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Textbook of Practical Microbiology
Textbook of Practical Microbiology
Textbook of
Practical
Microbiology
AHUJA
2 Textbook of Practical Microbiology
THE AUTHOR
Dr Subhash Chandra Parija MBBS, MD, PhD, FAMS, FICPath, FABMS, FICAI, FISCD and FIMSA is Director-Professor & Head,
Department of Microbiology, in the Jawaharlal Institute of Postgraduate Medical Education & Research, Pondicherry. Prof Parija completed his
MBBS at SCB Medical College, Cuttack, Utkal Unioversity, Orissa in 1977. He obtained his MD (1978-81) in Medical Microbiology from the
Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh. Prof Parija did his PhD from University of Madras in the year
1987 for his work on simple diagnostic methods in amoebiasis.
Prof Parija, after completion of the MD, began his carrier at the JIPMER, Pondicherry as senior resident in the year 1981. Subsequently He
became Professor of Microbiology in the year 1991, and Director-Professor of Microbiology in the year, 2004. During his tenure at JIPMER,
Pondicherry, Prof Parija was sent on deputation by Govt of India to set up and establish the departments of Microbiology & initiate an integrated
Clinical Laboratory Services at the B P Koirala Institute of Health Sciences, Dharan, Nepal, of which he was the founder head between 1995-98.
In recognition of his excellent contribution to the growth and development of the department of Microbiology, as well for the Institute, the
B P Koirala Institute of Health Sciences, Dharan, Nepal, conferred the most prestigious BP Koirala Internal Oration Award.
Prof Parija was awarded WHO fellowships for study of DNA probes, PCR and other molecular biological methods in the study of parasitic
diseases at the University of Aberdeen, UK. Prof. Parija is member of the ICMR task force on intestinal protozoal infections. He is also the
member of the Research Advisory Board of BP Koirala Institute of Health Sciences, Dharan, Nepal; Board of MD Examination in Parasitology,
Colombo University, Sri Lanka, and the visiting Professor of the College of Medicine & Health Sciences, Sultan Quaboos University, Muscat,
Oman.
Author of three books “Text Book of Medical Parasitology”, “Stool Microscopy ” and Sputum Microscopy: a Practical Manual”; editor of a book
“Review of Parasitic Zoonoses” and two monographs “Immunizing agents for tropics: success, failure and some practical issues” and “Kala-azar:
epidemiology, diagnosis and control in Nepal”; Prof Parija also has contributed several chapters for the books, compendium of lectures and
proceedings of scientific meetings. Prof Parija has published more than 137 papers both in the National and International journals of repute. Some
of his papers are quoted in text books and serial publications.
The development of simple, economical and rapid diagnostic tests in serodiagnosis of parasitic diseases such as amoebiasis and cystic
echinococcosis is the main field of his research. Prof Parija was the first to demonstrate excretion of hydatid antigen in urine and its detection for
diagnosis of cystic echinococcosis. He Developed for the first time the carbon-immunoassay (CIA) and staphylococci adherence test (SAT) as
two simple rapid diagnostic methods using a light microscope, and Co-agglutination (Co-A) and CIEP for the detection of antigen in the serum
in the patients with amoebic liver abscess and cystic echinococcosis, and for demonstration of antigen in the hydatid fluid and also in the urine for
the diagnosis of cystic echinococcosis. All these tests can be used in the field or in less equipped laboratories in the developing countries like India.
Prof Parija was first to report the use of LPCB and KOH in the wet mount preparation of stool for detection of intestinal parasites by light
microscopy.
Prof Parija is the recipient of the most prestigious Dr BC Roy National Award 2003 of the Medical Council of India in recognition of his
immense contribution to the development of Medical Microbiology. He was awarded the coveted Dr B P Pandey Memorial Oration Award of the
Indian Society for Parasitology, and BK Aikat Oration Award of the Indian Council of Medical Research for his research in diagnosis and
epidemiology of parasitic diseases. Other awards include Dr S.R.Memorial Award 2003 of the Bombay Veterinary College Alumni Association,
Dr SC Agarwal Oration Award 2001 of the Indian Association of Medical Microbiologists, Major General Saheb Singh Sokhey Award 1992 of the
Indian Council of Medical Research, Third Dr Datta Memorial Award 1999 of the Indian Association for the Advancement of Veterinary
Parasitology, IAPM (Orissa chapter) Oration Award 1993 of the Indian Association of Pathologists & Microbiologists (Orissa Chapter), Smt
Kuntidevi Malhotra Award 1990 of the Indian Association of Pathologists & Microbiologists, Dr S S Misra Memorial Award 1987 of the National
Academy of Medical Sciences, Young Scientist Award 1986 of the Indian Association of Medical Microbiologist and Best Scientific Paper Award 1987
of the JIPMER Scientific Society.
Professor Parija is the editor-in chief of the online journal Internet Journal of Parasitic Diseases, and member of the editorial editorial boards of
various journals both International (Parasitology International, BMC Infectious Diseases, BMC Clinical Pathology and Health Renaissance) and
National (Indian Journal of Medical Microbiology, Journal of Veterinary Parasitology, Journal of Parasitic Diseases and Indian Journal of Pathology &
Microbiology) .
He has been conferred with various fellowships such as FAMS, FICPath, FABMS, FICAI, FISCD and FIMSA by professional bodies. Prof
Parija has visited many countries, delivered invited talks in infectious diseases at universities, institutions conferences, seminars etc, and has
chaired scientific sessions in the international as well as national conferences. He has guided both MD and PhD students and has been examiner
for MBBS, MD and PhD of various universities of India and abroad.
Prof Parija was the former Secretary of the Indian Association of Medical Microbiologists and National Vice President of the Indian Association
of Pathologists and Microbiologists. He is currently the National Vice President of the Indian Association for Development of Veterinary Parasitolo-
gists . He is also life member and member of the executive council of many national and international scientific organizations.
1 / Running Head 1
Textbook of
Practical Microbiology
2 Textbook of Practical Microbiology
1 / Running Head 3
Textbook of
Practical Microbiology
Ahuja Publishers
Bangalore, New Delhi
4 Textbook of Practical Microbiology
Printed in India
ISBN
Published by
Ahuja Publishers
1 / Running Head 5
To my mother
Contents
Preface xi
Acknowledgments xii
Introduction 2
1 Compound Microscope 3
2 Darkground Microscopy 7
3 Measurement of Microorganisms 9
4 Hanging drop Preparation 11
5 Isolation of Pure Cultures 14
6 Simple Staining 20
7 Gram’s Staining 23
8 Acid Fast Staining 27
9 Albert’s Staining 31
10 Capsule Staining 34
11 Spore Staining 37
12 Negative Staining 40
20 Catalase Test 62
21 Oxidase Test 65
22 Coagulase Test 68
23 Urease Test 71
8 Textbook of Practical Microbiology
24 Indole Test 74
25 Methyl Red Test 76
26 Voges-Proskauer Test 78
27 Citrate Utilization Test 80
28 Triple Sugar Iron Agar (TSI) Test 82
29 Hydrogen Sulphide Test 85
30 Nitrate Reduction Test 88
31 Kirby-Bauer Method 92
32 Stoke’s Method 95
33 Agar Dilution Method 97
34 Broth Dilution Method 100
35 Epsilometer Test (E-test) 102
Introduction 106
36 Bacterial Agglutination Test 108
37 Blood Grouping 110
38 Latex Agglutination Test 112
39 Co-agglutination Test 114
40 Widal Test 116
41 Weil Felix Test 119
42 Anti-Streptolysin O (ASLO) Test 121
43 VDRL Test 123
44 Radial Immunodiffusion Test 126
45 Immunoelectrophoresis Test 128
46 Counter-current Immunoelectrophoresis Test 130
47 Indirect Haemagglutination Test 132
48 Immunofluorescence Test 135
49 Enzyme-linked Immunosorbent Assay 138
Introduction 144
50 Isolation of Plasmids 145
51 Polyacrylamide Gel Electrophoresis 148
52 Isolation of Antibiotic Resistant Mutant 152
53 Bacterial Conjugation 155
Introduction 188
64 Saline Wet Mount of Stool 189
65 Iodine Wet Mount of Stool 192
66 LPCB Wet Mount of Stool 195
67 Acid-fast Staining of Stool Smears 198
68 Leishman’s staining of Peripheral Blood Smears 201
69 Concentration of Stool for Parasites 205
70 Culture of Stool for Entamoeba histolytica 208
Introduction 212
71 Cultivation of Fungi 213
72 Gram’s Staining for Fungi 215
73 Lactophenol Cotton Blue (LPCB) Wet Mount of Fungi 217
74 Potassium Hydroxide Wet Mount of Fungi 219
75 Indian Ink Wet Mount Preparation 221
76 Slide Culture 223
77 Germ Tube Test 225
78 Urease Test 227
79 Carbohydrate Assimilation Test 229
80 Carbohydrate Fermentation Test 231
81 Identification of Common Fungi 233
Index 295
10 Textbook of Practical Microbiology
1 / Running Head 11
Preface
Textbook of Practical Microbiology, is a performance–based text designed for use by students of medicine,
microbiology, medical laboratory technology, allied sciences; by laboratory workers and by others who are interested
in study of practical microbiology.
The intent of the book is to provide recent information and explain in detail the routine diagnostic methods performed
in a Microbiology laboratory. Every effort has been made to incorporate all aspects of practical microbiology. A
sincere effort is made to provide the essential underlying principles of practical microbiology, to help students to
perform various practicals, and to learn and apply the knowledge of practical microbiology in clinical medicine.
Textbook of Practical Microbiology consists of 15 learning units. Each unit contains many practical exercises.
Each exercise contains learning objectives, theoretical aspects of the practical, principle of the test, and experimental
procedure in detail. Important points of the practical experiment are highlighted, possible questions with answers are
provided and finally, useful additional informations are provided as box items. The book is profusely illustrated with
diagrams, and photomicrographs both black and white, and colour.
I owe a special debt of profound gratitude to my mother late Smt. Nishamani Parija and father Shri Mana Govinda
Parija without whose encouragement the book would not have been possible.
I welcome readers views and suggestions for further improvement of the book in future editions.
Acknowledgements
I gratefully acknowledge all my colleagues and friends for their valuable advice and assistance in preparation of the
manuscript. It is my pleasure to thank my niece Er(Ms) Kukumina Parija, son-in-law Er. Subhasis Ray, nephew Er.
Rajkumar Parija, daughter-in-law Mrs Smriti Parija, and daughters Dr. Madhuri Parija and Miss Mayuri Parija for
their untiring secretarial help towards the preparation of the manuscript.
I am very much thankful to Ahuja Publishers, New Delhi who have been very supportive of this venture.
I
Microscope and Basic
Microbiological Techniques
Introduction
Lesson 1 Compound Microscope
Lesson 2 Darkground Microscopy
Lesson 3 Measurement of Microorganisms
Lesson 4 Hanging Drop Preparation
Lesson 5 Isolation of Pure Cultures
2
UNIT
Introduction
Microscope is the instrument, the most important characteristic of microbiology laboratories. The
magnification, it provides, enables us to see microorganisms and their structures otherwise invisible
to the naked eye. The magnitudes attainable by microscopes range 100X- 400000X. A microscope
may be defined as an optical instrument, consisting of a lens or a combination of lenses, for making
enlarged or magnified images of minute objects. (Micro: small; scope: to view).
Antony Von Leeuwenhoek is considered to be the first person who has seen a micro organism
through a simple microscope made by him with a magnification of 270-480 times. He described the
size, shape, movements of bacteria, protozoa and algae. These findings were later confirmed after
the development of compound microscope by Robert Hooke. The characteristic morphological
studies enabled by the discovery of powerful microscopes, helped the scientists to classify
microorganisms. Later improvements in the compound microscopes were made and Amici
discovered oil immersion lens, which enabled the scientists to study the characteristics more minutely.
Microscopes are continuously improved to enable us to have higher magnifications and better
resolutions.
Microscopes are of two categories: Light or optical microscopes and Electron microscopes
depending upon the principle on which the magnification is based. Light microscopy, in which the
magnification is obtained by a system of optical lenses uses light waves .The light microscopy
includes bright field microscopy, dark-field microscopy, fluorescence microscopy and phase contrast
microscopy. On the other hand the electron microscopy uses a beam of electrons in place of light
waves for visualization of objects .This includes transmission electron microscopy and scanning
electron microscopy.
Textbook of Practical Microbiology 3
LESSON
Compound Microscope
1
LEARNING OBJECTIVES Microscope stand
After completing this practical you will be able to: It is the main framework of the microscope. It consists of :
1 Become familiar with the principle, various parts of the a Main tube.
compound microscope and its usage in microbiology b Body tube.
laboratory. c An arm, which supports the main tube, body tube and the
2 Visualize the cellular morphology from stained slide stage.
preparation by using compound microscope. d A substage, and
e A foot or base upon which the whole instrument rests.
INTRODUCTION Main tube: The main tube primarily holds the objective and
eyepiece. The eyepiece, also known as ocular piece, is present
at the top of the main tube. The eyepiece is fitted loosely into
The commonly used microscope in microbiology laboratory is the upper end of the tube. This has a standard diameter so that
called compound microscope. In compound microscopy, the all the eyepieces are interchangeable. The lower end of the
microscopic field or area observed is brightly lighted and the tube contains objectives, which are screwed into what is known
objects being studied appear dark because they absorb some as a revolving nosepiece. A number of objectives of lenses of
of the light. Ordinarily microorganisms do not absorb much different magnifications are screwed into the nosepiece of the
light but staining them with a dye greatly increases their light microscope. These objectives can be revolved to increase or
absorbing ability resulting in greater contrast and color decrease the magnification of specimen being examined.
differentiation. Generally microscopes of this type produce a
useful magnification of about 1000x to 2000x. At magnifications Body and arm: The tube is attached to the microscope by the
greater than 2000x, the image becomes hazy. These component of the microscope called the body. The body of the
microscopes are provided by a coiled filament tungsten lamp. microscope and the tube attached to it are supported at the
The glowing filaments are prevented from causing glare by correct height by firm arm, which may also provide a lifting
focusing their light on the sub-stage condenser, rather than on handle for the microscope.
the object. This is known as Köhler illumination. Closing the
aperture of the condenser slightly may aid in detection of certain Substage: The substage lies immediately below the stage. This
organisms such as protozoa, fungi, etc. because the light will holds a condenser lens with an inbuilt diaphragm and a holder
be hitting the edges of the object at a sharper angle, increasing for light filter and stops.
contrast.
Foot: The microscope rests firmly on the laboratory bench
with the base called foot. This may be U-shaped or rectangular.
Parts of the compound microscope
Stage: A fixed platform with an opening in the center allows for
A microscope mainly consists of the passage of light from an illuminating source below to the
1 Microscope stand. lens system. It provides surface for the placement of a slide
2 Stage, and over the central opening. Stage can be of fixed or mechanical.
3 Microscope optics. Mechanical stage can be moved vertically or horizontally by
4 Compound Microscope
means of rack and pinion movement. Stage also contains clips The light source
on its surface to hold the slide.
A good source of light is needed to examine specimens
Microscope optics correctly. This may be daylight or electric light. Whatever the
source of light, it should fill field of view. It should also fill the
These include objectives, eye pieces, condenser and whole of the back lens of the objective regularly with light, if
illuminating source. not the image will not be clear. While using electric light, a blue
filter is placed between the source of illumination and the
Mechanical adjustment of a microscope substage condenser. In some other microscopes, one may find
that mirrors are also provided with artificial light. In such case,
It is being carried out to focus the specimen examined by the the flat side of the mirror should be used. When daylight is the
microscope. This adjustment includes coarse and fine focusing natural source of light, the concave mirror should be used
adjustments and condenser adjustments. without the substage condenser.
brings the real image to focus at the plane of the diaphragm. This examine for higher magnification. Focus the 100X objective
is within the focal length (f) of the eye lens. Then the eye lens using the fine adjustment. The condenser may be raised
produces the virtual magnified image that is seen by the eye. completely upward for obtaining better illumination.
12 Put off illumination and carefully clean all objective lenses
Importance of numerical aperture and eyepieces with lens paper.
13 Replace the microscope in its box.
The numerical aperture of a microscope objective is a measure
of its ability to gather light and resolve fine specimen detail at
a fixed object distance. All modern microscope objectives have OBSERVATIONS
the numerical aperture value inscribed on the lens barrel, which
allows determination of the smallest specimen detail resolvable Carefully observe the morphology and colour (after staining)
by the objective and an approximate indication of the depth of of bacteria present in the smear and also for uniformity in
field (Box 1-1). staining, size, shape, Gram’s reaction arrangement.
KEY FACTS
1 One should remember that proper use and care increases the life of a microscope many fold.
2 Microscope should be kept away from dust, moisture and direct sunlight.
3 After finishing work a cover should be put on the microscope.
4 Care must be taken while handling different parts of microscope.
5 Examination of the slide should always begin with the low power objective (10x).
6 Attempt should never be made to repair microscope by oneself.
VIVA
1 Classify microscopes.
2 What is Köhler illumination?
Ans Compound microscopes are provided by a coiled filament tungsten lamp. The glowing filaments are prevented from
causing glare by focusing their light on the sub-stage condenser, rather than on the object. This is known as Köhler illumination.
3 List the parts of a compound microscope.
4 Define magnification, numerical aperture and resolution.
FURTHER READINGS
1 Brooks GF, Butel JS and Morse SA. Jawetz, Melnick and Adelberg. Medical Microbiology. 23rd ed. (McGrow Hill, USA.) 2004.
2 Duddington CL. The Microscope. (Museum Press, London) 1964.
3 Evans EGV, Killington RA, Heritage J. Introductory Microbiology. (Cambridge University Press, London.) 1996.
4 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC (Eds.). Color Atlas and Textbook of Diagnostic Microbiology.
5th ed. (Lippincott Williams and Wilkins, USA.) 1997.
Textbook of Practical Microbiology 7
LESSON
Darkground Microscopy
2
LEARNING OBJECTIVES PROCEDURE
After completing this practical you will be able to: 1 Take a clean lens wiping paper and carefully clean all the
lenses.
1 Observe the motility of microorganisms by dark ground 2 Place a drop of oil on the depression present on top lens of
microscopy. the condenser.
3 Keep the wet mount preparation on the stage.
4 Raise the condenser slowly so that oil touches the bottom
INTRODUCTION of the slide.
5 Place the low power objective in position in the light path
Visibility of an object by a microscope depends upon contrast and focus.
between the object and its background. This can be improved 6 Using the centering screws adjust the light to fall on the
by dark ground microscopy than a compound microscope. center of the microscopic field. The bright spot of the light
must be in the center of the field. If a bright spot is not
obtained, slightly raise or lower the condenser to get the
PRINCIPLE bright spot.
7 Place the dry high power objective in the light path, focus
In dark-ground microscopy, the object under examination is and examine the slide.
illuminated not directly but very obliquely (Box 2-1). This is 8 Remove the high power objective from the light path. Place
done by opening the aperture of the condenser completely a drop of oil carefully over the cover slip.
and inserting a funnel stop below the condenser, hence all 9 Now place the oil immersion objective in the light path,
rays from the condenser are made to pass outside the objective. focus and observe the slide.
The rays are reflected or diffracted by the object in the specimen. 10 Observe the entire field for the motile bacteria, and also
Hence field appears dark and object is illuminated. observe the type of motility of bacteria.
11 Record the observations in the note book.
REQUIREMENTS
OBSERVATIONS
I Equipments
Compound light microscope with dark ground condenser. Brightly illuminated motile bacteria are observed under dark
background.
II Reagents
Cedar wood oil, lens wiping paper.
RESULTS AND INTERPRETATION
III Specimen
Freshly prepared wet mount preparation of bacteria suspension. The wet mount preparation shows motile bacteria.
8 Darkground Microscopy
KEY FACTS
1 Most of the dark ground condensers have fixed focus and must be use with thin slides (1 mm thick) and cover slips (0.1 mm
thick).
2 Oil must be used below and above the slide.
3 The wet mount preparation must be thin and should not be dense.
Dark ground microscopy involves the alteration of microscopic technique rather than the use of dyes or stains to achieve the
contrast. By the dark field method, the condenser does not allow light to pass directly through the specimen but directs the
light to hit the specimen at an oblique angle.
Only light that hits objects, such as microorganisms in the specimens will be deflected upward into the objective lens for
visualization. All other light that passes through the specimen will miss the objective, thus making the background a dark field.
VIVA
Dark ground microscope also is commonly used for demonstrating motility of the trophozoites of protozoa such as Trichomonas
vaginalis, Entamoeba histolytica, etc.
FURTHER READINGS
1 Brooks GF, Butel JS and Morse SA. Jawetz, Melnick and Adelberg. Medical Microbiology. 23rd ed. (McGrow Hill, USA.) 2004.
2 Duddington CL. The Microscope. (Museum Press, London) 1964.
3 Evans EGV, Killington RA, Heritage J. Introductory Microbiology. (Cambridge University Press, London.) 1996.
4 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC (Eds.). Color Atlas and Textbook of Diagnostic Microbiology. 5th
ed. (Lippincott Williams and Wilkins, USA.) 1997.
Textbook of Practical Microbiology 9
LESSON
Measurement of
3 Microorganisms
LEARNING OBJECTIVES counting the number of spaces occupied by the organism and
second by multiplying this number by the calculated calibration
After completing this practical you will factor for one ocular division.
III Specimen
PRINCIPLE Heat fixed smear of Escherichia coli
KEY FACTS
1 Carefully clean the eyepiece and objective lenses before starting experiment.
2 Expert technician help must be taken for inserting ocular micrometer.
3 There must be proper coincidence between the lines of stage micrometer and ocular micrometer.
VIVA
1 Explain the method for the measurement of the size of a microorganism by microscopy.
2 Describe the principle involved in the measurement of the size of microorganisms by microscopy.
FURTHER READINGS
1 Brooks GF, Butel JS and Morse SA. Jawetz, Melnick and Adelberg. Medical Microbiology. 23rd ed. (McGrow Hill, USA.) 2004.
2 Duddington CL. The Microscope. (Museum Press, London) 1964.
3 Evans EGV, Killington RA, Heritage J. Introductory Microbiology. (Cambridge University Press, London.) 1996.
4 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC (Eds.). Color Atlas and Textbook of Diagnostic Microbiology. 5th
ed. (Lippincott Williams and Wilkins, USA.) 1997.
Textbook of Practical Microbiology 11
LESSON
Hanging Drop
4 Preparation
LEARNING OBJECTIVES Spirochaetes are the examples of bacteria which are motile
but without presence of any external flagella .
After completing this practical you will be able to: The list of motile and non-motile bacteria are summarized in
the table 4-1.
1 Observe motility, and also observe natural sizes and shapes
of the cells by hanging drop preparation.
REQUIREMENTS
INTRODUCTION
I Equipments
Hanging drop preparation is one of the easiest method to Compound light microscope.
observe motility in a clinical microbiological laboratory. This is
carried out by putting a loop full of bacterial suspension on II Reagents and glass wares
the cover slip and placing it over a cavity slide and observing Normal saline, inoculating loop wire, staining tray, cavity slides
it under a microscope. Advantage of this method is that by and cover slips.
this method live bacteria can be observed. Examination of
living organisms is useful to observe cell activities, viz. III Specimen
motility, binary fission, and also to observe natural sizes and Log phase broth culture of E. coli
shapes of the cells.
Motility of bacteria can also be demonstrated by i. Craigie ’s
tube method , ii. swarming of the bacteria on a non inhibitory PROCEDURE
medium ( e.g, blood agar ) and iii. by dark ground microscopy.
Capillary tube method is a useful method for demonstrating 1 Take a clean grease free cavity slide.
the motility of anaerobic bacteria (Box 4-1). 2 Take a clean coverslip, apply paraffin to four corners of
coverslip.
3 Place a drop of broth culture on the coverslip with the help
PRINCIPLE of inoculating loop.
4 Place the cavity slide (cavity down) over the coverslip so
Microorganisms such as bacteria, because of their small size that the drop is placed in center.
and a refractive index that closely approximates that of water, 5 Invert the slide, and observe under microscope.
do not lend themselves readily to microscopic examination in a 6 First observe under low power (10x), locate the edge of the
living, unstained state. drop, shift the focus to high power (40x) and observe.
Bacteria are motile due to the presence of flagella .Depending 7 Record the observation in a notebook.
on the location of the attachment of the flagella , bacteria can
be classified as i. monotrichous (single polar flagellum at one
end) e.g, Vibrio cholerae , ii. amphitrichous (single flagellum QUALITY CONTROL
at both the ends) e.g Pseudomonas aeruginosa,
iii. lophotrichous (tufts of flagella at one or both ends ) e.g, The test wet mount preparation is compared with known motile
Spirilla, and iv.peritrichous ( fagella arranged all around the culture of P. aeruginosa for appropriate details such as motility,
cell) e.g, Escherichia coli, Salmonella sp , etc . and natural sizes and shapes of the bacteria.
12 Hanging drop Preparation
Capillary tube method is a useful method for demonstrating the motility of anaerobic organisms. A Robertson’s cooked meat
medium or thioglycollate broth culture, inoculated and incubated with anaerobic bacteria is taken. A broth culture of anaerobic
bacteria is prepared and is made to fill into the open capillary tube by capillary action. Once the tube is filled up, it is sealed at
both ends with clay to maintain anaerobiosis. The tube is then observed, under the high power objective of the light microscope
for the presence of motile anaerobic bacteria.
KEY FACTS
VIVA
FURTHER READINGS
1 Brooks GF, Butel JS and Morse SA. Jawetz, Melnick and Adelberg. Medical Microbiology. 23rd ed. (McGrow Hill, USA.) 2004.
2 Duddington CL. The Microscope. (Museum Press, London) 1964.
3 Evans EGV, Killington RA, Heritage J. Introductory Microbiology. (Cambridge University Press, London.) 1996.
4 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC (Eds.). Color Atlas and Textbook of Diagnostic Microbiology.
5th ed. (Lippincott Williams and Wilkins, USA.) 1997.
14
LESSON
Isolation of
5 Pure Cultures
III Specimen colony to a fresh agar plate with the help of sterile loop.
Mixed bacterial culture (24-48 hour nutrient broth cultures), Repeat the spread plate procedure.
exudates, stool and other clinical specimens. 11 After incubation confirm the purity of isolated colony.
1 Take 2 trypticase soy agar plates and mark as test and control. For streak plate method
2 Take L shaped 2 glass rods, dip the lower bent portion into
70% ethanol. 1 Observe all the plates for discrete colonies after 24, 48 and
3 With a sterile pipette, transfer 0.1 ml of culture from tube 1 to 72 hours after first incubation.
the first agar plate that has been placed on the turntable. 2 Search the entire plate for colonies present outside the streak
Replace the cover. lines, if so discard the plate and repeat the experiment.
4 Remove the glass rod from ethanol and pass it through the
Bunsen burner flame, with the bent portion of the rod
pointing downward to prevent the burning alcohol from For spread plate method
running down to arm. Allow the alcohol to burn off the rod
completely. Cool the rod for 10-15 seconds. 1 Observe the control and test plates after 24, 48 and 72 hours
5 Remove the Petri dish cover and spin the turntable. after first incubation.
6 While the turntable is spinning, lightly touch the sterile bent 2 Note the results and colony morphology of different
rod to the surface of the agar and move it back and forth. colonies.
7 Remove the glass rod, dip in alcohol and re flame.
8 Stop the turntable. Replace the cover on agar plate.
For pour plate method
9 Remove the agar plate from turntable, and incubate in an
inverted position at 37°C for 48-72 hours.
1 Check the control plates for isolated similar looking colonies
10 Carefully observe the colonies and transfer a well-discreet
only.
16 Isolation of Pure Cultures
2 Note the colony characters of well- discrete colony of all For spread plate method
types of colonies (after both incubations).
3 Preserve the colonies for further characterization. 1 Check the control plates for isolated same colonies only.
2 Note the colony characters of well- discrete colony of all
types of colonies (after both incubations).
RESULTS AND INTERPRETATION 3 Preserve the colonies for further characterization.
VIVA
Chemical methods
Use of a special carbon or nitrogen source
Use of dilute media
Use of inhibitory or toxic chemicals
Physical methods
Heat treatment
Incubation temperature
pH of the medium
Biological methods
Animal experiments
A culture that contains only one type of microorganisms is known as pure culture. Pure culture is also called axenic culture.
Separation of particular microorganism from a mixed population that exists in nature is called isolation.
KEY FACTS
FURTHER READINGS
1 Brooks GF, Butel JS and Morse SA. Jawetz, Melnick and Adelberg. Medical Microbiology. 23rd ed. (McGrow Hill, USA.) 2004.
2 Duddington CL. The Microscope. (Museum Press, London) 1964.
3 Evans EGV, Killington RA, Heritage J. Introductory Microbiology. (Cambridge University Press, London.) 1996.
4 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC (Eds.). Color Atlas and Textbook of Diagnostic Microbiology.
5th ed. (Lippincott Williams and Wilkins, USA.) 1997.
18
Textbook of Practical Microbiology 19
UNIT
II
Bacterial Staining
LESSON
Simple Staining
6
LEARNING OBJECTIVES Preparation of polychrome methylene blue: This is prepared
by allowing Loeffler’s methylene blue to ‘ripen’ slowly and
After completing this practical you will be able to: shaking it at intervals to aerate the contents thoroughly. The
slow oxidation of the methylene blue forms a violet compound
1 Stain smears by simple staining method. that gives the stain its polychrome properties. The ripening
2 Compare various morphological forms and arrangement of takes 12 months or more to complete. The stain maybe ripened
bacterial cells. quickly by addition of 1% potassium carbonate to the stain.
This stain gives the acidic cell structures of the bacteria, a
red-purple hue.
INTRODUCTION
III Specimen
Simple staining employs staining of bacterial smears with a Pus, CSF, aspirations.
single staining reagent. The commonly used simple stains are
the basic stains such as methylene blue, crystal violet and
carbol fuchsin. They provide good colour contrast and impart PROCEDURE
the same colour to the stained bacteria (Box 6-1).
1 Make a thin exudate smear on a slide.
2 Heat fixes the smear by passing the slide 2–3 times gently
PRINCIPLE over the Bunsen flame with the smear side up.
3 Pour Loeffler’s methylene blue over the smear and allow it to
In simple staining, the bacterial smear is stained with a single stand for 3 minutes.
reagent. Basic stains with chromophore are used in simple 4 Wash the stained smear with water and air dry it.
staining methods.Bacterial nucleic acids and certain cell wall 5 Observe the smear first under low power (10x) objective, and
components carry a negative charge that strongly attracts and then under oil immersion (100x) objective.
binds to the cationic positively charged chromogen, and imparts 6 Record the observations in the note book.
same colour to all bacteria.
QUALITY CONTROL
REQUIREMENTS
The morphology of the cellular components and bacteria in the
I Equipments known methylene blue stained smear (positive control smears)
Compound light microscope. are compared with that of the blue stained structures in the test
II Reagents and glass wares smear.
Loeffler’s methylene blue, polychrome methylene blue, carbol
fuchsin, water, microscopic glass slides and Bunsen burner.
Preparation of Loeffler’s methylene blue stain: This is OBSERVATIONS
prepared by dissolving 30 ml of saturated solution of methylene
blue in alcohol, in 100 ml of distilled water containing 0.1 ml of 1 Blue coloured spherical cells, approximately 0.5–1 µm size
potassium hydroxide (0.01%). seen arranged in clusters (Fig. 6-1).
Textbook of Practical Microbiology 21
Loeffler’s methylene blue: It demonstrates morphology of polymorphs, lymphocytes and other cells more clearly.
Polychrome methylene blue: It demonstrates the presence of bacteria in smear or wet mount preparation in addition to the
morphology of polymorphs and lymphocytes. It is also employed to demonstrate the acidic capsular material of the anthrax
bacilli as seen in the Mc Fadyean reaction.
KEY FACTS
VIVA
FURTHER READINGS
1 Evans EGV, Killington RA, Heritage J. Introductory Microbiology. (Cambridge University Press.) 1996.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5 th ed.
(Lippincott Williams and Wilkins.) 1997.
4 WHO. Guidelines on Standard Operating Procedures for Microbiology. Chapter 4: Staining Techniques.
5 WHO. Manual of Basic Techniques for a Health Laboratory, 1980.
Textbook of Practical Microbiology 23
LESSON
Gram’s Staining
7
LEARNING OBJECTIVES In the case of Gram-negative bacteria, the alcohol, being a lipid
solvent, dissolves the outer lipopolysaccharide membrane of
After completing this practical you will be able to: the cell wall and also damages the cytoplasmic membrane to
which the peptidoglycan is attached. As a result, the dye-iodine
1 Stain smears by Gram’s staining method. complex is not retained within the cell and permeates out of it
2 Differentiate between Gram positive and Gram negative during the process of decolourisation. Hence when a counter
bacteria. stain is added, they take up the colour of the stain and appear
pink.
Different modifications of Gram’s stainings are summarized
INTRODUCTION in the box7-1.
III Specimen Note: Decolourisation is a critical procedure. Hold the slide with
hand on one corner in a slant position and add drop by drop
Preparation of bacterial smear: (From liquid culture) of absolute alcohol till a faint colour comes out of the smear.
1 Take clean, and grease free glass slides for making the smears. 6 Rinse the smear again gently under tap water.
Note: Wipe the slide with a cotton swab dipped in alcohol 7 Cover the smear with dilute carbol fuchsin (counter stain)
and then holding its end with forceps roast it free from grease for 30 seconds to 1 minute.
by passing 6 – 12 times through a blue Bunsen flame. 8 Rinse the smear again gently under tap water and air dry it.
2 Take one or two loopfuls of the bacterial cell suspension 9 Observe the smear first under low power (10x) objective, and
and place on the clean slide with a bacteriological loop. then under oil immersion (100x) objective.
3 Then with circular movement of the loop, spread the cell 10 Record the observations in the note book.
suspension into a thin area. Note: List of Gram positive and Gram negative bacteria are
4 Allow the smear to air dry. summarized in the table 7-1.
5 Heat fix the smear while holding the slide at one end, and by
quickly passing the smear over the flame of Bunsen burner
two to three times. QUALITY CONTROL
Preparation of bacterial smear: (From solid media) On the same slide, smears of Staphylococcus aureus (Gram
1 Take clean and grease free glass slides for making the smears. positive bacteria) and Escherichia coli (Gram negative bacteria)
2 Take a loopful of 0.85% saline and place it on the centre of are made. The slide with control and test smears is stained by
the slide. Gram’s staining. The appearance of purple coloured Gram
3 With a straight wire touch the surface of a well isolated positive bacteria and pink coloured Gram negative bacteria in
colony from the solid media and emulsify in the saline drop the control smears indicate proper staining technique and
forming a thin film. stained test smear is compared with it.
4 Allow the smear to air dry.
5 Heat fix the smear while holding the slide at one end, and by OBSERVATION
quickly passing the smear over the flame of Bunsen burner
two to three times. Presence of 0.5–1 µm sized purple coloured spherical bacteria
arranged in clusters ( Fig. 7-1) and 1–2 mm sized pink coloured
rod shaped bacteria seen ( Fig. 7-2).
PROCEDURE
1 Cover the smear with the methyl violet (basic dye). Allow it RESULTS AND INTERPRETATION
to stand for one minute.
2 Rinse the smear gently under tap water. The stained smear contains mixture of Gram positive cocci
3 Cover the smear with Gram’s iodine (mordant) and allow it to arranged in grape-like clusters (eg. Staphylococcus species)
stand for one minute. and Gram negative bacilli (eg. E. coli).
4 Rinse the smear again gently under tap water.
5 Decolourise the smear with 95% alcohol. Various uses of Gram’s staining are summarized in the box 7-2.
FIGURE 7-1 Gram stained smear of Staphylococcus aureus, x 1000. FIGURE 7-2 Gram stained smear of Gram negative bacilli, x 1000.
1 Urine specimens
Identifies urine specimens that contain bacteria greater than 105 cfu/ml of urine.
Presence of at least 1 organism/ oil immersion field correlates with significant bacteriuria ( > 105 cfu/ml).
Presence of polymorphonuclear (PMN’s) in uncentrifuged urine correlates with number of PMN’s excreted per hour. In
patients with >400,000 PMN excreted into urine/hour are likely to be infected.
2 Stool specimens
Useful to detect
Candida.
Clostridium difficile.
3 Sputum specimens
Useful to detect
Streptococcus pneumoniae.
4 Pus specimen
Useful to detect
Staphylococcus aureus in pus from abscesses.
Streptococcus species in exudates from tonsillitis, pharyngitis and impetigo.
Gram negative rods in pus from cases of chronic otitis media and post operative wound infections.
26 Gram’s Staining
KEY FACTS
1 Gram staining is a differential stain, most commonly employed for diagnostic identification of bacteria in clinical specimens.
2 Gram staining differentiates bacteria into two categories: purple coloured Gram positive and pink coloured Gram negative
bacteria.
3 Tissue cells, leucocytes and the debris of inflammatory exudates all stain pink in Gram’s stained smears.
4 Gram positive bacteria have a thicker and denser peptidoglycan layer in the cell walls, which make them less permeable to the
stain, in comparison to those of the Gram negative bacteria.
5 The bacterial smear should not be overheated during heat fixation or over decolourised with alcohol, as it can cause in Gram
positive bacteria losing the dye-iodine complex and appearing Gram negative.
6 The age of culture can also influence the Gram stain reaction i.e. cultures more than 24 hours old can lose ability to retain
dye-iodine complex and appear pink coloured Gram-negative.
VIVA
1 Define differential stain. What are the other differential stains used for staining?
Ans. Differential stain is defined as those that contain two different coloured dyes, such that when the staining is completed,
different structures will take up different colours and can be differentiated.
The other differential stain that can be used for staining is acid fast stain and Albert’s stain.
3. Describe differences between a Gram positive and Gram negative cell wall.
Ans. The important differences between Gram positive and Gram negative cell wall are as follows:
i) Gram positives have a thick peptidoglycan layer in the cell wall when compared to Gram negatives which possess a
thinner peptidoglycan layer.
ii) Teichoic acid is present in Gram positive cell wall while it is absent in Gram negative cell wall.
iii) Lipopolysaccharide is absent in Gram positive cell wall and present in Gram negative cell wall.
