found in Z.N. stain & culture may be done in L.J. medium.

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54
S .T .D
Sexually Transmitted Disease
Q. Enumurate the cause of Sexual Transmitted Disease. Describe the
laboratory diagnosis STD?

ANSWER
Definition:-
These are group of communicable disease which are transmitted
predominantly or entirely by sexual contact & caused by bacteria, viruses,
protozoa & fungi may cause Genital ulcers, genital discharge or no genital
lesions.

Classification according to clinical manifestations:-

(A). Painless genital ulcers:-

(i) Syphilis – T. pallidum.
(ii) Lymphogranuloma venereum – Chlamydia trachomatis.
(iii) Donovanosis – Calymmatobacterium granulomatis.

(B). Painful genital ulcers:-

(i) Chancroid – Haemophilus ducreyi.
(ii) Herpes genitalis – Herpes simplex type 1&2.
(C). Urethral discharge:-
(i) Gonorrhoea – N.gonorrheae.
(ii) Non gonococcal urethritis –
- Ch.trachomatis (type D-K)
- Ureaplasma urealyticum.
- Mycoplasma genitalium.
- Mycoplama hominis.
(D) Vaginal discharge:-
(1) Gonorrhoea.

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(2) N.G.U.
(3) Trichomoniasis – T. vaginalis.
(4) Vaginitis - Gardnerella vaginalis, mobiluncus sp.
(5) Vulvo-vaginal candidiasis – C.albicans.
(E) Genital wart:-
Human papilloma viruses.
(F) No genital lesions:- Only systemic manifestations :-
 HIV 1&2.
 Hepatitis B.
 Hepatitis C.
(G) Miscellaneous:-
 Group B Streptococci.
 Molluscum contagiosum virus.
 Cytomegalo virus.
 Shigella sp.
 Campylobacter sp.
 Giardia lamblia.
 E.histolytica.
 Phthris pubis.
 Sarcoptes scabei.

Laboratory Diagnosis:-
Based on clinical manifestations & clinical examination.

(A) Painless genital ulcer

Sample collection:-
 Before antibiotic treatment.
 Serum or exudates from the lesion.
 Smear from the urethral lesion.
 Blood for serological test.

(1) Dark ground Examination:-

 T. Pallidum is recognized by its characteristic slender structure
& motility seen as white against black back ground.
 Identification of a single characteristic motile organism is
sufficient for diagnosis.
 For positive microscopy the no. of treponemes must be 104 or
more/ml of exudate.
(2) Giemsa staining :-
(a) Bipolar condensation of chromatin resembles safety pin
appearance evident of Donovan bodies for the diagnosis of
Donovanosis.
(b) Reniform inclusion bodies surrounding the nucleus in

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C.trachomatis.
(c) Iodine preparation – These inclusion bodies posses a glycogen
matrix hence can be seen in iodine preparation.
(3) Immunofluorescence staining:-
Using specific monoclonal antibody labelled with fluorescent dye.
Culture
Chlamydiae may be isolated in mice, yolk sac of 6-8 days old embryo
& in cell culture.
Serological test :-
(a) Syphilis is mostly diagnosed by serological tests.
Positive results are obtained about two weeks after the
appearance of primary sore.
Tests are:-
(i) Non specific or Non treponemal tests:-
(Standard test for syphilis)
 Very sensitive
 Positive in 70% primary syphilis and 100% in secondary
syphilis.
 Commonly use screening test
 To determine response to treatment.
 After the secondary stage titre ↓ & about one third of patient
with late syphlils are serongenative
E.g.:- VDRL Test
RPR Test
TRUST Test.
 A titre of 1:8 or more is considered significant.
 These tests become negative 6-18 months after effective
treatment.
(2). Specific treponemal test :- To confirm positive VDRL / RPR Test.
(i) Using Live treponem – T.P.I.Test.
(ii) Using Killed treponem – FTA-Absorption test.
(iii) Using extract of treponem – T.P.H.A. test.
Lymphogranuloma venerum;-
(1) Compliment Fixation Test for antibody detection
(2) Frei test – Skin test using heat inactivated LGV Shows
delayed type of hypersensitivity.
(3) ELISA
(4) PCR

(B) Painful Genital ulcers

Sample collection:-
(i) Exudate from the lesion.
(ii) Scrapping from base of the lesion.
(a). Gram’s Stain : -

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 May show Gram negative ovoid bacilli.

 “ Bacteria of fish ” (croud of fish ) appearances in
case of chancroid.
(b). Giemsa Stain:-

 Characteristic multinucleated giant cells with Eosinophilic

intranuclear inclusion bodies in herpes genitalis.
(c). Toludine blue Stain :-
 Stain with 1% solution of toluidine blue for 15 second shows
multinucleated giant cells with homogenously stained ground glass
chromatin K/a Tzanck cells in Herpes genitalis.
(d). Immonofluorescence -
Using specific monoclonuclear antibodies.
(e). Electron microscopy:-
Virions can be demonstrated.

Culture:-
For chancroid :-
A enriched with isovilatex calf serum & vancomycin & incubated at 370 C
under 10% CO2 atmosphere with high humidity shows small, gray, translucent
colonies.
Gram’s stain show – Gram negative cocobacilli.
For Herpes:-
Tissue culture on human diploid fibroblast cells shows cytopathic effect in 2
hrs.
Antibodies can be detect by Compliment Fixation test, ELISA and RIA .

(C) Urethral, Vaginal discharge

Sample collection:-
 Urethral discharge.
 Vaginal or cervical swab.
 After Prostatic massage in male.
 In suspected swab (not high vaginal) or Endocervical exudates
can be collected.
(1) Wet film Exam :-
Freshly collected specimen may show characteristic motile
Trichomonas vaginalis.
Clinically– T.vaginalis produces copious, frothy, yellow or yellow green
discharge.
(2) 10% KOH Exam:- May show yeast cells.
Discharge in candidiasis is thick & curd like.
(3) Gram’s stain:- shows
 Large number of pus cells.

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 In gonococcal infection – Large number of
intracellular, kidney shaped, Gram negative,
intracytoplasimc diplococci seen.
(4) Giemsa Stain:-
Presence of intra cytoplasmic inclusion bodies suggestive of
C. trachomatis.
(5) Saline microscopy:-
In bacterial vaginosis, vaginal discharge contains clue cells.
(Squamous epithelial cells coated with coccobacillary organisms)
Leucocytes absent
(6) Amine test:-
When vaginal fluid is mixed with 10% KOH-Fishy odour is immediately
liberated. It is due to volatile amines present in vaginal fluid.

Culture:-
Inoculate the culture media plates as per finding of smear examination.
(a) Chocolate agar- (or Thayer-Martin medium) for:-
Gonococci- Translucent colonies
(b) Fineberg’s medium-
For Trichomonas will show motile trophozoite after 5 days of
inoculation.
(c) Sabouraud’s dextrose agar:- For candida
 Creamy white smooth colony
 Budding Gram positive yeast cells
 Germ tube formation present
 Chlamydospore formation on cornoneal agar
(d) MacCoy or Hela cell tissue culture:- For C. trachomatis

(D) No gential lesions & only systemic manifestations

Serological test is performed for detection of HIV 1 & 2, HBV and HCV.

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55
Respiratory Infecton
Respiratory Infection
Q. Enumerate the causes of sore throat. Discuss the laboratory diagnosis of
sore throat?

ANSWER
Definition:
Infection of Throat & Pharynx is known as sore throat.
Causes of sore throat divided into various syndromes.
Syndrome Organism
(a) Sore throat Viruses- 2/3 cases
Bacteria-
Streptococcus pyogenes
Staphylococcus aureus
H. influenza
(b) Diphtheria Corynebacteruim diptheriae
(c) Vincent angina Borrelia vincenti & Fusobacterium
(d) Candidosis Candida albicans
(e) Infectious mononucleosis Ebstein- Barr virus

Pseudo membrane is form by following organism:

C. diptheriae
Candida albicans
Streptococcus pyogenes
Treponema vincenti
Staphylococcus aureus
Micro organism causing Upper Respiratory Infection
(a) Sinusitis- Streptococcus pneumonae, H influenza,
Streptococcus pyogenes, Staphylococcus
aureus.
(b) Otitis media:- S. pneumoniae , H. influenzae, S. pyogenes,
S. aureus, M. catarrhalis.
(c) Epiglottitis:- H. influenza type b

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Micro organism causing Lower Respiratory Infection

(a) Tracheo bronchitis:- (whooping cough)
B. pertussis, B. parapertussis
(b) Pneumonias:-
S. Pneumonae H. influenzae
S. aureus Klebsiella Pneumoniae
Mycoplasma Pneumoniae Chlamydia Pneumoniae
Chlamydia psittaci
(c) Lung abscess: -
M. tuberculosis S. pneumoniae
S. aureus
Laboratory diagnosis of sore throat:
Specimen:-
Posterior Pharyngeal swab,
Posterior nasal swab
Pseudo membrane swab
Transport specimen as soon as possible, If delay, keep in refrigerator 40C.