4 Describe the conditions, which may result in a Gram-positive bacteria turning as Gram negative.
Ans. The conditions that can result in Gram positive bacteria appearing Gram negative are:
i) Excess decolourisation by ethanol/ acetone.
ii) Loss of cell wall due to action of lysozyme, penicillin, changes in the pH and aged cultures.
5 Give 2 examples each of Gram positive and Gram negative cocci and bacilli.
FURTHER READINGS
1 Evans EGV, Killington RA, Heritage J. Introductory Microbiology. (Cambridge University Press.) 1996.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th ed.
(Lippincott Williams and Wilkins.) 1997.
4 WHO. Guidelines on Standard Operating Procedures for Microbiology. Chapter 4: Staining Techniques.
5 WHO. Manual of Basic Techniques for a Health Laboratory, 1980.
Textbook of Practical Microbiology 27
LESSON
Acid-Fast Staining
8
LEARNING OBJECTIVES stain (methylene blue) appearing blue, while the acid-fast cells
retain the red colour of primary stain (carbol fuchsin).
After completing this practical you will be able to :
Different modifications of acid-fast staining is summarized in
1 Stain smears by acid-fast staining method. the box 8-1.
2 Become familiar with the chemical basis of the acid-fast stain
REQUIREMENTS
INTRODUCTION
I Equipments
The acid fast staining method is a modification of Ehrlich’s Compound light microscope.
(1882) method. Ehrlich suggested the method for the differential
staining of tubercle bacilli and other acid-fast bacilli with aniline- II Reagents and glass wares
gentian violet followed by strong nitric acid. The ordinary Bunsen flame/ torch soaked in methylated spirit, loop wire,
aniline dye solutions do not readily penetrate the substance of glass slides, slide rack, strong carbol fuchsin, acid-alcohol (3
the tubercle bacillus and therefore is unsuitable for staining. ml HCl + 97 ml ethanol) (decolourising agent), and Loeffler’s
The Ziehl-Neelsen acid-fast staining method has proved to be methylene blue (counter stain).
most useful for staining acid fast bacilli belonging to the genus
Mycobacterium especially Mycobacterium tuberculosis and Preparation of strong carbol fuchsin: This solution is prepared
Mycobacterium leprae, and also for Nocardia. These acid fast by dissolving 5 grams basic fuchsin powder in 25 grams
bacilli have a high concentration of the lipid-mycolic acid in crystalline phenol by placing them in a 1 litre flask. The flask
their cell walls. When stained they appear pink against a blue containing solution is kept over a boiling water-bath for about
background. 5 minutes, shaking the contents from time to time. When the
solution is complete, 50 ml of 95% alcohol or 100% ethanol is
added to the solution and mixed thoroughly. Then 500ml of
PRINCIPLE distilled water is added to it and the mixture is filtered before
use.
Acid fastness of acid-fast bacilli is attributed to the presence
of large quantities of unsaponifiable wax fraction called mycolic Preparation of 20% sulphuric acid : 800ml of water is collected
acid in their cell wall and also the intactness of the cell wall. in a large flask. The 200ml concentrated sulphuric acid (about
The degree of acid fastness varies in different bacteria. 98% or 1.835g / ml)) is poured slowly down the side of the flask
In this staining method, application of heat helps the dye into the water, about 50 ml at a time. The mixture becomes hot.
(a powerful staining solution containing carbol fuchsin and Remainder of acid is added in same manner.
phenol) to penetrate the tubercle bacillus. Once stained, the Note: The acid must be added to the water. It is dangerous to
stain cannot be easily removed. The tubercle bacilli resist the add the water to the acid. Great care must be taken to avoid
decolourizing action of acid-alcohol which confers acid fastness spilling the acid on skin, clothing or elsewhere.
to the bacteria. The other microorganisms, which are easily
decolourised by acid-alcohol, are considered non-acid fast. Preparation of 95% alcohol : This is prepared by adding 95
The non-acid fast bacilli readily absorb the colour of the counter ml of ethanol and adding water to it to make 100ml.
28 Acid Fast Staining
Preparation of acid-alcohol decolouriser: This solution 8 Rinse the smears again under tap water and air dry it.
contains 75 ml concentrated hydrochloric acid (HCl) and 25 ml 9 Observe the smear first under low power (10x) objective,
of industrial methylated spirit. Methylated spirit is poured into and then under oil immersion (100x) objective.
a large flask. The flask is placed in 5–8 cmm of cold water in the Note: The smear should be examined following a zig-zag
sink. Then hydrochloric acid is added slowly and the top of the pattern for at least 10 minutes or 300 fields, before declaring
flask is covered to stop the fumes from escaping. It is left for 10 the smear negative.
minutes. It is then decanted into a labeled bottle for use. The 10 Record the observations in the note book. Findings are
final concentration of HCl is 3%. recorded, together with grading of the positive smear.
III Specimen
Sputum smear positive for tubercle bacilli / culture smear of QUALITY CONTROL
Mycobacterium species.
Note: Frequently examined specimens for the detection of A positive control sputum smear from a known case of
Mycobacterium tuberculosis are summarized in the box 8-2. tuberculosis patient, stained with Z-N stain is compared with
the stained test smear for appropriate morphology and staining
appearance.
PROCEDURE With appropriate staining, the acid-fast bacillus appears
pink against blue background of pus cells.
1 Heat fixes the smears by passing the slide 2–3 times gently
over the flame with the smear side up. Allow the smear to
be air dried. OBSERVATION
2 Put the smears on a slide rack and cover the smears with
strong carbol fuchsin. Allow it to stain for 5 minutes. Presence of 3 x 0.3 µm sized pink coloured slender rod shaped
3 During this period, heat the slides from below intermittently structures are seen with curved ends, and are scattered amidst
by Bunsen flame or torch soaked in methylated spirit blue coloured round cells with darkly stained multilobed
without boiling the solution, until the steam rises. Do not nucleus.
allow the stain to dry on the slide, and if necessary add
more carbol fuchsin to cover the smear.
4 Rinse the smears gently under tap water. RESULTS AND INTERPRETATION
5 Cover the smear with 20% sulphuric acid for at least 10
minutes for decolourisation. The stained smear contains pink coloured acid fast bacilli seen
6 Wash the slides thoroughly with water to remove all traces among the blue coloured multilobed pus cells.
of acid. The smear is positive for acid fast bacilli.
Note: Decolourisation with 95% alcohol for 2 minutes is Probably, the smear contains Mycobacterium tuberculosis
only optional and may be omitted. (Fig. 8-1).
7 Cover the smear with Loeffler’s methylene blue for 15–20 Note: List of other acid–fast structures are provided in the
seconds. table 8-1.
FIGURE 8-1 Acid fast stained smear of Mycobacterium FIGURE 8-2 Acid fast stained smear of Mycobacterium leprae, x 1000
tuberculosis, x 1000.
Textbook of Practical Microbiology 29
BOX 8-1 DIFFERENT MODIFICATIONS OF ACID FAST STAIN AND THEIR USES
Pulmonary tuberculosis
Sputum.
Bronchial or laryngeal washings.
Gastric lavage (when sputum is swallowed as in children).
Miliary tuberculosis
Bone marrow.
Liver biopsy.
Tuberculous meningitis
Cerebrospinal fluid.
Renal tuberculosis
Urine.
KEY FACTS
1 Ziehl-Neelsen stain is intended for the differential staining of tubercle bacilli from other acid fast bacilli.
2 Acid fast bacilli appear pink coloured in stained smears.
3 At the end of the process of decolourisation by sulphuric acid, the smear will appear faintly pink.
4 Acid fastness of the bacteria is attributed to presence of mycolic acid in high concentration in the cell walls of tubercle bacilli
and also to the intactness of the cell wall.
5 Positive sputum smear is graded only under oil-immersion as follows:
3–9 AFB in entire smear = 1+
10 or > AFB in entire smear = 2+
10 or > AFB in each oil immersion field = 3+
30 Acid Fast Staining
VIVA
4. List another method that can be used for detecting acid fast bacteria in a smear?
Ans. Fluorescent staining methods using fluorochrome dyes such as auramine O and rhodamine is another method that can
be used for detecting acid fast bacteria in a smear. By this method acid fast bacteria fluoresces bright yellow or orange
against a greenish background.
5. What are the various specimens obtained in the laboratory for the diagnosis of tuberculosis?
FURTHER READINGS
1 Evans EGV, Killington RA, Heritage J. Introductory Microbiology. (Cambridge University Press.) 1996.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th ed.
(Lippincott Williams and Wilkins.) 1997.
4 WHO. Guidelines on Standard Operating Procedures for Microbiology. Chapter 4: Staining Techniques.
5 WHO. Manual of Basic Techniques for a Health Laboratory, 1980.
Textbook of Practical Microbiology 31
LESSON
Albert’s Staining
9
LEARNING OBJECTIVES II Reagents and glass wares
Bunsen flame, loop wire, glass slides, Albert’s stain I and II.
After completing this practical you will be able to:
Preparation of Albert’s stain I: This stain is composed of 1.5
1 Stain smears by Albert’s staining to demonstrate the presence grams toluidine blue, 2 grams malachite green, 10 ml glacial
of metachromatic granules in the diphtheria bacillus. acetic acid, 10 ml alcohol (95% ethanol) and 1 litre distilled
water. Toluidine blue and malachite green are dissolved in the
alcohol and then added to the water and acetic acid. The stain
INTRODUCTION is then allowed to stand for one day and then filtered.
The diphtheria bacillus, Corynebacterium diphtheriae has well Note: Toluidine blue stains the granules bluish black due to
developed granules within their bacterial cytoplasm. These metachromatic effect and malachite green stains the bacilli
granules are known as Volutin granules, Babes Ernst granules, green.
polar bodies or metachromatic granules. These granules are
made up of polymetaphosphate and are seen in unstained wet Preparation of Albert’s stain II: Albert’s II (also known as
preparations as round, refractile bodies within the bacterial Albert’s iodine) is composed of 6 gram iodine, 9 gram potassium
cytoplasm. With basic dyes, granules tend to stain more iodide and 900 ml distilled water. The solution is made by first
strongly than the rest of the bacterium. With toluidine blue or dissolving 2 gram potassium iodide in 10 ml distilled water and
methylene blue, they stain metachromatically, and appear then 1 gram of iodine is further added to it with dissolution.
reddish purple in colour. These granules are clearly Then 290 ml of distilled water is added and final volume is made
demonstrated best by special stains such as Albert’s, Neisser’s up to 300 ml.
or Puch’s stain. With Albert’s staining, the bacilli appear green
with bluish-black metachromatic granules. III Specimen
Exudate smear collected directly from pseudomembrane
obtained using a throat swab / culture smear of C. diphtheriae.
PRINCIPLE
7 Record the observations in the note book. Findings are The smear is positive for bacilli showing bluish-black
recorded, together with grading of the positive smear. metachromatic granules.
Probably, the smear contains Corynebacterium
diphtheriae.
QUALITY CONTROL
OBSERVATION
RESULTS AND INTERPRETATION FIGURE 9-1 Albert’s stained smear of Corynebacterium diphtheriae
showing volutin granules, x 1000.
The stained smear contains malachite green stained bacilli
showing bluish-black metachromatic granules.
Direct fluorescent antibody testing for Corynebacterium diphtheriae is a rapid diagnostic method like that of Albert’s
staining. This method involves the use of specific antibodies raised against C. diphtheriae conjugated with a fluorescent dye
to detect directly the C. diphtheriae antigen in the throat swab or smear taken from pseudomembrane. The smear is viewed by
immunofluorescent microscopy. The method is highly specific as well as sensitive.
VIVA
1. What is metachromasia?
Ans. When a substance is stained with a particular coloured dye, and if a change in original colour is observed, this phenomenon
is called metachromasia. This phenomenon is observed with C. diphtheriae. The granules present in the bacteria are called
metachromatic granules because the blue colour of the stain is changed to bluish-black by those granules.
2 Mention the other names for metachromatic granules.
3 What is the composition of metachromatic granules?
Ans. Metachromatic granules are composed of polymetaphosphate, ribonucleic acid and protein.
4 What is the significance of metachromatic granules?
Ans. The significance of metachromatic granules is that they represent storage depots of materials needed to form high-
energy phosphate bonds. They are not found during active growth period and are depleted under starvation conditions.
Their presence in thin slender bacilli helps to distinguish C. diphtheriae from short, thick, plumpy, non- pathogenic
diphtheroids which lack them.
5 What is the typical arrangement of C. diphtheriae? What is the reason for such an arrangement?
Ans. The typical arrangement of C. diphtheriae is Chinese letter pattern or Cuneiform arrangement, (bacilli are seen
arranged at various angles to each other resembling the letters V or L. This is due to incomplete separation of the daughter
cells after longitudinal binary fission.
6 Mention the other stains used for the detection of C. diphtheriae.
Textbook of Practical Microbiology 33
KEY FACTS
FURTHER READINGS
1 Evans EGV, Killington RA, Heritage J. Introductory Microbiology. (Cambridge University Press.) 1996.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company,
St. Louis) 2002.
3 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic
Microbiology. 5th ed. (Lippincott Williams and Wilkins.) 1997.
4 WHO. Guidelines on Standard Operating Procedures for Microbiology. Chapter 4: Staining Techniques.
5 WHO. Manual of Basic Techniques for a Health Laboratory, 1980.
34
LESSON
Capsule Staining
10
LEARNING OBJECTIVES the capsular materials are water soluble and may be dislodged
and removed with vigorous washing. Bacterial smears should
After completing this practical you will be able to : not be heated, because the resultant cell shrinkage may create
a clear zone around the organism, an artifact that can be mistaken
1 Demonstrate capsule of the bacteria present in animal tissues, for capsule.
blood, serous fluids and pus and artificial cultures , by The capsule is non-ionic, so that the dyes commonly used
positive staining and negative staining methods. will not bind to it. Two dyes, one acidic and one basic, are used
2 Differentiate between capsulated and non-capsulated to stain the background and the cell wall, respectively.
bacteria.
REQUIREMENTS
INTRODUCTION
I Equipments
Some bacteria secrete chemical substances that accumulate on Compound light microscope.
the outer surfaces of the cell wall and form capsules. When the
capsule has a diameter of 20 nanometer or more and seen under II Reagents and glass wares
light microscope, it is referred to as macrocapsule. When the Bunsen flame, inoculating loop, staining tray, glass slides, 1%
diameter is less than 20 nm and seen under electron microscope, crystal violet and 20% copper sulphate (CuSO4 5H2O) for
it is called microcapsule. Capsules may be seen in stained or positive staining; and India ink or Nigrosin stains for negative
unstained preparations as a clear zone around the bacteria. staining. Nigrosin staining is prepared by adding 0.03 grams of
Two types of staining procedures can be employed to nigrosin in 100 ml of distilled water.
demonstrate the capsule. They are the positive and negative
staining procedures. In the positive staining technique, the III Specimen
capsule is stained and coloured whereas in the negative staining Culture of Streptococcus pneumoniae (A capsulated
procedure, the background is stained and the capsule is seen bacterium).
as unstained hallow around the organism.
The best method for staining capsules on bacteria in either PROCEDURE
liquid or solid media is the wet-film India ink method. Dry-film
negative staining methods using India ink, nigrosin or eosin For positive staining of smears
are somewhat less reliable, since occasionally shrinkage spaces 1 Make a smear from colony of S. pneumoniae on a clean
give the appearance of capsules around bacteria that are non- grease free glass slide, and allow it to air dry.
capsulated. Note: The smear is not heat fixed.
2 Put the smear on a slide rack and flood smear with crystal
violet. Allow it to stain for 5-7 minutes.
PRINCIPLE 3 Wash the smear with 20% copper sulphate solution and blot
it dry.
Chemically, the capsular material is a polysaccharide, a 4 Observe the smear first under low power (10x) objective, and
glycoprotein or a polypeptide. Capsule staining is more difficult then under oil immersion (100x) objective.
than other types of differential staining procedures because 5 Record the observations in the note book.
Textbook of Practical Microbiology 35
QUALITY CONTROL
OBSERVATION
KEY FACTS
1 Capsule of the bacteria, Bacillus anthracis in the stained blood smears is demonstrated by McFadyean reaction which uses
polychrome methylene blue.
2 Capsules of the bacteria can be demonstrated by two methods: positive staining and negative staining procedures.
3 In the positive staining technique, the capsule is stained and coloured. In negative staining, the background is stained, and
the capsule is seen as an unstained halo around the organism.
4 The wet India ink preparation can also be employed for demonstration of slime which are irregular masses of amorphous
material seen lying between the bacteria and outside the capsules of capsulate ones.
5 Modified India ink preparation using 2% mercurochrome helps clearly demonstrate the internal structure of capsulated
budding yeast.
6 India ink film should have appropriate thickness if the film is too thick, the capsule will be obscured by overlying ink and if
too thin, the capsules get crushed or flattered and the India ink background becomes too pale to give a good contrast.
36 Capsule Staining
VIVA
FURTHER READINGS
1 Evans EGV, Killington RA, Heritage J. Introductory Microbiology. (Cambridge University Press.) 1996.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th ed.
(Lippincott Williams and Wilkins.) 1997.
4 WHO. Guidelines on Standard Operating Procedures for Microbiology. Chapter 4: Staining Techniques.
5 WHO. Manual of Basic Techniques for a Health Laboratory, 1980.
Textbook of Practical Microbiology 37
LESSON
Spore Staining
11
LEARNING OBJECTIVES stain (malachite green) and counter stain (0.5% safranine or
0.05% basic fuchsin).Ordinary tap water acts as decolourising
After completing this practical you will be able to: agent.
Unlike most of the vegetative cells that are stained by
1 Stain smears for bacterial endospores by malachite green stain. common procedures, the spore, because of its impervious coats,
2 Differentiate between bacterial spore and vegetative cell are not stained by the primary stain easily. The application of
forms in the bacterial smear. heat facilitates penetration of the primary stain, malachite green.
After the primary stain is applied and the smear is heated, both
the vegetative cell and spore appear green.
INTRODUCTION Once the spore is stained with the malachite green, it cannot
be decolourised by tap water, which removes only the excess
Spore production is a very important characteristic of some primary stain. The spore remains green. On the other hand,
bacteria such as members of anaerobic genera Clostridium water removes the stain from the vegetable cells, because the
and aerobic genus Bacillus. They are highly resistant and stain does not demonstrate a strong affinity for the vegetative
metabolically inactive forms. They occur when environmental cell components and these vegetable cells therefore become
conditions become unfavorable for continuing vegetative colourless.
cellular activities, particularly with the exhaustion of nutritional Red coloured-safranine as counterstain is used as the
carbon source. Because of the chemical composition of spore second reagent to colour the decolourised vegetative cells,
layers, the spore is resistant to deleterious effects of excessive which will absorb the counterstain and appear red. The spores
heat, freezing, radiation, desiccation, and chemical agents, as retain the green of the primary stain.
well as to the commonly employed microbiological stains.
The morphology of the bacterial endospores is best
observed in unstained wet films under the phase-contrast REQUIREMENTS
microscope, where they appear freely or within the bacterial
cells as large, refractile, oval or spherical bodies. I Equipments
Different staining techniques are available for staining of Compound light microscope.
spores. These are malachite green stain (Schaeffer and Fulton
method), modified Ziehl-Neelsen stain using 0.25% sulphuric II Reagents and lab wares
acid as decolouriser, toluidine blue stain and also Gram’s stain. Bunsen burner, beaker of boiling water, staining tray, glass
With the Gram’s stain, the body of the bacillus appears slides, inoculating loop, malachite green and safranine.
deeply stained, whereas the spore is unstained and appears as
a clear area in the organism. The spores on staining with Preparation of malachite green stain: This stain is prepared
modified Ziehl-Neelsen stain appear red and bacilli blue. by dissolving 5 gram of malachite green in 100 ml of distilled
water.
III Specimen
Smear collected from 48 hours to 72 hours nutrient agar slant
OBSERVATION
culture of Bacillus cereus/ thioglycollate culture of Clostridium
butyricum. On a clean glass slide, a smear from the culture is Bacterial endospores stain green, and vegetative bacilli stain
made in saline, then air dried and fixed with heat. red. (Fig. 11-1)
1 Heat fix the smears by passing the slide 2–3 times gently over A 2 – 3 µm red coloured rod-shaped structure seen along with
the flame with the smear side up. Allow the smear to be air dried. an intracellular 0.5 µm sized spherical green coloured structure.
2 Put the slide with the smear over a beaker of boiling water, It represents red coloured vegetative bacilli with green coloured
resting it on the run with the bacterial film upper most. spores by the malachite green staining method. May be spore-
3 When, within several seconds, large droplets have bearing bacilli (eg. Bacillus species or Clostridium species)
condensed on the underside of the slide, flood the smear
with 5% acqueous solution of malachite green and allow to
act for 1 minute, while the water continues to boil.
3 Wash the smears with cold water.
4 Then cover the smear with 0.5% safranine or 0.05% basic
fuchsin. Allow it to act for 30 seconds.
5 Rinse the smears again under tap water and blot those dry.
6 Observe the smear first under low power (10x) objective ,
and then under oil immersion (100x) objective.
7. Record the observations in the note book.
QUALITY CONTROL
VIVA
KEY FACTS
1 Spores are highly resistant, metabolically inactive forms occurring when environmental conditions are unfavorable.
2 The morphology of spores is best observed in unstained wet films under the phase-contrast microscope.
3 The staining methods available for demonstrating spores are Gram’s staining, modified acid fast staining using 0.25%
sulphuric acid as decolouriser, toluidine blue staining and malachite green staining (Schaeffer-Fulton Method).
4 With malachite green staining vegetative bacilli appear red and spores green.
5 With toluidine blue staining , vegetative bacilli appear light blue and spores dark blue.
Textbook of Practical Microbiology 39
FURTHER READINGS
1 Evans EGV, Killington RA, Heritage J. Introductory Microbiology. (Cambridge University Press.) 1996.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th ed.
(Lippincott Williams and Wilkins.) 1997.
4 WHO. Guidelines on Standard Operating Procedures for Microbiology. Chapter 4: Staining Techniques.
5 WHO. Manual of Basic Techniques for a Health Laboratory, 1980.
40
LESSON
Negative Staining
12
LEARNING OBJECTIVES REQUIREMENTS
OBSERVATION
The nigrosin-stained smear contains capsulated bacteria. e.g. FIGURE 12-1 Nigrosin-stained smear showing capsulated
K. pneumoniae. K. pneumoniae, x 400.
Bacteria Fungus
Streptococcus pneumoniae Cryptococcus neoformans
Klebsiella pneumoniae
Haemophilus influenzae
Group B streptococci
Group D streptococci
KEY FACTS
1 Negative staining procedure is so called because the background gets stained and the organism remains colourless.
2 In negative staining method only background is stained, the organism and capsule remained unstained and refractile.
VIVA
1 List the different stains that can be employed for negative staining.
Ans: The different stains employed for negative staining are: India ink, modified India ink (using 2% mercurochrome) and
10% nigrosin.
2 List the capsulated organisms and bacteria that can be demonstrated by negative staining.
FURTHER READINGS
1 Evans EGV, Killington RA, Heritage J. Introductory Microbiology. (Cambridge University Press.) 1996.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th ed.
(Lippincott Williams and Wilkins.) 1997.
4 WHO. Guidelines on Standard Operating Procedures for Microbiology. Chapter 4: Staining Techniques.
5 WHO. Manual of Basic Techniques for a Health Laboratory, 1980.
42
Textbook of Practical Microbiology 43
UNIT
III
Cultivation of Bacteria
LESSON
Media for Routine
13 Cultivation of Bacteria
LEARNING OBJECTIVES 2 Enriched medium: These are solid selective media. These
media, in addition to basal nutrients also contain nutritional
After completing this practical you will be able to: supplements like blood, serum, etc., which favour the growth
of fastidious bacteria. e.g. blood agar (Fig. 13-2), chocolate
1 Know the following commonly used culture media as well as agar (Fig. 13-3) , Löwenstein- Jensen medium (Fig. 13-4), etc.
their uses in a clinical microbiology laboratory: 3 Enrichment media: These are liquid selective media. They
favor the growth of some bacteria by extending the lag phase
a. Basal medium of others eg. Selenite F broth.
b. Differential medium 4 Selective media: These media contain ingredients that
c. Selective medium selectively enable the growth of some species, while
d. Enriched, and inhibiting others eg. Deoxycholate citrate agar (DCA) medium.
e. Enrichment media This medium is a selective medium for growth of Salmonella
spp. present in stool which contains a mixed bacteria flora.
This medium inhibits Escherichia coli and other Gram-
INTRODUCTION negative bacteria.
5 Differential media: These media differentiates between
Cultivation of bacteria is often the first step in the diagnosis of species of bacteria depending on a specific property.
infectious disease. Since bacteria have varied growth Example: MacConkey (Fig. 13-5) agar is a differential medium.
requirements, a wide range of media is available. The choice of This medium is used to demonstrate lactose fermenting
most appropriate media depends on many factors including properties, and differentiate between lactose and non-lactose
nutritional and growth requirements of the bacteria, biochemical fermenting bacteria.
properties and many other properties of the bacteria.
PRINCIPLE
QUALITY CONTROL
REQUIREMENTS
I Equipments
Bacteriological incubator. FIGURE 13-4 Lowenstein-Jenson’s medium.
III Specimen
24 hour broth cultures of Staphylococcus aureus, E. coli,
Proteus mirabilis and Salmonella spp.
PROCEDURE
Table 13-1 List of different type of media commonly used for isolation of bacteria
KEY FACTS
1 Bacteria have different nutritional requirements. Most of the bacteria grow on enriched media.
2 S. aureus can grow on MacConkey medium.
3 Media serve many purposes such as culture and isolation of the bacteria from clinical specimens, demonstration of growth
and biochemical characteristics of bacteria, and for storage of bacterial isolates, etc.
VIVA
FURTHER READINGS
1 Bhattacharya S, Vijayalakshmi N, Parija SC. Uncultivable bacteria: Implications and recent trends towards identification. Indian J Med
Microbiol. 2002: 20; 174-177.
2 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995.
3 Isenberg HD. (Ed) Clinical Microbiology Procedures Handbook. American Society for Microbiology, Washington DC, 1992.
4 PHLS Standard Operating Procedures. Inoculation of Culture Media. No B.SOP 54 Version: 1, 1998.
5 WHO. Guidelines on standard operating procedures for Microbiology. Blood safety and clinical technology. Chapter 6: Cultivation of
bacteria on laboratory media. (World Health Organisation, Geneva) 1997.
Textbook of Practical Microbiology 47
LESSON
Temperature
14 Requirement
for Growth of Bacteria
LEARNING OBJECTIVES bacteria prefer to grow in the bodies of warm blooded hosts
and include most bacterial pathogens that cause diseases in
After completing this practical you will be able to understand: humans.
3 Thermophilic bacteria: These bacteria can grow above 35°C.
1 Effect of temperature on growth of the bacteria in culture They are either facultative with an optimum temperature of
media. 45°C -60°C but capable of growth at 37°C or obligate, growing
only at temperature above 50°C.
Table 14-1 Examples of bacteria showing different temperatures for their growth
KEY FACTS
1 Temperature requirement for optimal growth and enzyme activity are often different.
2 Different bacteria require different optimal temperature for their growth.
3 Most of the medically important bacteria grow at 35°C -37°C.
4 Campylobacter grows at 42°C.
VIVA
FURTHER READINGS
1 Bhattacharya S, Vijayalakshmi N, Parija SC. Uncultivable bacteria: Implications and recent trends towards identification. Indian J Med
Microbiol. 2002: 20; 174-177.
2 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995.
3 Isenberg HD. (Ed) Clinical Microbiology Procedures Handbook. American Society for Microbiology, Washington, DC, 1992.
4 PHLS Standard Operating Procedures. Inoculation of Culture Media. No B.SOP 54 Version: 1, 1998.
5 WHO. Guidelines on standard operating procedures for Microbiology. Blood safety and clinical technology. Chapter 6: Cultivation of
bacteria on laboratory media. (World Health Organisation, Geneva) 1997.
Textbook of Practical Microbiology 49
LESSON
pH Requirement for
15
15 Growth of Bacteria
After completing this practical you will be able to understand: 1 Using sterile pipettes, inoculate 0.1 ml of each organism into
1 The pH requirement of bacteria. nutrient broth tubes of pH, 3, 5, 7 and 9.
2 Incubate the tubes for 24-48 hr.
3 Check for growth of bacteria in these tubes.
INTRODUCTION
Like temperature, pH also plays an important role in the growth QUALITY CONTROL
and reproduction of bacteria. Each species of bacteria can grow
within a particular pH range, and maximum growth occurs in an 1 Nutrient broth tube inoculated with Candida spp as quality
optimum pH range. This is a reflection of this natural environment control for growth at acidic pH.
eg. enteric bacteria such as Escherichia coli have a broad pH 2 Nutrient broth tube inoculated with E. coli as quality control
range, similar to that of the gut. Generally, bacteria grow best at for growth at pH 7.2
a pH between 5.5-8 optimum pH being 6.5-7.5 Fungi thrive in an 3 Nutrient broth tube inoculated with A. faecalis as quality
acidic environment of about pH 4-6. Most laboratory media control for growth at alkaline pH.
have a neutral pH which suits nearly all organisms.
OBSERVATIONS
PRINCIPLE
1 Reproduction of bacteria is maximal at optimal pH.
Since pH changes can occur during growth of the bacteria 2 Ability to tolerate and grow at a particular pH will be seen as
due to the accumulation of metabolic byproducts, buffers are turbidity in culture tubes.
incorporated into the medium to prevent pH change. These
may include natural buffers like proteins, peptone and amino
acids which retard the shift because of their amphoteric nature. RESULTS AND INTERPRETATION
Salts of weak acids and weak bases may also be added.
1 All 3 microorganisms (E. coli, A. faecalis and Candida spp)
show growth and turbidity at pH 7.
REQUIREMENTS 2 No microorganism shows any growth at pH 3.
3 E. coli and A. faecalis do not show any growth at pH 3 or pH 5.
I Equipments 4 Only A. faecalis shows growth and turbidity at pH 9.
Bacteriological incubator. 5 Only Candida spp shows growth and turbidity at pH 5.
II Reagents
The ability to grow at different pH varies among bacteria.
Nutrient broth tubes, 3 each of pH, 3, 5, 7 and 9.
Most pathogens show an optimum pH close to that of their
III Specimen preferred habitat.
A 24 hour broth culture of Escherichia coli, Alcaligenes
faecalis and Candida spp.
50 pH Requirement of Growth of Bacteria
KEY FACTS
VIVA
FURTHER READINGS
1 Bhattacharya S, Vijayalakshmi N, Parija SC. Uncultivable bacteria: Implications and recent trends towards identification. Indian J Med
Microbiol. 2002: 20; 174-177.
2 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995.
3 Isenberg HD. (Ed) Clinical Microbiology Procedures Handbook. American Society for Microbiology, Washington, DC, 1992.
4 PHLS Standard Operating Procedures. Inoculation of Culture Media. No B.SOP 54 Version: 1, 1998.
5 WHO. Guidelines on standard operating procedures for Microbiology. Blood safety and clinical technology. Chapter 6: Cultivation of
bacteria on laboratory media. (World Health Organisation, Geneva) 1997.
Textbook of Practical Microbiology 51
LESSON
Oxygen Requirement
16 for Growth of Bacteria
PRINCIPLE I Equipments
Bacteriological incubator and water bath.
On the basis of oxygen requirement, bacteria can be classified
into 5 groups as follows: II Reagents and media
Nutrient broth tubes and brain heart infusion (BHI) agar tubes.
1 Aerobes: These bacteria can grow only in the presence of
free oxygen. Enzyme systems present in these bacteria require III Specimen
oxygen to be the final electron (hydrogen) acceptor A 24 hour nutrient broth culture of Escherichia coli,
especially for the oxidative breakdown of high energy Pseudomonas aeruginosa, and 48 hour thioglycollate broth
molecule like glucose. culture of Clostridium sporogenes.
2 Microaerophiles: These bacteria require small amount of
oxygen for their growth and survival. An excess of oxygen
is inhibitory to their growth.
3 Obligate anaerobes: These bacteria cannot survive in the
presence of free oxygen, hence other molecule act as the
final electron acceptor.
4 Aerotolerant anaerobes: They do not use oxygen as a final
electron acceptor but possess enzymes like superoxide
dismutase and catalase, which can prevent the accumulation
of toxic metabolites produced in the presence of oxygen.
Therefore, oxygen is not lethal to them.
5 Facultative anaerobes: These bacteria can grow in the
presence or absence of free oxygen. They generally follow
an aerobic respiratory pathway, an anaerobic respiratory
FIGURE 16-1 Candle jar used for incubation and culture of bacteria..
52 Oxygen Requirement for Growth of Bacteria
KEY FACTS
1 Oxygen is both beneficial and toxic to living organisms. The benefits are because of their ability to act as a final electron
acceptor in the respiratory pathway with the liberation of much energy. However it also results in the accumulation of toxic
byproducts.
2 Bacteria may possess enzyme system to remove these toxic substances and thus help in their survival in the presence of
oxygen.
3 Anaerobic bacteria can be cultured by the exclusion of air using anaerobic jars, prereduced media, etc.
VIVA
1 Give example of obligate aerobes, microaerophiles, obligate anaerobes, aerotolerant anaerobes and facultative anaerobes.
FURTHER READINGS
1 Bhattacharya S, Vijayalakshmi N, Parija SC. Uncultivable bacteria: Implications and recent trends towards identification. Indian J Med
Microbiol. 2002: 20; 174-177.
2 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995.
3 Isenberg HD. (Ed) Clinical Microbiology Procedures Handbook. American Society for Microbiology, Washington, DC, 1992.
4 PHLS Standard Operating Procedures. Inoculation of Culture Media. No B.SOP 54 Version: 1, 1998.
5 WHO. Guidelines on standard operating procedures for Microbiology. Blood safety and clinical technology. Chapter 6: Cultivation of
bacteria on laboratory media. (World Health Organisation, Geneva) 1997.
Textbook of Practical Microbiology 53
LESSON
Culture of
17
17 Anaerobic Bacteria
There are different ways of creating anaerobic conditions II Reagents and media
suitable for the growth of obligate anaerobes. Deep nutrient Blood agar plates and thioglycollate broth culture.
agar tubes are the simplest method. The tubes are inoculated
while still molten, cooled rapidly and incubated. Anaerobes III Specimen
grow in the depths of the medium, and the number of colonies A 48 hour thioglycollate broth culture of anaerobic bacteria
becomes fewer towards the surface. Strict anaerobes will not (Clostridium sporogenes, Bacteroides spp), and 24 hour
grow within a centimeter of the surface. cultures of facultative anaerobes (Escherichia coli), and
Alternatively, reducing agents like 0.5-1% glucose, 0.1% obligate aerobes (Pseudomonas aeruginosa).
ascorbic acid, 0.1% cysteine, 0.1% thioglycollate can be added.
Cooked meat particles also act as a good reducing agent
Example. Robertson Cooked meat medium (Fig. 17-1). PROCEDURE
For culture of anaerobes, oxygen must be excluded either
by combustion or by replacing it with an inert gas. 1 Divide each plate into 4 quadrants.
In many laboratories, combustion involves the combining 2 Inoculate a loopful of each organism into a quadrant.
of oxygen with hydrogen to form water in the presence of a 3 Stack the plates into the anaerobic jars, introduce the catalyst
catalyst like palladium or palladinized asbestos. Anaerobic jars and quickly seal the lid.
are a constant feature of anaerobic culture. They include the Note: Anaerobic condition should be checked by alkaline
McIntosh and Fildes jar (Fig. 17-2) , which has inlets to admit methylene blue indicator.
hydrogen and carbon dioxide, a vacuum pump for evacuating 4 Incubate the plates at 37ºC for 48 hours.
oxygen, and a catalyst fitted into the lid. 5 Incubate one plate aerobically.
A simpler but more expensive technique is the Gaspak 6 Remove the plates from the jars and examine for growth.
system. This utilizes a transparent polycarbonate jar with a lid
54 Culture of Anaerobic Bacteria
FIGURE 17-1 Robertson’s cooked meat medium. FIGURE 17-2 Macintosh Filde’s jar.
QUALITY CONTROL Cl. sporogenes and Bacteroides are strict anaerobes that cannot
grow if oxygen is present.
1 Blood agar inoculated with P. aeruginosa, the bacteria that
does not grow anaerobically E. coli will grow on all the plates, either incubated aerobically
2 Thioglycollate broth culture inoculated with Cl. sporogenes, or anaerobically. E. coli is a facultative anaerobe and can grow
the bacteria that does not grow aerobically either in the presence or absence of oxygen.
VIVA
KEY FACTS
FURTHER READINGS
1 Bhattacharya S, Vijayalakshmi N, Parija SC. Uncultivable bacteria: Implications and recent trends towards identification. Indian J Med
Microbiol. 2002: 20; 174-177.
2 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995.
3 Isenberg HD. (Ed) Clinical Microbiology Procedures Handbook. American Society for Microbiology, Washington, DC, 1992.
4 PHLS Standard Operating Procedures. Inoculation of Culture Media. No B.SOP 54 Version: 1, 1998.
5 WHO. Guidelines on standard operating procedures for Microbiology. Blood safety and clinical technology. Chapter 6: Cultivation of
bacteria on laboratory media. (World Health Organisation, Geneva) 1997.
56
LESSON
Sterilization of
18 Commonly Used
Culture Media
LEARNING OBJECTIVES Chamber land),b) asbestos filters (e.g., Seitz filter (Fig. 18-1), c)
sintered glass filter (Fig. 18-2), and d) cellulose membrane filters.
After completing this practical you will be able to become Of these cellulose membrane filter is most extensively used now
familiar with: a days.
Different methods of sterilization of various substances are
1 The commonly used techniques for the sterilization of culture summarized in the table 18-1.
media.
REQUIREMENTS
INTRODUCTION
I Equipments
Sterilization is the process by which an article is freed of all Autoclave (Fig. 18-3), hot air oven (Fig. 18-4) and Seitz filter.
living organisms, including spores. This is vital for isolation
and maintenance of microbes. The common methods used for II Reagents
sterilization of media is heat and filtration. Sterile nutrient broth, Browne’s tube no. 3, and filter paper strips
impregnated with spore of Clostridium tetani and filter paper
strips impregnated with spores of Bacillus stearothermophilus.