Steps of Examination
(A). Microscopic examination of specimen
Gram’s staining:
Gram positive cocci in chain: Streptococci
Gram Positive cocci in cluster: Staphylococci
Gram positive bacilli in cuneiform: C. Diphthriae
Gram positive yeast: Candida
(B) Culture:-
Sample is inoculated in below mentioned media: -
1. Blood agar (BA),
2. Chocolate agar (CA)
3. Mac Conkeys agar (MCA)
4. Loeffellers serum slope (for Diphtheria)
5. Sabourauds dextrose agar
6. Lowenstein Janson media (L.J. Media)
Incubate for 24 hrs at 370C and Loeffellers serum slope is observed after
6-8 hrs. of incubation.
(a) Blood Agar:- For haemolytic bacteria.
Observe for hemolysis & do grams stain, if GPC then
suspect for -
With α – Haemolysis With β Haemolysis
Strepto. Pneumonae Staph. Aureus
Strepto. Viridans Strepto. Pyogenes
(b) Chocalate Agar:- For H. Influenza.
(b) Mac-Conckey Agar:-

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For lactose & non lactose fermenting colonies.

For Klebsiella pneumonae & other Enterobacteriaceae.
For pseudomonas.
(c) Loefflers serum slope:-
For C. diphtheriae colonies:
White creamy colonies developed.
Cuneiform letter arrangement of light green coloured bacilli
with dark green coloured granules seen in albert’s stain.

Bacteria Identification characteristics

Staph. aureus Golden yellow, opaque, pin head colonies
Catalase test: Positive
Coagulase test: Positive
MSA test: Positive
Strep. pyogenes Pin point, β Hemolytic colonies
Bacitracin sensitivity: Positive
ASO test: Positive
S. pneumoniae - Optochin sensitivity Positive,
Bile solubility Positive,
Gram positive diplococcic with thick capsule
Quellung’s reaction Positive.
H. influenzae - Gram negative coccobacilli
Satellitism positive
Klebsiella pneumoniae Mucoid colony
Biochemical reaction
Indole(–ve), MR(–ve),VP(+ve), Citrate(+ve)
Pseudomonas Non Lactose Fermanting colonies
Oxidase test: Positive
Gram negative bacilli
Actively motile
Candida Smooth white colony
Budding yeast cell seen
Pseudohyphae seen
Germ tube present
Chlamydiophore seen
M. Tuberculosis Rough, tough and buff colonies
Acid fast Bacilli seen
Bordetella pertussis Mercury drop appearance colony on Bordet Gengou
Agar

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56
Urinary Infecton
Urinary Tract Infection
Q. Describe the etiological agent and laboratory diagnosis of U.T.I.?
Q. Write Short note on the U.T.I.?

ANSWER
Type of Urinary Tract Infection
Lower Urinary Tract Infections Upper Urinary Tract Infections
1. Urethritis 1.Acute pyelitis
2. Cystitis 2.Acute pyelonephritis
3. Prostatitis
Factors limiting multiplication of organisms in urinary tract
1 A high rate of urine flow.
2 Regular complete bladder emptying.
3 Constant dilution of residual urine in the bladder by inflow
from kidney.
4 Mucosal defenses act by rapid clearing of bacteria from the
mucosa.
5 Local antibody, complement and lysozyme may also give
protection.
6 Secretions of prostate and periurethral glands possess
antibacterial activity.
Factors favouring multiplication of organisms
1 pH 6.0-7.0
2 Lowered osmolality
3 Glucose
4 Obstruction
Clinical feature
1 Asymptomatic UTI or covert bacteria
Adult women 5%
Girls 1-3%
Boys 0.3%

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2 Symptomatic UTI
Dysuria, frequency, suprapubic pain along with loin pain and
tenderness
There may be fever and rigors
Fever is associated with pyelonephritis
More commen in women then men.
3% at age of 20, increasing by 1% in subsequent decade.
50% women suffer from UTI during adult life.
In men incidenc is high in person over 60 yrs age due to enlarged
prostate.
Predisposing factor
1 Female urethra is in proximity to anus
2 Short length of urethra( about 4 cm)
3 Termination of urethra beneath the labia
4 Obstruction to flow of urine due to tumour, stone,
stricture, BPH
5 Neurogenic bladder dysfunction e.g. spine cord injury,
multiple sclerosis
6 Vesico-urethral reflex
7 Bacterial virulence
8 Pregnancy due to
a Dilatation of ureters and renal pelvis
b Pressure of the enlarged pregnant uterus on the
ureter at the brim of pelvis
c Atony due to inhibitory effect of progesterone
d Temporary incompetence of vesico-urethral valves.