PRINCIPLE
III Specimen
Sterilization by heat Spore suspensions of Bacillus spp and suspension of
Escherichia coli
This can be performed by two methods as mentioned below:
Dry heat: Sterilisation by dry heat kills the bacteria by oxidizing
PROCEDURE
essential cell components of the cell. The hot air oven is commonly
used in a microbiology laboratory for sterilization of laboratory
1 Soak strips of filter paper in the spore suspensions of
glassware, oils, powders etc. In a hot air oven, temperatures
Bacillus spp and dry them in a Petri dish in the incubator.
maintained at 160°C, for an hour is effective for sterilization.
2 Take 7 test tubes and place a dried strip in each of them.
Moist heat: Moist heat kills the microorganisms by coagulat-
3 Place the first three tubes in a hot air oven at 160°C for 20, 30
ing their proteins and denaturing their enzymes. Moist heat has
and 60 minutes respectively.
greater penetrating power than the dry heat and so relatively lower
4 Autoclave the 4th, 5th and 6th tubes for 10, 20, and 30 minutes
temperature is required for sterilization by this method. Pasteuri-
respectively.
sation is an example of sterilization by moist heat (Box 18-1).
5 After cooling, aseptically add 5 ml of nutrient broth to each
tube and incubate for 24 hours at 37°C.
6 The 7th tube acts as control.
Sterilization by filtration 7 Inoculate a drop of the E.coli suspension onto a nutrient
agar plate.
Heat labile fluids such as serum, antibiotics, etc. are sterilized by 8 Filter the suspension through a Seitz filter.
passing them through special filters. There are different types 9 Aseptically inoculate the filtrate onto a nutrient agar plate
of filters. These include a) earthenware candles (e.g., Berkefield, and incubate both plates at 37°C for 24 hours.
Textbook of Practical Microbiology 57
OBSERVATIONS
If the spores have been killed by the heat process there will be
no growth as demonstrated by a lack of turbidity. Similarly, if
the bacteria have been held back by the filter there should be
no growth in the sample of filtrate.
Pasteurization is a method of sterilisation by moist heat. It is sufficient to kill heat labile bacteria, but not spores. Two methods
are available: holder method (60°C for 30 minutes) and flash method (72° C for 15 seconds). Some pathogens such as Coxiella
burnetii is not destroyed, so this is not a very efficient method of sterilization.
Dry heat
Flaming Inoculating wires, loops.
Hot air oven Glassware, oils, powders, paraffin .
Incineration Biohazardous material- used gloves, needles, etc.
Moist heat
Pasteurization Serum, milk.
Inspissation Löwenstein Jensen media, Loeffler’s serum slope.
Boiling Needles, glass syringes.
Tyndallization Sugar solution.
Autoclaving Heat stable media such as nutrient agar.
Filtration Heat labile media, serum, antibiotics, etc.
KEY FACTS
1 Various factors influence sterilization by heat. These include time, temperature, number of microorganisms and spores and
their type, nature of material, etc.
2 It is important not to overfill the media container.
3 The load should be properly plugged or wrapped to ensure sterility.
4 The load should be cooled before being removed from the autoclave/hot air oven.
5 Filters should be assembled and autoclaved prior to use.
VIVA
3 How would you sterilize a) sera, b) Löwenstein Jensen medium, c) nutrient agar and d) paraffin?
FURTHER READINGS
1 Bhattacharya S, Vijayalakshmi N, Parija SC. Uncultivable bacteria: Implications and recent trends towards identification. Indian J Med
Microbiol. 2002: 20; 174-177.
2 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995.
3 Isenberg HD. (Ed) Clinical Microbiology Procedures Handbook. American Society for Microbiology, Washington, DC, 1992.
4 PHLS Standard Operating Procedures. Inoculation of Culture Media. No B.SOP 54 Version: 1, 1998.
5 WHO. Guidelines on standard operating procedures for Microbiology. Blood safety and clinical technology. Chapter 6: Cultivation of
bacteria on laboratory media. (World Health Organisation, Geneva) 1997.
Textbook of Practical Microbiology 59
LESSON
Antiseptics and
17
19 Disinfectants
PROCEDURE
INTRODUCTION
1 With a sterile pipette, transfer 1 ml of the used disinfectant
Disinfection is defined as the destruction of microorganisms into 9 ml sterile nutrient broth.
not including bacterial spores. The process reduces 2 Also mix 1 ml of fresh disinfectant with 9 ml of E coli culture.
microorganism to a level acceptable for a defined purpose. A 3 Inoculate this mixture onto 10 different areas of two well
chemical agent that disinfects a substance is called a dried nutrient agar plates each.
disinfectant. A disinfectant that can be safely applied to living 4 Incubate one plate for 3 days at 37°C and the other for 7
tissue is called an antiseptic. days at room temperature.
Table 19-1 summarises the list of commonly used
disinfectants and antiseptics.
QUALITY CONTROL
Disinfectants Antiseptics
Lysol 70% ethyl alcohol
Zephiran Chlorhexidine
Glutaraldehyde Iodine
Sodium hypochlorite Dettol
1 Phenols (e.g. lysol, chloroxylenol): They act by a number of mechanisms such as disruption of cells, precipitation of
protein inactivation of enzyme, etc. Phenol is the standard disinfectant against which other disinfectants are compared.
2 Alcohols (e.g. ethyl alcohol, usually at a concentration of 70%): They can also be used as antiseptics and are active
against viruses as well. They act by denaturing proteins damaging lipid complexes and dehydration.
3 Halogens (eg. iodine, chlorine and sodium hypochlorite): Iodine is commonly used as an antiseptic. It acts by oxidation.
Chlorine is a widely used disinfectant. The compounds act by the liberation of nascent oxygen and are effective against
most bacteria and viruses.
4 Heavy metals and their compounds (e.g. copper sulphate): They act by inactivating cellular protein.
5 Synthetic detergents (e.g. sodium lauryl sulfate): They help in mechanical removal of microorganism.
6 Quaternary ammonium compound (e.g. zephiran): They act on the cell membrane of bacteria.
KEY FACTS
1 Disinfectant does not guarantee sterility. They merely ensure that an object is relatively free of microbial contamination.
2 Antiseptics can be used for decontamination of living tissue.
3 Disinfectants are used for decontamination of non-living tissue.
4 It is essential to test disinfectant from time to time to detect loss of activity.
VIVA
FURTHER READINGS
1 Bhattacharya S, Vijayalakshmi N, Parija SC. Uncultivable bacteria: Implications and recent trends towards identification. Indian J Med
Microbiol. 2002: 20; 174-177.
2 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995.
3 Isenberg HD. (Ed) Clinical Microbiology Procedures Handbook. American Society for Microbiology, Washington, DC, 1992.
4 PHLS Standard Operating Procedures. Inoculation of Culture Media. No B.SOP 54 Version: 1, 1998.
5 WHO. Guidelines on standard operating procedures for Microbiology. Blood safety and clinical technology. Chapter 6: Cultivation of
bacteria on laboratory media. (World Health Organisation, Geneva) 1997.
Textbook of Practical Microbiology 61
UNIT
IV
ENZYMATIC AND BIOCHEMICAL
ACTIVITIES OF BACTERIA
LESSON
Catalase Test
20
LEARNING OBJECTIVES REQUIREMENTS
After completing this practical you will be able to: I Reagents and glass wares
3% hydrogen peroxide, glass slides, test tubes, glass rod /
1 Demonstrate the presence of catalase, an intracellular enzyme platinum loop / plastic loop and other standard lab wares.
in the bacteria.
2 Distinguish the bacteria based on the catalase activity. II Specimen
Tube method
Gas bubbles are released when colonies are introduced into BOX 20-1 USES OF CATALASE TEST
the hydrogen peroxide in the test tube.
The catalase test is useful to differentiate:
It helps to differentiate tubercle bacilli from atypical mycobacteria. Most atypical mycobacteria are strongly catalase positive,
while tubercle bacilli are weakly positive.
Most strains of mycobacteria, except certain strains of Mycobacterium tuberculosis complex (some isoniazid resistant
strains) and M. gastri, produce the intracellular enzyme catalase.
The test is performed by mixing equal volumes of 30% hydrogen peroxide and 0.2% catechol in distilled water and then
adding it to 5ml of a mycobacterial test culture. It is allowed to stand for a few minutes. Effervescence indicates catalase
production.
a) Relative activity of the enzyme catalase determined by the height of the column of oxygen bubbles formed by the action of
untreated enzyme produced by the organism (Semi quantitative catalase test). On the basis of the semi quantitative
catalase test, mycobacteria are classified in to 2 groups: i. those producing <45mm column height of bubbles, and ii.those
producing >45mm column height of bubbles.
b) By the ability of the enzyme catalase to remain active after heating, a measure of the heat stability of the enzyme (Heat
stable catalase test). When heated to 68°C for 20 minutes, the catalase of M. tuberculosis. M. bovis, M. gastri and M.
haemophilum becomes inactivated.
64 Catalase Test
KEY FACTS
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS (Eds). Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis)
2002.
3 Introduction to Clinical Microbiology. University of Texas- Houston Medical School, DPALM Medic. 1995.
4 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC (Eds). Color Atlas and Textbook of Diagnostic Microbiology. 5th
ed. (Lippincott Williams and Wilkins, USA). 1997.
Textbook of Practical Microbiology 65
LESSON
Oxidase Test
17
21
LEARNING OBJECTIVES dihydrochloride (1%), and dimethyl – p – phenylene diamine
dihydrochloride (1%).
After completing this practical you will be able to: Wood stick/platinum loop/glass rod, and filter paper.
1 Demonstrate the presence of oxidase, an intracellular enzyme
in the oxidase-positive bacteria. II Specimen
2 Distinguish the bacteria based on the cytochrome oxidase Young culture of bacteria to be tested, preferably less than 24
activity. hours old, growing on an agar plate or agar slant.
INTRODUCTION PROCEDURE
The enzyme oxidase plays a vital role in the operation of the The test is performed by following two methods:
electron transport system during aerobic respiration.
Cytochrome oxidase catalyzes the oxidation of a reduced 1 Direct plate technique, and
cytochrome by molecular oxygen, resulting in the formation of 2 Indirect paper strip procedure.
water or hydrogen peroxide.Aerobic bacteria, as well as some
facultative anaerobes and microaerophiles exhibit oxidase Direct plate technique
activity.
1 Take a nutrient agar plate with colonies of bacteria to be tested.
2 Add 2 to 3 drops of reagent (tetramethyl p-phenylene diamine
PRINCIPLE hydrochloride or dimethyl-p- phenylene diamine
dihydrochloride) directly to the bacterial colonies growing
The cytochromes are iron containing haemoproteins that act on medium in the plate.
as the last link in the chains of aerobic respiration by 3 Note the change of colour of the colonies.
transferring electrons (hydrogen) to oxygen, with the formation
of water. The cytochrome oxidase test uses certain reagent Indirect filter paper strip procedure
dyes such as p-phenylene diamine dihydrochloride which acts
as a substitute for oxygen as artificial electron acceptors. This 1 Take a filter paper strip.
enzyme oxidises the reagent N-N tetramethyl para-phenylene 2 Moisten the filter paper strip with freshly prepared 1% oxidase
diamine hydrochloride (a colour less reagent in reduced form) reagent.
to indophenol blue, a purplish blue coloured product. Note: Oxidase reagent is freshly prepared in distilled water
List of oxidase positive and negative bacteria is presented every day.
in the table 21-1. 3 Pick up the colonies to be tested with the help of a glass rod
or plastic loop or platinum wire.
4 Smear the colonies into the reagent zone of the filter paper.
REQUIREMENTS 5 Note the change in colour if any within 10 seconds.
QUALITY CONTROL Table 21-1 List of oxidase positive and negative bacteria
VIVA
KEY FACTS
1 The dye, p-phenylene diamine dihydrochloride is the substitute for oxygen as artificial election acceptors.
2 In reduced state the dye is colourless and in oxidized state deep purple in colour.
3 Colour change must be noted within 10 seconds.
4 Tetramethyl derivative of p-phenylene diamine is recommended because the reagent is i more stable in storage, ii more
sensitive to the detection of cytochrome oxidase, and iii. less toxic than dimethyl derivative.
5 Nichrome or stainless steel inoculating loops or wires should not be used for performing the test because surface oxidation
products formed during the process of sterilization by flaming may result in false positive reaction.
6 Always freshly prepared reagent should be used.
7 Colonies for testing should not be taken from blood agar.
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS (Eds). Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis)
2002.
3 Introduction to Clinical Microbiology. University of Texas- Houston Medical School, DPALM Medic. 1995.
4 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC (Eds). Color Atlas and Textbook of Diagnostic Microbiology. 5th
ed. (Lippincott Williams and Wilkins, USA). 1997.
68
LESSON
Coagulase Test
22
LEARNING OBJECTIVES by tube coagulase test. In this method, a suspension of coagulase
producing staphylococci is prepared in plasma in a test tube,
After completing this practical you will be able to: and incubated at 37°C for 3-6 hours. In a positive test, the enzyme
1 Demonstrate the presence of coagulase, an intracellular coagulase secreted by S. aureus is liberated to the medium, which
enzyme in the bacteria, especially Staphylococcus aureus. reacts with fibrinogen to produce a visible fibrin clot.
2 Distinguish the bacteria based on the coagulase activity. List of coagulase positive bacteria is presented in the
table 22-1.
INTRODUCTION
REQUIREMENTS
The enzyme, coagulase, produced by a few Staphylococcus
species, is a key feature of pathogenic Staphylococcus. The I Reagents and lab wares
enzyme causes coagulation of blood, allowing the organism to Rabbit plasma with EDTA anticoagulant, saline, glass slides,
“wall” its infection off from the host’s protective mechanisms test tubes, glass rod/platinum loop/plastic loop and other
rather effectively. standard lab wares.
Coagulase is a protein having a prothrombin-like activity
capable of converting fibrinogen into fibrin, which results in II Specimen
the formation of visible clot. In the laboratory, the coagulase Pure growth of S. aureus from solid media preferably from non-
test is used to identify S. aureus and differentiate it from the blood agar plates (Examples: nutrient agar, Muller-Hinton agar).
other species of coagulase-negative Staphylococcus.
PROCEDURE
PRINCIPLE
Slide test
S. aureus produces the enzyme coagulase in 2 forms: a. bound 1 Take a clean glass slide.
coagulase and b. free coagulase. 2 Mark it into two halves by a glass marking pencil.
3 Add two drops of sterile saline on two halves of the glass
Bound coagulase slides.
4 Pick up the colonies of S. aureus to be tested from agar
Bound coagulase is also known as clumping factor. It is bound to culture and gently emulsify with drops of saline.
the bacterial cell wall and is not present in culture filtrates. Presence 5 Add a drop of undiluted plasma to the bacterial suspension
of this enzyme is tested by slide coagulase test. Fibrin strands are and mix with a wooden applicator sticks.
formed between the bacterial cells when suspended in plasma 6 Place another drop of saline in other half of the slide as a control.
(fibrinogen), causing them to clump into visible aggregates. 7 Rock the slide, back and froth, and observing for the prompt
clumping of the bacterial suspension within 10-15 seconds.
Free coagulase
Tube test
Free coagulase is a thrombin-like substance present in S. aureus 1 Take 0.5 ml of rabbit plasma (diluted 1 in 5 with saline) in a
culture filtrates (Box 22 -1). Presence of free coagulase is tested test tube.
Textbook of Practical Microbiology 69
QUALITY CONTROL
1 Staphylococcus aureus.
RESULTS AND INTERPRETATION 2 Staphylococcus schleiferi
4 Staphylococcus felis
5 Staphylococcus lutrae
In slide test, Positive reaction will be detected within 10–15 6 Staphylococcus intermedius
seconds of mixing the plasma with the suspension by the 7 Staphylococcus hyicus
formation of a white precipitate and agglutination of the 8 Peptostreptococcus hydrogenalis
Free coagulase is an extracellular enzyme produced by Staphylococcus aureus. It activates a coagulase reacting factor (CRF)
normally present in the plasma to clot by the conversion of fibrinogen to fibrin. Coagulase does not clot plasma of guinea pigs
because they lack CRF. There are 8 antigenic types of coagulase (designated as A to H).Most human strains of S. aureus
produce coagulase A.
All coagulase producing staphylococci are S. aureus and as a result coagulase production is considered the best laboratory
evidence for the potential pathogenicity of Staphylococcus.
Coagulase may act to coat the bacterial cells with fibrin rendering them resistant to opsonization and phagocytosis.
KEY FACTS
1 Two types of coagulase are produced by S. aureus. These are bound coagulase, and tube coagulase.
2 Bound coagulase is tested by slide coagulase test.
3 Free coagulase is tested by tube coagulase test.
4 In the tube coagulase test, on continued incubation, the clot already formed may be lysed by fibrinolysin secreted by some strains.
5 Rabbit plasma with EDTA is used in both tests.
6 Pooled human plasma can be used after checking with a standard strain of S. aureus.
70 Coagulase Test
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS (Eds). Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis)
2002.
3 Introduction to Clinical Microbiology. University of Texas- Houston Medical School, DPALM Medic. 1995.
4 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC (Eds). Color Atlas and Textbook of Diagnostic Microbiology. 5th
ed. (Lippincott Williams and Wilkins, USA). 1997.
Textbook of Practical Microbiology 71
LESSON
Urease Test
23
LEARNING OBJECTIVES II Reagents and glass wares
Inoculating wire, Christensen’s urea agar, and 12 × 100 mm test
After completing this practical you will be able to: tubes.
INTRODUCTION
PROCEDURE
Certain bacteria and fungi possess the enzyme urease that
hydrolyzes urea releasing ammonia into the medium. This 1 Pick up the colonies of P. mirabilis from the culture on
produces a change in the pH of the medium that can be detected nutrient agar.
by the color change in the indicator dye. This test can be used 2 Inoculate Christensen’s urea agar slope with these bacterial
to differentiate different groups of bacteria and fungi. colonies.
3 Incubate the tube at 37°C for 18 hours.
4 Observe any change of colour in the inoculated medium.
PRINCIPLE
Urea is a diamide of carbonic acid. Urease, the enzyme produced BOX 23-1 CHRISTENSEN’S UREA AGAR
by the bacteria and fungi, hydrolyses urea and releases ammonia
and carbon dioxide. Ammonia reacts in solution to form Composition
ammonium carbonate, which is alkaline leading to an increase Peptone – 0.1 gm.
in pH of the medium. Phenol red that is incorporated in the Glucose – 0.1 gm.
medium changes its color from yellow to red in alkaline pH, NaCl – 0.5 gm.
thus indicating the presence of urease activity. The composition Monopotassium phosphate – 0.2 gm.
and preparation of Christensen’s urea agar is described in the Phenol red (1.2%) – 1.0 ml.
box 23-1. Agar – 2 gm.
List of urease producing microorganisms is summarized in Distilled water – 100 ml.
the Table 23-1. pH – 6.8.
Preparation
REQUIREMENTS Prepare the base, sterilize by autoclaving at 121°C for 15
min. Cool to 50°C in a waterbath and then add 5 ml of filter
I Equipments sterilized 40% urea solution. Mix, distribute in 2–4 ml amounts
Incubator. in 12×100 mm test tubes. Allow the medium to solidify in a
slanting position in such a way to get half inch butt and one
inch slant.
72 Urease Test
Brucella species
Helicobacter pylori
Proteus species
Morganella species
Klebsiella species
Enterobacter species
Cryptococcus neoformans
Trichophyton mentagrophytes
FIGURE 23-1 Urease negative and positive test.
QUALITY CONTROL
The uninoculated medium is colour less. In a positive test, after
Positive control: P. mirabilis (urease positive bacteria). incubation, the colour of the medium changes to purple pink.
Negative control: Escherichia coli (urease negative bacteria).
An uninoculated medium is incubated along with the test to
compare the colour change. RESULTS AND INTERPRETATION
KEY FACTS
1 Certain bacteria and fungi possess the enzyme urease that hydrolyzes urea releasing ammonia into the medium.
2 Phenol red that is incorporated in the medium changes its color from yellow to red in alkaline pH, thus indicating the
presence of urease activity.
3 Control strains should be used for interpretation of results.
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS (Eds). Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis)
2002.
3 Introduction to Clinical Microbiology. University of Texas- Houston Medical School, DPALM Medic. 1995.
4 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC (Eds). Color Atlas and Textbook of Diagnostic Microbiology. 5th
ed. (Lippincott Williams and Wilkins, USA). 1997.
74
LESSON
Indole Test
24
LEARNING OBJECTIVES Kovac’s reagent consists of para-dimethyl amino
benzaldehyde, 5.0 gm; isoamyl alcohol, 75.0 ml; and
After completing this practical you will be able to: concentrated hydrochloric acid, 25.0 ml.
1 Determine the ability of bacteria to degrade the amino acid Ehrlich’s reagent consists of p-dimethyl amino
tryptophan. benzaldehyde, 4.0 gm; absolute ethyl alcohol, 380.0 ml; and
2 Distinguish the bacteria based on the indole activity. concentrated hydrochloric acid, 80.0 ml.
III Specimen
24 hours to 48 hours peptone water culture of Escherichia coli
INTRODUCTION
incubated at 37°C.
Tryptophan is an essential amino acid that can undergo
oxidation by enzymatic activities of some bacteria. Conversion PROCEDURE
of tryptophan into metabolic products is mediated by the
enzyme tryptophanase. The metabolic end products are indole, 1 Take 0.5 ml of 24 hours to 48 hours peptone water cultures of
skatole and indole acetic acid. The ability to hydrolyse E. coli in a small test tube.
tryptophan with the production of indole is not a characteristic 2 Add 0.2 ml of Kovac’s reagent to the peptone water and shake.
of all bacteria.Only some bacteria produce indole. 3 Allow it to stand for few minutes and read the result.
Tryptophan is an amino acid.This is present in peptone water Positive control: E. coli (indole positive bacteria).
of the culture medium, and when acted upon by the enzyme Negative control: Klebsiella pneumoniae (indole negative
tryptophanase, it is converted into indole, skatole and indole bacteria).
acetic acid. The indole reacts with aldehydes to produce a red
coloured product. The aldehyde used in the test is para dimethyl
amino benzaldehyde.
OBSERVATION
List of indole positive and negative bacteria are presented
in the Table 24-1 In a positive test, a red-violet ring develops within minutes on
addition of Kovac’s reagent. In a negative test a yellow ring
appears.
REQUIREMENTS
RESULTS AND INTERPRETATION
I Equipments
Incubator. Positive indole test is indicated by the appearance of red-violet
ring on adding the reagent. Negative reaction is indicated by
II Reagents and lab wares developing a yellow ring (Fig. 24-1). E. coli colonies tested are
Peptone water / tryptone broth, Kovac’s reagent, or Ehrlich’s an indole producing bacteria. K.pneumoniae does not produce
reagent, glass tubes and inoculating wire. the indole
Textbook of Practical Microbiology 75
KEY FACTS
1 End product of tryptophan metabolism by tryptophanase is indole, skatole, and indole acetic acid.
2 Indole reacts with p-dimethyl amino benzaldehyde to form Quinoidal red-violet compound.
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS (Eds). Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby
Company, St. Louis) 2002.
3 Introduction to Clinical Microbiology. University of Texas- Houston Medical School, DPALM Medic. 1995.
4 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC (Eds). Color Atlas and Textbook of Diagnostic
Microbiology. 5th ed. (Lippincott Williams and Wilkins, USA). 1997.
76
LESSON
Methyl Red Test
25
LEARNING OBJECTIVES The methyl red test is a quantitative test for acid production,
requiring positive organisms to produce strong acids (lactic
After completing this practical you will be able to: acid, acetic acid, formic acid) from glucose through the mixed
acid fermentation pathway. Because many species of the
1 Determine the ability of bacteria to oxidise glucose with the Enterobacteriaceae may produce sufficient quantities of strong
production of high concentrations of acidic end products acids that can be detected by methyl red indicator during the
by methyl red test. initial phase of incubation, only organisms that can maintain
2 Differentiate between all glucose oxidizing enteric bacteria this low pH after prolonged incubation (48–72 hours)
particularly Escherichia coli and Enterobacter aerogenes. overcoming the pH buffering system of the medium can be
called methyl red positive.
List of MR positive and negative bacteria is presented in
INTRODUCTION the table 25-1.
PRINCIPLE
PROCEDURE
Methyl red is a pH indicator with a range between 6.0 (yellow)
and 4.4 (red). The pH at which methyl red detects acid is 1 Take 0.5 ml of broth cultures of E. coli in a small test tube.
considerably lower than the pH of other indicators used in 2 Add five drops of 0.04% solution of methyl red directly to
bacteriologic culture media. Thus to produce a colour change, the broth culture and mix well.
the test organism must produce large quantities of acid from 3 Note any change in the colour of medium at once.
the carbohydrate substrate being used.
Textbook of Practical Microbiology 77
QUALITY CONTROL
Look for the development of stable red colour on adding methyl FIGURE 25-1 IMViC Test.
red indicator.
KEY FACTS
1 MR test detects the products of stable high concentration of acidic end products.
2 The test differentiates between E. coli and E. aerogenes.
3 Methyl red is the indicator used in the test.
4 Methyl red is yellow in alkaline pH and red in acidic pH.
5 Development of red colour on addition of methyl red to broth culture of bacteria is considered positive.
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS (Eds). Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis)
2002.
3 Introduction to Clinical Microbiology. University of Texas- Houston Medical School, DPALM Medic. 1995.
4 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC (Eds). Color Atlas and Textbook of Diagnostic Microbiology. 5th
ed. (Lippincott Williams and Wilkins, USA). 1997.
78
LESSON
Voges-Proskauer Test
26
LEARNING OBJECTIVES small amount of acetyl methyl carbinol present in the medium
is converted to diacetyl, which reacts with the peptone of the
After completing this practical you will be able to: broth to produce a red colour.
List of VP positive and negative bacteria is presented in the
table 26-1.
1 Perform Voges-Proskauer test.
REQUIREMENTS
INTRODUCTION
I Equipments
Voges-Proskauer is a double eponym, named after two Incubator.
microbiologists working at the beginning of the 20th century.
They first observed the red colour reaction produced by II Reagents and lab wares
appropriate culture media after treatment with potassium
hydroxide. It was later discovered that the active product in Inoculating loop.
the medium formed by bacterial metabolism is acetyl methyl VP broth. It consists of polypeptone, 7 gm; glucose,5 gm;
carbinol, a product of the butylene glycol pathway. dipotassium phosphate, 5 gm and distilled water, 1 litre at a pH
of 6.9.
5% a naphthol. It consists of a naphthol, 5 gm; and absolute
PRINCIPLE ethyl alcohol, 100 ml. It serves as the colour intensifier.
40% potassium hydroxide. It consists of 40 gm potassium
The Voges-Proskauer test determines the capability of some hydroxide in 100 ml distilled water. It serves as the oxidising
bacteria to produce non-acidic or neutral end products such as agent.
acetyl methyl carbinol from the organic acids produced as a
result of glucose metabolism. Pyruvic acid, the pivotal III Specimen
compound formed in the fermentative degradation of glucose Culture of Escherichia coli, Enterobacter aerogenes and
is further metabolised through various metabolic pathways, Klebsiella pneumoniae in glucose phosphate medium
depending on the enzyme systems possessed by different incubated at 30°C for five days or 37°C for 48 hours.
bacteria.
One such pathway results in the production of acetoin
(acetyl methyl carbinol), a neutral-reacting end product. Enteric PROCEDURE
bacteria such as members of the Klebsiella-Enterobacter-
Hafnia-Serratia group produce acetoin as the chief end 1 Take 1 ml of broth cultures of E. coli in a small test tube.
products of glucose metabolism and form smaller quantities of 2 First add 40% KOH and then add 0.6 ml of a 5% solution of
mixed acids. a naphthol in ethanol to the broth culture and shake gently.
The test depends on the production of acetyl methyl It is essential that the reagents are added in this order.
carbinol from pyruvic acid, as an intermediate product in its 3 Note any change in the colour of medium within 2-5 minutes.
conversion to 2: 3 butylene glycol. In the presence of
atmospheric oxygen and alkali (40% potassium hydroxide), the
Textbook of Practical Microbiology 79
QUALITY CONTROL hour.The test should not be read after standing for over 1 hour
because negative VP test may produce a copper-like colour,
Positive control: E. aerogenes. leading to a false positive interpretation.
Negative control: E. coli.
KEY FACTS
1 The VP test shows the presence of non acidic neutral end product acetyl methyl carbinol.
2 A positive test is represented by the development of a pink colour 15 minutes or more after addition of the reagents,
deepening to magenta or crimson in half an hour.
3 A negative test is indicated by colour less reaction for half an hour.
4 Some times prolonged incubation of cultures is required before doing the test.
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS (Eds). Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis)
2002.
3 Introduction to Clinical Microbiology. University of Texas- Houston Medical School, DPALM Medic. 1995.
4 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC (Eds). Color Atlas and Textbook of Diagnostic Microbiology. 5th
ed. (Lippincott Williams and Wilkins, USA). 1997.
80
LESSON
Citrate Utilisation Test
27
LEARNING OBJECTIVES REQUIREMENTS
KEY FACTS
1 Simmon’s citrate medium contains sodium citrate which acts as a sole source of carbon.
2 Bromothymol blue is the indicator used in Simmon’s citrate medium.
3 Development of deep blue colour is taken as positive.
4 A negative test is indicated by no change of colour of the citrate medium.
VIVA
1 What are the constituents of Simmon’s citrate medium?
2 What is the principle behind citrate utilisation test?
3 What is the positive control and negative controls used in the citrate utilisation test?
4 What are the organisms that are citrate utilisation positive and negative?
5 How will you interpret the results of citrate utilisation test?
Ans: A positive test is represented by the development of a deep blue colour within 24 hours to 48 hours indicating that the
test organisms has been able to utilize citrate contained in the medium, with the production of alkaline products.
6 Mention different types of citrate utilization tests.
Ans: Simmon’s citrate utilization test and Koser’s citrate utilization test.
7 List differences between Koser’s citrate and Simmon’s citrate media.
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS (Eds). Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis)
2002.
3 Introduction to Clinical Microbiology. University of Texas- Houston Medical School, DPALM Medic. 1995.
4 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC (Eds). Color Atlas and Textbook of Diagnostic Microbiology. 5th
ed. (Lippincott Williams and Wilkins, USA). 1997.
82
LESSON
Triple Sugar Iron (TSI)
28 Agar Test
LEARNING OBJECTIVES to yellow and the whole medium appears yellow in colour. After
further incubation as the glucose is fully exhausted, the bacteria
After completing this practical you will be able to: begin to oxidatively degrade the amino acid present in the
medium. Since oxygen is exposed only to the slant portion,
1 Differentiate among the members of the family oxidative degradation occurs only in the slant portion. This
Enterobacteriaceae by the TSI test. oxidative degradation results in production of alkali products,
which reverts the colour of the slant to red colour. In the deeper
part of the tube, amino acid degradation is insufficient to
INTRODUCTION overcome the acid formed, so medium in the butt part remains
yellow in colour.
The triple sugar iron (TSI) agar is an example of a composite If the TSI medium is inoculated with lactose fermenting
medium used widely for the identification of bacterial organism, then even after the glucose is completely used up in
isolates.This medium is convenient and economical, because first 8–12 hours, fermentation continues as the organism is able
as a single composite medium different properties of the bacteria to use lactose which is present in concentration 10 times that of
which otherwise would have required the use of many separate glucose. So the acid production continues to occur even after
media could be used. 18–24 hours and both the slant and the butt appear yellow.
The TSI agar is designed to differentiate among different For the detection of H2S which is a colourless gas, medium
groups or genera of the family Enterobacteriaceae. The latter must include an indicator to detect the H2 S. Sodium thiosulfate
are the Gram Negative bacilli capable of fermenting glucose is the source of sulfur atoms. Ferrous sulfate is the indicator
with the production of acid. The differentiation can be made on used for the detection of the H2S which is indicated by the
the basis of differences in carbohydrate fermentation and production of insoluble black precipitate.
hydrogen sulfide production by various intestinal bacteria.
The TSI agar has glucose, lactose, and sucrose as the sources
of carbohydrates. The slant contains lactose and sucrose in the REQUIREMENTS
concentration of 1% and glucose in the concentration of
0.1%.Phenol red is the acid base indicator incorporated in the I Equipments
medium.This indicator helps to detect carbohydrate fermentation Incubator.
that is indicated by a change in colour of the medium from orange
red to yellow in the presence of acid. II Reagents and lab wares
Inoculating loop and triple sugar iron agar (It contains beef
extract, 3g; yeast extract, 3g; peptone, 15g; proteose peptone,
PRINCIPLE 5g; lactose, 10g; sucrose, 10 gm; glucose, 1 gm; ferrous sulfate,
0.2 gm; sodium chloride, 5g; sodium thiosulfate, 0.3g; agar, 12
The TSI agar is distributed in the tube which contains a slant gm; phenol red, 0.024 g; and distilled water to equal 1 litre) at
and a butt.TSI medium indicates whether the bacteria ferments pH 7.4.
glucose only, or lactose and sucrose also with or without
production of gas. The medium can detect production of III Specimen
hydrogen sulphide (H2S) as well as other bacteria which utilizes Culture of test organisms such as Escherichia coli, Proteus
only glucose (but not lactose or sucrose). Due to the acid mirabilis and Klebsiella pneumoniae in glucose phosphate
production, the colour of the phenol red (indicator) is changed medium incubated at 37°C for 48 hours.
Textbook of Practical Microbiology 83
PROCEDURE
QUALITY CONTROL
VIVA
KEY FACTS
1 Beef extract, yeast extract, peptone, and proteose peptone makes the medium nutritionally rich.
2 Glucose and lactose; and sucrose are added in the ratio of 1: 10 in the TSI agar.
3 Oxidative degradation of amino acids in the medium occurs only in the slant.
4 Ferrous sulfate is the indicator for the production of the hydrogen sulfide.
5 Sodium thiosulfate is the source of sulfur.
6 Phenol red is the indicator used to detect acid /alkaline changes in the medium.
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS (Eds). Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby
Company, St. Louis) 2002.
3 Introduction to Clinical Microbiology. University of Texas- Houston Medical School, DPALM Medic. 1995.
4 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC (Eds). Color Atlas and Textbook of Diagnostic Microbiology. 5th
ed. (Lippincott Williams and Wilkins, USA). 1997.
Textbook of Practical Microbiology 85
LESSON
Hydrogen Sulfide Test
29
LEARNING OBJECTIVES reduction of inorganic sulfur compounds such as the
thiosulfates (S2O3), sulfates (SO4) or sulfites (SO3). The medium
After completing this practical you will be able to: contains sodium thiosulfate, which are reduced to sulfite by
certain microorganisms with the liberation of hydrogen sulfide.
1 Determine the ability of certain bacteria to produce hydrogen The sulfur atoms act as hydrogen acceptors during oxidation
sulfide from substrates such as the sulfur containing amino of the inorganic compound. Liberated H2S combines with
acids or inorganic sulfur compounds. indicator like ferrous sulfate (Fe SO4) to produce black insoluble
precipitate (ferrous sulfide).
List of media used for detecting production of hydrogen
INTRODUCTION sulphide is presented in the box 29-1.
List of bacteria producing hydrogen sulphide is presented
Some bacteria liberate sulfur from sulfur containing amino acids in the table 29-1.
or other sulfur containing compounds. The sulfur is used as
final hydrogen acceptor leading to the formation of hydrogen
sulphide (H2S). Sulfur containing amino acids such as REQUIREMENTS
methionine, cystine, cysteine or inorganic compounds such as
this sulphates, etc. should be present in the medium to detect I Equipments
the presence of the H2 S. H2S being a gas, will escape from the Incubator.
medium. So, the indicators such as heavy metal ions are added
to the medium, which support the growth of the organism. II Reagents and lab wares
Bacteria which are tested for the production of H2 S must contain Sulphur containing medium such as Kligler iron agar, TSI agar
enzyme system which release sulfide from the sulfur source. or lead acetate agar, etc. and inoculating loop.
Sulfides combine with hydrogen ion to form H2 S. H2 S combines
with heavy metals to form insoluble black precipitate. III Specimen
Soy broth cultures of test bacteria such as Enterobacter
aerogenes, Proteus vulgaris and Salmonella Typhimurium
PRINCIPLE incubated at 37°C for 24- 48 hours.
BOX 29-1 LIST OF MEDIA USED FOR Table 29-1 List of H2S positive bacteria
DETECTING PRODUCTION OF
1 Citrobacter freundii
HYDROGEN SULFIDE
2 Salmonella Arizona
List of media 3 Salmonellae spp (except S. Paratyphi A)
1. Bismuth sulfite agar 4 Proteus mirabilis
2. Citrate sulfide agar 5 Proteus vulgaris
3. Deoxycholate citrate agar 6 Edwardsiella tarda
4. Lysine iron agar. 7 Edwardsiella hoshinae
5. Kligler iron agar 8 Shewanella putrefaciens
6. TSI agar 9 Campylobacter sputorum
7. Lead acetate agar 10 Brucella abortus
8. Salmonella-Shigella agar 11 Brucella suis.
9. Sulphur indole motility medium 12 Erysiphilothrix rhusiopathiae
10. Xylose lysine deoxycholate agar
11. Hektoen enteric agar
12. Peptone water, lead acetate paper inserts. RESULTS AND INTERPRETATION
Note: Source of sulfur and H2S indicators are different for
each medium. Black coloration along the streak line or throughout the medium
indicates H2S production. If black colour is not produced then
Sources of sulfur H2S is not produced (Fig. 29-1).
1. Sulfite
2. Peptone
3. Sodium thiosulfate
H2S indicator
1. Ferrous sulfate
2. Ferric ammonium citrate
3. Lead acetate
4. Peptonised iron.
QUALITY CONTROL
VIVA
1 What are the conditions required for the production and demonstration of H2S by bacteria?
2 What are the sequence of events occurring in the production and detection of H2S?
3 What is the source of sulfur in the media?
4 What are the indicators of H2S production?
5 What are the sulfur containing amino acids?
6 How will you pick up the colonies for inoculation?
7 What are the media that can be used in H2S production detection?
8 Name some H2S producing bacteria.
Textbook of Practical Microbiology 87
KEY FACTS
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS (Eds). Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis)
2002.