Possible pathogens of U.T.I.

BACTERIA
Gram positive Gram negative
Staphylococcus Escherichia coli
saprophyticus Proteus species
Haemolytic streptococci Pseudomonas aeruginosa
Klebsiella strains
*Salmonella Typhi
*Salmonella Paratyphi
*Neisseria gonorrhoeae
*These species are not primarily pathogens of the urinary tract, but may be
found in urine.
Other bacteria can also cause UTI
Mycobacterium tuberculosis,
Leptospira interrogans,
Chlamydia,
Mycoplasma and

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Candida species.
PARASITES
Schistosoma haematobium,
Trichomonas vaginalis
Enterobius vermicularis
Wuchereria bancrofti
Onchocerca volvulus
Finding intestinal parasites in urine indicates faecal contamination.
The presence of bacteria in urine is called bacteriuria. It is usually regarded as
significant when the urine contains 105 organisms or more per ml (108/L) in
pure culture.
Infection of the bladder is called cystitis.
Infection of the kidney is called pyelonephritis. .
E. coli is the commonest urinary pathogen causing 60–90% of infections.

UTIs caused by
 Pseudomonas, Proteus, Klebsiella species and S. aureus, are associated
with hospital-acquired infections, often following catheterization or
gynaecological surgery.
 Proteus infections are also associated with renal stones.
 Staphylococcus saprophyticus infections are usually found in sexually
active young women.
 Candida urinary infection is usually found in diabetic patients and those
with immuno-suppression.
 M. tuberculosis is usually carried in the blood to the kidney from another
site of infection. It is often suspected in a patient with chronic fever
when there is pyuria but the routine culture is sterile.
 Pyuria with a negative urine culture may also be found when there is
infection with Chlamydia trachomatis, Ureaplasma, or N. gonorrhoeae,
or when a patient has taken antimicrobials.
 S. Typhi and S. Paratyphi can be found in the urine of about 25% of
patients with enteric fever from the third week of infection.
 Typhoid carriers may excrete S. Typhi in their urine for many years.
 Carriers are common in schistosomiasis endemic areas.
COLLECTION AND TRANSPORT OF URINE
Midstream urine passed by the patient at the beginning of the day
should be sent for examination.
LABORATORY EXAMINATION OF URINE
Examine the specimens microscopically
Urine is examined microscopically to detect:
 Significant pyuria, i.e. WBCs in excess of 10 cells/HPF (106/L) of urine
 Red cells
 Casts
 Yeast cells

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 T. vaginalis motile trophozoites

 S. haematobium eggs
 Bacteria (providing the urine is freshly collected)
Detecting bacteria in uncentrifuged (fresh) urine indicates urinary infection,
i.e. bacteriuria in excess of 105/ml

Culture the specimen

Specimen can be inoculated on the different culture medium.
Nutrient Agar
Blood Agar
Mc Conkey Agar
Cystine lactose electrolyte-deficient (CLED) agar
Estimating bacterial numbers
It is necessary to estimate the approximate number of bacteria in urine
because normal specimens may contain small numbers of contaminating
organisms, usually less than 10,000 (104) per ml of urine.
Urine from a person with an untreated acute urinary infection usually
contains 100 000 (105) or more bacteria per ml.
Appearance of some urinary pathogens on CLED agar
 E. coli: Yellow (lactose-fermenting) opaque colonies often with slightly
deeper coloured centre.
 Klebsiella species: Large mucoid yellow or yellow-white colonies.
 Proteus species: Transluscent blue-grey colonies.
 P. aeruginosa: Green colonies with rough periphery (characteristic
colour).
 E. faecalis: Small yellow colonies.
 S. aureus: Deep yellow colonies of uniform colour.
 S. saprophyticus and other coagulase negative staphylococci: Yellow

Report the bacterial count as:

Less than 10,000 organisms/ml (104/ml):- Insignificant.
10000–100000/ml (104–105/ml):- Doubtful significance
More than 1,00,000/ml (105/ml):- Significant bacteriuria.