3 Introduction to Clinical Microbiology. University of Texas- Houston Medical School, DPALM Medic. 1995.
4 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC (Eds). Color Atlas and Textbook of Diagnostic Microbiology. 5th
ed. (Lippincott Williams and Wilkins, USA). 1997.
88
LESSON
Nitrate Reduction Test
30
LEARNING OBJECTIVES extract, 3 gm; peptone,5 gm; potassium nitrate,1 gm; agar,12
gm, and distilled water, 1 L).
After completing this practical you will be able to: Reagent A (a naphthylamine, 5 g; acetic acid (5N) 30%, 1 L).
1 Determine the ability of certain bacteria to reduce nitrates Reagent B (sulphanilic acid, 8 g; acetic acid (5N) 30%, 1 L).
(NO3–) to nitrites (NO2–) or beyond the nitrite stage. Inoculating loop.
III Specimen
INTRODUCTION Cultures of test bacteria such as Escherichia coli, Acinetobacter
baumannii, etc. in a broth containing 1% potassium nitrate
Nitrates serve as a source of nitrogen for many bacteria. They broth (KNO3) incubated at 37°C for 5 days.
can also act as final electron acceptor. Many bacteria can be
differentiated and are identified by their capacity to reduce
nitrates to nitrites. Most of the bacteria belonging to the family PROCEDURE
Enterobacteriaceae reduce nitrates. This character is also useful
for the identification of species in the genera Neisseria, 1 Mix an equal volume (0.5 ml) of reagent A with 0.5 ml of
Haemophilus and Branhamella. Some Pseudomonas and non- reagent B just before use.
fermenters reduce nitrate to nitrite and further down to N2 and 2 Add 0.1 ml of the test reagent to 1 ml of the culture broth of
molecular nitrogen. This is called denitrification. the bacteria to be tested.
3 Observe for any change of colour immediately within few
minutes.
PRINCIPLE
Bacteria demonstrating nitrate reduction have the capability of ex- QUALITY CONTROL
tracting oxygen from nitrates to form nitrite and other reduction
products. The chemical reaction is NO3–+ 2e– + 2H+®NO2– + H2O. Positive control: E. coli.
The presence of nitrites in the test medium is detected by Negative control: A. baumannii.
the addition of a-naphthylamine and sulphanilic acid, with the
formation of a red diagonium dye, p-sulfobenzena-azo-a-
naphthylamine. OBSERVATION
I Equipments
Incubator.
RESULTS AND INTERPRETATION
II Reagents and lab wares The development of a red colour within 30 seconds. after adding
1% potassium nitrate broth (KNO3) or nitrate agar slant (beef the test reagents indicates the presence of nitrites and
represents positive reaction (Fig. 30-1).
Textbook of Practical Microbiology 89
KEY FACTS
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS (Eds). Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis)
2002.
3 Introduction to Clinical Microbiology. University of Texas- Houston Medical School, DPALM Medic. 1995.
4 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC (Eds). Color Atlas and Textbook of Diagnostic Microbiology. 5th
ed. (Lippincott Williams and Wilkins, USA). 1997.
90
Textbook of Practical Microbiology 91
UNIT
V
Antimicrobial
Sensitivity Tests
LESSON
Kirby–Bauer Method
31
LEARNING OBJECTIVES II Reagents and lab wares
0.5 McFarland standard, Mueller Hinton agar plates (pH 7.2-
After completing this practical you will be able to: 7.4), peptone water , filter paper discs impregnated with
appropriate concentration of antibiotics, sterile cotton swabs,
1 Determine antibacterial sensitivity of bacterial isolates by millimeter ruler, forceps and inoculating wire.
Kirby –Bauer disc diffusion method. Preparation of 0.5 McFarland standard: Solution A is
prepared by adding barium chloride (BaCl 2, 2H2O) to 100ml
distilled water. Solution B is prepared by adding 1ml of
INTRODUCTION sulphuric acid (H2S04 (0.36N) to 100 ml of distilled water. Then
0.5 ml of solution A is added to 99.5 ml of solution B, mixed well
Due to emergence of many antibiotic resistant strains of and distributed in test tubes with a screw cap. The cap is closed
bacteria, antimicrobial susceptibility testing is done in order to tightly to avoid evaporation. The mixture is stored in the dark.
determine which antimicrobial agent to use against a specific The solution is agitated vigorously before using it.
strain of bacteria. The available chemotherapeutic agents vary
in their scope of antimicrobial activity. Some have a limited III Specimens
spectrum while others have a wide spectrum of activities against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC
bacteria. The bacterial strains isolated from clinical samples 25922, Enterococcus faecalis ATCC 29212, and Pseudomonas
should be tested for antimicrobial sensitivity because it gives aeruginosa ATCC 27853.
the clinician an idea as to what antimicrobial therapy should be Preparation of suspension of bacteria: Approximately, 4-5
started to the patients (Box 31-1). well isolated colonies of the bacterial strain to be tested are
inoculated into 5 ml of peptone water, and is incubated at 37 °C
for 3-4 hours. The turbidity of the suspension is adjusted to
PRINCIPLE match 0.5 McFarland standards. If the density is more it is
diluted with sterile saline. The comparison is made against a
Kirby – Bauer method is a method of determination of antibiotic white back ground with a contrasting black line.
sensitivity of the bacteria by disc diffusion method. In this
method, a standard suspension of bacteria to be tested are
inoculated on the surface of Mueller Hinton agar plates. Filter PROCEDURE
paper discs containing specific concentration of antimicrobial
agents are pressed on to the surface and incubated at 35°C 1 After standardisation of bacterial suspension, immerse a
overnight (18-24 hr.). After incubation, the zone of inhibition sterile cotton swab in it and rotate the swab several times
of growth of bacteria around each disc is measured and the with firm pressure on the inside wall of the tube to remove
susceptibility is determined. excess fluid.
2 Prepare a Mueller Hinton agar (MHA) plate (pH 7.2-7.4) with
a depth of 4 mm.
REQUIREMENTS 3 Inoculate the dried surface of the MHA agar plate by
streaking the swab three times over the entire agar surface.
I Equipments It is streaked in three directions by rotating the plate 60°
Incubator. after each streak.
Textbook of Practical Microbiology 93
OBSERVATIONS
Antibiotics: It is a substance produced by microorganisms or a similar substance produced wholly or partially by chemical
synthesis that inhibits the growth or causes death of other organisms in low concentrations.
Antimicrobial agent: It is a chemical substance inhibiting the growth or causing death of microorganism.
Bacteriostatic drug: Certain antimicrobial agents inhibit the growth by preventing the multiplication of organisms. They do
not cause death. These are called bacteriostatic agents. eg. tetracycline, erythromycin, and sulphonamides.
Bactericidal drug: The drugs that cause irreversible damage to bacteria resulting in death are called bactericidal drugs.
e.g. aminoglycosides, penicillin and quinolones.
KEY FACTS
1 Depth of agar in medium should be 4 mm as some antibiotics show decreased zone size with increased depth while others
show slight increase.
2 Standardization of the bacterial inoculum is important. It should be such that it gives rise to a semi confluent growth, as
growth denser than this or lighter than this give problem while reading zone size.
3 Proper storage of the antimicrobial discs, so that they retain their potency.
94 Kirby-Bauer Method
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 94-96, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th
Edition. 1997. pp. 1395. Lippincott Williams and Wilkins.
4 Lalitha M.K. Manual on Antimicrobial Susceptibility Testing. Christian Medical College, Vellore, 2004, pp 43.
5 WHO. Guidelines on Standard Operating Procedures for Microbiology. Blood safety and Clinical Technology. Chapter 7: Antimicrobial
Susceptibility Testing.
Textbook of Practical Microbiology 95
LESSON
The Stokes Method
32
LEARNING OBJECTIVES III Specimens
Control strains: Staphylococcus aureus NCTC 6571,
After completing this practical you will be able to: Escherichia coli NCTC 10414 and Pseudomonas aeruginosa
1 Determine antibacterial sensitivity of bacterial isolates by NCTC 10662.
Stokes method. Preparation of suspension of bacteria: Approximately, 4-
5 well isolated colonies of the bacterial strain to be tested are
transferred to Tryptic soy broth or BHI broth. The turbidity
INTRODUCTION of the suspension is adjusted to match 0.5 McFarland
standards.
Stokes method is an example of the disc diffusion test and is
another method used for routine antibiotic sensitivity testing of
bacterial strains. The method makes use of in-built controls against PROCEDURE
many variables and therefore provides dependable results. A set
of standard strains are used as control strains depending on the 1 The inoculation plates are dried with lids open so that there
bacterium to be tested. The control strains are Escherechia coli are no droplets of moisture on the surface.
NCTC 10414 for testing coliform bacilli from urinary tract. 2 The control culture is applied in two bands on either side of
Pseutomonas aeruginosa NCTC 10662 against aminoglycosides. the plate leaving a central band uninoculated with the help
Kirby-Bauer and Stokes methods are compared in the of sterile swab.
table 32-1. 3 The test organism is applied in the central portion without
touching the either sides.
4 Antibiotic discs are applied with forceps on the line between
PRINCIPLE the test and control organisms and pressed gently to ensure
even contact with the medium. There should be a minimum
In this test antibiotic discs are applied between the standard and distance of 2 cm between two discs. Four discs can be
test inocula, so that zones of inhibition formed around each disc accommodated on an 85 mm circular plate.
are composed of standard and test bacteria. The diffusion of 5 For inoculation, a rotatory plating method can also be used
antibiotic takes place and thus the susceptibility of those wherein the control strain is applied on the outer periphery
organisms to the antibiotic are known by measuring zone size. and the test strain is applied in the central portion. In such a
method 6 discs can be put on an 85 mm circular plate.
6 The plates are then incubated overnight at 35°C –37°C.
REQUIREMENTS
OBSERVATIONS
Zone sizes are measured from the edge of the discs to the edge
of the zone. Comparison of the zones of inhibition between the
standard and test bacteria indicates the sensitivity of the test
bacteria. If the test zones are obviously larger than the control
or give no zone of inhibition at all; there is no need to perform
any measurement with calipers or a millimeter scale.
1. Test and control strains have 1. Test and control strains tested on the same
to be tested on separate plates. 2. 4 discs on 85mm plate and 6 discs by rotatory method.
2. 6 discs applied on 85mm plate. 3. Zone diameter measured from edge of disc to edge of zone
3. Zone diameter is measured including disc diameter. of inhibitation the disc.
KEY FACTS
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 94-96, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th
Edition. 1997. pp. 1395. Lippincott Williams and Wilkins.
4 Lalitha M.K. Manual on Antimicrobial Susceptibility Testing. Christian Medical College, Vellore, 2004, pp 43.
5 WHO. Guidelines on Standard Operating Procedures for Microbiology. Blood safety and Clinical Technology. Chapter 7: Antimicrobial
Susceptibility Testing.
Textbook of Practical Microbiology 97
LESSON
Agar Dilution Method
33
LEARNING OBJECTIVES II Reagents and lab wares
0.5 McFarland standard, sterile Mueller Hinton agar (pH 7.2-
After completing this practical you will be able to: 7.4), sterile Mueller Hinton broth, antibiotic powder, sterile test
1 Determine antibacterial sensitivity of bacterial isolates by tubes, pipettes, screw capped flat bottomed bottles (25 ml
agar dilution method. capacity) and Petri dishes (90 mm diameter).
These also include sterile saline (0.85 %) and stock solution
of antibiotic.
INTRODUCTION Preparation of stock solutions of antibiotics: The required
dilutions of the antibiotics are made as per the table 33-1. Prepare
Agar dilution is a quantitative method for determining the a stock solution containing 2000 µg / ml of the antibiotic to be
minimum inhibitory concentration of the antibiotics against tested. For example weigh 200 mg of the antibiotic powder and
bacteria to be tested. It is mainly useful in testing isolates from dissolve in 5 ml of distilled water / appropriate solvent. Mix 0.5
serious infections like bacterial endocarditis or to verify ml of this solution with 9.5 ml distilled water (working solution
equivocal results (e.g. intermediate susceptibility of contains antibiotics at a strength of 200 µg / ml-solution A)
ciprofloxacin against Salmonella Typhi).
Diffusion tests used to determine the susceptibility of III Specimens
organisms isolated from clinical specimens have their own Preparation of suspension of bacteria: Approximately, 4-5 well-
limitations, when equivocal results are obtained, or in prolonged isolated colonies of the bacterial strain to be tested are
serious infections eg. bacterial endocarditis. In these cases, the transferred to Tryptic soy broth or BHI broth. The turbidity of
quantitation of antibiotic against pathogen needs to be more the suspension is adjusted to match 0.5 McFarland standards
precise So when in doubt about the sensitivity of pathogen the (106 organisms/ml).
way to a precise assessment is to determine the minimum inhibitory
concentration (MIC) of the antibiotic to the organisms concerned.
PROCEDURE
Table 33.1 System for preparing dilutions for agar dilution method
Antibiotic Solution + Sterile Water (Vol) = Intermediate conc Final conc at 1:10 in
(µg / ml) in tubes agar plates (µg / ml)
Volume µg/ml
2 160 - E 2 80 - F 8
1 160 - E 3 40 - G 4
1 160 - E 7 20 - H 2
2 20 - H 2 10 - I 1
1 20 - H 3 5 - J 0.5
1 20 - H 7 2.5 - K 0.25
KEY FACTS
1 Agar dilution method is a quantitative method for determining the minimum inhibitory concentration (MIC) of the antibiotic
against bacteria to be tested.
2 The method is carried out on Muller Hinton agar.
3 It is mainly useful in testing isolates from serious infections like bacterial endocarditis or to verify equivocal results (e.g.
intermediate susceptibility of ciprofloxacin against Salmonella Typhi).
4 Selective media should not be used for performing agar dilution method.
5 Electrolyte deficient media also should not be used because it will give false results due to variations in the salt content on
action of many antibiotics.
Textbook of Practical Microbiology 99
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 94-96, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th
Edition. 1997. pp. 1395. Lippincott Williams and Wilkins.
4 Lalitha M.K. Manual on Antimicrobial Susceptibility Testing. Christian Medical College, Vellore, 2004, pp 43.
5 WHO. Guidelines on Standard Operating Procedures for Microbiology. Blood safety and Clinical Technology. Chapter 7: Antimicrobial
Susceptibility Testing.
100
LESSON
Broth Dilution Method
34
LEARNING OBJECTIVES II Reagents and lab wares
0.5 McFarland standard, sterile Mueller Hinton broth, antibiotic
After completing this practical you will be able to: powder, sterile test tubes. sterile pipettes of 10ml, 5 ml, 2 ml and
1 ml, sterile capped tubes and test tube rack.
1 Determine antibacterial sensitivity of bacterial isolates by These also include stock solution of antibiotic.
broth dilution method. Preparation of stock solutions of antibiotics: The required
dilutions of the antibiotics are made as per the table 34-1. Prepare
a stock solution containing 2000 µg / ml of the antibiotic to be
INTRODUCTION tested. For example weigh 200 mg of the antibiotic powder and
dissolve in 5 ml of distilled water / appropriate solvent. Mix 0.5
Broth dilution is also known as tube dilution method. It is ml of this solution with 9.5 ml distilled water (stock solution
another quantitative method for determining the minimum contains antibiotics at a strength of 200 µg / ml-solution A)
inhibitory concentration (MIC) of the antibiotic against a
bacteria to be tested. In this method, serial dilutions of the III Specimens
antibiotics are taken in test tubes and a standardise suspension Preparation of suspension of bacteria: Approximately, 4-5 well
of the bacterium is inoculated. After incubating overnight , the isolated colonies of the bacterial strain to be tested are trans-
MIC of the antibiotics is determined by observing the lowest ferred to Tryptic are soy broth or BHI broth. The turbidity of
concentration of antibiotics that inhibits growth of the bacteria. the suspension is adjusted to match 0.5 McFarland standards.
The minimum bactericidal concentration (MBC) can also be
estimated by this method by subculturing from the lowest
concentration of drug that kills the bacteria. PROCEDURE
KEY FACTS
1 Minimum inhibitory concentration (MIC) is defined as the highest dilution which inhibits growth of the bacteria. It is noted
by lack of turbidity in the tube.
2 Because very faint turbidity may be given by the inoculum itself, the inoculated tube kept in the refrigerator overnight may
be used as the standard for the determination of complete inhibition,
3 Standard strain of known MIC should be tested as control to check the reagents and conditions.
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 94-96, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th
Edition. 1997. pp. 1395. Lippincott Williams and Wilkins.
4 Lalitha M.K. Manual on Antimicrobial Susceptibility Testing. Christian Medical College, Vellore, 2004, pp 43.
5 WHO. Guidelines on Standard Operating Procedures for Microbiology. Blood safety and Clinical Technology. Chapter 7: Antimicrobial
Susceptibility Testing.
102
LESSON
Epsilometer Test (E-test)
35
LEARNING OBJECTIVES REQUIREMENTS
After completing this practical you will be able to: I Reagents and lab wares
1 Determine antibacterial sensitivity of bacterial isolates by Commercially available E-test strips, 0.5 McFarland standards,
Epsilometer test (E-test), an automated system for measuring sterile Mueller Hinton agar plates (150 or 90 mm with a depth of
the minimum inhibitory concentration (MIC) of the bacteria. 4 mm). In a 90 mm plate, a single antibiotic strip can be tested.
In a 150 mm plate, at least 4 antibiotic strips can be tested.
These also include foreceps, 0.85% saline for inoculum
INTRODUCTION preparation, and sterile swabs
2 Ensure that the agar surface is dry before swabbing it. Dip a
swab in the inoculum, remove excess fluid and swab the
entire agar surface evenly in 3 directions.
3 Allow the agar surface to dry for 10 minutes to 15 minutes on
the bench or in the incubator.
4 Open the E-test package and place the strips in a dry petri dish.
5 Apply the strips to the agar surface with a forceps. Always
apply the strip with the MIC scale facing the opening of the
plate. Do not apply it upside down.
Note: Be firm when applying the strip. Once applied, do not
move the strip.
6 Use templates to position 4 to 6 strips on a 150 mm plate or
one to two strips on a 90 mm plate. FIGURE 35-1 E-Test.
7 Place the handle of the strip closest to the rim of the plate.
Note: Always store unused strips in airtight containers at -
20°C or -70°C. RESULTS AND INTERPRETATION
8 Incubate at 37°C for 18-24hours.
Read plates after the recommended incubation period only if
sufficient growth is seen and the inhibition eclipse is clearly
QUALITY CONTROL visible.
Read the MIC where the ellipse intersects the scale.
Package labels for each antibiotic will carry performance and Always read the end point at complete inhibition of all
reproducibility data. NCCLS QC ranges and interpretive guidelines. growth including hazes and isolated colonies.
Since E- test comprises a continuous gradient, MIC values
in between two – fold dilutions can be obtained.
Always round up these values to the next two-fold dilution
OBSERVATIONS
before interpretation. For example: If ampicillin breakpoints are
given as S=1, I = 2, R=4 µg/ml, then an E-test MIC of 1.5 µg/ml
After incubation an elliptical zone of growth inhibition is seen
is rounded up to 2 µg/ml and the category reported as
around the strip. The MIC is read from the scale at the
Intermediate (I)
intersection of the zone with the strip (Fig. 35-1).
KEY FACTS
1 The E-test strips have to be placed in proper orientation. Placing strips upside down on the agar will alter the results.
2 Diffusion of antibiotics begins immediately after placement of the strips, which cannot be moved once it has touched the agar.
3 Read plates after the recommended incubation period only if sufficient growth is seen and the inhibition eclipse is clearly
visible.
4 Read the MIC where the ellipse intersects the scale.
5 Always store unused strips in airtight containers at -20°C or -70°C.
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 94-96, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th
Edition. 1997. pp. 1395. Lippincott Williams and Wilkins.
4 Lalitha M.K. Manual on Antimicrobial Susceptibility Testing. Christian Medical College, Vellore, 2004, pp 43.
5 WHO. Guidelines on Standard Operating Procedures for Microbiology. Blood safety and Clinical Technology. Chapter 7: Antimicrobial
Susceptibility Testing.
104
Textbook of Practical Microbiology 105
UNIT
VI
Immunology
Introduction
Lesson 36 Bacterial Agglutination Test
Lesson 37 Blood Grouping
Lesson 38 Latex Agglutination Test
Lesson 39 Co-agglutination Test
Lesson 40 Widal Test
Lesson 41 Weil Felix Test
Lesson 42 Anti-Streptolysin O (ASLO) Test
Lesson 43 VDRL Test
Lesson 44 Radial Immunodiffusion Test
Lesson 45 Immunoelectrophoresis Test
Lesson 46 Counter-current Immunoelectrophoresis Test
Lesson 47 Indirect Haemagglutination Test
Lesson 48 Immunofluorescence Test
Lesson 49 Enzyme-Linked Immunosorbent Assay
106
Introduction
Serological tests are widely used for diagnosis of many infectious diseases including bacterial, viral, fungal and parasitic. These
tests may be agglutination, precipitation, neutralization, etc. with a variation in their sensitivity and specificity. List of few
common serological tests are mentioned in the table.
Slide agglutination tests. Bacterial agglutination test. For confirmation of identification of the bacterial
isolates by using specific antisera.
Shigella agglutination.
Salmonella agglutination.
Vibrio agglutination.
Streptococcus Lance field’s grouping.
Blood grouping. For blood group antigen detection A, B, AB and O.
Widal test. For antibody detection against
Salmonella Typhi
S. Paratyphi A
S. Paratyphi B
S. Paratyphi C.
Tube agglutination tests. Standard agglutination test. For antibody detection against
Brucella species.
Cold agglutination test. For antibody detection against
Mycoplasma pneumoniae.
Indirect haemagglutination test. For antibody detection in:
Amoebiasis
Lymphatic filariasis
Echinococcosis
Toxoplasmosis
Rickettsial infection.
Passive agglutination tests. Latex agglutination test. For antigen detection in infections caused by:
Streptococcus pneumoniae
Haemophilus influenzae
Cryptococcus neoformans
Echinococcus granulosus.
Co-agglutination test. For antigen detection in:
Cryptococcosis
Echinococcosis
Lymphatic filariasis
Textbook of Practical Microbiology 107
Precipitation tests Ring test. For antigen detection in infections caused by:
Brucella (Milk ring test)
B.anthracis (Ascoli’s test)
Tube test. Toxoid precipitation for diphtheria.
VDRL test. Slide flocculation test for demonstration of reaginic
antibodies in syphilis.
Gel diffusion test. For detection of:
a-Fetoprotein
N. meningitidis antigen
HBs antigen.
Immunofluorescence test Direct immunofluorescence test. For antigen detection in infections caused by:
Respiratory syncytial virus
Measles
Mumps
Rabies
Influenza
Indirect immunofluorescence test. For antibody detection in:
Toxoplasmosis
Amoebiasis.
108
LESSON
Bacterial Agglutination
36 Test
LEARNING OBJECTIVES only small quantities of culture are available. The slide
agglutination tests have many uses. They are used for
After completing this practical you will be able to: confirmatory identification of Salmonella, Shigella and Vibrio
isolates, identification of Bordetella pertussis and typing of
1 Demonstrate the application of slide agglutination reaction streptococci (e.g. Streptococcus Lance field’s grouping) and
for the identification of bacteria. pneumococci. The slide agglutination test is rapid and
convenient.
INTRODUCTION
PRINCIPLE
Agglutination is an example of antigen-antibody reaction in
which antigen is particulate or insoluble in nature. The soluble In the slide agglutination test a drop of the appropriate specific
antigens can be tested in agglutination reaction by coating antiserum is added to a smooth, uniform suspension of a
them with carrier particles such as red blood cells, bacteria or bacterial isolate from the clinical specimens resulting in
inorganic particles such as the latex. When a particulate antigen agglutination. A positive reaction is identified by the clumping
is mixed with its antibody in the presence of electrolytes at a together of bacteria and clearing of the drop. The reaction is
suitable temperature and pH, the particles are clumped or facilitated by mixing of the bacterial colony and the antiserum
agglutinated. with a loop. The agglutination reaction occurs instantly or
Antibodies cause agglutination by binding to antigens on within seconds.
the particles; they help to neutralize the slight negative charge
that particles in solution normally carry (the zeta potential).
This allows the particles to approach each other. IgM REQUIREMENTS
immunoglobulins actually function as a bridge between two
particles, with one of the five subunits binding to one particle, I Reagents and glass wares
and another subunit binding to another particle. As in the case Glass slides, bacteriological loop, glass marking pencil, saline
with precipitation reaction, more quantity of antibody can (0.85%), specific antiserum against the bacterium(e.g., antiserum
actually inhibit agglutination. In this case, each particle will be against Salmonella Typhi, Shigella flexneri or Vibrio cholerae)
completely covered with antibodies, and they will not clump to be tested.
together. This phenomenon is known as prozone reaction.
Not all antibodies can agglutinate particles. Antibodies, which II Specimen
cannot cause agglutination, are called “incomplete” antibodies. Pure 24 hour growth of bacteria (e.g., Salmonella Typhi, Shigella
This is probably because of restricted movement in the hinge flexneri or Vibrio cholerae) from solid media preferably from
region of the immunoglobulin. IgG4 antibodies are an example non-blood agar plates (Examples : nutrient agar, Muller-Hinton
of incomplete antibodies. agar).
The slide agglutination is a frequently used procedure for
the identification of many bacterial isolates from clinical
specimens. This method is also used for blood grouping and PROCEDURE
cross matching. The test is performed on a glass slide, hence
called slide agglutination test. This method is useful where 1 Take a clean glass slide.
Textbook of Practical Microbiology 109
2 Mark it into two halves by a glass marking pencil and label the test reagent as compared to control reagent is indicative of
them as test and control. a positive reaction. Absence of reaction with a test reagent
3 Place a drop of saline in both the halves. indicates a negative reaction irrespective of any reaction with
4 Pick up the colonies of Salmonella to be tested from agar control organism.
culture and gently emulsify with drops of saline in both the
halves by loop.
5 Add a drop of specific antisera to the bacterial suspension RESULTS AND INTERPRETATION
in the half labeled as test and mix.
6 Place another drop of saline in the half of the slide labeled as Positive agglutination: Clumping in the test half and no clumping
control, and mix. in the control half. It identifies specific bacteria (Fig. 36-1).
7 Gently rock the slide, back and froth for 2 minutes. Negative agglutination: No clumping either in the test half or
8 Observe the clumping of the bacterial suspension in the test. control half. It shows either antiserum is not good or bacteria
and the antisera are not specific to each other.
Auto agglutination: Clumping in both test and control halves.
QUALITY CONTROL Test is fallacious, hence discard the result.
OBSERVATIONS
KEY FACTS
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworth, London, 94-96, 1995.
2 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th
Edition. Lippincott Williams and Wilkins. 1997; pp. 1395.
3 Stites DP. Terr AI and Parslow TG. Medical Immunology. 10th Edition. 2001. pp. 902.
110
LESSON
Blood Grouping
37
LEARNING OBJECTIVES PROCEDURE
After completing this practical you will be able to: 1 Take three different slides and label the slides as A, B, and D.
1 Demonstrate blood grouping by agglutination reaction. 2 Clean middle finger of the left hand with the spirit and allow
it to dry.
3 Prick the finger with sterile lancet.
INTRODUCTION 4 Collect 3 drops of blood on three different slides labeled as
A, B, and D.
The ABO system contains four blood groups and is determined 5 Add a drop of antiserum A to A and anti B to B anti D to D.
by the presence or absence of two distinct antigens, A and B, on 6 Mix with an applicator stick. Mix the samples on the slide by
the surface of erythrocytes. Red cells of group A carry antigen A, gentle rocking for about two minutes.
cells of group B antigen B and cells of group AB have both A and 7 Examine each zone for agglutination of RBCs.
B antigens, while group O cells have neither A nor B antigen. The 8 Record result of the test immediately before the drop dries out.
four groups are also distinguished by the presence or absence of
two distinct isoantibodies in the serum. The serum contains the
isoantibodies specific for the antigen that is absent on the red cell. QUALITY CONTROL
Blood group antigens are inherited according to Mendelian
laws. Their synthesis is determined by allelomorphic genes A, B and Blood group antisera (Anti-A, anti-B, anti-D or Rh antiserum)
O. Genes A and B give rise to the corresponding antigens, but O is must be checked with known blood before to test.
an amorph and does not produce any antigen. Group O is the
commonest group and group AB is the rarest. In India the distribu-
tion is O – 40%, A – 22%, B – 33%, and AB – 5%. O group population OBSERVATIONS
is called universal donors and AB is called universal recipients.
Observe the test mixture for clumping.
Check for autoagglutination of test RBCs.
PRINCIPLE
When a drop of anti A / anti B or anti Rh antibody is added to RESULTS AND INTERPRETATION
a drop of blood, the antibody binds with its specific antigen
present on the RBCs and causes agglutination of the RBCs.
A clear agglutination indicates positive result.
If the test sample is agglutinated with anti A antibody then
REQUIREMENTS the blood is Group A.
If the test sample is agglutinated with anti B antibody then
the blood is Group B.
I Reagents and lab wares
If the test sample is agglutinated with anti A and anti B
Blood group antisera (Anti-A, anti-B, anti-D or Rh antiserum),
antibodies both, then the blood is Group AB.
glazed white ceramic slide, applicator stick and lancet.
If the test sample is not agglutinated with anti A and anti B
II Specimen antibodies, then the blood is Group O.
Blood with anticoagulant or finger prick blood.
Textbook of Practical Microbiology 111
KEY FACTS
VIVA
1 What exactly does it tell you about a person’s blood cells if you know that they are of blood type A, B, AB, or O?
Ans: It shows the presence or absence of two distinct antigens, A and B, on the surface of erythrocytes.
2 Explain what blood types a person of type A, B, AB, or O can receive and why?
Ans :Red cells of group A carry antigen A, cells of group B carry antigen B and cells of group AB have both A and B
antigens, while group O cells have neither A nor B antigen. The serum contains the isoantibodies specific for the antigen
that is absent on the red cell. Immune isoantibodies may develop following ABO incompatible transfusion. Red cells also
carry another antigen that reacts with rabbit serum to Rhesus monkey erythrocytes called Rhesus or Rh factor. Rh-negative
population contains anti Rh antibodies in their blood. Hence a person should not receive blood, which contains isoantibodies
to own RBC. O group population is called universal donors since their blood does not contain any blood group antigens (A,
B and AB). AB group is called universal recipients due to absence of isoantibodies.
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworth, London, 94-96, 1995.
2 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th
Edition. Lippincott Williams and Wilkins. 1997; pp. 1395.
3 Stites DP. Terr AI and Parslow TG. Medical Immunology. 10th Edition. 2001. pp. 902.
112
LESSON
Latex Agglutination Test
38
LEARNING OBJECTIVES of the latex particles and form visible agglutination or clumping
of the particles.
After completing this practical you will be able to:
1 Demonstrate the application of latex agglutination test for
detection of soluble antigen. REQUIREMENTS
KEY FACTS
VIVA
1 List application of the LAT for diagnosis of infectious diseases by demonstration of antibodies in the serum.
2 List application of the LAT for diagnosis of infectious diseases by demonstration of antigens in the serum.
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworth, London, 94-96, 1995.
2 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th
Edition. Lippincott Williams and Wilkins. 1997; pp. 1395.
3 Stites DP. Terr AI and Parslow TG. Medical Immunology. 10th Edition. 2001. pp. 902.
114
LESSON
Co-Agglutination Test
39
LEARNING OBJECTIVES PROCEDURE
After completing this practical you will be able to: 1 Take a clean glass slide.
1 Demonstrate the application of Co-agglutination test for 2 Mark it into two halves by a glass marking pencil and label
detection of soluble antigen. them as test and control.
3 Add a drop of test CSF in the test half, and a drop of saline
in the control half.
INTRODUCTION Note: The CSF specimen has to be absorbed with stabilized
SAPA cells (not coated with specific pneumococcal antibody)
The Co-agglutination (Co-A) test is a simple slide agglutination to prevent nonspecific agglutination with human IgG.
test. The test is being used for detection of specific antigen in 4 Add a drop of pneumococcal antibody-coated Co-A reagent
the serum, cerebrospinal fluid (CSF), urine and other body fluids. to the serum in the half labeled as test and mix.
Commercial Co-A kits are now available for detection of 5 Place another drop of pneumococcal antibody-coated Co-A
haemophilus, meningococcal and pneumococcal antigens in reagent in the other half of the slide labeled as control, and mix.
the CSF, and salmonella antigen in the serum. The kits are also 6 Gently rock the slide, back and froth for 2 minutes.
available for identification of Neisseria gonorrhoeae and sero 7 Observe the agglutination of the bacteria in the test.
grouping of Staphylococcus aureus.
In this exercise you will perform the Co-A for detection of
pneumococcal antigen in the CSF for diagnosis of meningitis. QUALITY CONTROL
REQUIREMENTS Agglutination of the Co-A reagents with the CSF , and absence
of any agglutination in the control half indicates the Co-A to
I Reagents and lab wares be positive and shows the presence of pneumococcal antigen
Co-agglutination reagent, glass slides, and applicator stick. in the CSF.
Absence of agglutination either in the test half or in the
II Specimen control half indicates the test to be negative and shows the
CSF sample to be tested. absence of pneumococcal antigen in the CSF.
Textbook of Practical Microbiology 115
KEY FACTS
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworth, London, 94-96, 1995.
2 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th
Edition. Lippincott Williams and Wilkins. 1997; pp. 1395.
3 Stites DP. Terr AI and Parslow TG. Medical Immunology. 10th Edition. 2001. pp. 902.
116
LESSON
Widal Test
40
LEARNING OBJECTIVES REQUIREMENTS
5 In the row labeled as TO, mix and transfer 0.2 ml of serum The last tube showing agglutination is considered as the
from 2nd tube to the 3rd, then from 3rd to 4th, and so on end point.
through the 6th tube. Discard 0.2 ml from the 6th tube. The reciprocal of the dilution is considered as the titre (e.g.,
6 Follow the same dilutions in the rows labeled as TH, AH If the dilution of the last tube showing agglutination is 1: 200,
and BH. then the titre is 200).
7 Add 0.2 ml of TO antigens to tubes from 1st to 7th in the
row TO.
8 Add 0.2 ml of TH antigens to tubes from 1st to 7th in the RESULTS AND INTERPRETATION
row TH.
9 Add 0.2 ml of AH antigens to tubes from 1st to 7th in the 1 A progressive rise in the titer between first and third week
row AH. after onset of fever is highly significant.
10 Add 0.2 ml of BH antigens to tubes from 1st to 7th in the 2 A positive or a negative result in a single test is not significant.
row BH. 3 Since the antibodies are detected only after 7 days to 10
Note: The final dilution of the sera after addition of antigen days of illness, test should be done later.
are 1:25, 1;50, 1;100, 1:200, 1:400, 1:800. 4 The serum of some uninfected subjects causes agglutinations
11 Incubate the racks in water bath at 37 °C for 18 hrs. at dilutions of about 1:50, so titers are considered significant
when agglutination occurs in serum dilution above 100.
5 H agglutinins tend to persist longer than O agglutinins.
QUALITY CONTROL 6 O antibody titre rise indicates recent infection.
7 Persons immunized with TAB vaccine may show high titers
7th tube in each row acts as a negative antigen control. of antibodies to all the antigens and so only a marked rise in
The same dilutions of a known positive serum is tested with titer is considered significant.
TO, TH, AH and BH antigens. 8 Early treatment with antibiotics will alter antibody response.
OBSERVATIONS
VIVA
KEY FACTS
1 In India enteric fever is caused commonly by S. Typhi, and S. Paratyphi A, rarely by S. Paratyphi B.
2 Since “ O” antigens are related to each other, the O antigen of S. Typhi is only tested, while H antigens of S. Typhi,
S. Paratyphi A and S. Paratyphi B are tested separately.
3 The last tube showing agglutination is considered as the end point.
4 The reciprocal of the dilution is considered as the titre(e.g., If the dilution of the last tube showing agglutination is 1: 200,
then the titre is 200).
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworth, London, 94-96, 1995.
2 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th
Edition. Lippincott Williams and Wilkins. 1997; pp. 1395.
3 Stites DP. Terr AI and Parslow TG. Medical Immunology. 10th Edition. 2001. pp. 902.
Textbook of Practical Microbiology 119
LESSON
Weil-Felix Test
41
LEARNING OBJECTIVES II Reagents and glass wares
Small test tubes, test tube racks, and pipettes.
After completing this practical you will be able to: Saline (0.85 % NaCl), and P. vulgaris OX-19, OX-2 and
P. mirabilis OX-K antigens. The antigens are stored at 4°C
1 Demonstrate the cross-reacting antibodies to Rickettsial before use.
antigens using Proteus strains OX-19, OX-2 of Proteus
vulgaris and OX-K of P. mirabilis by Weil-Felix test. III Specimen
Serum sample to be tested.
INTRODUCTION
PROCEDURE
Weil-Felix is a serological test used for diagnosis of Rickettsial
infection by demonstration of antibodies in the serum. In this Preparation of master dilution of the serum
test, certain strains of Proteus are used instead of specific
rickettsial pathogens as antigens. This test was developed 1 Add 2.7 ml of saline in a test tube.
from the observation that certain strains of Proteus isolated 2 Add 0.3 ml of serum to the test tube which makes the master
from the urine of patients with epidemic typhus were aggluti- serum dilution as 1: 10.
nated by the sera of the typhus patients. This test has been
used for presumptive serological evidence of Rickettsial Performance of the test
disease.
1 Arrange 3 rows of test tubes, each row containing 7 test tubes.
2 Add 1 ml of saline to all the tubes.
PRINCIPLE 3 Add 1 ml of diluted serum from master dilution into the first
tubes in all the three rows of the tubes.
It is an agglutination test for cross-reacting antibodies. It is 4 Make doubling dilutions in the first row from the first
based on the principle that many patients infected with one of tube(serum dilution 1:20) and discard 1 ml from the 6th tube
the Rickettsia produce antibodies that can agglutinate certain (serum dilution 1:640).
strains of bacteria of genus Proteus because of the presence Note: Leave the 7th tube as control without serum.
of a common alkali stable carbohydrate antigen. 5 In the same way, make doubling dilutions in the second and
Rickettsia prowazaki and R. mooseri share alkali stable third row tubes.
carbohydrate antigens with P. vulgaris OX 19, R. tsutsugamushi 6 Add 0.5 ml of concentrated P. vulgaris OX-19 in to all 7
with P. mirabilis OX K and R.rickettsi, and R.conori with tubes in the first row of tubes.