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57
Differences
Difference between Prokaryotic Cells and Eukaryotic Cells
Q. Write difference between Prokaryotic Cells and Eukaryotic Cells? (B.Sc.
Nur.- 2007)

ANSWER
Structure Prokaryotic cells Eukaryotic Cells
Nucleus

Genetic material Free in the cytoplasm Within a membrane-

bound Nucleus
Nuclear membrane Absent Present
Nucleolus Absent Present
Chromosome Absent Present
Deoxyribonucleo- Absent Present
Protein
Nuclear division By Binary fission Mitosis
Extrachromosomal Present in the In mitochondria
DNA cytoplasm
Cytoplasm
Cell wall Present; Imparts Usually absent except
rigidity in fungi
Mitochondria Absent Present
Golgi apparatus Absent Present
Endoplasmic Absent Present
Reticulum
Lysosomes Absent Present
Pinocytosis Absent Present
Protein production site Ribosomes( free in the Rough endoplasmic
cytoplasm) consisting reticulum with
of a 50 S and 30 S ribosomes
subunit consisting of a 60S and
40S subunit

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Chemical structure
Sterol Absent Present
Muramic acid Present Absent
Diaminopimelic acid Usually Present Absent

Difference between Gram positive & Gram Negative cell

wall
Q. Write difference between Gram positive & Gram Negative cell wall?
(B.Sc. Nur.- 2010), (B.Sc. Nur.-2007)

ANSWER
Character Gram positive Gram Negative
Peptidoglycan 50-90% of the dry 5-10% of the dry weight
weight of the cell wall of the cell wall
Thickness Thicker Thinner
Teichoic acid Present Absent
Lipid Absent / Scanty Present
Periplasmic space Absent Present
Lipoprotein Absent Present
Structure Simple Complex
Aromatic & sulphur Absent Present
aminoacid

Difference between flagella and Pili

Q. Write short note on the difference between flagella and Pili?

ANSWER
Character Flagella Pili
Size Large Small
Thickness +++ +
Appearance Straight Never straight
Attached to cell wall No Yes
Origin Cytoplasmic membrane Cell Wall
Organ of movement Yes No
Organ of adhesion No Yes
Required for No Yes
conjugation
Chemical composition Flagellin Pilin

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Difference between Protoplasts and Spheroplasts

Q. Write difference between Protoplasts and Spheroplasts?

ANSWER
Protoplast Spheroplast
Seen in gram positive treated with Seen in gram negative treated with
lysozyme lysozyme EDTA
Consist cytoplasmic membrane and Consist of remnants of complex cell
its contents wall
Metabolize and grow in size but do Multiply by fission or budding
not multiply
Osmatically more sensitive Osmatically less sensitive
Can not revert parent form Can revert parent form
Resemble to L-form Resemble to unstable L- form

Difference between Light and Electron Microscope

Q. Write difference between Light and Electron Microscope?

ANSWER
Character Electron microscope Light microscope
Source Electron Photon
Wavelength 0.005nm 500nm
Resolving power 100,000 times more 100,000 times less
than light microscope than electron microscope
Resolution 0.3-0.5 nm (>100 times) 100 times less
Numerical aperture Small Large
Beam focused by Electromagnets Lenses

Difference between Exotoxin & Endotoxin

Q. Write difference between Exotoxin & Endotoxin. (B.Sc. Nur.-2011), (B.Sc.
Nur.- 2010), (B.Sc. Nur.-2009) , (B.Sc. Nur.-2008) ,(MBBS-2007), (MBBS-
2006), (MBBS-2004)

ANSWER
Properties Exotoxin Endotoxin
Composition Protein Lipopolysaccharide
complex
Effect of heat Heat labile except toxin Heat stable

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of Clostridium
botulinum
Nature Secreted by living cell Integral part of cell wall
Antigenicity Strong Weak
Produced by Gram positive Gram negative bacteria
Some gram negative
Toxicity Highly toxic Moderate toxic
Filterability Filterable Not filterable
Convertibility to toxoid Yes No
Fever Don’t produce Usually produce
Enzyme action Yes No
Pharmacological effect Specific Nonspecific