P. vulgaris OX 2. 7 Add 0.5 ml of concentrated P. vulgaris OX-2 in to all 7 tubes
in the second row of tubes.
8 Add 0.5 ml drop of concentrated P. mirabilis OX-K in to all
REQUIREMENTS 7 tubes in the third row of tubes.
9 Incubate the racks in water bath at 37°C for 2 hr., followed by
I Equipments reincubating at 4°C for 18 hours.
Water bath.
120 Weil Felix Test
KEY FACTS
1 Since the Weil-Felix antigens also react with Proteus antibodies, false positive reactions may occur in urinary tract infections
with Proteus, in leptospirosis, in Borrelia infections and in severe liver disease.
2 Proper precautions should be taken for standardization of antigens. It should not be standardized against sera from rabbits
immunized with homologous strain of Proteus species, but with sera derived from patients infected with rickettsiae.
3. The test is not helpful for the detection of antibodies in rickettsial pox, trench fever or Q fever as these persons do not
develop Proteus agglutinins.
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworth, London, 94-96, 1995.
2 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th
Edition. Lippincott Williams and Wilkins. 1997; pp. 1395.
3 Stites DP. Terr AI and Parslow TG. Medical Immunology. 10th Edition. 2001. pp. 902.
Textbook of Practical Microbiology 121
LESSON
Anti-Streptolysin O (ASLO)
42 Test
INTRODUCTION
PROCEDURE
Str. pyogenes produces several exotoxins and enzymes, which Serum dilution
contribute to its virulence. Streptococci produce two
haemolysins, namely streptolysin O and streptolysin S. 1 The initial serom dilutions of 1:10, 1:60 and 1:85 are prepared in
Streptolysin O is oxygen labile and heat labile. It is inactive test tubes. Subsequent dilutions are carried out in microtitre plates.
in the oxidized form but may be reactivated by treatment with 2 Both serum and buffer should be placed at room temperature
mild reducing agents. It is lethal on intravenous injection into when preparing dilution.
animals and has a specific cardiotoxic and leucotoxic activity. 3 Standard serum of know titer is included in each days run to
Streptolysin O is antigenic and elicits the production of serve as positive control.
specific antibodies against the antigen in an infected human
host. This antibody is known as antistreptolysin, it regularly Test procedure
appears in sera following streptococcal infection. Hence,
1 Label microtiter plate so that each specimen is alligned in
demonstration of antistreptolysin in the serum is an indirect
two rows (1:60 row and 1:85 row) with six wells per row.
indicator of infection. Many methods are available for detection
2 Prepare serial two fold dilutions through sixth well. Serum
of antistreptolysin in the serum. Streptolysin S is not antigenic,
dilutions for each serum sample are first row - 1:60, 1:120,
hence do not elicit production of any antibodies.
1:240, 1:480, 1:960, 1:1920; second row - 1:85, 1:170, 1:340,
1:680, 1:1360, 1:2760.
3 Add 0.025ml of antigen to all tubes, gently agitate, and
PRINCIPLE incubate at 37°C for 15 minutes.
4 Add 0.025 ml of cold 2.5% red blood cells to all wells and
When streptolysin O in its reduced form is added to red blood incubate in water bath at 37° C for one hour.
cells hemolysis occurs. If a patient’s serum contains
antistreptolysin O antibodies, antigen-antibody reaction takes
place and no hemolysis takes place. QUALITY CONTROL
A cell control, antigen control and a standard serum control
always should be used with the test.
REQUIREMENTS
I Equipments
OBSERVATIONS
Water bath. Observe the microtitre plates for presence or absence of hemolysis.
122 Anti-Streptolysin
KEY FACTS
1 Streptolysin O is antigenic and elicits the production of specific antibodies against the antigen in an infected human host.
2 Streptolysin S is not antigenic; hence do not elicit production of any antibodies.
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworth, London, 94-96, 1995.
2 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th
Edition. Lippincott Williams and Wilkins. 1997; pp. 1395.
3 Stites DP. Terr AI and Parslow TG. Medical Immunology. 10th Edition. 2001. pp. 902.
Textbook of Practical Microbiology 123
LESSON
VDRL Test
43
LEARNING OBJECTIVES test. When cardiolipin antigen combines with reaginic antibodies
in patient’s serum, it forms visible floccules. This test is also
After completing this practical you will be able to: used to detect antibodies in the CSF.
The biological false positive reaction is a noted problem
1 Perform the VDRL test. associated with the VDRL test (Box 43-2).The advantages and
2 Demonstrate the presence of reaginic antibodies in patient’s disadvantages of the VDRL test are listed in the table 43-1.
serum.
REQUIREMENTS
INTRODUCTION
I Equipments
When soluble antigen and specific antibodies in the serum are Water bath and VDRL shaker.
combined in the right proportions, they will bind to each other
to form an insoluble complex which results in visible II Reagents and glass wares
precipitations. The precipitates settle at the bottom of the VDRL antigen (It contains cardiolipin, 0.3%, lecithin, 0.24%
container. The precipitation reaction can be demonstrated in and cholesterol, 0.9%), buffered saline solution, saline, glass
the liquid medium as well as in the solid media such as in gels. stoppered bottles, VDRL glass slides (2”X3”) with depressions
The process of precipitation in the gel can be hastened by of 14 mm diameter each, tubes, pipettes and racks.
migration of antigens and antibodies under an electric field. Preparation of buffered saline solution: This is prepared by
Temperature, pH and concentration of sodium chloride in the mixing 0.5 ml of formaldehyde, 3.037 gm of disodium hydrogen
medium influence the reaction of precipitation. phosphate (Na 2HPO 4, 2H 2O), 0.170 gm of potassium
Often the precipitate fails to settle down at the bottom of dihydrogen phosphate (KH2PO4), and 10 gm of sodium chloride
the container but remains suspended as floccules, such reaction (NaCl), in 1000 ml of distilled water. pH is adjusted to 6.0+ 0.1.
is known as flocculation. Venereal Disease Research Laboratory Each antigen pack usually consists of 10 ampoules of 5 ml
(VDRL) test is an example of flocculation test to detect reaginic buffered saline solution.
antibodies in serum of patients suffering from syphilis. Preparation of un buffered saline solution: This is prepared
VDRL –ELISA is a modification of the VDRL test (Box 43-1). by adding 1 gm of sodium chloride to 100 ml of distilled water.
III Specimen
Serum and CSF.
PRINCIPLE
PROCEDURE
The VDRL slide flocculation test is a simple, rapid convenient
and economical test for serodiagnosis of syphilis. This is an Preparation of serum
example of standard test of syphilis in which cardiolipin antigen
is used to detect non-specific reaginic antibodies in the serum. 1 Inactivate the serum in water bath at 56°C for 30 minutes.
Cardiolipin antigen is an alcoholic extract of beef heart tissue Note: After removal from the water bath, all the sera are
to which cholesterol and lecithin are added. This is a non- examined and those found to contain particulate debris are
treponemal test because no treponemal antigen is used in this recentrifuged. The quantity of serum used in each test is 0.05 ml.
124 VDRL Test
1 Pipette 0.4 ml of buffered saline to the bottom of a 1 oz reagent Quantitative test is performed on all reactive serum samples
bottle with flat or concave inner bottom surface. and on all samples showing weakly reactive or rough in the
2 Add 0.5 ml of antigen, drawn from an ampoule in to a 1.0 ml qualitative test. Different dilutions of the serum are prepared in
pipette graduated to tip, directly on to the saline while rotating tubes as successive twofold dilutions (in the range of 1:2, 1:4,
the bottle on a flat surface. The antigen is added drop by 1:8, 1:16, etc.) with buffered saline solutions. Each dilution of the
drop but rapidly so that it takes approximately 6 seconds to serum is treated as an individual serum. These serum dilutions
complete the delivery. are tested as described above, in qualitative serum test.
3 Blow the last drop of antigen and continue rotation of the
bottle for 10 more seconds. Quality control
4 Then add 4.1 ml of buffered saline from a 5.0 pipette stopper
Known reactive (with known titre) and non-reactive sera are
the bottle and shake it vigorously for approximately 10 seconds.
included.
Note: Temperature of the buffered saline solutions and
antigen should be preferably between 23°C to 29°C during
the preparation of antigen emulsion. OBSERVATIONS
Maturation of the antigen: It increases the sensitiveness and Observe for the formation of floccules immediately after rotation
this is almost complete in 15 minutes to 30 minutes. The antigen under a microscope with low power objective (10 x
emulsion prepared ought to be used during the same day. magnification). The antigen particles are seen as small fusiform
Preliminary testing of antigen emulsion: Each batch of prepared needles, which remain more or less evenly, disperse in a non-
antigen emulsion should first be examined by testing known reactive serum, whereas these aggregate into clumps in reactive
reactive and non-reactive sera. This test should present sera. Zone reactions are recognized by the irregular clumping,
typically reactive and non-reactive results, and the number which are not compact. In such cases the results are reported
and distribution of antigen particles per microscopic field in on the basis of quantitative reaction done on the same serum.
the negative sera should be optimum.
Advantages Disadvantages
An automated VDRL-ELISA test has been developed using cardiolipin antigen, which can measure IgG and IgM antibodies
separately and is suitable for large scale testing of sera. The newest of the nontreponemal tests, VISUWELL Reagin, is based
on the Pedersen method. In the indirect ELISA procedure, VDRL antigen coats the wells of a microtiter plate. This test has a
sensitivity of 97% in untreated syphilis and specificity of 97%. The reactivity of this test disappears with treatment of the
patient. The disadvantage of this test is the inability to quantitate the reactivity of a patient’s serum to an end point titre in
order to assess the efficacy of treatment. Several hundred sera can be tested by VISUWELL Reagin test in a day.
Biological false positive (BFP) reactions are defined as positive reactions obtained in tests using cardiolipin antigen, with
negative results in specific treponemal tests, in the absence of past or present treponemal infections, and not caused by
technical faults.
As cardiolipin antigen is present both in T. pallidum and in mammalian tissues, reaginic antibodies may be induced by
treponemal or host tissue antigens. This accounts for BFP reactions. They represent the nontreponemal cardiolipin antibody
responses. BFP reactions may occur in about one percent of normal sera. Clinically, BFP reactions may be classified as acute
or chronic.
Acute BFP reactions last only for a few weeks or months and are usually associated with acute infections, injuries or
inflammatory conditions. Chronic BFP reactions persist for longer than six months and are typically seen in SLE and other
collagen diseases. Leprosy, malaria, relapsing fever, infectious mononucleosis, hepatitis and tropical eosinophilia are examples
of other conditions associated with BFP reactions.
KEY FACTS
1 VDRL is an example of slide flocculation test and is an example of standard test of syphilis.
2 Cardiolipin antigen is used to detect non-specific reaginic antibodies in the serum.
3 Maturation of the antigen increases sensitiveness of the test.
4 Observe for the formation of floccules immediately after adding the antigen to the serum in a VDRL slide.
5 Quantitative test is performed on all reactive serum samples and on all samples showing weakly reactive or rough in the
qualitative test.
6 Zone reactions are recognized by the irregular clumping, which are not compact.
7 VDRL test shows biological false positives in a variety of conditions.
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworth, London, 94-96, 1995.
2 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th
Edition. Lippincott Williams and Wilkins. 1997; pp. 1395.
3 Stites DP. Terr AI and Parslow TG. Medical Immunology. 10th Edition. 2001. pp. 902.
126
LESSON
Radial
44 Immunodiffusion Test
LEARNING OBJECTIVES Analytical balance, water bath, dark ground illuminating box,
moist chamber, microscope slides, glass plates, beakers,
After completing this practical you will be able to: syringes, micropipettes, Pasteur pipettes, measuring cylinder,
1 Quantitate the protein antigens in a test serum by single measuring template and test tubes.
radial immuno diffusion.
III Specimen
Serum.
INTRODUCTION
Observe the gels for precipitin lines after 24 hours under oblique
illumination for ring precipitates.
KEY FACTS
1 Circular well with a clean edge is very important for a well-defined ring precipitate.
2 Since there is a batch-to-batch variation the reference graph should be plotted for every new batch.
3 The slope of the calibration graph is influenced by antibody concentration in the gel.
4 The size of the ring is proportional to the amount of antigen in the well.
VIVA
1 What is the principle of gel diffusion test?
2 What are the advantages of gel diffusion?
Ans. The advantages are as follows:
a The reaction is visible as distinct band of precipitation.
b Precipitin band is stable and can be stained for preservation.
c The number of different antigens in the reacting mixture can be readily observed.
3 Classify gel diffusion methods?
Ans.
Immunodiffusion
Single diffusion in one dimension (Oudin procedure).
Double diffusion in one dimension (Oakley Fulthorpe procedure).
Single diffusion in two dimensions (Radial immunodiffusion).
Double diffusion in two dimensions (Ouchterlony procedure).
Immunoelectrophoresis.
Electroimmunodiffusion
Countercurrentimmunoelectrophoresis.
Rocket electrophoresis.
3 What are the uses of gel diffusion test?
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworth, London, 94-96, 1995.
2 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th
Edition. Lippincott Williams and Wilkins. 1997; pp. 1395.
3 Stites DP. Terr AI and Parslow TG. Medical Immunology. 10th Edition. 2001. pp. 902.
128
LESSON
Immunoelectrophoresis
45 Test
III Specimen
INTRODUCTION Patients serum. Human serum (antigen) and antihuman serum
(antibodies)
When mixtures, such as those in body fluids, are being analyzed
in diffusion experiments like the double-diffusion system, it is
difficult to distinguish the individual antigenic components PROCEDURE
from each other. Immunoelectrophoresis in principle is a method
of combination of electrophoresis and diffusion. By these 1 Take a clean glass slide, To coat the slide place two to
methods antigens present in a mixture are separated from each three drops of the molten agar and spread over the surface
other by agar gel electrophoresis. Subsequently these antigenic of the slide.
components react specifically with their antibodies to form 2 Put the coated slide on a horizontal work bench.
antigen-antibody precipitates that are formed in the gel. 3 Pipette 6 ml of agar to the slide, spread it uniformly and
Immunoelectrophoresis is useful for demonstration of normal allow it to set at room temperature, and then at 4°C in a
and abnormal serum proteins such as the myeloma proteins. refrigerator for 1 hour to set completely.
4 Remove the slide from the refrigerator.
5 Place the slide on a graph paper. Punch two wells at about
PRINCIPLE one third of its length. Suck out the agar plugs from wells
and discard.
Immunoelectrophoresis techniques is carried out on a glass Note: These two wells should be 2 mm in diameter and I
slide layered with semisolid gel. An aliquot of the antigen cm apart.
mixture is kept in the well cut out of the gel. Electrophoresis is 6 Fill the two wells with the serum.
carried out for one hour under electric current, this procedure Note: the serum is mixed with the indicator dye (bromthymol
permits separation of proteins. A rectangular trough is then blue)with a pipette.
cut parallel to the line of these separated proteins. Antiserum 7 Keep the slide in the electrophoresis chamber. Connect it
is placed in this trough. Diffusion is allowed to occur for 18-24 to the buffer with Whatman 3 filter paper strips.
hours. The antiserum will diffuse laterally and react with the Note: The slide should be kept in such a way that the well
separated components. Individual precipitation lines develop containing antigen will be near the cathode. This will make
with each separated components of the antigen mixture. the components move toward the anode.
8 Fill the electrophoresis chamber with the buffer.
9 Connect it to the power supply and run the electrophoresis
REQUIREMENTS for 60-90 min at 10-15 mA/slide till the bromthymol blue
(indicator dye) spreads to the end of the slide.
I Equipments 10 Remove the slides after turning off the power. Cut a trough
Electrophoresis power supply, and electrophoresis chamber. between the two wells running parallel to the direction ofthe run.
Textbook of Practical Microbiology 129
11 Remove the agar and fill the trough with the antiserum.
12 Keep the slide in the moist chamber and allow the diffusion OBSERVATIONS
to take place at 4 °C for 24 hours.
13 Remove the slides and observe for the lines of precipitation. Observe the gels for precipitin lines after 24 hours at 4°C.
14 Wash the slide and stain the slide.
RESULTS AND INTERPRETATION
QUALITY CONTROL
1 The antigen components in the mixture is noted by observing
Known positive and negative samples are used in the test. different lines of precipitations.
2 The slide may be stained and preserved in a plastic bag for a
permanent record.
KEY FACTS
1. Immunoelectrophoresis is useful for demonstration of normal and abnormal serum proteins such as the myeloma proteins.
2. Keep the slide in the electrophoresis chamber in such a way that the well containing antigen will be near the cathode. This will
make the components move toward the anode.
3. Observe the gels for precipitin lines after 24 hours at 4°C.
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworth, London, 94-96, 1995.
2 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th
Edition. Lippincott Williams and Wilkins. 1997; pp. 1395.
3 Stites DP. Terr AI and Parslow TG. Medical Immunology. 10th Edition. 2001. pp. 902.
130
LESSON
Counter-current
46 Immunoelectrophoresis
Test
LEARNING OBJECTIVES the cathodic side and the well containing antibodies will be on
the anodic side, so that during electrophoresis antibodies move
After completing this practical you will be able to: towards the cathode and antigens move towards the anode. A
1 Perform counter-current immunoelectrophoresis (CIEP). line of precipitation visible to the naked eye is formed at a point
2 Demonstrate antibodies or antigens in serum and other body between the antigen and antibodies. The test takes 30 minutes.
fluids by counter-current immunoelectrophoresis (CIEP). The lines of precipitation in the slide can be stained by Amido
black or 1% Coomassie brilliant blue stains.
In this chapter the CIEP to detect hydatid antigen in the
INTRODUCTION serum will be described.
2 Put the coated slide on a horizontal work bench. 15 After staining, the lines of precipitation stains dark blue.
3 Pipette 2.5 ml of 1% agar to the slide, spread it uniformly
and allow it to set at room temperature, and then at 4°C in a
refrigerator for 1 hour to set completely. QUALITY CONTROL
4 Remove the slide from the refrigerator.
5 Place the slide on a template. Punch out parallel rows of Known hydatid antigen-positive and - negative serum samples.
wells 4mm in diameter on the slides at distance of 3 mm With every slide known hydatid antigen is placed in the wells
from each other on the cathodic side and hydatid antibodies in the wells on the
Note: Nine pairs of wells are punched out on each slide. anodic side as control.
6 Fill each well with 10µl of the patients serum to be tested
and polyclonal hydatid antibodies.
7 Keep the slide in the electrophoresis chamber. Connect it OBSERVATIONS
to the buffer with Whatman 3 filter paper strips.
Note: The slide should be kept in such a way that the well Observe the lines of precipitation after 30 min.
containing test sera will be near the cathode and the wells First observe for the lines of precipitation in between the wells
containing the hydatid antibodies will be near the anodic side. containing known hydatid antigen and known hydatid
8 Fill the electrophoresis chamber with the buffer. antibodies (Control row).
9 Connect it to the power supply and run electrophoresis for Then observe for the lines of precipitation in the test sera.
30 min at 10-15 mA/slide.
10 Remove the slides after turning of the power off.
11 Remove the slides and observe for the lines of precipitation.
RESULTS AND INTERPRETATION
Note: The slides can be kept at 4 °C for 24 hr and the result
read and stained.
1 The line of precipitation is present in the control row : The
12 To stain the slides wash the slides first in saline for several
test is performed correctly.
hours. Remove the salts by washing the slides several times
2 Line of precipitation is formed between the test serum and
in distilled water for one hour.
hydatid antibodies wells: It indicates the patient serum is
13 Stain the slides by immersing the slides in 1% Amido black
positive for hydatid antigen by the CIEP test.
in 7% acetic acid for 15-30 minutes.
3 No line of precipitation is formed between the test serum
14 Destain the slides with 7% acetic acid till the background
and hydatid antibodies wells: It indicates the patient serum
stain is removed.
is negative for hydatid antigen by the CIEP test.
KEY FACTS
1 The CIEP test is based on the movement of the antibodies toward the cathode while the antigens move in the opposite
directions (toward the anode) in a layer of agarose on a glass slide under electric current.
2 The CIEP test has the advantages of being simple, and rapid test, the result can be obtained within 30-45 minutes of
performing the test.
3 After staining by Amido black, the lines of precipitation stains dark blue.
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworth, London, 94-96, 1995.
2 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th
Edition. Lippincott Williams and Wilkins. 1997; pp. 1395.
3 Stites DP. Terr AI and Parslow TG. Medical Immunology. 10th Edition. 2001. pp. 902.
132
LESSON
Indirect
47 Haemagglutination Test
KEY FACTS
1 The red blood cells from different sources have been used by various workers in the IHA test.
2 The red blood cells act as carrier particles for the antigen (against which antibodies will be demonstrated).
3 The RBCs sensitized with antigen are added to the patients sera to demonstrate specific antibodies in serum.
4 The IHA test is carried out in a microtiter plate.
5 The lowest amount of antigen which shows the maximum haemagglutination with the known positive control and negative
reaction with the negative control is taken as OSD of the antigen for that batch.
6 The serum showing a titre of 1: 128 and above is considered positive for hydatid disease.
134 Indirect Haemagglutination Test
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworth, London, 94-96, 1995.
2 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th
Edition. Lippincott Williams and Wilkins. 1997; pp. 1395.
3 Stites DP. Terr AI and Parslow TG. Medical Immunology. 10th Edition. 2001. pp. 902.
Textbook of Practical Microbiology 135
LESSON
Immunofluorescence
48 Test
LEARNING OBJECTIVES In this experiment you will use the method to demonstrate
unknown bacterial antigens. The fluorescein labeled antibody
After completing this practical you will be able to: is tagged to alcohol or acetone fixed bacterial smear. This is
followed by washing the smear with physiologically buffered
1 Demonstrate bacterial antigen by immunofluorescence test. saline. During this step any uncombined fluorescent antibody
will be washed away. In a positive test, the final reaction will be
observed by a green fluorescence when observed under a
INTRODUCTION fluorescent microscope.
Advantages and disadvantages of the immunofluorescence
Immunofluorescence assays involve the use of either antigen test are summarized in the table 48-1.
or antibody labeled with a fluorescent substance. The
fluorescent dyes have the property of absorbing the light of
one wavelength (e.g., ultra violet light) and reflect back the REQUIREMENTS
light of a different wavelength (visible light). These reflected
lights are visualized by a fluorescent microscope under ultra I Equipments
violet radiation.. Fluorescein isothiocyanate (FITC) is the most Fluorescent microscope.
common dye used in the test. The dye emits a greenish/yellow
light. Rhodamine (red/orange), dansyl (yellow), and II Reagents and lab wares
phycoerythrin are the other dyes used in the test. Acetone, phosphate buffered saline (pH 7.2), buffered glycerol
Direct fluorescent antibody test is employed to detect the (pH 7.2): 10% glycerol in PBS, Evan’s blue counter stain
specific antigens of bacteria, viruses, parasites or other antigens (1:10000), and bovine serum albumin.
in the serum, CSF, urine, faeces, tissues and other body fluids. Glass slides, Petri dishes, U-shaped glass rods to fit into
This test is most frequently used to detect rabies virus antigen petri dishes, filter paper, and Coplin jar and glass marking pencil.
in the tissue collected from the skin on the nape of the neck or Commercially available fluorescent antibody Streptococcus
face. This is also used to detect Corynebacterium diphtheriae, group A and fluorescent antibody Enterococcus group D.
Neisseria gonorrhoeae, measles and mumps in various clinical
specimens.
III Specimen
Broth cultures of Group A Streptococcus pyogenes and Group
D Enterococcus faecalis incubated at 35°C for 24 hours.
PRINCIPLE
Unknown mixed broth cultures of Group A S. pyogenes/
Escherichia coli and Group D E.faecalis / E. Coli
In this test specific antibodies raised against the antigen to be
detected (e.g., anti-rabies antibodies to detect rabies antigen)
is labeled with a fluorescent dye, and is used to detect unknown
antigen (e.g., rabies antigen) in the specimen. If antigen is
PROCEDURE
present in the specimen, the same will bind the fluorescein 1 Take three clean glass slides.
labeled antibodies, and the antibody-bound fluorescein will 2 With glass marking pencil label first slide as S. pyogenes,
emit a fluorescence, which will be observed by a fluorescent and second slide as E. faecalis. Divide the third slide in
microscope using ultra violet radiations. half and label as mixed unknowns.
136 Radial Immunodiffusion Test
Advantages Disadvantages
KEY FACTS
1 Immunofluorescence assays involve the use of either antigen or antibody labeled with a fluorescent substance.
2 Direct fluorescent antibody test is employed to detect the specific antigens.
3 Slides should be cleaned before use.
4 The smears should not be allowed to dry at any stage.
5 To minimize non-specific binding of proteins to cells or tubes, all dilutions and washings should be done in PBS containing
0.1 – 1% BSA.
6 1-10 µg/ml of phenylene diamine could be added to the mounting medium to prevent the fading of fluorescence of fluorescein.
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworth, London, 94-96, 1995.
2 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th
Edition. Lippincott Williams and Wilkins. 1997; pp. 1395.
3 Stites DP. Terr AI and Parslow TG. Medical Immunology. 10th Edition. 2001. pp. 902.
138
LESSON
Enzyme-linked
49 Immunosorbent Assay
After completing this practical you will be able to: 1. The sandwich ELISA for antigen: This method to detect
antigen is very sensitive. The walls of the microtiter plates are
1 Know the importance of various steps involved in performing coated with specific antibody against the antigen to be
enzyme-linked immunosorbent assay (ELISA). detected. The specimen such as serum to be tested are added
2 Perform the ELISA test and interpret the results. to the coated wells. If antigen is present in the serum, then it
will combine with the coated antibody. Antibody conjugated
with enzyme is added to detect the antigen-antibody reactions.
INTRODUCTION The conjugated antibody combines with antibody that has
combined with the antigen. A substrate is added to detect the
Enzyme-linked immunosorbent assay (ELISA) is a widely used binding of conjugated antibody to antigen-antibody complex.
method for demonstration of specific antigens or antibodies in In a positive test, the positive reaction is tested by the change
the serum and other body fluids. They are used extensively for of colour, which can be read by spectrophotometer or ELISA
diagnosis of a variety of human diseases including many viral reader.
(AIDS, hepatitis, rubella, respiratory syncytial infection, etc.), 2. The indirect ELISA for antibodies: This is a frequently used
parasitic (amoebiasis, cysticercosis, leishmaniasis, lymphatic method to detect antibodies. Polystyrene tubes or wells in
filariasis, etc.), bacterial (syphilis, brucellosis, salmonellosis, polystyrene plates are coated with the antigen solution. An
etc.) and fungal (histoplasmosis, aspergillosis, etc.). The aliquot of the serum to be tested is added to each tube and is
principle of ELISA in essence is similar to that of the incubated, followed by washing. To detect the reaction a goat
fluorescence, the only difference being that an enzyme is used antihuman immunoglobulin antibody conjugated with enzyme
instead of fluorescent dye. The most commonly used enzymes is added. The enzyme conjugated antihuman immunoglobulin
are alkaline phosphatase, horseradish peroxidase and ß- binds to the antibody. A substrate is added to detect the binding
galactosidase. Enzyme activity is generally determined by the and in a positive reaction the enzyme acts on the substrate to
change of colour, which can be read by spectrophotometer or produce a change of colour.
ELISA reader. Then the enzyme-linked antiglobulin is added. If antibodies
ELISA is a heterogenous enzyme assay that uses solid are present in the serum, they will have bound to the antigen,
phases e.g. plastic tubes, polyvinyl chloride plates, beads, which has bound to the plate, and now the antiglobulin will
microplate, and membranes such as cellulose acetate or cellulose bind to the primary antibodies. Add the substrate for the
nitrate. The enzyme immunoassay can be broadly of three types: enzyme. If the reaction occurs (for example, if the solution
a. the sandwich ELISA for antigen, b. the indirect ELISA for changes color), this means that the enzyme-linked antiglobulin
antibodies, and c. competitive ELISA for antibodies. was bound, which means that the serum did, in fact, contain
Depending on the nature of the antigen used, the ELISA the antibodies of interest. The color change can be quantified
can also be classified as 1st, 2nd and 3rd generation ELISA as using a spectrophotometer.
used in HIV infections (Box 49-1). 3. Competitive ELISA for antibodies: This involves the use of
Enzymes and substrates used in the ELISA are listed in the two specific antibodies. One antibody is conjugated with enzyme;
table 49-1. where as the other antibody is present in the serum to be tested.
Competition occurs between these two antibodies for the same
antigen, hence is called the competitive ELISA. This is used for
demonstration of HIV antibodies in the serum.
Textbook of Practical Microbiology 139
The Dot ELISA is a rapid visually read microassay form of 7 Add 100 µl of conjugate to each well and incubate at 37°C
the ELISA (Box 49-2). The advantages and disadvantages of for 1 hour.
ELISA tests are mentioned in the table 49-2. 8 Wash the wells thrice and add 100 µl of substrate solution
In this experiment indirect ELISA to detect antibodies and and incubate for 30 minutes at 37°C in dark.
sandwich ELISA to detect antigen will be described. 9 Stop the reaction with 50µl of 3N sulphuric acid (H2SO4) to
each well.
10 Read the results with naked eye or at 492 nm in an ELISA reader.
INDIRECT ELISA TO DETECT ANTIBODIES
QUALITY CONTROL
REQUIREMENTS
Appropriate controls, such as controls with positive and
negative sera, buffer control, conjugate control and substrate
I Equipments control should be tested along with every plate.
Micro titer plate, ELISA reader and ELISA washer.
III Specimen
PROCEDURE Serum, CSF, urine etc.
4 Wash thrice with wash buffer and remove all residual fluid.
5 Add 100µl of diluted serum samples and incubate for 1 RESULTS AND INTERPRETATIONS
hour at 37°C.
6 Wash the wells with washing buffer and remove all residual Development of yellow colour indicates the test serum contains
fluid. antigens to tested antibody. Development of no colour
7 Add 100µl of conjugate to each well and incubate at 37°C inducates negative reaction.
for 1 hour.
8 Wash the wells thrice and add 100µl of substrate solution
and incubate for 30 minutes at 37°C in dark.
9 Stop the reaction with 50µl of 3N H2S04 to each well.
10 Read the results with naked eye or at 492 nm in an ELISA reader.
QUALITY CONTROL
OBSERVATIONS
Observe the microtiter plate for the development of colour. FIGURE 49-1 ELISA test.
Observe control (negative, positive and blank) wells.
1st generation ELISA: It is an ELISA in which crude antigen is used for the detection of antibodies.
2nd generation ELISA: It is an ELISA in which semi-purified antigen is used for the detection of antibodies.
3rd generation ELISA: It is an ELISA in which recombinant antigens or synthetic peptides are used for the detection of antibodies.
Dot ELISA is a rapid visually read microassay. The principle is similar to that of ELISA where enzyme is used as a marker or
label to detect the binding of antigen and antibody. The test is performed on a nitrocellulose membrane. Enzyme converts
colourless substrate to a coloured product, which indicates the presence of antigen-antibody binding. When the test serum
is layered on to the membrane, specific antibodies if present, will bind to corresponding dot of the antigen. Addition of a
labeled serum antibody (conjugate) and the subsequent development of the colour allow the detection of the presence of
antibodies based on the pattern of the antigen sites. Dot-ELISA can be used for the detection of antibodies as well as antigen.
Enzymes Substrates
Advantages Disadvantages
KEY FACTS
1 Preliminary chequer board titrations are carried out for standardizing antigen and antibody concentration.
2 Read all plates at a standard time after addition of the substrate because colour is not stable.
3 Substrate should be made just before use as it decomposes spontaneously and is unstable.
4 OPD should be stored at 4°C in the dark. It is carcinogenic.
VIVA
1 Expand ELISA.
Ans. Enzyme-linked immunosorbent assay.
2 What is the principle of enzyme immuno assay?
3 What are different steps in the ELISA procedure?
Ans.
a Coating solid surface with antigen/antibody.
b Binding antibody/antigen from test sample.
c Binding conjugate.
d Addition of substrate.
e Observe the colour change and read the O.D. values.
4 What are various types of the ELISA?
Ans. Competitive ELISA, sandwich ELISA, indirect ELISA, membrane bound ELISA, cassette ELISA, biotin-avidin ELISA,
protein-A ELISA, etc.
5 What are the enzymes and substrates used in the ELISA?
6 What do you understand by the first, second and third generation ELISA?
7 What are the advantages and disadvantages of the ELISA?
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworth, London, 94-96, 1995.
2 Koneman EW, Allen SD, Janda WM, Schreckenbergu PC and Winn Jr. WC. Color Atlas and Textbook of Diagnostic Microbiology. 5th
Edition. Lippincott Williams and Wilkins. 1997; pp. 1395.
3 Stites DP. Terr AI and Parslow TG. Medical Immunology. 10th Edition. 2001. pp. 902.
142
Textbook of Practical Microbiology 143
UNIT
VII
Microbial Genetics and
Molecular Techniques
Introduction
Lesson 50 Isolation of Plasmids
Lesson 51 Polyacrylamide Gel Electrophoresis
Lesson 52 Isolation of Antibiotic Resistant Mutant
Lesson 53 Bacterial Conjugation
144
Introduction
The objectives of the study are to demonstrate the genetic FREQUENTLY USED TERMINOLOGY IN
methods such as isolation of plasmids, polyacrylamide gel GENETICS
electrophoresis (PAGE), isolation of antibiotic resistant mutants
and conjugation in bacterial systems.
Phenotype: Phenotype refers to “the observable outward
appearance of an organism, which is controlled by the
Genetic information is stored in DNA of the bacteria as a
genotype and its interaction with the environment”.
sequence of nucleotide bases (adenine, cytosine, guanine,
Genotype: Genotype refers to “the genetic makeup of an
thymine, abbreviated as A, C, G, T respectively) read sequentially
individual organism”.
in a 5' to 3' direction (or in RNA, with uracil, abbreviated U,
Transcription: The “central dogma” of molecular biology
replacing thymine). The most common form of DNA (present
describes the transcription of genetic information from a
in all cellular genomes, as well as many viral genomes) is double
DNA nucleotide triplet code to an RNA triplet code, followed
stranded. The 5' to 3' polarity of the two strands is opposite,
by translation to specific amino acid sequences in protein.
and they are held together by hydrogen bonding between
Prototrophs: A wild type strain that has minimal
nucleotide base pairs, A to T and G to C. The sense strand
requirements for exogenously supplied nutrients is referred
carries the coded genetic information. The antisense strand
to as a prototroph.
consists of a complementary sequence of bases oriented in the
Auxotrophs: A mutant strain that has lost the ability to
opposite 5' to 3' direction. During DNA replication, the two
synthesize its own supply of a particular nutrient, such as
strands separate and each is used as a template for synthesis
histidine or adenine or thiamine is called an auxotroph.
of a new complementary strand. This allows genetic information
Recombinant DNA technology: Recombinant DNA in this
to be replicated with a high level of precision. Because
context refers to the creation of a new combination of DNA
replication is bidirectional, new DNA can only be synthesized
segments that are not found together naturally. Such
in a 5' to 3' direction, the overall pattern of replication is rather
technology is now widely used in many practical applications
complex.
ranging from basic research on control of gene expression
to forensic medicine to biotechnology.
Genetic information is transcribed from DNA to RNA, with the
Restriction endonucleases: An endonuclease is an enzyme
antisense strand of the DNA serving as a template for
that can cleave the phosphodiester bonds of a nucleic acid at
synthesis of RNA with the same base sequence (5' to 3') as the
an internal site (as opposed to cleavage by an exonuclease,
sense strand of the double helical DNA, except that uracil (U)
which can only remove nucleotides from one of the ends of a
replaces thymine (T). Genetic information contained in
nucleic acid). Example: Eco RI G|AATTC.
messenger RNA (mRNA) is translated into a sequence of amino
Hybridization probes: Complementary strands of DNA, RNA,
acids in a polypeptide chain during protein synthesis
or DNA plus RNA hybridize readily to form double stranded
(translation). A redundant nucleotide triplet code, read 5' to 3'
helical structures when placed under suitable annealing
on the mRNA (and on the sense strand of the DNA), specifies
conditions. This property is used extensively in molecular
the amino acid sequence of the protein, read from N-terminal to
genetics to identify specific nucleic acid sequences. A probe
C-terminal.
consisting of radioactively labeled DNA (or RNA) is hybridized
to denatured DNA (or naturally single-stranded RNA)
The study of genetic materials helps to a). demonstrate mutations,
immobilized on a support, such as a nitrocellulose membrane.
b). study the gene transmission among progeny or generations,
Hybridization is normally done at a temperature about 25°C
c) treat genetic disorders, and d) in diagnosis and research.
below the melting (denaturation) temperature for the DNA.
Textbook of Practical Microbiology 145
LESSON
Isolation of Plasmids
50
LEARNING OBJECTIVES II Reagents and lab wares
Ethidium bromide solution, Luria-Bertani medium,
After completing this practical you will be able to: micropipettes, Eppendorf tubes, and Bunsen burner.
1 Isolate the plasmid from bacteria. Preparation of ethidium bromide stock solution: The solution
is prepared by dissolving 10 mg of ethidium bromide powder in
1 ml of double distilled water.
INTRODUCTION Preparation of ethidium bromide working solution: The
solution is prepared bu adding 10µl ethidium bromide stock
Bacterial plasmids are small, circular, extra chromosomal DNAs solution in 100 ml of agarose solution.
that are capable of autonomous replication within bacterial cells. Preparation of Luria-Bertani medium (LB medium): The
Plasmids have their own origins of replication, such that they medium is prepared by adding the following ingredients: Bacto-
can multiply independently within the bacterial cells. They tryptone, 10 g; Bacto yeast extract, 5 g; sodium chloride (NaCl),
usually contain only a few thousand base pairs of DNA and 10 g; and distilled water, 1000 ml. The pH is adjusted to 7.0.
carry only a few genes, often for antibiotic resistance. Plasmids III Specimen
that carry appropriate genes are capable of making bacteria Bacterial strain: Plasmid containing strain PHSV 106 or
resistant to antibiotics(Box 50-1), which makes it possible to equivalent strain is grown in a medium containing 20µg/ml
select for bacteria that have taken up the plasmids. ampicillin. The medium contains the following solutions:
Solution A
PRINCIPLE 50 mM glucose.