Differences between T-lymphocyte and B-lymphocyte

Q. Write differences between T-lymphocyte and B-lymphocyte?

ANSWER
T- lymphocyte B-lymphocyte
Mature in thymus Mature in bone marrow
Bring about cell-mediated immunity Bring about humoral immunity
Produce Iymphokines Produce antibodies
Bind to sheep erythrocytes forming Bind to sheep erythrocytes coated
rosettes by CD2 antigen with antibody
and complement fanning rosettes due
to the
presence of C3 receptors on the cell
surface.
Have thymus specific antigen on Do not possess such antigen
their surface
Have CD3 receptor on their surface Have immunoglobulins on their
surface
Viewed under the scanning Have an extensively filamentous
microscope, T- cells are generally surface with
free of cytoplasmic surface numerous microvilli
projections

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Difference between Streptococci pneumoniae and
Streptococci viridans
Q. Write difference between Streptococci pneumoniae and Streptococci
viridans.(B.Sc. Nur.-2011) , (B.Sc. Nur.-2009) , (BDS-2006), (MBBS-2005)

ANSWER
Streptococci Streptococci viridans
pneumoniae
Morphology Capsulated, lanceolate, Non-capsulated, oval or
diplococci (in pairs) round cocci in chains
Colonies on blood agar Initially dome shaped Dome shaped with α—
with α -haemolysis, later haemolysis
'draughtsman' colonies
Colonies in liquid Uniform turbidity Granular turbidity,
medium powdery
Deposit
Bile solubility Positive Negative
Inulin fermentation Positive Negative
Optochin sensitivity Positive Negative
Animal pathogenicity Fatal infection Non-pathogenic

Difference between Acute Rheumatic Fever and Acute

Glomerulonephritis
Q. Write difference between Acute Rheumatic Fever and Acute
Glomerulonephritis?

ANSWER
Acute Rheumatic Acute Glomerulonephritis
Fever
Primary site of Throat Throat or skin
infection
Prior sensitization Essential Not necessary
Immune response Marked Moderate
Complement level Unaffected Lowered
Course Progressive or static Spontaneous resolution
Prognosis Variable Good
Hereditary tendency Present Not known
Penicillin prophylaxis Essential Not indicated

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Difference between free coagulase and bound coagulase enzyme

Q. Write difference between free coagulase and bound coagulase enzyme?

ANSWER
Free coagulase Bound Coagulase
Extracellular It is constituent of cell wall
Secreted into the culture Bound within cell wall
Heat labile Heat stable
Requires cooperation of CRF Independent from CRF
Detected by tube test Detected by slide test

Difference between ELTor and Classic Vibrio

Q. Write difference between ELTor and Classical Vibrio? (MBBS-2006)
ANSWER

Test Classical Vibrio ELTor Vibrio

Haemolysis of sheep Negative Positive

Erythrocytes
Agglutination of chick Negative Positive
Erythrocytes
Voges-Proskauer (VP) Negative Positive
reaction
Polymyxin B sensitivity Positive Negative
Susceptibility to Positive Negative
Mukherjee
Phage IV

Difference between Actinomyces and Nocardia

Q. Write difference between Actinomyces and Nocardia?

ANSWER
Character Actinomyces Nocardia
Acid Fastness Non acid fast Weakly acid fast
Oxygen requirement Anaerobic Aerobic
Culture media Enriched media Ordinary media
Infection Endogenous Exogenous

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Difference between Injectable(IPV) & Oral Polio Vaccine(OPV)
Q. Write difference between IPV and OPV?

ANSWER
IPV OPV
Nature of vaccine Killed Live attenuated
Route of administration Intramuscular Oral
Humoral IgG Present Present
Local IgA Absent Present
Prevention of paralysis Present Present
Reinfection with wild Absent Present
virus
Control of epidemic Absent Present
Commercial production Difficult Easy
Cost Expensive Economical
Shelf life Longer Shorter
Sensitivity to heat Less Extreme
Vaccine associated Absent Present
paralysis

Difference between Fertilised egg and Unfertilised egg

Q. Write difference between Fertilised egg and Unfertilised egg?

ANSWER
Fertilised egg Unfertilised egg
Round and oval More elliptical and longer than a
fertilised egg
Yellow-brown (bile stained) Darker (brownish) in colour (bile
stained).
Consist three layers – The shell is thinner with the outer -
The outer - coarsely mamillated Mamillary coat scanty and irregular.
albuminoid coat,
The middle - a thick transparent
middle layer, and
The inner - lipoidal vitelline
Membrane
Unsegmented ovum with a clear Contains a small atrophied ovum
crescentic area at each pole
Floats in saturated solution of egg does not float in saturated salt
common salt solution

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Differentiation of amoebic and bacillary dysentery

Q. Write difference between amoebic and bacillary dysentery? (B.Sc. Nur.-
2009), (B.Sc. Nur.-2008)

ANSWER
Character Amoebic dysentery Bacillary dysentery
Clinical
Onset Gradual Acute
Fever and No Fever and usually
vomiting vomiting
Faeces (Fresh)
Macroscopic
Odour Offensive Odourless
Nature Blood and mucus with Blood and mucus without
faeces faeces
Reaction Acid Alkaline
Colour Dark red (Altered blood) Bright red (Fresh Blood)
Consistency Not adherent to container Adherant to container
Microscopic
RBC In clump Discrete in rouleaux
Pus Scanty Numerous
Macrophage Nil or very few Large in number
Ghost cells Nil Numerous
Trophozoite Present Nil
Bacteria Present Nil
C.L Crystal Present Nil

Difference between of indirct and direct coombs test

Q. Write difference between of indirct and direct coombs test?

ANSWER
Direct Coombs' test Indirect Coombs' test
Object It detects presence of It detects incomplete
incomplete antibodies antibody present in
adsorbed onto RBC serum.
surface.

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Indications of tests Autoimmune haemolytic Detection of anti Rh

anaemia, antibody in serum of Rh
erythroblastosis foetalis negative pregnant
women of Rh positive
husband
Test procedure Red cells washed in Patient's serum mixed
saline 3 times by with saline washed Rh
centrifugation to remove positive group (or that
serum proteins except of same group of
globulin. patient) red cells and
When washed RBCs incubated at 37 oC for
mixed with Anti human 30 minutes.
gamma globulin in Red cells washed in
presence of bovine saline three times.
albumin clumping of Washed cells mixed
RBCs occur. with Coombs' serum.
Agglutination occurs in
positive cases