25 mM Tris chloride (ph 8).
This method utilizes the molecular characteristics of covalently 10mM EDTA (pH 8).
closed circular deoxyribonucleic acid (DNA) that is released Solution B
from cells under conditions that denature DNA by using alkaline 0.2 N sodium hydroxide (NaOH).
sodium dodecyl sulfate. Under these conditions, chromosomal 1% SDS.
DNA concentrations are reduced or eliminated. The DNA is
freed of RNA and proteins by RNase and protease treatments. Solution C
Just before extraction, the plasmid DNA is released from the 5M potassium acetate, 60 ml.
folded chromosomal complex by a shearing step or by RNase Glacial acetic acid, 11.5 ml.
treatment. The plasmid DNA can be resolved as covalently Distilled water, 28.5 ml.
closed circular molecules by centrifugation in ethanol
containing ethidium bromide. The clarified extract is used
directly for electrophoretic analysis.
PROCEDURE
Extraction of plasmid
REQUIREMENTS
1 Inoculate a single bacterial colony into 2 ml of LB medium
I Equipments containing antibiotic ampicillin in a 15 ml test tube and
Centrifuge and inoculating chamber. incubate over night with vigorous shaking.
146 Isolation of Plasmids
2 Take 1.5 ml of culture into a Microfuge tube and centrifuge 6 Remove the gel carefully and place on UV transilluminator
at 12, 000 g for 2 min at 4°C and discard the supernatant. and examine for the bands.
3 Resuspend the pellet in 100 µl of ice-cold solution A and
vortex vigorously.
4 Add 200 µl of solution B and mix the contents by inverting QUALITY CONTROL
the tube rapidly several times.
5 Add 150 µl of ice-cold solution C and mix the contents by A parallel procedure should be carried out using standard strain
inverting the tube. of bacteria.
6 Store the tube in ice for 5 minutes. Molecular weight marker must be used in the analysis.
7 Centrifuge at 12, 000 g for 5 minutes at 4°C and transfer the
supernatant to a fresh tube.
8 Precipitate the DNA with two volumes of 100% ethanol at OBSERVATIONS
room temperature. Mix well and allow it to stand for 2
minutes. Observe the stained gel under UV illumination for DNA band.
9 Centrifuge at 12000 g for 5 minutes at 4°C and remove Observe the bands of molecular weight marker.
supernatant.
10 Resuspend the pellet in 50 µl of TE (pH 8) containing
RNAse (20 µg/ml).
RESULTS AND INTERPRETATION
11 Run in agarose gel containing ethidium bromide and view
under UV transilluminator.
Plasmid appears as a sharp orange band.
Undigested RNA appears as hazy opaque area along the lanes.
Electrophoresis on agarose gel
KEY FACTS
1 Plasmid is small, circular, extra chromosomal DNA that is capable of autonomous replication within bacterial cells.
2 Care must be taken at every step of the procedure for extraction of the plasmid DNA and electrophoresis on agarose gel.
3 Care must be taken while decanting the supernatant after centrifugation.
VIVA
FURTHER READINGS
1 Collier L, Balows A, Sussman M. Topley and Wilson’s Microbiology and Microbial Infections. 9th Edition. Systemic Bacteriology. Volume
2.. Arnold publishers. pp. 2002, pp 1501.
2 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995. pp 94-96.
3 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. 1996. pp. 921.
148
LESSON
Polyacrylamide
51 Gel Electrophoresis
6 Electrophoresis / Running Buffer (1x) : 1000ml. 11 Connect the electrodes to the power pack and make pre-
3g Tris : 25mM. run at a constant current of 20mA for 10-15minutes.
14.4g glycine : 192mM. 12 Switch off power supply and load the wells with your protein
1g SDS : 0.1%. samples (10-50µl) diluted in sample buffer along with a
Distilled water to make 1000ml molecular weight marker (5-10µl) using a micropipette/
syringe.
7 Sample buffer : 10ml. 13 Run the electrophoresis at a constant current of 20mA till
0.6ml 1M Tris-HCl (pH 6.8) : 60mM. the dye front reaches the separating gel and then increases
5ml 50% glycerol : 25%. it to 25mA.Stop the run when the dye front reaches the
2ml 10% SDS : 2%. bottom of the gel.
0.5ml 2-mercaptoethanol : 14.4mM. 14 Now remove the side spacers from the glass plates and
1ml 1% Bromophenol blue : 0.1%. carefully scoop the gel using a spatula. The gel may be
0.9ml distilled water stained and destained for visualization or, used as such in
blotting experiments without staining.
8 Staining solution : 1000ml.
15 The gel may be stained using the Coomassie staining
1.0g Coomassie blue R-250.
solution for 1-2hrs and then destained using the destaining
450ml methanol.
solution for overnight in a rocking shaker. An alternate
450ml distilled w.ater
silver staining procedure too can be followed who is
100ml glacial acetic acid.
sensitive but a little expensive.
9 Destaining solution : 1000ml.
100ml methanol.
100ml glacial acetic acid. QUALITY CONTROL
800ml distilled water.
Known protein ladder is loaded in each run.
III Specimen
Protein suspension to be separated.
OBSERVATIONS
PROCEDURE Observe the control protein bands.
Observe and analyze the test protein bands.
1 Mix protein sample (20µl) with sample buffer (5µl) in an
Eppendorf tube and heat it at 75-100°C for 2- 10 minutes
depending on the nature of the sample. RESULTS AND INTERPRETATION
2 Assemble the clean glass plates by placing the spacers-
two on either sides, or one along the bottom edge and fix 1 The protein bands separated according to the molecular
the whole assembly tightly with clamps or gel casting stand. weight and charge are visualized directly.
3 Seal the glass plate assembly with 2% agar or, melted wax 2 Check out for the desired bands by comparing with a
on all three sides leaving the topside so that the assembly standard marker.
is leak proof. 3 Interpret the desired band based on experimental and
4 Prepare and cast the separating gel using a small beaker. literature analysis by either visually or using a gel
5 Mix the components of the separating gel mixture without documentation system with appropriate software.
TEMED and de aerate. 4 The bands can also be transferred by blotting and confirmed
6 Add TEMED finally to the mixture and pour immediately by coupling with appropriate antisera containing
between glass plates upto 2cm below the notch and allow complimentary antibodies for the antigen (electro-immuno
the gel to polymerize for 30-60 minutes at room temperature. transfer blot).
7 Overlay the acrylamide with n-butanol, which helps to keep
the gel surface flat. After polymerization remove the butanol
and rinse with water. Table51-1 List of other types of electrophoresis in gels
8 Prepare the stacking gel and cast over the separating gel as
mentioned above and insert the comb. Electroimmunodiffusion.
9 Allow to polymerize for 15-30minutes. Then remove the Counterimmunoelectrophoresis.
comb carefully and rinse the wells with distilled water. Rocket electrophoresis.
10 Now remove the casted gel plate from the gel casting stand/ Southern blotting.
clamps and detach the bottom spacer. Place the gel assembly Northern blotting.
into electrophoresis chamber with the notched plates facing
inside and fill the upper and lower tanks with running buffer.
Note: Remove air bubbles in the wells if any, using a syringe.
150 Polyacrylamide Gel Electrophoresis
The gel concentration percentage, i.e.: whether 7.5, 10, 12.5 or 15 %, depends on ones experimental need and the desired quantity of stock
solutions to cast the gel are calculated using the following formula.
Acrylamide stock x /3 ml
Stacking / separating gel buffer 2.5ml
Distilled water (7.5-x /3) ml
10% APS 50µl
TEMED 5µl (10µl if x < 8%)
Total volume 10 ml
10% Separation gel 5% Stacking gel
Acrylamide stock 3.3 ml. Acrylamide stock 1.6 ml. Separating gel buffer 2.5ml Stacking gel buffer 2.5ml
Distilled water 4.2ml. Distilled water 5.9ml.
10% APS 50µl. 10% APS 50µl.
TEMED 5µl. TEMED 5µl.
Total volume 10 ml. Total volume 10 ml.
Advantages
Efficient tool for characterizing proteins.
Highly sensitive.
Rapid method.
Protein bands are stable and can be stained for preservation.
The number of different antigens can be readily observed.
Disadvantages
Chemicals are neurotoxic.
Very expensive.
Trained professional and well equipped laboratory is required.
VIVA
KEY FACTS
1 Care must be taken for spillage or direct exposure to reagent while casting a gel.
2 Avoid introducing air bubbles in to the gel.
3 Always freshly prepared buffers and reagents should be used.
4 Observe for complete circuit of electricity (use sufficient levels of buffer) throughout the procedure.
FURTHER READINGS
1 Collier L, Balows A, Sussman M. Topley and Wilson’s Microbiology and Microbial Infections. 9th Edition. Systemic Bacteriology. Volume
2.. Arnold publishers. pp. 2002, pp 1501.
2 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995. pp 94-96.
3 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. 1996. pp. 921.
152
LESSON
Isolation of Antibiotic
52 Resistant Mutant
INTRODUCTION I Equipments
Micro centrifuge.
Mutation is any change in genetic information relative to a reference
“wild-type” genome, including changes that affect expression of II Reagents and lab wares
genes without altering their coding sequences and changes that Eppendorf tubes, micropipettes, two 10 ml trypticase soy agar
do not cause any detectable phenotypic difference (silent in 20 ml test tubes and streptomycin solution (10 mg per 100 ml
mutations). In a complex organism, mutation can occur at many distilled water).
different structural levels and can be classified in many different
ways. Mutations are mainly classified into two types: Point III Specimen
mutations and large-scale mutations (Box 52-1).Mechanisms of Pure growth of Escherichia coli from solid media preferably
mutations are many (Box 52-2). from non-blood agar plates (Examples: nutrient agar, Muller-
Hinton agar), to be tested for antibiotic resistance is used as
The importance of mutation specimen.
In classical genetics, a point mutation was originally defined as a change in an inherited trait that was not accompanied by any
chromosomal change that could be seen with a light microscope. Point mutation can result in missense (amino acid substitution),
nonsense (insertion of a stop codon), or frameshift (either positive or negative).
Missense mutations
Most base pair substitutions change the amino acid specified by the codon in which they occur. Such mutations are described
as missense mutations because they cause an amino acid substitution in the coded protein. Depending on the nature of the
amino acid substitution and its location within the protein, missense mutations may have a variety of effects, ranging from
complete loss of biological activity to reduced activity or temperature-sensitive activity or no functional effect at all.
Nonsense mutations
Base pair substitutions that generate an in-frame stop codon within a previously functional protein coding sequence cause
premature termination of translation of the protein in question and are referred to as nonsense mutations.
Silent mutations
In some cases, base pair substitutions generate a different codon for the same amino acid, with no biological effect whatsoever.
This is most likely to happen in the third position (wobble base) of redundant codons for the same amino acid. Such changes
are considered to be mutations because they alter the genetic code. However, because they have no phenotypic effect, even
at the level of protein amino acid sequence, they are called silent mutations.
Frameshift mutations
Addition or deletion of a single base pair in the middle of a coding sequence will result in out-of-frame translation of all of the
downstream codons, and thus result in a completely different amino acid sequence, which is often prematurely truncated by
stop codons (UAG, UAA, UGA) generated by reading the coding sequence out-of-frame. Such mutations, which are a special
subclass of point mutations, are referred to as frameshift mutations. Deletion of a single base pair results in moving ahead one
base in all of the downstream codons, and is often referred to as a positive frameshift. Addition of one base pair (or loss of two
base pairs) shifts the reading frame behind by one base, and is often referred to as a negative frameshift.
Tautomerization
Spontaneous mutations that involve base pair substitutions are caused primarily by configurational changes within the
individual bases that result in mispairing. These changes, which are called tautomeric shifts, involve momentary expression of
rare alternative molecular configurations that exist in equilibrium with the more common forms. Specifically, proton shifts can
convert the amino groups in adenine and cytosine to imino groups, and the keto groups in guanine and thymine to enol
groups.
Transitions
A tautomeric shift in any of the four DNA bases can lead to mispairing of A to C or G to T. The tautomeric state can occur either
in the template base or the incoming base. During the next round of DNA synthesis, the mispaired base will pair with its normal
partner, resulting in a transition, in which an AT base pair replaces a GC or a GC replaces an AT, with no change in the purine:
pyrimidine polarity of the base pair. Transitions are the most common type of mutation resulting from spontaneous mispairing
due to tautomerization.
Transversions
To achieve a transversion, in which the positions of purine and pyrimidine are reversed in the DNA double helix, two events
are thought to be involved, tautomerization of one of the bases and rotation of the other to yield a purine: purine pairing. The
frequency of spontaneous transversions, which is lower than that of transitions, appears to be consistent with this interpretation.
A second possible mechanism for transversions is the formation of an apurinic site, which can result in replacement of the
original purine with any of the four bases.
Genes are stable repositories of the information needed for synthesis of all of the RNA and proteins in a living organism.
Survival and stability of each species is dependent on faithful replication of genetic information for use by each new generation.
However, a low level of mutational change is highly desirable. Over an extended period of time, mutational changes provide the
ability for species to adapt to changing conditions and challenges, and thus serve as the raw material for selective survival and
the evolution of more advanced and efficient species, as well as the development of biological diversity.
KEY FACTS
VIVA
FURTHER READING
1 Collier L, Balows A, Sussman M. Topley and Wilson’s Microbiology and Microbial Infections. 9th Edition. Systemic Bacteriology. Volume
2. Arnold publishers. pp. 2002, pp 1501.
2 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995. pp 94-96.
3 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. 1996. pp. 921.
Textbook of Practical Microbiology 155
LESSON
Bacterial Conjugation
53
LEARNING OBJECTIVES Differences between F+ strain and Hfr strain are listed in the
Table 53-1.
After completing this practical you will be able to:
Prepare control plates of parental Hfr and F– and aseptically add 0.1 Presence of colonies in the test medium indicates the transfer
ml of each strain to its agar plate and spread with a bent gloss rod. of genetic material.
The DNA transfer that occurs in conjugation begins as a single strand break in the donor chromosome (or plasmid), with only
one strand transferred through the F– pilus to the recipient cell. The single strand left behind in the donor and the one that is
transferred to the recipient are both converted into double strands, restoring the donor chromosome and generating double
stranded donor DNA in the recipient. As a result of receiving new DNA from the donor, the recipient is temporarily partially
diploid. Recombination can then integrate parts of the transferred DNA into the recipient genome. Any DNA that is not
integrated is soon destroyed.
Transformation
Transformation refers to the ability of extracellular DNA to enter a bacterial cell and recombine with the bacterial genome,
thereby giving the bacterial cell new genetic properties.
Transduction
Transduction refers to a genetic exchange in which bacteriophages carry bacterial genes from one bacteria cell to another.
There are two classes of transduction, specialized and generalized. In specialized transduction, a lysogenic phage undergoes
recombination with the host genome and later when it is excised to become an independent phage genome, it carries one or
more host genes with it, which can be transduced into a new host cell and recombined into the genome of that cell. In
generalized transduction, fragments of host DNA are mistakenly packaged into a bacteriophage in place of its own DNA.
1. F factor usually present in the form of a plasmid. 1. F factor has become integrated into the bacterial chromosome.
2. Low frequency of recombination. 2. High frequency of recombination.
3. Only F factor transfer occurs in conjugation. 3. Transfer of chromosomal DNA from the donor to the
recipient F– cell begins adjacent to the integrated F factor
and can progress around the entire bacterial chromosome if
the process is not interrupted.
KEY FACTS
VIVA
FURTHER READINGS
1 Collier L, Balows A, Sussman M. Topley and Wilson’s Microbiology and Microbial Infections. 9th Edition. Systemic Bacteriology. Volume
2. Arnold publishers. pp. 2002, pp 1501.
2 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 1995. pp 94-96.
3 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. 1996. pp. 921.
158
Textbook of Practical Microbiology 159
UNIT
VIII
Bacteriology
LESSON
Normal Microbial Flora
54 of the Mouth
PRINCIPLE OBSERVATIONS
Saliva is collected from the mouth and inoculated onto the 1 Look for the presence of haemolysis on blood agar plate by
surface of the agar plates. The colonies grown on these plates viewing the plate under transmitted light.
are studied further for their identification. 2 Observe all the colonies on both plates.
3 Record your observations.
4 Perform Gram stain. Observe and record morphology of the
REQUIREMENTS bacteria.
I Equipments
Microscope and CO2 incubator.
Textbook of Practical Microbiology 161
KEY FACTS
1 There are many microorganisms which are present as part of the normal flora in the mouth and teeth.
2 Saliva sample should be collected in a sterile bottle.
3 Saliva sample should be processed immediately after collection.
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 94-96, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Jawetz, Melnick and Adelberg. Medical Microbiology. 23rd Edition. McGaw Hill. 2003.
4 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. 1996.
5 Murray PR, Rosenthal KS, Kobayashi GS and Pfaller ME. Medical Microbiology. 5th Edition. Mosby Inc. 2002.
162
LESSON
Normal Microbial Flora
55 of the Throat
OBSERVATIONS
PRINCIPLE
1 Look for the presence of hemolysis on blood agar plate by
Specimen taken from the throat is inoculated onto the surface viewing the plate under transmitted light.
of the media plates. The colonies grown on these plates are 2 Perform oxidase test for the colonies grown on chocolate agar.
studied further for their identification. 3 Look for the presence of black colored colonies on potassium
tellurite agar.
4 Record your observations.
REQUIREMENTS 5 Perform Gram stain. Observe and record morphology of the
bacteria.
I Equipments
Microscope and CO2 incubator.
RESULTS AND INTERPRETATIONS
II Reagents and lab wares
Bunsen burner, tongue depressor, sterile cotton swabs, glass 1 On blood agar, streptococci produce pinpoint a-hemolytic
slides and pencil, Gram staining reagents (methyl violet, Iodine, colonies where as staphylococci produce pinhead colonies
95% alcohol and dilute control fuchsin), filter paper containing with b-hemolysis.
1% tetramethyl paraphenyline diamine dihydrochloride, two 2 On Gram stain, streptococci are identified based on the
blood agar plates, one potassium tellurite agar plate, and one arrangement, they are Gram positive cocci arranged in chains.
5ml sterile saline tube. Staphylococci are arranged in clusters.
Textbook of Practical Microbiology 163
3 On chocolate agar, colonies grown are tested for the presence paper. Oxidase negative bacteria do not produce color when
of the enzyme oxidase (refer chapter 21). Neisseria are oxidase the colonies are streaked on oxidase paper.
positive. They turn the oxidase paper into deep purple in 4 Diphtheroids when grown on potassium tellurite agar
color when they are streaked on the surface of the filter produce pinpoint black colored colonies.
KEY FACTS
1 There are many microorganisms which are present as part of the normal flora in the throat and upper respiratory tract.
2 Throat swab should be collected in a sterile bottle.
3 Throat swab should be processed immediately after collection.
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 94-96, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Jawetz, Melnick and Adelberg. Medical Microbiology. 23rd Edition. McGaw Hill. 2003.
4 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. 1996.
5 Murray PR, Rosenthal KS, Kobayashi GS and Pfaller ME. Medical Microbiology. 5th Edition. Mosby Inc. 2002.
164
LESSON
Normal Microbial Flora
56 of the Skin
LEARNING OBJECTIVES dextrose agar plate, lactophenol cotton blue and reagents for
Gram stain.
After completing this practical you will be able to:
1 Demonstrate various microorganisms which are present as III Specimen
part of the normal flora on the skin. Skin swab.
INTRODUCTION PROCEDURE
Skin as part of its normal flora, normally harbours many bacteria. 1 Collect the swab from surface of the skin by rubbing the
It contains 102 to 104 organisms per cm2. Staphylococcus cotton swab after moistening the swab in sterile saline.
epidermidis and diphtheroids are predominant bacteria. The 2 Place the swab in a tube containing sterile saline and mix it.
microbial flora present in the pharynx and trachea are similar to 3 Inoculate a loopful suspension on one plate each of blood
that present in the mouth. Streptococcus viridans, micrococci, agar, mannitol salt agar and Sabouraud’s dextrose agar
peptostreptococci, propionibacterium, b-haemolytic 4 Incubate the Sabouraud’s agar plate for 48 hours at 25°C
streptococci, enterococci, diphtheroids, Candida species, and the remaining plates for 48 hours at 37°C.
Malasezia furfur , etc. are the other microorganisms.
OBSERVATIONS
PRINCIPLE
1 Look for the presence of hemolysis on blood agar by viewing
Swab is collected from surface of the skin and is inoculated on the plates under transmitted light.
agar plates and incubated. The colonies are studied further for 2 Look for the presence of mold like or moist growth on
their identification. Sabouraud’s agar plate.
3 Look for the presence of yellow color of the medium
surrounding colonies grown on mannitol salt agar medium.
REQUIREMENTS Yellow color colony is suggestive of S. aureus.
I Equipments
Microscope and incubator. RESULTS AND INTERPRETATION
VIVA
KEY FACTS
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 94-96, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Jawetz, Melnick and Adelberg. Medical Microbiology. 23rd Edition. McGaw Hill. 2003.
4 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. 1996.
5 Murray PR, Rosenthal KS, Kobayashi GS and Pfaller ME. Medical Microbiology. 5th Edition. Mosby Inc. 2002.
166
LESSON
Identification of
57 Staphylococcus aureus
LEARNING OBJECTIVES
demonstrate the presence of Gram positive cocci arranged in
After completing this practical you will be able to: clusters along with pus cells (Fig. 57-1).
1 Know about the clinical importance of staphylococci.
2 Know the procedures used to identify Staphylococcus 2 Culture
aureus and differentiate it from other staphylococcal species.
Clinical specimens are inoculated onto nutrient agar, blood
agar and MacConkey agar plates and kept for aerobic incuba-
INTRODUCTION tion at 37°C. S. aureus produces opaque, circular colonies with
butyrous consistency. Golden yellow pigment is demonstrated
Staphylococcus species are Gram positive spherical shaped on nutrient agar (Fig. 57-2). On blood agar, they produce b-
bacteria measuring 0.8 µm to 1 µm in size arranged in clusters. haemolytic golden yellow or white colonies (Fig. 57-3). On
They are catalase positive, non-spore forming bacteria MacConkey agar they produce minute pink colored colonies.
Staphylococci are part of the indigenous flora of skin surfaces,
mucous membranes and upper respiratory tract. They usually 3 Coagulase test
cause suppurative lesions such as pustule, furuncle or carbuncle
and can also involve muscles and bones. They are less Coagulase test is most important test used for identification of
commonly associated with systemic infection. However they S. aureus (refer chapter 22).
produce a variety of exotoxins causing various toxin-mediated
diseases such as food poisoning, toxic shock syndrome and 4 Deoxyribonuclease test
staphylococcal scalded skin syndrome.
Laboratory tests for differentiation of staphylococcal Coagulase positive S. aureus also produce the enzyme
species are listed in the table 57-1. deoxyribonuclease enzyme. Detection of the presence of the
enzyme, deoxyribonuclease is used to reconfirm the
identification of S. aureus.
SPECIMENS The test organism is streaked onto the DNA agar medium
(0.2% DNA). Then the DNA plate is incubated overnight at
Clinical specimens like pus swab, and wound swab. 37°C. After incubation, 3.6% of hydrochloric acid (1N HCl) is
In this chapter the following tests will be described which added to the medium to determine the DNase activity. DNase
are employed routinely for identification of S. aureus. positive S. aureus hydrolyze the DNA resulting in production
of halo around the growth. Absence of halo around the growth
indicates DNase-negative S. aureus which are also coagulase
TESTS FOR THE IDENTIFICATION OF negative.
STAPHYLOCOCCUS AUREUS
5 Mannitol salt agar
1 Direct examination This is a selective medium used to isolate staphylococci from
clinical specimens. It contains nutrient agar with 1% mannitol,
Gram stain and 7.5% sodium chloride. Phenol red is used as an indicator.
Gram stain of smears of clinical specimens is carried out to The medium is used to test mannitol fermenting ability of
Textbook of Practical Microbiology 167
S. aureus. It also tests ability of S. aureus to tolerate the high The plate is then incubated overnight at 37°C. The presence of
salt concentration in the medium and grow readily. zone of inhibition (17 mm) around the disc indicates novobiocin-
S. aureus produces yellow colonies on the medium while sensitive S. aureus and S. epidermidis and no zone of inhibition
other staphylococcal species do not. The yellow colour is seen around the disc indicates novobiocin-resistant S. saprophyticus.
due to the fermentation of mannitol with production of acid by
S. aureus, thereby reducing pH of the medium to the acidic. In
acidic pH the indicator, phenol red, produces yellow colour.
6 Novobiocin sensitivity
Growth on
Mannitol salt agar + - -
Colonial pigmentation Golden yellow White White
Coagulase test + - -
DNAase test + - -
Hemolysis on blood agar Beta - -
Novobiocin sensitivity Sensitive Sensitive Resistant
FIGURE 57-2 Staphylococcus aureus colonies on nutrient agar. FIGURE 57-3 Staphylococcus aureus colonies on blood agar.
168 Identification of Staphylococcus aureus
KEY FACTS
1 Yellow color pigmentation can also be produced by micrococci which can be distinguished from staphylococci by various
biochemical tests such as modified oxidase and sensitivity for novobiocin, bacitracin and furazolidone.
2 Some coagulase negative staphylococci can produce beta hemolysis on blood agar similar to coagulase positive
staphylococci, hence should not be confused with that of S. aureus.
3 Some species of staphylococci produce only bound coagulase (clumping factor) such as S.hydenensis and S.schleifer
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 94-96, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Jawetz, Melnick and Adelberg. Medical Microbiology. 23rd Edition. McGaw Hill. 2003.
4 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. 1996.
5 Murray PR, Rosenthal KS, Kobayashi GS and Pfaller ME. Medical Microbiology. 5th Edition. Mosby Inc. 2002.
Textbook of Practical Microbiology 169
LESSON
Identification of
58 Streptococcus pneumoniae
A blood agar plate is divided into two halves by glass marking 5 Inulin fermentation
pencil. One half is labeled as S. pneumoniae while the other half
is labeled as S. mitis. Using a sterile cotton swab, test strains are S. pneumoniae is capable of fermenting the sugar, inulin whereas
inoculated onto the surface of blood agar, followed by application other alpha hemolytic streptococci do not ferment inulin.
of 0.05 units Optochin disc over the inoculated surface by using The test strain is inoculated into the inulin sugar medium
forceps. The plate is incubated aerobically at 37°C under 10% (serum sugar) and is incubated over night at 37°C. Positive test
CO2 environment for 24 hours. Development of zone of inhibition is indicated by change of color of the media from red to yellow.
of 15 mm and more shows the test to be positive. Zone of Negative test is indicated by no color change, and medium
inhibition less than 15 mm shows the test to be negative. continues to appear as red.
FIGURE 58-1 Streptococcus pneumoniae on blood agar. FIGURE 58-2 Optochin sensitivity test.
1. Bile solubility + -
2. Optochin sensitivity + -
3. Inulin fermentation + -
4. Quellung reaction + -
5. Mouse virulence + -
KEY FACTS
1 Streptococcus pneumoniae are Gram positive cocci arranged in pairs or as short chains and individual coccus is lanceolate
shaped.
2 Gram stain of the CSF from a case of meningitis shows both intracellular as well as extra cellular cocci.
3 Str. pneumoniae is bile solubility test positive.
4 Str. pneumoniae are sensitive to Optochin.
5 Str. pneumoniae is capable of fermenting the sugar, inulin whereas other alpha hemolytic streptococci do not ferment inulin.
Textbook of Practical Microbiology 171
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 94-96, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Jawetz, Melnick and Adelberg. Medical Microbiology. 23rd Edition. McGaw Hill. 2003.
4 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. 1996.
5 Murray PR, Rosenthal KS, Kobayashi GS and Pfaller ME. Medical Microbiology. 5th Edition. Mosby Inc. 2002.
172
LESSON
Identification of
b-haemolytic Streptococci
59
LEARNING OBJECTIVES streptococci cause neonatal infections such as septicemia and
meningitis. Group D streptococci may cause urinary tract
After completing this practical you will be able to: infections and wound infections.
All members of b-haemolytic streptococci are Gram positive TESTS FOR IDENTIFICATION OF
and fastidious. They are cocci arranged in chains. They appear STREPTOCOCCUS PYOGENES
as circular translucent pinpoint colonies on blood agar.
Streptococci, both aerobic and anaerobic are classified on the 1 Direct examination
basis of their haemolysis on blood agar. On blood agar three
types of haemolysis are observed. These are:
Gram stain
a) a hemolysis: It is an incomplete form of hemolysis where a Gram stain of smears from clinical specimens like throat swab is
green zone is produced around the colonies. carried out to demonstrate the presence of Gram positive cocci
b) b hemolysis: It is a complete form of hemolysis. This appears arranged in short chains (Fig. 59-1).
as a clear zone around the colonies. b-haemolytic streptococci
are frequently associated with pathogenicity, and 2 Culture
c) g haemolysis: It is described as absence of any hemolysis
around the colony. g-haemolytic streptococci are avirulent. The specimens are inoculated onto 5% sheep blood agar and
kept for aerobic incubation at 37°C for 24 hours in the presence
b-haemolytic streptococci produce a clear zone of of C02. Str.pyogenes produces pinpoint colonies which are
haemolysis on the blood agar surrounding the colonies, within surrounded by a large zone of b-hemolysis (Fig. 59-2).
which erythrocytes are completely lysed. The lysis is caused
by two haemolysins, streptolysin O and streptolysin S. 3 Bacitracin sensitivity test
b-haemolytic streptococci on basis of their carbohydrate C
antigen are classified in to 20 groups (from A to V, excepting I Bacitracin test is a frequently used test to identify and
and J). This classification is known as Lancefield classification. differentiate group A streptococci from non- Group A
Group A streptococci, also known as Streptococcus streptococci. Bacitracin disc when placed on the surface of
pyogenes is the important species causing most human blood agar plate streaked with test organism inhibits its growth
infections. S. pyogenes causes a wide variety of suppurative and forms a zone of inhibition.
infections of the respiratory tract, skin, genital and other deep The test organism is streaked on to the surface of the blood
infections. It also causes non-suppurative infections such as agar. Then a bacitracin having strength of 0.04 units is placed
rheumatic fever and acute glomerulonephritis. Group B on the inoculated plate. The plate is incubated at 37°C for
Textbook of Practical Microbiology 173
overnight. Positive test is indicated by a zone of inhibition when reacts with iron salts in the medium causes brown to
around the bacitracin disc. The negative test is indicated by no black discolouration of medium following incubation.
zone of inhibition around the bacitracin disc. Test strain is inoculated onto the surface of the bile aesculin
Most strains of S. pyogenes are bacitracin sensitive. agar and kept for aerobic incubation at 37°C for 24–48 hr. Positive
test is indicated by change of the brown colour of the medium
4 CAMP (Christie, Atkins and Munch-Peterson) in to black. Negative test is indicated by no brown or black
test discoloration of medium.
FIGURE 59-1 Streptococci in short chains, x 1000. FIGURE 59-3 CAMP Test.
VIVA
KEY FACTS
1 b-haemolytic streptococci produce a clear zone of haemolysis on the blood agar surrounding the colonies, within which
erythrocytes are completely lysed.
2 Bacitracin test is a frequently used test to identify and differentiate group A streptococci from non- Group A streptococci.
3 Blood agar plate for CAMP test should be incubated in the environment of 5–10% CO2.
4 CAMP test should be done with parallel positive and negative control strains.
5 Bile aesculin test is carried out to identify Group D streptococci, heat resistant test and salt tolerance test.
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 94-96, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Jawetz, Melnick and Adelberg. Medical Microbiology. 23rd Edition. McGaw Hill. 2003.
4 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. 1996.
5 Murray PR, Rosenthal KS, Kobayashi GS and Pfaller ME. Medical Microbiology. 5th Edition. Mosby Inc. 2002
Textbook of Practical Microbiology 175
LESSON
Identification of
60 Corynebacterium diphtheriae
KEY FACTS
1 C. diphtheriae causes natural infection only in man.
2 Toxin detection is important to prove the role of C. diphtheriae in causation of disease than just identification of
C. diphtheriae.
3 Throat swab should be processed immediately without delay. If any delay is anticipated Amie’s transport medium can be
used to transport the specimen.
4 Presumptive identification of C. diphtheriae is made based on the presence of metachromatic granules by Albert’s stain.
5 Loeffler’s serum slope and potassium tellurite agar are the two media frequently used for culture of C. diphtheriae.
6 C. diphtheriae ferments glucose, maltose and sucrose but do not ferment lactose, mannitol and trehalose.
7 C. diphtheriae do not hydrolyse urea and gelatin whereas non-pathogenic C. ulcerans hydrolyses urea.
VIVA
1 Name other staining methods used to demonstrate volutin granules.
Ans. Neisser’s and Ponder’s stains.
2 Why serum sugars are used for testing acid production by C. diphtheriae?
Ans. These organisms are fastidious. They cannot grow in ordinary sugar media.
3 Name other organisms, which have metachromatic granules.
Ans. Bordetella and Brucella.
Textbook of Practical Microbiology 177
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 94-96, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Jawetz, Melnick and Adelberg. Medical Microbiology. 23rd Edition. McGaw Hill. 2003.
4 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. 1996.
5 Murray PR, Rosenthal KS, Kobayashi GS and Pfaller ME. Medical Microbiology. 5th Edition. Mosby Inc. 2002
178
LESSON
Identification of
61 Lactose Fermenting
Enterobacteriaceae
SPECIMENS
Stool, urine, wound swab, sputum, etc.
In this chapter, following tests will be described which are
routinely used for identification of E.coli and Klebsiella spp.
+ + – –
FIGURE 61-2 Mc Conkey agar with lactose fermenting pink mucoid FIGURE 61-4 IMViC test for Escherichia coli.
colonies of Klebsiella pneumoniae.
– – + +
FIGURE 61-3 CLED Medium with lactose fermenting yellow FIGURE 61-5 IMViC test for Klebsiella pneumoniae.
colonies of Escherichia coli.
KEY FACTS
1 Enterobacteriaceae is the part of the normal intestinal flora of animals and humans.
2 Lactose fermenters such as Enterobacter, Klebsiella and Citrobacter and enterotoxigenic E. coli causes watery diarrhoea.
3 Antibiotic susceptibility testing should be done for the identified organism which will give the important information of the
susceptibility pattern of the organism.
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 94-96, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Jawetz, Melnick and Adelberg. Medical Microbiology. 23rd Edition. McGaw Hill. 2003.
4 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. 1996.
5 Murray PR, Rosenthal KS, Kobayashi GS and Pfaller ME. Medical Microbiology. 5th Edition. Mosby Inc. 2002
Textbook of Practical Microbiology 181
LESSON
Identification of
62 Vibrio cholerae
Gram stain
Gram negative curved or straight rods.
Biochemical tests
Catalase test: Positive. + + –
Oxidase test: Positive. FIGURE 62-1 Lysine and ornithine decarboxylase and arginine
dihydrolase tests for Vibrio cholerae.
Nitrate reduction test: Reduced to Nitrite.
Kligler’s iron agar medium – K/A.
Fermentation of sugars (glucose, sucrose and mannose
fermented: Acid only, lactose not fermented).
Indole test: Positive.
Citrate utilization test: Positive.
Urease test: Negative.
Lysine decarboxylation test: Positive.
Ornithine decarboxylation test: Positive.
Arginine dihydrolase test: Negative.
Confirmation of V. cholerae isolates is done by serotyping
with specific O antisera (slide agglutination test) using
O1 Ogawa and, O1 Inaba antisera. FIGURE 62-2 Polymyxin B sensitivity of Vibrio cholerae.
Table62-1 Differences between V. cholerae biotype classical and V. cholerae biotype El Tor
KEY FACTS
1 Subculture from the incubated alkaline peptone water to be done within 6 hours of incubation.
2 Looking for the presence of darting motility is not recommended for the diagnosis of cholera because this type of motility can
also be observed in other bacteria.
3 Biochemical tests like Voges Proskauer, polymyxin B sensitivity, sheep RBC hemolysis, chick cell agglutination are usually
carried out to differentiate between V. cholerae biotypes Classical and Eltor.
Textbook of Practical Microbiology 183
VIVA
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 94-96, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11th ed. (The CV Mosby Company, St. Louis) 2002.
3 Jawetz, Melnick and Adelberg. Medical Microbiology. 23rd Edition. McGaw Hill. 2003.
4 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. 1996.
5 Murray PR, Rosenthal KS, Kobayashi GS and Pfaller ME. Medical Microbiology. 5th Edition. Mosby Inc. 2002.
184
LESSON
Identification of
63 Pseudomonas aeruginosa
After completing this practical you will be able to Pus swab is inoculated on to blood agar, MacConkey agar and
1 Know the clinical importance of Pseudomonas aeruginosa. nutrient agar and the plates are incubated at 37°C overnight.
2 Perform the tests to identify P. aeruginosa. Moist hemolytic colonies are produced on blood agar (Fig. 63-1).
Greenish blue pigmented colonies are produced on nutrient
agar (Fig. 63-2). P. aeruginosa colonies produce earthy or grape
INTRODUCTION juice like odour.
Pus swab.
4 Biochemical tests
In this chapter the following tests will be described which are
employed routinely for identification of P. aeruginosa. They are non-fermenters. They break down glucose oxidatively
with production of acid only (Fig. 63-3).
1 Direct examination Antibiotic susceptibility testing is carried out for the identified
bacteria which provide the important information of the
susceptibility pattern of the organism (refer chapter 31).
Gram stain
Gram stain of the pus swab reveal thick Gram negative bacilli
measuring 1.5–3 µm by 0.5 µm in size.
Textbook of Practical Microbiology 185
– +
FIGURE 63-1 Non hemolytic Pseudomonas colonies on blood agar. FIGURE 63-3 OF test showing oxidative utilization of glucose.
KEY FACTS
1 P. aeruginosa are non-fermentative slender Gram negative bacilli.
2 They utilise carbohydrates oxidatively.
3 P. aeruginosa produces greenish blue pigmented colonies on nutrient agar.
4 P. aeruginosa is an oxidase positive bacteria.
VIVA
1 Name the selective media used for the isolation of P. aeruginosa.
Ans. Cetrimide agar.
2 Name different pigments produced by P. aeruginosa?
FURTHER READINGS
1 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 94-96, 1995.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Jawetz, Melnick and Adelberg. Medical Microbiology. 23rd Edition. McGaw Hill. 2003.
4 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. 1996.
5 Murray PR, Rosenthal KS, Kobayashi GS and Pfaller ME. Medical Microbiology. 5th Edition. Mosby Inc. 2002.
186
Textbook of Practical Microbiology 187
UNIT
IX
Parasitology
Introduction
Introduction
Parasitology traditionally includes the study of three major groups of animal parasites: parasitic protozoa, parasitic helminths
(worms), and those arthropods that directly cause disease or act as vectors of various pathogens. Infections of humans caused
by parasites number in the billions and range from relatively innocuous to fatal. The diseases caused by parasites constitute
major human health problems throughout the world.
The incidence of many parasitic diseases (e.g. schistosomiasis, malaria) have increased rather than decreased in recent years.
Other parasitic illnesses have increased in importance as a result of the AIDS epidemic (e.g., cryptosporidiosis, Pneumocystis
carinii pneumonia, and strongyloidiasis). The unicellular parasites (protozoa) and multicellular parasites (helminths, arthropods)
are antigenically and biochemically complex, as are their life histories and the pathogenesis of the diseases they cause. The basis
for effective treatment and cure of a patient is the rapid diagnosis of the disease and its causative agent, which is based on the
analysis of the clinical symptoms in combination with laboratory tests. As we are in the 21st century, clinicians are becoming
increasingly able to diagnose and treat diseases at the molecular level.
During past few years, there has been an increased awareness of the importance of trained and qualified personnel to perform
diagnostic procedures. It becomes even more important to provide well-written lab protocols and to standardize test methods for
consistency. Therapy based on patient history and symptoms is not generally recommended in cases of parasitic infections.
Thus understanding of characteristics of any parasitic infection and the use of appropriate diagnostic procedures accompanied
by a complete understanding of the limitations of each procedure become very important.
Textbook of Practical Microbiology 189
LESSON
Saline Wet Mount
64 of Stool
REQUIREMENT OBSERVATIONS
I Equipments 1 A 10–15 µm sized round refractile, colourless structure seen
Compound light microscope. with four nuclei, though not distinct.
190 Saline Wet Mount of Stool
2 A 15–30µm sized, round refractile, colourless structure seen 3 Cyst of Giardia intestinalis (Fig. 64-2).
with at least eight nuclei, though not distinct. 4 Hook worm egg (Fig. 64-3).
3 A 6–10µm sized oval shaped colourless structure seen with 5 Fertilized egg of Ascaris lumbricoides (Fig. 64-4).
distinct wall and faint axostyle. 6 Egg of Trichuris trichiura. (Fig. 64-5).
4 A 60µm × 40µm sized, oval shaped, non-bile stained,
colourless structure seen with transparent hyaline shell
membrane and segmented ovum with blastomeres. A clear
space is visible between the thin hyaline shell membrane
and blastomeres.
5 A 60–75µm × 40–50µm sized, round oval shaped, bile stained
structure seen with thick translucent shell with an
albuminous coat. It contains a very large conspicuous
unsegmented ovum with a clear space at each pole.
6 A 25µm × 50µm sized, bile coloured, and barrel shaped
structure seen with visible mucus plugs at each pole. It has a
double layered egg shell enclosing a visible unsegmented ovum.
FIGURE 64 -1 Cyst of Entamoeba histolytica/dispar, x 400. FIGURE 64 -4 Fertilized egg of Ascaris lumbricoides, x 100.
FIGURE 64 -2 Cyst of Giardia intestinalis, x 400. FIGURE 64 -5 Eggs of Trichuris trichiura, x 400.
Textbook of Practical Microbiology 191
KEY FACTS
1 A stool wet mount preparation should always first be screened under the low power objective (10x) and then under the high
power (40x) for identification.
2 A wet mount preparation should neither be too thick nor thin. The preparation should be such that, a printed letter should
be read through it. The wet mount preparation must not overflow.
3 Trophozoites and larvae are visualised best in the saline wet mount.
4 Bile staining property of the parasitic egg can be appreciated.
VIVA
FURTHER READINGS
1 Garcia LS. Diagnostic Medical Parasitology. ASM press, Washington D.C. 4th Edition. 2003.
2 Parija SC. Textbook of Medical Parasitology. All India Publishers and Distributors. 3rd Edition. 2006.
3 Parija SC. Stool Microscopy. BPKIHS, Dharan, Nepal, 1998.
192
LESSON
Iodine Wet Mount
65 of Stool
OBSERVATIONS
FIGURE 65- 4 Fertilized egg of Ascaris lumbricoides, x 400. FIGURE 65 –5 Eggs of Trichuris trichiura, x 400.
KEY FACTS
VIVA
Disadvantages
a Trophozoites are killed by iodine, hence cannot be demonstrated in iodine wet maint.
b The bile staining property of the helminthic eggs cannot be made out.
c Chromatoidal bars in protozoan cysts are not clearly demonstrable.
FURTHER READINGS
1 Garcia LS. Diagnostic Medical Parasitology. ASM press, Washington D.C. 4th Edition. 2003.
2 Parija SC. Textbook of Medical Parasitology. All India Publishers and Distributors. 3rd Edition. 2006.
3 Parija SC. Stool Microscopy. BPKIHS, Dharan, Nepal, 1998.
Textbook of Practical Microbiology 195
LESSON
LPCB Wet Mount
66 of Stool
OBSERVATION
FIGURE 66 - 1 Cyst of E. histolytica / dispar, x 400. FIGURE 66 - 4 Fertilzed egg of Ascaris lumbricoides, x 100.
Textbook of Practical Microbiology 197
KEY FACTS
1 LPCB wet mount of stool is always examined at least 30 minutes after preparation of the wet mount.
2 A wet mount preparation should neither be too thick nor thin.
3 LPCB kills the trophozoites of Entamoeba, Giardia and Trichomonas, hence can not be demonstrated by LPCB.
4 In LPCB preparation, both bile-stained and non bile-stained helminthic eggs are stained blue.
VIVA
FURTHER READINGS
1 Garcia LS. Diagnostic Medical Parasitology. ASM press, Washington D.C. 4th Edition. 2003.
2 Parija SC. Textbook of Medical Parasitology. All India Publishers and Distributors. 3rd Edition. 2006.
3 Parija SC. Stool Microscopy. BPKIHS, Dharan, Nepal, 1998.
198
LESSON
Acid-fast Staining
67 of Stool Smears
PROCEDURE
INTRODUCTION
1 Make a smear of stool on a clean glass slide.
Intestinal coccidian parasites, namely Cryptosporidium parvum, 2 Heat fix the smears by heating at 70°C for 10 minutes.
Cyclospora cayetanensis and Isospora belli cause infection of 3 Put the smears on a slide rack and flood the smear with
the gastrointestinal tract of humans. The infection causes carbol fuchsin.
diarrhoea, which may be serious in the immunocompromised 4 Heat the slides from below intermittently by Bunsen flame
patients. Oocysts, the diagnostic morphological form of the until the steam rises. Do not allow the stain to dry on the
parasite, are usually excreted in the human faeces. These oocysts slide, and if necessary add more carbol fuchsin to cover
are demonstrated by the modified acid fast staining technique the smear. The slide is allowed to stain for 9 minutes.
as pink-coloured acid fast structures against a blue background. 5 Wash the smears with tap or distilled water.
6 Cover the smear with 5% aqueous sulphuric acid, as
decolouriser, for 30 seconds.
PRINCIPLE 7 Wash the slides with water to remove all traces of acid.
8 Cover the smear with methylene blue, as counter stain, for
The principle of modified acid fast staining is based on the fact 1 minute.
that the oocyst of these coccidian parasites are acid fast and 9 Rinse the smears again under tap water and air dry it.
retain the basic dye (dilute carbol fuchsin)appearing pink. They 10 Observe the smear first under low power (10x) objective,
do not get decolourised with the acid alcohol and hence do not and then under oil immersion (100x) objective.
take up the counter stain methylene blue. The stool material in Note: The smear should be examined following a zig-zag pattern
the background gets decolourised easily and take up the for at least 10–30 minutes, before declaring the smear negatives.
counter stain appearing blue. Both hot and cool modified acid 11 Record the observations in the note book. Findings are
stains have been used with equal sensitivity. Acid fast parasites recorded, together with grading of the positive smear.
and parasitic components are listed in the box 67-1.
In this chapter hot modified acid fast technique will be described.
QUALITY CONTROL
OBSERVATIONS
FIGURE 67- 1 Oocyst of C. parvum, x 1000. FIGURE 67- 3 Oocyst of C. cayatenensis, x 1000.
KEY FACTS
1 Modified acid fast staining is a widely used method for the demonstration of the oocysts of intestinal coccidian parasites
in stool.
2 It is an example of permanent staining of stool.
3 Stool specimens should be handled carefully, as infection is caused by ingestion of oocysts from hands.
4 Oocysts of Cryptosporidium and Isospora are consistently acid fast in nature while Cyclospora is variably acid fast.
5 Cyclospora oocysts are twice the size of the Cryptosporidium oocysts.
6 Cryptosporidium oocysts are to be distinguished from yeast cells which are non acid fast and show budding and variable
sizes.
200 Acid-fast Staining of Stool Smears
VIVA
FURTHER READINGS
1 Garcia LS. Diagnostic Medical Parasitology. ASM press, Washington D.C. 4th Edition. 2003.
2 Parija SC. Textbook of Medical Parasitology. All India Publishers and Distributors. 3rd Edition. 2006.
3 Parija SC. Stool Microscopy. BPKIHS, Dharan, Nepal, 1998.
Textbook of Practical Microbiology 201
LESSON
Leishman’s Staining of
68 Peripheral Blood Smears
LEARNING OBJECTIVES Thin blood films are used primarily for identification of
species of malarial parasites. Thin blood films are of monolayer
After completing this practical you will be able to: thickness, allows better identification of species. The
disadvantage of thin blood smear is that as less amount of the
1 Perform Leishman’s staining of peripheral blood smear for blood is examined, the number of parasites per field too are
malaria parasites and microfilariae. much less than that of thick blood smear.
2 Detect and identify important parasites of blood such as The procedure followed for staining thick smears are
malarial parasites (various morphological forms) and essentially the same as for thin smears except that the initial
microfilaria of filarial worm. steps of fixation smears by methanol is not done. The absence
of fixation of the smears by methanol allows lysis of red blood
cells and dehaemoglobinisation by aqueous stain solution.
INTRODUCTION Thick blood smear is primarily used as a screening procedure.
It is a sensitive procedure for detection of parasites compared
Haemoparasites are found in the peripheral blood smears of to thin smear, as it allows examination of larger amount of blood.
many parasitic diseases. These include i). protozoal infections Disadvantage of thick blood smear is that species identification
such as malaria, babesiosis, Chagas’ disease, African of parasites cannot be made. Thick blood smears are several
trypanosomiasis and leishmaniasis; and ii) helminthic infections layers thick, allows only detection of parasites.
such as lymphatic filariasis, and loiasis. The parasites found in the peripheral blood smear are listed
Examination of permanent stained blood smears is essential in the box 68-1.
for detection and specific identification of blood parasites. Thick
and thin blood smears stained with Romanowsky’s stains are
the permanent staining methods widely used for demonstration REQUIREMENTS
of blood parasites. These blood smears, although simple and
easy to prepare require experienced eyes for the screening, I Equipments
detection and identification of these haemoparasites. Compound light microscope.
Microfilariae although can be identified in wet mount preparation
of fresh blood, on basis of their shape and motility, their correct II Reagents and lab wares
and accurate identification is possible only after permanent Bunsen flame and clean glass slides, Leishman’s stain, EDTA
staining of blood smear. For malarial parasites, blood films help anticoagulated blood, 70% ethyl alcohol, buffered distilled
quantitate parasitaemia, in addition to species identification. water and methyl alcohol.
Preparation of EDTA anticoagulated blood: It is prepared by
dissolving 5 grams of EDTA in 100 ml of distilled water. Then
PRINCIPLE 0.4 ml is aliquoted into tubes and the water is evaporated. Blood
can then be added to the anticoagulant or 20 mg of dry EDTA
As a rule thin blood smears are fixed by alcohol or other fixatives too can be added per tube. Then blood can be added to it.
before staining. Two types of stains are used. One type of stains Preparation of Leishman’s stain: Leishman’s stain is prepared
(e.g. Leishman’s, Wright’s, etc.) has both fixatives and staining by dissolving 0.15 gram of Leishman’s dry powder in 100 ml of
reagents. The other type of reagents (e.g. Giemsa, Field, JSB absolute methyl alcohol in a bottle. The bottle is shaken until
stains) has only staining reagents. Therefore, the thin films need the powder is dissolved and allowed to stand for 48 hours with
prior fixation before staining with any of these stains. frequent shaking in between.
202 Leishman’s staining of Peripheral Blood Smears
III Specimen b The thin film only must be fixed in absolute methanol before
Fresh blood obtained by finger prick (or) EDTA anticoagulated staining.
blood. c Both the thick and thin blood films should be stained
simultaneously.
PROCEDURE
Leishman’s staining
Preparation of thin blood smear
1 Flood the blood smear with 5–10 drops of the Leishman’s
1 Take an alcohol cleaned and grease free slide. stain and allow to stand for 2 minutes.
Note: If old slides are to be used, they should be first cleaned 2 After 2 minutes, dilute the stain by adding twice as many
with detergent and then with 70% ethyl alcohol. drops of buffered distilled water. Allow the solution to mix
2 Place a small drop of fresh blood on the clean microscopic well.
slide, about 1.5 cm from the end of the slide. 3 Allow the solution to stand for 15–20 minutes.
3 Touch the drop of blood with the edge of another slide and 4 Wash the slide with buffered distilled water, and rinse it dry.
allow the blood to spread along the edge. 5 Examine the stained slide under the oil immersion objective
4 Push the second slide or the spreader across at about a 30° (100 x) of the microscope.
angle, along the surface of the horizontal slide to the far end
forming a ‘tongue shaped’ thin film.
Note: The thin, feathered end should be at least 2 cm long, QUALITY CONTROL
and occupy the central area of the slide, with free margins on
both sides. A known positive control, Leishman stained thick and thin
5 Allow the thin blood film to dry, and then stain the smear blood films for malarial parasites and microfilaria, is compared
with Leishman’s stain. with the stained test blood films for similar morphology of the
7 First screen the film with the low-power objective of the parasites.
microscope for detection of microfilaria and examine at least
two hundred microscopic fields using 100 x objectives for
the presence of malarial parasites. OBSERVATIONS
Preparation of thick blood smear 1 The blood film shows the presence of RBC’s identified as
pale round cells with dense periphery and lighter core. Some
1 Place two or three small drops of fresh blood (without any of the normal sized RBC’s show blue ring-shaped parasite
anticoagulant) on a grease-free alcohol cleaned slide. cytoplasm surrounding a central vacuole with a red coloured
2 Spread and mix the drops of blood with the corner of another nucleus (1 chromatin dot) present at its centre. Often 2 or
slide, over an area of about 2 cm in diameter. The mixing is more ring forms of the parasite are found inside a single RBC.
continued for about 30 seconds to prevent formation of any
fibrin strands which may conceal the parasites in a stained a Neutrophils in the blood films are identified by their
smear. multilobed nucleus.
Note: If blood containing anticoagulant is used, 2 or 3 drops b Lymphocytes are identified as having big nucleus pushing
maybe spread over an area of about 1 cm in diameter. the cytoplasm to the periphery.
3 Allow the thick blood film to air-dry at room temperature in a c Monocytes have kidney shaped nucleus.
dust free area.
4 After drying, lyse the thick blood film with distilled water for 2 The blood film shows the presence of normal RBC’s,
dehaemoglobinisation. neutrophils, lymphocytes and monocytes. In addition, typical
5 After the step of dehaemoglobinisation, stain the film with crescent (banana) shaped structures are seen with dark blue
Leishman’s staining. cytoplasm, compact nucleus, pigment and central chromatin.
6 After staining, air-dry the film in a vertical position. These structures have rounded or pointed ends and are
7 Examine the stained smear under oil-immersion objective about one and half-time larger than the RBC.
(100 x) for detection of malarial parasites and microfilaria. 3 The blood film shows the presence of RBC’s, neutrophils,
lymphocytes and monocytes. In addition, some of the
Preparation of combined thick and thin films enlarged RBC’s show within it, large amoeboid coarse
haemazoin pigments occupying the entire RBC. 12 to 24 darkly
In field surveys, it may be helpful to prepare slides with both stained merozoites are found within the RBC’s.
the thick and thin film on the same slides. 4 The Leishman stained thick blood smear shows the presence
Note: With this type of preparation: of coiled structure with a sheath and the absence of nuclei in
a Sufficient time should be allowed for the thick portion of the tail end. In addition, RBC’s, neutrophils, lymphocytes
the smear to dry before staining. and monocytes are also seen.
Textbook of Practical Microbiology 203
FIGURE 68-1 The blood film shows the presence of ring forms of FIGURE 68-4 Leishman stained thick blood smear with microfilaria
Plasmodium falciparum, x1000. of Wuchereria bancrofti, x1000.
KEY FACTS
1 Always thin blood films should be fixed by alcohol or other fixatives before staining.
2 The stains used for staining blood films are of two types: i) One type of stain which has both fixatives and staining reagents
e.g., Wright’s, Leishman stains, and ii) Other type of stain which has only staining reagents e.g. Giemsa, Field’s, Jaswant
Singh Bhattacharjee stain. Hence with the use of these stains, the thin films should be fixed before staining.
3 Thick blood films should not be fixed because fixation with methanol prevents lysis of RBC’s and dehaemoglobinisation.
4 Thick blood films helps in detection of microfilaria and malarial parasites. It does not serve the purpose of species identification.
5 Thin blood films allows identification of malarial parasite to the species level.
204 Leishman’s staining of Peripheral Blood Smears
VIVA
Protozoal infections
Malaria.
Babesiosis.
Chagas disease.
African trypanosomiasis.
Leishmaniasis.
Helminthic infections
Lymphatic filariasis.
Loiasis.
FURTHER READINGS
1 Garcia LS. Diagnostic Medical Parasitology. ASM press, Washington D.C. 4th Edition. 2003.
2 Parija SC. Textbook of Medical Parasitology. All India Publishers and Distributors. 3rd Edition. 2006.
3 Parija SC. Stool Microscopy. BPKIHS, Dharan, Nepal, 1998.
Textbook of Practical Microbiology 205
LESSON
Concentration of
69 Stool for Parasites
11 Discard all the fluid into the discarding jar by one firm
PROCEDURE swing, leaving behind one or two drops of fluid with the
sediment.
Saturated salt solution flotation method 12 Mix the sediment with the fluid using an applicator stick.
Then with a Pasteur pipette, aspirate a little of the sediment
1 Take a walnut sized stool (approx. 1 gram) in a 30 ml glass fluid and examine by making the saline and iodine wet mount
vial. preparations.
2 Add a few ml of saturated sodium chloride solution to the 13 Examine the wet mount preparation first under low power
stool and mix it with a broomstick. objective (10 x) of the microscope for parasite eggs and cysts.
3 When the stool suspension is smooth, add a few more ml Then focus under the high power objective (40x) to study
of salt solution and mix. the morphology and identification of the egg and cysts.
Note: The steps 2 and 3 are repeated, until the container is
nearly full with continuous stirring.
4 Remove any coarse material found floating by a broomstick
5 Add more salt solution little by little using a Pasteur pipette, QUALITY CONTROL
until a convex meniscus is formed at the top of the container.
6 Then place a broad cover slip over the glass vial, so that it Wet mounts are prepared from a stool specimen known to be
is in contact with the top of the meniscus. positive for ova and cysts and compare with the wet mounts
7 Allow it to stand for 20 minutes to 30 minutes. obtained after the formol ether sedimentation technique for
8 Lift the cover slip from the meniscus with a steady but similar morphology of eggs and cysts.
rapid movement and place on a clean microscope slide.
9 Make a wet mount preparation with one or two drops
adherent to the cover slip.
10 Examine the wet mount preparation first under low power OBSERVATIONS
objective (10x) of the microscope for parasite eggs. Then
focus under the high power objective (40x) to study the 1 A 10-15µm sized spherical structure seen possessing 1-4
morphology and identification of the egg. nuclei.
2 A 15-30µm sized spherical structure seen possessing 1-8 nuclei.
Formalin –ether sedimentation method 3 A 6-10µm size oval ellipsoidal shaped structure seen with 4
nuclei remains of axoneme and flagella.
1 Take a half teaspoon of stool in a 15ml centrifuge tube
4 A 60µm × 40µm size oval shaped, non bile stained structure
containing 10 ml of 10% formalin, and allow it to stand for
seen with a thin transparent hyaline shell membrane
30 minutes.
enclosing 7-8 blastomeres, and clear space between the shell
2 Filter the faecal suspension through two layers of gauze in
membrane and blastomeres.
a funnel into a 15 ml centrifuge tube. Add saline to the tube
5 A 60-75µm × 40-50µm size rounded, bile stained structure
to bring the fluid level within several millimeters of the rim
seen with a thick translucent shell with an albuminous coat
of the tube.
containing a large conspicuous unsegmented ovum with a
3 Centrifuge the tube at 500 g for 10 min.
clear space at each pole.
4 Discard the supernatant. Suspend the sediment in saline,
nearly filling up to the brim of the tube.
5 Centrifuge the tube again for 10 min at 500 g.
6 Resuspend the sediment in 7 ml of 10% formalin, and 3 ml RESULTS AND INTERPRETATION
of ether.
7 Close the tube with a stopper and shake it well for 30
seconds. 1. Cyst of Entamoeba histolytica /dispar.
Note: Hold the tube in such a way that the stopper is held 2. Cyst of Entamoeba coli.
away from the face. 3. Cyst of Giardia intestinalis.
8 Remove the stopper carefully. 4. Egg of hook worm.
9 Centrifuge the tube at 500 g for 10 minutes. Allow the tube
to stand 5 min. 5. Fertilized egg of Ascaris lumbricoides.
Note: After centrifugation, four layers are formed namely,
i) first sediment layer at the bottom of the tube containing Table 69-1 Other floatation methods for concentration of
parasitic cysts or eggs, ii) second layer of formol saline, iii) stool
third layer of faecal debris on the top of the formol saline
layer and iv) lastly a top layer of ether. Zinc sulphate floatation method.
10 Remove the plug of faecal debris by piercing all around Magnesium sulphate floatation method.
with an applicator stick, taking care not to disrupt the debris. Sheather’s sucrose floatation method.
Textbook of Practical Microbiology 207
Disadvantages
Not suitable for demonstration of unfertilized eggs of Ascaris, operculated eggs of trematodes, larvae of Strongyloides and
eggs of Taenia species.
High specific gravity of the fluid may cause distortion of the morphology of the parasitic eggs and cysts.
Formalin-ether sedimentation method
Advantages
More sensitive method.
Good method for both cysts and ova.
Morphology of eggs and cysts preserved.
Disadvantages
Technically cumbersome.
Ether is highly inflammable and not easily available.
KEY FACTS
1 Concentration of stool is the method of choice, when the number of parasites in the stool specimens are less and cannot be
demonstrated in stool wet mount preparation.
2 Trophozoites of protozoa cannot be concentrated by any of the concentration methods.
3 Stool concentration by salt floatation and formol ether sedimentation are the two reliable methods of concentration of
parasites in stool. It increases sensitivity of stool microscopy.
4 Salt floatation is a better method for concentration of helminthic eggs.
5 In formol ether procedure, formol saline is used to fix the cysts of protozoa and eggs, thus unaltering morphology of these
structures. These also kill the parasitic eggs and cysts. Ether is used to dissolve and extract the fat and other debris in the stool.
VIVA
FURTHER READINGS
1 Garcia LS. Diagnostic Medical Parasitology. ASM press, Washington D.C. 4th Edition. 2003.
2 Parija SC. Textbook of Medical Parasitology. All India Publishers and Distributors. 3rd Edition. 2006.
3 Parija SC. Stool Microscopy. BPKIHS, Dharan, Nepal, 1998.
208
LESSON
Culture of Stool for
70 Entamoeba histolytica
1 Inoculate a loopful of the stool sample or liver pus on to the A known positive culture of E. histolytica already maintained
slant of the two medium tubes. by serial sub cultivation in the culture medium.
2 Incubate the tubes in an incubator at 37°C for 24 hours to 48
hours. OBSERVATIONS
3 Observe the cultures regularly at 2, 3 and 4 days by examining
Presence of 8–30 µm structure, actively motile with hyaline,
0.1 ml of sediment under light microscope for characteristic
finger-shaped pseudopodia, clearly differentiated cytoplasm
motility of the trophozoites.
into ectoplasm and endoplasm, and cytoplasmic inclusions
Note: Although the initial culture may appear negative, such as red blood cells, leucocytes and tissue debris.
subcultures may reveal amoebae.
4 Examine the growth of amoebae by a wet mount preparation
of culture fluid. RESULTS AND INTERPRETATION
Trophozoites of E. histolytica is grown in the culture.
KEY FACTS
1 Cultivation of E. histolytica can be done using both polyxenic and axenic culture medium.
2 In polyxenic culture medium (polybacterial culture) bacterial flora serve as rich source of nutrients for the feeding amoebae.
3 In axenic culture medium (bacteria-free culture), addition of special vitamin mix provides a rich source of nutrition.
4 Axenic culture medium is not used routinely for culture and isolation of the amoebae from stools specimens.
5 Axenic culture medium is employed to study pathogenicity of amoeba, in vitro testing of efficacy of anti-amoebic drugs and
preparation of axenic amoebic antigen for immunodiagnosis of amoebiasis.
VIVA
FURTHER READINGS
1 Garcia LS. Diagnostic Medical Parasitology. ASM press, Washington D.C. 4th Edition. 2003.
2 Parija SC. Textbook of Medical Parasitology. All India Publishers and Distributors. 3rd Edition. 2006.
3 Parija SC. Stool Microscopy. BPKIHS, Dharan, Nepal, 1998.
210
Textbook of Practical Microbiology 211
UNIT
X
Mycology
Introduction
Lesson 71 Cultivation of Fungi
Lesson 72 Gram’s Staining for Fungi
Lesson 73 Lactophenol Cotton Blue (LPCB) Wet Mount
Lesson 74 Potassium Hydroxide Wet Mount
Lesson 75 India Ink Preparation
Lesson 76 Slide Culture
Lesson 77 Germ Tube Test
Lesson 78 Urease Test
Lesson 79 Carbohydrate Assimilation Test
Lesson 80 Carbohydrate Fermentation Test
Lesson 81 Identification of Common Fungi
212
Introduction
The fungi are now recognized as significant causes of morbidity and mortality. They have emerged as important etiological
agents of opportunistic infections and full-fledged diseases. Today, the incidence of the fungal infections has increased
enormously, due to underlying predisposing factors such as immunocompromised situations. The prognosis among these
patients is very poor and therefore an early diagnosis and treatment is essential. The symbiotic relation of the fungi, like other
organisms, can be divided into three modes of their existence namely mutualism, commensalisms and parasitism.
The aged patients, whose life span has been extended by treatment of cancer or other debilitating diseases, are more susceptible
to secondary fungal infections than the young individuals. In addition many factors directly or indirectly decrease the accuracy
of data on fatal mycoses cases. The fungal infections are not usually transmitted sexually like those of the viral, bacterial or
parasitic diseases. Till date there is no ideal vaccine available to control any fungal infections. Simultaneously, the reported
deaths from the fungal infections have maintained an approximately constant numerical ratio to the general increase in the
population.
The nosocomial fungal infections have been recognized as a significant ground for adverse patient outcome and a major
public health problem. Nosocomial blood stream infections are particularly serious and there is evidence to suggest that these
infections are becoming more common. Nosocomial mycoses have developed especially in association with or as a consequence
of the extraordinary progress in the management of seriously ill patients. However, despite this increase, there has been
comparatively little progress in understanding the pathogenesis of nosocomial fungal infections or in their prevention, diagnosis
and treatment.
The fungal diseases can be classified according to the primary site of infection as follows:
1 Superficial mycoses: The infection is limited to the outer most layers of the skin and its appendages. The immune response is
rarely induced.
2 Cutaneous mycoses: The infection extends deeper into the epidermis and it also invades hair and nails. It evokes a high
inflammatory response in the host.
3 Subcutaneous mycoses: The infection is due to the pathogenic organism of low virulence and usually following traumatic
injury. It involves the dermis, subcutaneous tissues, muscles and fasciae.
4 Systemic mycoses: The infection originates primarily at one site and disseminate systemically to other body sites.
5 Besides these a fifth group opportunistic has come into focus because of increasing use of immunosuppressive therapy or
AIDS epidemic. These infectious agents are of low pathogenic potential and produce disease only under unusual circumstances,
mostly involving host debilitation.
Fungal infections can be diagnosed by their demonstration, isolation and final identification from clinical specimens. Diagnosis
of fungal infections is made by direct and indirect methods. Direct methods include the demonstration of fungi or their components
in body tissues or fluids. Wet mount of clinical specimens is a very useful method for demonstration of fungi in clinical
specimens. Various wet mount preparation used in a mycology laboratory include lactophenol cotton blue (LPCB) wet mount,
potassium hydroxide (KOH) wet mount and India ink preparation.
Textbook of Practical Microbiology 213
LESSON
Cultivation of Fungi
71
LEARNING OBJECTIVES III Specimen
Skin and nail scrapings, hair, and exudate from lesions, urine,
After completing this practical you will be able to: sputum, bronchial washings, biopsied materials and CSF.
Observe both the test tubes and control tubes everyday for
REQUIREMENTS upto 30 days.
Table 71-1 List of media used for fungal culture BOX 71-1 SABOURAUD’S DEXTROSE
AGAR
Sabouraud’s dextrose agar (SDA)
Sabouraud’s dextrose agar with antibiotics Sabouraud’s dextrose agar
Ascospore medium It consists of 40 gm of glucose / dextrose, 10 gm of
Bennett’s agar for Nocardia neopeptone and 35 gm of agar in 1000 ml of distilled water.
Blood agar base pH is 5.5 – 6.0.
Casein agar base for identification of Trichophyton species
Sabouraud’s dextrose agar with antibiotics
Casein agar for Nocardia It consists of 20 gm of glucose/dextrose, 10 gm of neopeptone
Casitone medium of Howell and Pine for Actinomyces and 20 gm of agar in 1000 ml of distilled water. pH is 7.0.
Carbohydrate broth for Candida Sterilised by autoclaving. It contains 40 mg of
Czapek – Dox medium chloramphenicol and 40 mg of cycloheximide (actidione).
Gelatin medium for Nocardia
Malt – Yeast extract agar (Wicker ham)
Medium 199 for Nocardia munutissima
Mohapatra and Pine medium for Nocardia
Potato glucose agar
Rice agar with Tween 80
Rice medium for Microsporum species
Salvin’s YP medium for yeast phase of Histoplasma
capsulatum
Tarshis’ penicillin blood Agar
Trypticase soy agar
Tyrosine agar
Xanthine Agar
FIGURE 71-1 SDA with Candida albicans colonies.
KEY FACTS
1 Choose appropriate medium based upon clinical history of the patient.
2 Always inoculate in duplicate tubes and incubate one at room temperature and another at 37°C.
3 Media should be sterile.
4 pH should be appropriate.
VIVA
FURTHER READINGS
1 Collier L, Balows A, Sussman M. Topley and Wilson’s Microbiology and Microbial Infections. 9th Edition. Mycology. Volume 4. pp. 711.
Arnold publishers.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Jagadish Chander. A Text Book of Medical Mycology. Interprint. 2000.
Textbook of Practical Microbiology 215
LESSON
Gram’s Staining for Fungi
72
LEARNING OBJECTIVES Sabouraud’s dextrose agar and place on the clean slide with
a bacteriological loop.
After completing this practical you will be able to: 3 Then with circular movement of the loop, spread the cell
suspension into a thin area.
1 Stain fungal smears by Gram’s staining method. 4 Allow the smear to air dry.
5 Heat fix the smear while holding the slide at one end, and
quickly passing the smear over the flame of Bunsen burner
INTRODUCTION two to three times.
Gram stain was devised by Christian Gram in 1884 as a method of II Staining Procedure
staining bacteria in tissues. Fungal material appears Gram positive.
1 Heat fixes the smear by passing the slide 2–3 times gently
over the flame with the smear side up.
PRINCIPLE 2 Cover the smear with the methyl violet. Allow it to stand
for one minute.
The Gram reaction is dependent on the permeability of the 3 Rinse the smear gently under tap water.
fungal cell wall, to the dye-iodine complex like those of bacteria 4 Cover the smear with Gram’s iodine and allow it to stand
(refer chapter 7). for one minute.
5 Rinse the smear again gently under tap water.
6 Decolourise the smear with 95% alcohol for 15 to 20 seconds.
REQUIREMENTS 7 Rinse the smear again gently under tap water.
8 Cover the smear with dilute carbol fuchsin for 30 seconds
I Equipments to 1 minute.
Compound light microscope. 9 Rinse the smear again gently under tap water and air dry it.
10 Observe the smear first under low power (10x) objective,
II Reagents and lab wares and then under oil immersion (100x) objective.
Bunsen flame, loop wire, methyl violet (basic dye), Gram’s iodine 11 Record the observations in the note book.
(mordant), 95% ethanol (decolourising agent), and 1% safranine
or dilute carbol fuchsin (counter stain).
QUALITY CONTROL
III Specimen
Candida albicans culture on Sabouraud’s dextrose agar. On the same slide, at one end a thin control smear of mixture of
Staphylococcus aureus (Gram positive bacteria) and
Escherichia coli (Gram negative bacteria) is made and at other
PROCEDURE end of the slide test smear is made. The slide with control and
test smears is stained by Gram’s staining. The appearance of
I Preparation of fungal smear: purple coloured Gram positive bacteria and pink coloured Gram
negative bacteria in the control smear indicates proper staining
1 Take clean, and grease free glass slides for making the smears. technique and stained test smear is compared with it.
2 Take one or two loopful of C. albicans culture on
216 Gram’s Staining for Fungi
OBSERVATION
KEY FACTS
1 Gram staining is a differential stain, which can also be used for detection of fungi in clinical specimens.
2 Gram staining gives preliminary indication of infection.
3 Tissue cells, leucocytes and the debris of inflammatory exudates all stain pink in Gram’s stained smears.
FURTHER READINGS
1 Collier L, Balows A, Sussman M. Topley and Wilson’s Microbiology and Microbial Infections. 9th Edition. Mycology. Volume 4. pp. 711.
Arnold publishers.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Jagadish Chander. A Text Book of Medical Mycology. Interprint. 2000.
Textbook of Practical Microbiology 217
LESSON
Lactophenol Cotton
73 Blue (LPCB)
Wet Mount
Identification of filamentous fungi is made by their characteristic 1 On a clean glass slide, place one drop of LPCB.
microscopic morphology such as shape, size, arrangement of 2 Touch the adhesive side of the tape of transparent scotch
spores and hyphae. There are three different preparations of tapes on the surface of the colony at a point intermediate
LPCB mounts as mentioned below: between its centre and periphery.
1 Scotch tape preparation. 3 Fix the adhesive side of the tape over an area on the glass
2. Tease mount preparation, and slide containing the LPCB.
3 Slide culture preparation. 4 Examine the preparation under 10x and 40x of a light microscope.
OBSERVATIONS
KEY FACTS
1 The fungal culture should be first is teased well and then spread in LPCB for better results.
VIVA
FURTHER READINGS
1 Collier L, Balows A, Sussman M. Topley and Wilson’s Microbiology and Microbial Infections. 9th Edition. Mycology. Volume 4. pp. 711.
Arnold publishers.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Jagadish Chander. A Text Book of Medical Mycology. Interprint. 2000.
Textbook of Practical Microbiology 219
LESSON
Potassium Hydroxide
74 Wet Mount
After completing this practical you will be able to: 1 Emulsify the specimen in a drop of 10% KOH on a
microscopic slide with the help of a loop.
1 Prepare potassium hydroxide (KOH) wet mount of clinical 2 Apply gentle heat by passing the slide over a Bunsen flame
specimens. for 3–4 times.
2 Demonstrate the presence of fungal elements in the given 3 Cover the smear with the cover slip.
clinical specimen by KOH wet mount preparation. 4 Leave it for 5–10 min.
5 Examine the slide under low (10x) and high power (40x)
magnifications
INTRODUCTION 6 Examine the slide for 15–20 min. for demonstration of shining
fungal elements.
The potassium hydroxide (KOH) wet mount preparation is very
useful for the presumptive diagnosis of the type of fungal
infection. The procedure also helps in the selection of appropriate QUALITY CONTROL
culture media for the isolation of etiological fungal agent.
1 10% KOH should be prepared at the right concentration.
2 Emulsification of specimen should be homogenous in KOH
PRINCIPLE solution.
REQUIREMENTS
RESULTS AND INTERPRETATIONS
I Equipments
Microscope. Different fungi will have different morphological forms (yeasts,
cells with pseudo hyphae, budding, septate, and aseptate
II Reagents and lab wares hyphae, granules, etc.) which can be clearly seen in a KOH wet
Glass Petri dishes, slide, cover slip, straight/bent wire, needle, mount.
Bunsen flame and 10% KOH. Interpretation of results should be done by critical analysis
of the type, size and color of fungal elements which will be
III Specimen different for different fungi.
Pus from draining sinuses, aspirate from nasal sinuses,
respiratory specimen, skin scrapings, nail scrapings, hair,
corneal scrapings, material from external ear, etc.
220 Potassium Hydroxide Wet Mount
KEY FACTS
1 The KOH clears out the background scales or cell membranes that may be confused with fungal hyphal elements in
microscopy of clinical specimens.
2 The KOH should be prepared at the right concentration (10%).
3 Emulsification of specimen should be homogenous.
VIVA
FURTHER READINGS
1 Collier L, Balows A, Sussman M. Topley and Wilson’s Microbiology and Microbial Infections. 9th Edition. Mycology. Volume 4. pp. 711.
Arnold publishers.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Jagadish Chander. A Text Book of Medical Mycology. Interprint. 2000. pp. 232.