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 A
INDEX
Chancroid 135
Actinomycosis 154, 155 Chickenpox 175
Acid fast staining 11 Chikungunya 193
Agglutination63 Chlamydia trachomatis 160
AIDS 200 Cholera 128
Albert’s staining 13, 100 Chromomycosis 211
Amoebic dysentery 222 Clostridium
Amoebic ulcer222 Perfringens 108
Anaerobiosis 115 Tetanus 111
Ankylostoma 263 Botulinum 112
Anthrax 104 Difficile 115
Antigenic Drift 183 Coagulase 87
Antigenic shift 183 Conjugation 37
Arbo viruses 191 Coombs test 66
ART 205 Corynebactrium diphtheria 98
Ascaris lumbricoides 260 Coxeilla 160
Aspergillosis 216 Cryptococcosis 213
Australia Antigen 197 Cultivation of virus 166
Autoclave 27 Cysticercosis 248
B Cytomegalovirus 176
Babes-Ernest granules 99 D
Bacterial growth curve 22 Dark ground microscope 8
Bacteriphage 170 Dengue fever 191
Bence Jones Protein 58 Dengue hemorrhagic fever 192
Biomedical Waste 284 Dengue shock Syndrome 192
Bird flu 184 Dermatophyte 209
Blood culture 124 Diarrhoea 294
Black water fever 236 Disinfectant 31
Bordetella pertusis 136 Diphtheria 98
Brill-Zinsaar disease 158 DNA Probe 42
Bubonic plague 132 Dracunculus medinensis 272
C E
CAMP test 93
Candidiasis 215 Electron microscope 7
Capsule16 Electroimmunodiffusion 62
Capsular swelling reaction 93 Elek’s gel precipitation 100
Casoni test 252 Elephantiasis 270
Cell wall 14 ELISA 60
Cerebral malaria 227 Entamoeba histolytica 219
Enteric fever 122 Hydrophobia 188
Esch. coli 118 Hypersensitivity reaction76
Endemic typhus 158 Cell mediated 76
Epidemic typhus 158 Delayed 76
Epstein-Barr (EB) virus 177 Immediate76
F Humeral 76
Fasciola Hepatica 253 Type I 76
Filariasis 271 Type II 77
Fimbriae 19 Type III 78
Flagella 17 Type IV 79
Flocculation reaction 60 I
Fluorescence microscope 6 Inspissations30
Forsemann antigen 51 Immunity 45
Frei’s test 163 Innate 45
G Acquired 47
Gas gangrene 108 Active 48
Gel diffusion 61 Herd 49
Gene transfer 36 Passive 48
Genetic engineering 41 Immune diffusion 61
Giardia lamblia 223 Immunoelectrophoresis 61
Gram negative 10, 15 Immunoflouroscence 67
Gram positive 10, 15 Immunization 80
Gram’s staining 10 Immunoglobulin 54
Gonorrhoea 94 IgA 56
Griffith typing 89 IgD 57
Guinea worm 272 IgE 58
H IgG 55
HAART 205 IgM 56
HACEK 50 Influenza virus 182
Hapten 50 Inclusion bodies 169
Haemophilus ducreyi 135 J
Hard chancre 147 J chain 56
Herpes virus 173 K
Herpes simplex virus 174 Kala azar 240
Heterophil antigen 51 Kinyoun acid fast stain 13
Hepatitis Viruses 196 Koch’s postules 3
Histoplasmosis 212 Koch’s phenomenon 3
HIV 200 Koplic’s spots 186
Hook worm 263 L
Hot air oven 29 Lancefield classification 89
Human leucocyte antigen 52 Larva migrans 265
Hydatid cyst 250 Cutaneous 265
 Visceral 266 Pasteurization 2, 30
Leeuwenhoek 4 Paul Bunnel test 52, 178
Leishmania 240 PCR 72
Leprosy 144 Phase contrast microscope 7
Lepromin test 145 Penicillosis 217
Leptospirosis 152 Pertusis 136
Louis Pasteur 1 Petroff method 140
Lymphogranulum venerum 162 Pili 19
Lysogenic conversion 38, 103 Plague 132
M Pneumocytosis 218
Major histocompatibility Polio Virus 180
complex 52 Precipitation reaction 59
Malaria 227 Prions 189
MALT 84 Prozone phenomenon 65
Mc Intosh & Field’s Jar 116 Pulse Polio Immunization82, 181
Measles 186 Q
Meningitis 303 Q-fever 160
Montoux test 142 Quellung reaction 93
MOTT 142
Mumps 185 R
Mutation 40 Rabies virus 187
Mycetoma foot 154 RCM 117
Mycobacterium leprae 144 Respiratory infection 311
Mycobacterium tuberculosis 138 Reverse CAMP test 110
N Rhabdovirus 187 r
Nagler’s reaction 109 Rhinosporidiosis 212
National immunization Rickettsiae 158
programme 81 Robert Koch 2
Negri,s bodies 188 Round worm 260
Neisseria 94 RPR 149, 151
Neil Mooser reaction 159 Rubella Virus 194
Neurocysticercosis 248 Rota virus 195
Neural vaccines 83 S
Nocardia 156 Salmonella 122,
Non tuberculous Satallitism 134
mycobacterium 142 Schick test 101
O Schistosomiasis 256
Ophthalmia neonatorum 162 Shigella 121
Opportunistic infection 203 Shingles 175
Opportunistic mycoses 214 Sleeping sick ness 237
P Sporotrichosis 212
Paragonimus 254 Spore 20
Sporulation 21 Rabies 81, 82, 182
Soft chancre Varicella-zoster virus 175
Staphylococci 86 VDRL 149, 150
Sterilization 25 Vibrio cholera 128
STD 306 Volutin granule 99
Streptococci 89 W
Super antigen 53 Weil’s disease 152
Swine flu 182 Weil-felix reaction 52, 158
Syphilis 146 Whooping cough 136
T Widal test 125
Taenia solium 246 Wucheria Bancrofti 268
Taenia sagineta 243 Y
Tissue culture 167 Yersinia pestis 132
Trachoma 162
Transduction 37 Z
Transposon 35 Ziehl-Neelsen Staining 11
Transformation 36 Zone phenomenon 65
Travellers diarrhea 120
Treponema pallidum 146
Trichomonas 225
Trypnosomiasis 237
Tuberculosis 138
Tuberculin test 142
Typhoid fever 123
Typhus fever 158
Tyndalization 31

U
UTI 314
Universal precaution 281
V
Vaccine 80
BCG 80
DPT 81, 101, 112
Killed 81
Live attenuated 80
Measles 80
MMR 80
Polio 80
Sabin 80, 181
Salk 81, 180