Textbook of Practical Microbiology 221
LESSON
India Ink Preparation
75
LEARNING OBJECTIVES PROCEDURE
After completing this practical you will be able to: 1 Put a drop of CSF on the microscopic slide.
2 Put a drop of India ink to the CSF on the microscopic slide.
1 Prepare India ink wet mount of cerebrospinal fluid (CSF). 3 Emulsify the specimen with India ink on the slide.
2 Demonstrate the presence of capsulated yeast, Cryptococcus 4 Place a cover slip over the preparation, taking care not to
neoformans in the CSF. trap air bubbles in the preparation.
5 Blot dry the excess fluid.
6 Examine the slide under low (10x) and high power (40x)
magnifications
INTRODUCTION
Capsule is a protective layer found around some bacteria and QUALITY CONTROL
some fungi like C. neoformans. Hence demonstration of capsule
by India ink preparation, especially in an emergency conditions, 1 India ink preparation in distilled water should be made exactly
in cerebrospinal fluid (CSF) is a very useful procedure for to 0.5%.
diagnosis of meningitis caused by C. neoformans. An early 2 Care should be taken not to trap air bubbles which will mimic
diagnosis will help for prompt treatment of the condition. capsules of yeast cells.
OBSERVATIONS
PRINCIPLE
The India ink preparation is observed under microscope and
India ink is used as a negative stain preparation. When used in noted for presence of clear halo around yeast cells (Fig. 75-1).
wet mount preparation of the CSF, the background appear black,
and the unstained capsule of C. neoformans appears as a white
halo around the yeast cells in microscopy. RESULTS AND INTERPRETATION
Since India ink stains the background and leaves a clear halo
around the cells, preparations with such appearance can be
REQUIREMENTS confirmed to have yeast cells with capsules and the organism
may be identified as C. neoformans.
I Equipments
Microscope.
III Specimen
Cerebrospinal fluid (CSF).
FIGURE 75-1 India ink preparation showing Cryptococcus
neoformans with capsule, x 400.
222 Indian Ink Wet Mount Preparation
KEY FACTS
A clear distinction should be made between capsules and air bubbles, trapped between the cover slip and slide.
VIVA
FURTHER READINGS
1 Collier L, Balows A, Sussman M. Topley and Wilson’s Microbiology and Microbial Infections. 9th Edition. Mycology. Volume 4. pp. 711.
Arnold publishers.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Jagadish Chander. A Text Book of Medical Mycology. Interprint. 2000.
Textbook of Practical Microbiology 223
LESSON
Slide Culture
76
LEARNING OBJECTIVES (d) Wrap the preparation in Kraft 20 paper for sterilizing in
hot air oven. Several of these setups should be kept ready
After completing this practical you will be able to: on hand.
1 Perform slide culture of the fungal preparation. 2 Sterile test tube with rimless mouth and an inside diameter of
2 Mount the cover slip after the growth of fungus. approximately 15 mm.
3 Demonstrate fungal morphology without disturbing the aerial 3 Standard laboratory glassware.
hyphae and conidiophores, if present.
II Reagents
Sterile distilled water and Petri dish containing Sabouraud’s
INTRODUCTION agar (or other medium of choice) to a depth of 2 mm.
1 From the Petri dish containing Sabouraud’s agar cut out one
PRINCIPLE square cm block of agar for each slide culture to be
inoculated.
Slide culture is a very useful technique in identification of the 2 With the flat side of a sterile bacteriological loop, or with a
type of fungi. The fungal element that is to be identified will spatula, place an agar block in the centre of the slide in the
produce characteristic hyphae and spores, when incubated on slide culture set up.
a suitable growth medium. This can be visualized undisturbed 3 With a probe, inoculate around the periphery of the agar
using this technique. The mould that is to be cultured is block, three to four fragments of the mold to be cultured.
inoculated onto a small piece of an agar below a cover slip. The 4 With forceps, the tips of which have been flamed, place the
whole setup is kept in a Petri dish with moisture. The cover slip cover slip on the agar block.
after incubation is lifted, stained and observed under a 5 With a pipette, thoroughly moisten, but not to excess, the
microscope for identification of the fungi. filter paper with sterile distilled water.
6 Incubate the slide culture at room temperature.
7 Remove the slide culture from the Petri dish and dry the
REQUIREMENTS bottom of the slide with a tissue.
8 When growth appears peneath the cover slip. Take a slide
I Equipments place a drop of LPCB, place the cover slip removed from the
1 Slide culture set (Fig. 76-1). block on the LPCB.
(a) Into a Petri dish, place one piece of filter paper slightly 9 Place the slide on the microscope stage and examine. The
smaller in diameter than the Petri dish. aerial hyphae including the conidiophores will be seen to
(b) Place a V-shaped glass rod on the filter paper. grow along the undersurface of the cover slip
(c) Place a 1 by 3 inch glass slide and a 22 mm square, No. 1
glass cover slip on top of the filter paper.
224 Slide Culture
1 All the materials should be sterilized and checked for sterility Small spore bearing fungi make beautiful permanent mounts.
before use. Some large spore bearing organisms like Microsporum gypsum
2 Distilled water to be used should be checked for sterility . do not stain as well. With the type of hyphae, arrangement of
3 Components of the medium used should be adjusted conidiophores, staining characters etc., the final interpretation
according to standard procedure. of the fungal type can be made.
OBSERVATIONS
KEY FACTS
VIVA
FURTHER READINGS
1 Collier L, Balows A, Sussman M. Topley and Wilson’s Microbiology and Microbial Infections. 9th Edition. Mycology. Volume 4. pp. 711.
Arnold publishers.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Jagadish Chander. A Text Book of Medical Mycology. Interprint. 2000. pp. 232.
Textbook of Practical Microbiology 225
LESSON
Germ Tube Test
77
LEARNING OBJECTIVES II Reagents and glass wares
Standard laboratory glassware, and test tubes 12×75 mm,
After completing this practical you will be able to: human, foetal calf or rabbit serum.
1 Demonstrate the production of germ tube by Candida species.
III Specimens
24 hour culture of suspected fungal colony on Sabouraud’s
dextrose agar to be tested.
INTRODUCTION 24 hour culture of known strains of C. albicans and
C. parapsilosis colony on Sabouraud’s dextrose agar.
Candida species are usually found as normal flora of the oral
cavity and gastrointestinal tract of man. These may be isolated
from respiratory secretions, gastric washings, stool, vaginal PROCEDURE
secretions, urine, skin, nail, etc. Under normal conditions
Candida species are not pathogenic. However, in case of 1 Take three test tubes and label as 1, 2 and 3.
immunocompromised individuals, and certain other situations 2 Add 0.5ml. of serum to each of the test tube.
(Box 77-1), Candida species can cause a wide variety of 3 Take a half of a single colony to be tested by using a sterile
opportunistic infections. loop, and mix with serum in the test tube 1.
Germ tube test is a very simple and efficient test to distinguish 4 Similarly, take a half of C. albicans single colony by using a
pathogenic Candida from non-pathogenic ones. It is very widely sterile loop, and mix it with serum in the test tube 2.
used in diagnostic laboratories because of its reproducibility. 5 Similarly, take a half of C. parapsilosis single colony by using
a sterile loop, and mix it with serum in the test tube 3.
6 Incubate all the tubes at 37°C for a maximum of 1½ hrs.
PRINCIPLE 7 Place one drop of suspension from tube 1, 2, and 3 onto 3
different slides and place cover slips over the drops.
Germ tube is an initial hypha from a sprouting conidia, spore or 8 Examine the slide under low (10x) and high power (40x)
yeast. Formation of germ tube can be demonstrated by magnifications.
inoculating rabbit, fetal calf or human serum with a small quantity
of growth of Candida species. The suspension is then
incubated at 37°C for a minimum of 1½–2 hours, after which a
QUALITY CONTROL
drop is examined under the microscope for the germ tube. 1 Positive control for germ tubes: C. albicans.
Germ tubes are produced by C. albicans, C.stellatoidea 2 Negative control for germ tubes: C. parapsilosis.
and rarely C. tropicalis. At times, some strains of C. albicans 3 Human or rabbit serum should be checked for contamination
isolated from the patients with antifungal drugs or patients prior to use.
with cancer do not produce germ tubes. 4 Control organisms should be tested individually for
production of germ tubes.
REQUIREMENTS
OBSERVATIONS
I Equipments
Under the microscope, the whole field under the cover slip is
Microscope. examined for any cell showing production of germ tube (Fig. 77-1).
226 Germ Tube Test
Tube 2 will show production of germ tube and Tube 3 will not.
The drop from tube 1 should be read and compared with these
controls.
Tube 2 contains C. albicans and hence shows germ tube
production while tube 3 does not show production of germ
tube since it contains C. parapsilosis. Tube 1 should be
interpreted with care by observing for the presence or absence
of germ tube and should be compared with tube 2 and tube 3. FIGURE 77-1 Germ tube test, x 400.
BOX 77-1 PREDISPOSING FACTORS Table 77-1 Differences between germ tubes and
FOR CANDIDIASIS pseudohyphae
KEY FACTS
1 Germ tube is a useful test to identify C. albicans and few other species which are pathogenic.
2 Rabbit, foetal calf or human serum can be used for demonstrating germ tube formation.
3 Germ tubes are produced by C. albicans, C.stellatoidea and rarely C. tropicalis.
4 At times, some strains of C. albicans isolated from the patients with antifungal drugs or patients with cancer do not produce
germ tubes.
5 C. tropicalis may show germ tube formation after 3 hours with a constriction at the base of the germ tube.
VIVA
FURTHER READINGS
1 Collier L, Balows A, Sussman M. Topley and Wilson’s Microbiology and Microbial Infections. 9th Edition. Mycology. Volume 4. pp. 711.
Arnold publishers.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Jagadish Chander. A Text Book of Medical Mycology. Interprint. 2000.
Textbook of Practical Microbiology 227
LESSON
Urease Test
78
LEARNING OBJECTIVES III Specimen
C. neoformans culture.
After completing this practical you will be able to:
KEY FACTS
1 Certain fungi possess the enzyme urease that hydrolyzes urea releasing ammonia into the medium.
2 Phenol red that is incorporated in the medium changes its color from yellow to red in alkaline pH, thus indicating the
presence of urease activity.
3 Always check the sterility of the slants before inoculation.
4 An un inoculated medium must be incubated along with the test.
5 Observe the growth of inoculum irrespective of change in colour.
VIVA
FURTHER READINGS
1 Collier L, Balows A, Sussman M. Topley and Wilson’s Microbiology and Microbial Infections. 9th Edition. Mycology. Volume 4. pp. 711.
Arnold publishers.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Jagadish Chander. A Text Book of Medical Mycology. Interprint. 2000.
Textbook of Practical Microbiology 229
LESSON
Carbohydrate
79 Assimilation Test
After completing this practical you will be able to: 1 Pipette 2.0 ml. of sterile saline into a test tube.
2 With a sterile loop pick up few isolated colonies of the
1 Find out the pattern of assimilation of carbohydrates by organism from the SDA plate and emulsify to a turbidity
yeast and yeast-like fungi. equal to McFarland 4 units.
3 Cover the surface of yeast nitrogen base agar with the
suspension of the yeast cells.
4 Remove the excess fluid and allow the surface of the agar to dry.
INTRODUCTION
5 With sterile forceps place the carbohydrate disc onto the
surface of agar in such a way that at least 30 mm space is
Yeast and yeast-like fungi use carbohydrates as sources of present between each disc.
energy. Hence in this test, utilization of carbohydrate is used 6 Incubate the plate at 30°C or 37°C for 24–48 hours.
as a definitive diagnosis for yeast or yeast-like fungi. 7 At the end of the incubation period observe the plate for
growth around the disc.
PRINCIPLE
QUALITY CONTROL
Yeast and yeast-like fungi are identified by the pattern of
carbohydrate assimilation. They are inoculated on the 1 Carbohydrate discs and media should be checked using
carbohydrate-free yeast nitrogen base agar on which different standard control strains as follows:
filter paper discs containing various carbohydrates are placed. Candida albicans ATCC 14053
After incubation for appropriate time, growth around the discs C. guilliermondii ATCC 6260
is observed and the carbohydrate utilization pattern is assessed. C. pseudotropicalis ATCC 4135
2 Sterility of glassware and media should be ensured.
REQUIREMENTS OBSERVATION
I Equipments After incubation, growth of the fungi around the discs is
Incubator. observed and the carbohydrate utilization pattern is assessed.
Composition Preparation
Boric acid – 500 µgm All the above ingradients are to be sterilized by filtration and dispersed
Copper sulphate – 40 µgm aseptically.
Potassium iodide – 100 µgm Add. 10 ml. of the above solution to 90ml of 2% molten agar and pour into
Ferric chloride – 200 µgm the plates.
Manganese sulphate – 400 µgm
Sodium molybdate – 200 µgm
Zinc sulphate – 400 µgm
Biotin – 2 µg
Calcium pantothenate – 400 µgm
Folic acid – 2 µg
Inositol – 2000 µgm
Niacin – 400 µgm
p-aminobenzoic acid – 200 µgm
Pyridoxine hydrochloride – 400 µgm
Riboflavin – 200 µg
Thiamine hydrochloride – 400 µgm
L-Histidine monohydrochloride – 10.0 milligram
DL-Methionine – 20 mg
DL-Tryptophan – 20 mg
Magnesium sulphate – 500 mg
Sodium chloride – 100 mg
Ammonium chloride – 5 g
Monopotassium phosphate – 1 g
Distilled water – 1000 ml
KEY FACTS
1 Turbidity using the organism should be done in such a way that it is equal to McFarland Standard 4 units.
2 Sterility should be maintained for medium and carbohydrate discs.
3 Growth of fungi near carbohydrate discs should be confirmed after thorough examination.
VIVA
FURTHER READINGS
1 Collier L, Balows A, Sussman M. Topley and Wilson’s Microbiology and Microbial Infections. 9th Edition. Mycology. Volume 4. pp. 711.
Arnold publishers.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Jagadish Chander. A Text Book of Medical Mycology. Interprint. 2000. pp. 232.
Textbook of Practical Microbiology 231
LESSON
Carbohydrate
80 Fermentation Test
After incubation, the Durham’s tubes are observed for presence Presence of bubbles or drop in the fluid level in Durham’s tube
of any gas bubbles. indicates fermentation of sugars. Development of yellow color
is not a reliable indicator of fermentation and is ignored.
KEY FACTS
1 Carbohydrate fermentation test is a supplementary test only when there is difficulty in making a definitive identification
using carbohydrate assimilation test.
2 Development of yellow color is not reliable indicator of fermentation and hence production of air bubble in Durham’s tube
is to be noted.
VIVA
FURTHER READINGS
1 Collier L, Balows A, Sussman M. Topley and Wilson’s Microbiology and Microbial Infections. 9th Edition. Mycology. Volume 4. pp. 711.
Arnold publishers.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Jagadish Chander. A Text Book of Medical Mycology. Interprint. 2000.
Textbook of Practical Microbiology 233
LESSON
Identification of
81 Common Fungi
After completing this practical you will be able to: Lactophenol cotton blue wet mount preparation of the fungus
(refer chapter 73).
1 Know important characteristic morphological features of
different fungi.
2 Identify a given fungus. QUALITY CONTROL
Microscopy
Spores are oval, colorless and sometimes brown. Mycelium Aspergillus
non-septate, sporangiophores straight and terminate in black
sporangium containing sporangiospores and columella; root Usually infect plants and animals. More than 170 species are
like hyphae (rhizoids) penetrating the medium. known. Almost sixteen different species cause human disease.
They are:
Mucor
Aspergillus fumigatus
Common food contaminant. Aspergillus flavus
Aspergillus niger
Colony morphology
Resembles colony of Rhizopus. Aspergillus oryzae
Aspergillus amstelodami
Microscopy Aspergillus verscicolor
Oval spores, non-septate mycelium giving rise to single Aspergillus terreus
sporangiophores and terminate in globular sporangium Aspergillus nidulans
containing columella and sporangiospores. Aspergillus candidus
Aspergillus ustus
Alternaria Aspergillus carnens
Aspergillus arenaceus
Usually found on plants.
Aspergillus clavatus
Colony morphology Aspergillus caseillus
Grayish green or black colonies with grey edges and swarming Aspergillus restricutus
over entire plate with grayish white aerial hyphae.
The characteristics used to identify them are:
Microscopy A) Colour and shape of conidial head.
Conidia multi celled, pear-shaped and attached to single B) Number of sterigmata/phialids.
conidiophores arising from septate mycelium. C) Shape of vesicles.
D) Colour of conidiophore.
Fusarium E) Presence and absence of cleistothecia, and
F) Size and shape, and color of ascospore and conidia.
Usually found in soil.
Aspergillus fumigatus
Colony morphology
Woolly white colonies may change to pink, purple or yellow
Colonies
(Fig. 81-1).
Colonies are blue-green, velvety, surface powdery or granular,
sometimes radially folded (Fig. 81-2).
Microscopy
Conidia multi celled, oval or crescent shaped, attached to
conidiophores arising from a septate hypha. Conidial head
Columnar, compact.
Conidiophore
Smooth, terminate into club or flask shaped green.
Vesicle
Club or flask shaped, green.
Sterigmata
Produced on upper half of the vesicle, uniseriate, appears green
in color and crowded. Axis of sterigmata is roughly parallel to
that of conidiophore and conidia produced in chains on
sterigmata.
Conidia
FIGURE 81-1 SDA with colonies of Fusarium species.
Globose, green and echinulate.
Textbook of Practical Microbiology 235
FIGURE 81-2 SDA with colonies of Aspergillus fumigatus. FIGURE 81-4 SDA with colonies of Aspergillus flavus.
Aspergillus niger
Aspergillus flavus
Colonies
White, fluffy, reverse buff colored, covered with black spores Colonies
(Fig. 81-3). Rapidly growing, yellowish green and velvety colony (Fig. 81-4).
Penicillium species
Colonies
Initially white and fluffy, later turns to green and blue green,
with radial folds.
Hyphae
Septate, hyaline.
Conidiophores
Long with branching phialids.
Phialids
FIGURE 81-3 SDA with colonies of Aspergillus niger.
Flask shaped, branched and gives rise to brush like appearance,
producing sterigmata.
236 Identification of Common Fungi
Sterigmata Macroconidia
Long with tapering end, producing chains of conidia. String-like, bean shaped, septate, with characteristic rat tail
appearance, rarely produced.
Conidia
Long chains of conidia, spherical or oval in shape. Microconidia
Tear-shaped, rarely produced. Swollen antler-like hyphae
Cladosporium produced rarely, may produce chains of chlamydospores.
Colony Colony
Small, heaped, greenish black and powdery. Slow growth, port wine or deep violet colored, flat or heaped
up, surface waxy and glabrous, wrinkled loose pigment present.
Microscopy
Conidia develop at the end of complex conidiophores arising Macroconidia
from brown septate mycelium. Generally not present. Thick walled structures resembling
macroconidia of T. rubrum may be present.
Cephalosporium
Used in antibiotic production Microconidia
Generally not present, pyriform. Chains of chlamydospores
produced.
Colony
Rapidly growing compact, moist colonies, cottony, with gray
or rose colored aerial hyphae. Epidermophyton floccosum
Microscopy Colony
Velvety or powdery surface, surface folded in radiating furrows,
Single celled conical or elliptical conidia held together in clusters
reverse of colony yellow tan.
at the tips of conidiophores. Erect, unbranched conidiophores
arise from septate mycelium.
Macroconidia
Clavate, smooth, thick walled, septa 2–3, characteristics clusters
Trichophyton verrucosum of twos and threes.
Colony Microconidia
Slow grower, white glabrous heaped up colony, sometimes Not produced.
button like with velvety texture. No pigment produced on reverse.
Variants with yellow or grey white colonies, rugal folds seen. Chlamydospores
Produced, racquet hyphae and nodular bodies present.
KEY FACTS
1 Characters of aerial hyphae, septation, conidial shape, size and color are very important for identifying the fungus.
2 Colony characters, color, obverse and reverse should be kept in mind.
Textbook of Practical Microbiology 237
VIVA
FURTHER READINGS
1 Collier L, Balows A, Sussman M. Topley and Wilson’s Microbiology and Microbial Infections. 9th Edition. Mycology. Volume 4. pp. 711.
Arnold publishers.
2 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
3 Jagadish Chander. A Text Book of Medical Mycology.. Interprint. 2000.
238
Textbook of Practical Microbiology 239
UNIT
XI
Virology
LESSON
Cultivation of Viruses in
82 the Cell lines
LEARNING OBJECTIVES Many viruses kill the infected viral cells in which they grow
and bring about detectable changes in morphology of the cells.
After completing this practical you will be able to: These changes are collectively known as cytopathic effects.
Some viruses however do not produce any cytopathic effect
1 Know and understand the common cell lines used for routine (e.g., rubella virus).
maintenance of viruses in a virology laboratory. The most important precaution to be taken during
maintenance of cell lines is sterility. Contamination of cell lines
should be prevented and even cross contamination among cell
INTRODUCTION lines should be avoided.
PRINCIPLE PROCEDURE
Viruses infect healthy cells grown in the laboratory. When 1 Discard the trypsin versene mixture and add a small amount
susceptible cells are used for inoculation of viruses, they show of MEM with 10% FCS to the monolayer of cells.
pathological changes and the viruses can be harvested from 2 Count the cells with the medium in a hemocytometer for
the cells for further tests. The growth of viruses in the cell lines appropriate splitting.
can be known by a) cytopathic effects, b) immunofluorescence, 3 Inoculate the cells into sterile flasks or tubes for viral
c) haemagglutination and haemadsorption, and d) interference. inoculation.
Textbook of Practical Microbiology 241
QUALITY CONTROL
FIGURE 82-1 Inverted microscope.
1 Sterility precautions should be taken perfectly.
2 Susceptible cells to be selected for the appropriate virus.
OBSERVATIONS
The cell lines are observed for any cytological alterations that
are diagnostic of viral infections (Table 82-1).
Table 82-1 The cell lines and indications for the viruses and cytopathic effects they produce
Adenovirus Primary human embryonic kidney cells. Rounding and clustering of swollen cells.
HeLa cells.
Herpes simplex virus HEp 2 cells. Rounding and swelling of cells.
Varicella zoster virus Human kidney cells. Multinucleated giant cell formation.
Cytomegalovirus Human fibroblast cells. Giant cell formation.
Enterovirus Hep 2 cells. Rapid rounding of cells progressing to complete
Vero cells. cell destruction.
RD cells.
Paramyxovirus Primary human and monkey kidney cells. Syncytia formation.
LLC-MK2.
242 Cultivation of Viruses in the Cell lines
KEY FACTS
1 The most important aspect to be taken care of in cell culture is sterility. Hence precautions for sterility should be meticulously
followed.
2 Susceptible cells should be selected for the appropriate viral inoculation.
VIVA
1 Why is cell culture method, the most preferred method in a virology laboratory?
2 What precaution is to be taken during maintenances of cell lines?
3 What are the commonly used cell lines in virology laboratory?
FURTHER READINGS
1 Collier L, Balows A, Sussman M. Topley and Wilson’s; Microbiology and Microbial Infections. 9th Edition. Virology. Volume 1. Arnold
publishers.
2 Knipe DM, Howley PM. Field’s Virology. 4th Edition. Lippincott Williams and Wilkins. 2001.
3 Zuckerman AJ, Banatvala JE, Pattison JR, Griffiths PD and Schoub BD. Principles and Practice of Clinical Virology. 5th Edition. John
Willey and sons Ltd. 2004.
Textbook of Practical Microbiology 243
LESSON
Cultivation of Viruses in
83 Embryonated Egg
INTRODUCTION REQUIREMENTS
Prior to 1950s the technique of propagation of viruses in I Equipments
embryonated eggs was popular because of non-availability of Egg holders, candling lamp and hole puncher,
cell culture techniques during those times. The eggs are used
because they are inexpensive, easily available and much simpler II Reagents and lab wares
to handle than animals. The viruses can grow in different Syringes (1 tuberculin syringe preloaded with 0.1 ml of 10-3
compartments of the egg . Since eggs lack a well developed dilution of NDV in GLB), gauze, pencils, gloves, sterile forceps,
defence mechanism of their own, they do not interfere with Pasteur pipette with bulb, screw capped sterile vials, egg, melted
growth of viruses. paraffin wax and 70% ethanol.
However, many viruses fail to grow on primary inoculation The eggs are candled to determine the position of the
into eggs. Nevertheless, the embryonated eggs are still used embryo and its viability. Since viruses need living tissues to
for the isolation of avian viruses, influenza viruses and also for replicate, candling is important. When candled, healthy embryo
vaccine production. has an orange color with evident vasculature. Dead embryo shows
clear yellow with no vasculature. Black, green or brown color
indicates contamination. Dead embryos are promptly discarded.
PRINCIPLE
III Specimen
Usually 8-11 days old chick embryos are used. Age of the egg Virus isolate or specimen suspected to contain virus.
chosen depends on route of inoculation described since various
membranes and their contents vary in size as embryo matures.
These eggs are inoculated by one of the following routes: PROCEDURE
yolk sac, amniotic sac, allantoic cavity and chorioallantoic
membrane (CAM). Any of the viruses or specimen suspected 1 Disinfect the egg shell.
to contain viruses are inoculated. After inoculation, eggs are 2 Drill the egg shell.
incubated for 2-9 days. Viral growth is recognized by death of 3 Inoculate the specimen in the embryonated egg through
embryo, formation of pocks, or haemagglutination property of appropriate route.
embryonic fluid. Note: Age of the embryonated egg is chosen depending on
Yolk sac is mainly used for culture of some viruses, certain bacteria the route of inoculation since various membranes and their
(Chlamydia and Rickettsia) and parasite (Toxoplasma gondii). contents vary in size as embryo matures (Table 83-1).
Amniotic sac is used for the primary isolation of influenza 4 After 2–5 days post injection, viral growth in the egg is
virus. recognized by death of embryo, pocks, or hemagglutination.
244 Cultivation of Viruses in Embryonated Egg
1 Before inoculation, the eggs are candled to determine the Viral growth in the inoculated egg is recognized by death of
position of the embryo and its viability. the embryo, pock formation in CAM (Fig. 83-1),
2 Age of the embryonated egg is chosen depending on the haemagglutination, etc.
route of inoculation since various membranes and their
contents vary in size as embryo matures.
3 Sterile precautions should be taken throughout the
procedure.
OBSERVATIONS
Table 83-1 The routes of inoculation of the egg and the viruses isolated
KEY FACTS
1 Usually 8-11 days old chick embryos are used for cultivation of viruses.
2 Since viruses need living tissues to replicate, candling of eggs is important.
VIVA
FURTHER READINGS
1 Collier L, Balows A, Sussman M. Topley and Wilson’s; Microbiology and Microbial Infections. 9th Edition. Virology. Volume 1. Arnold
publishers.
2 Knipe DM, Howley PM. Field’s Virology. 4th Edition. Lippincott Williams and Wilkins. 2001.
3 Zuckerman AJ, Banatvala JE, Pattison JR, Griffiths PD and Schoub BD. Principles and Practice of Clinical Virology. 5th Edition. John
Willey and sons Ltd. 2004.
246
Textbook of Practical Microbiology 247
UNIT
XII
Microbiology of
Water, Milk and Air
LESSON
Microbiology of Water
84
LEARNING OBJECTIVES The total coliform count is widely regarded as the most
reliable indicator of potable water quality. However, the
After completing this practical you will be able to: presence of coliforms not necessarily indicate faecal or sewage
contamination, because, these organisms are present in large
1 Know various methods used for testing potable water. quantities in the environment.
2 Test the quality of the potable water for drinking.
Faecal or thermotolerant coliforms
INTRODUCTION These satisfy the criteria for coliforms but have the additional
property of the ability to grow at a higher temperature 44°C.
Drinking water is acceptable and fit for drinking when it is
clear, colourless, odourless and without disagreeable taste. Faecal Escherichia coli
Microscopically it should be free from pathogenic organisms.
Natural sources of water usually contain some saprophytic These are defined as thermotolerant coliforms which ferment
bacteria, such as Pseudomonas, Serratia, Flavobacterium, lactose (or) mannitol at 44°C with the production of acid and
Chromobacterium, Acinetobacter, Alcaligenes, etc. These gas within 24 hours and also form indole from tryptophan at
saprophytes are harmless. 44°C. The presence of E. coli is considered as the contamination
Water gets contaminated by pathogens which are of water with feces of human or animal origin.
introduced into water by sewage pollution. A wide varieties of
diseases are transmitted by contaminated water. There are some Faecal streptococci
indicator organisms, whose presence indicates the
contamination of water with fecal matter. These are: These are catalase negative, Gram positive cocci present in the
intestinal tract of man and animals. These organisms have the
1 Coliforms. Lancefield group D antigen, hydrolyse aesculin and can grow
2 Fecal (thermotolerant) coliforms. at 45°C, in the presence of azide and 40% bile. Such organisms
3 Faecal Escherichia coli. which can survive 60°C for 30 min and can grow at 10°C at pH
4 Faecal Streptococci, and 9.6 and in 6.5% of sodium chloride (NaCl) belong to the genus
5 Clostridium perfringens. Enterococcus (Enterococcus faecalis and E. faecium) .
Clostridium perfringens
Coliforms
The presence of this organism in water in the absence of other
Coliforms are defined as members of the family Entero- indicators of contamination of water implies remote or
bacteriaeceae which grow in the presence of bile salts and intermittent fecal pollution.
produce acid and gas from lactose within 48 hours at 37°C. In Viruses in water are destroyed by chlorination. When the
order to include anaerogenic bacteria and those which are non- concentration of free residual chlorine is at least 0.5 mg per
lactose fermenters, it has been modified as the members of the litre, for a minimum contact period of 30 minutes at pH below 8
Enterobacteriaeceae capable of growing galactosidase at 37°C and a turbidity of 1 nephelometric turbidity unit or less, protozoa
that normally posses. such as Entamoeba histolytica, Giardia species and
Textbook of Practical Microbiology 249
Balantidium coli may be present in the drinking water. Coliforms Differential coliform test
are not the reliable indicator of protozoal contamination. This is called Eijkman test. This test is usually employed to
find out whether the coliform bacilli detected in the presumptive
Collection of water samples test are E. coli or not.
After the usual presumptive test, subcultures are made from
Water is collected in heat sterilised bottles containing a all the bottles showing acid and gas to fresh tubes of single
sufficient volume of sodium thiosulphate to neutralise the strength MacConkey medium already warmed to 37°C. They
bactericidal effect of any chlorine or chloramine in the water. are incubated at 44°C for 24 hours. Those showing gas in
For collection from tap, water is allowed to run to waste for Durham’s tubes contain E. coli. From the number of positive
2–3 min. before collecting it into the bottle. When collecting tubes obtained, results are read off the probability tables.
water samples from lakes or streams the bottle is opened at a Further confirmation of the presence of E. coli is done by
depth of about 30 cm with its mouth facing the current. At least testing for indole production and citrate utilization tests.
100 ml of water to be tested is collected in each bottle.
After collecting the water in the bottle, the bottle is stoppered, Membrane filtration method
and sent to the laboratory as quickly as possible within 6 hours A measured volume of water is filtered through a Millipore
keeping it in a cool container and protecting it from light. filter. All the bacteria present are retained on its surface. This is
then placed on suitable media and incubated at the appropriate
temperatures. Colonies on the surface of the membrane are
PRINCIPLE counted. After 18 hours of incubation the presumptive coliform
counts and detection of E. coli can be directly made.
The following tests can be done for bacteriological analysis of water:
1 Plate count.
Detection of faecal streptococci
2 Detection of coliform bacteria and E. coli. Subcultures are made into tubes containing 5 ml of glucose
a Presumptive coliform count: multiple tube technique. azide broth from the positive bottles in the above test.
b Differential coliform test. Streptococcus faecalis if present produces acid in the medium
c Membrane filtration method. within 18 hours at 45°C.
3 Detection of faecal streptococci.
4 Examination of Cl. perfringens, and Examination for Cl. perfringens
5 Test for pathogenic bacteria. Water is incubated in litmus milk medium at 37°C for 5 days, if
positive, stormy fermentation occurs.
Plate count
Examination for pathognic bactrial cells test
This consists of inoculating the nutrient agar with water to be For other special pathogens like Salmonella, Vibrio, etc.
tested and incubating the agar aerobically in parallel at 37°C corresponding special media are used.
for 1–2 days and at 22°C for 3 days. After incubation number of In this chapter Presumptive coliform test and diffrential
the colonies formed in the agar are counted. Those which grow coliform test will be discussed.
at 37°C are associated with organic material of human or animal
origin and those growing at a lower temperature are mainly
saprophytes that normally inhabit water, soil, and vegetables. REQUIREMENTS
The agar count at 22°C gives an indication of the amount of
decomposing organic matter in the water available for bacterial I Equipments
nutrition. Though these are non-pathogenic the greater the amount Bacteriological incubator, autoclave and water bath.
of organic matter present, the more likely is the water to be
contaminated with parasitic and potentially pathogenic organisms. II Reagents
The agar count at 37°C is a more important index of dangerous MacConkey broth, brilliant green bile broth
pollution. Preparation of MacConkey broth: This is prepared by mixing
pancreatic digest of gelatin, 20 grams; lactose, 10 grams; bile
salt, 5 grams; bromocresol purple, 0.01 grams and final pH (at
Detection of coliform bacteria and E.coli
25°C) is adjusted at 7.3 ± 0.2.
Preparation of double strength medium: It is prepared by
Presumptive coliform test – Multiple tube suspending 35 grams in 500 ml of distilled water and heated to
technique dissolve the medium completely. 50 ml and 10 ml of the media are
The test is called presumptive, because of the presumption dispensed into screw capped bottles with inverted Durham’s tubes
that the reactions are due to coliforms organisms. The count is and are sterilized by autoclaving at 15 lbs (121°C for 15 minutes).
made by adding varying quantities of water (0.1 ml–50 ml) to Preparation of single strength medium: It is prepared by
bile salt lactose peptone water with an indicator for acidity. suspending 3.5grams in 100 ml of distilled water. 5 ml quantities
Acid and gas formation indicate the growth of coliform bacilli. of the medium are dispensed in screw capped bottles with
250 Microbiology of Water
KEY FACTS
1 Drinking water is acceptable and fit for drinking when it is clear, colourless, and odourless and without disagreeable taste.
Microscopically it should be free from pathogenic organisms.
2 Coliforms are defined as the members of the family Enterobacteriaeceae which grow in the presence of bile salts and produce
acid and gas from lactose within 48 hours at 37°C.
3 They are indicator of faecal contamination.
4 Coliforms are not the reliable indicator of protozoal contamination.
5 Eijkman test is usually employed to find out whether the coliform bacilli detected in the presumptive test are E. coli or not.
VIVA
FURTHER READINGS
1 Ananthanarayanan. R, Paniker. C.K, Ananthanarayanan and Paniker’s Textbook Of Microbiology; 7th Edition, Orient Longman, pp 603 –
609, 2005.
2 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. pp. 921, 1996.
252
LESSON
Microbiology of Milk
85
LEARNING OBJECTIVES number of viable bacteria in the milk. For un refrigerated milk
there is a consistent relationship between the bacterial count
After completing this practical you will be able to: and the dye reduction time.
I Equipments
INTRODUCTION Water bath.
Human infections may be caused by the ingestion of animal II Reagents and glass wares
milk which contains microorganisms derived either from the
animal (faeces), from the environment, or from milk handlers Sterile test tubes, sterile pipettes, sterile rubber stopper,
such as dairy workers. Certain precautions are taken to prevent methylene blue tablets and distilled water.
contamination. A wide number of bacteria are found in
contaminated milk (Table 85-1). III Specimen
The infections that can be transmitted from infected animals Milk to be tested.
to humans through contaminated milk are many. They include
tuberculosis, brucellosis, streptococcal and staphylococcal
infections, salmonellosis and Q fever. PROCEDURE
The routine bacteriological examination of milk to detect
bacterial contaminations can be done by the following methods: The time of reduction is affected by the temperature at which
the milk is held before testing. The test should not be done if
1 Viable count. the atmospheric shade temperature exceeds 21°C.
2 Test for coliform bacilli.
3 Methylene blue reduction test. 1 Prepare a 1 in 3,00,000 solution of methylene blue by
4 Phosphatase test. dissolving a standard methylene blue tablet in 200ml cold
5 Turbidity test, and sterile glass-distilled water and making upto 800ml with more
6 Examination for specific pathogens. of such water.
Note: This solution can be stored in dark in a refrigerator
Of these tests, methylene blue test is a rapid, simple, inexpensive and can be used for 2months.
and most widely used test for testing the milk. This method will 2 Mix thoroughly the milk sample.
be described in this chapter. 3 Aseptically transfer 10ml of sample of milk with a pipette to
a sterile test tube.
4 Add 1ml of methylene blue solution by a 1ml sterile pipette.
PRINCIPLE 5 Put a sterile rubber stopper to the test tube; invert it once or
twice to mix the contents.
Time taken for bacterial dehydrogenases to reduce methylene 6 Place these test tubes at 37°C in a thermostatically controlled
blue dye and decolourise it, is taken as an indicator of the water bath for 30 min.
Textbook of Practical Microbiology 253
Note: The water level must be above the top of the milk and
the bath covered with a lid to exclude the light.
RESULTS AND INTERPRETATION
A. Add 1ml methylene blue to 10ml milk that has been held at
Table 85-1 List of bacteria that can be found in contaminated milk
100°C for 3 min, to inactivate its reducing system.
1 Streptococcus lactis
B. Add 10 ml milk to 1 ml of tap water. 2 Streptococcus faecalis
3 Achromobacter
4 Clostridium perfringens
OBSERVATIONS 5 Clostridium butyricum
6 Bacillus subtilis
After incubation, compare the test mixture with control A to 7 Bacillus cereus
see whether there is any decolourization in the former and with 8 Proteus vulgaris
9 Staphylococcus aureus
control B to see whether decolourisation is complete.
10 Gaffkya tetragena
KEY FACTS
1 Tuberculosis, brucellosis, streptococcal and staphylococcal infections, salmonellosis and Q fever are some of the infections
transmitted by contaminated milk.
2 Methylene blue test is rapid, simple, inexpensive and most widely used test for testing the microbial contamination of the
milk.
3 The time of reduction is affected by the temperature at which the milk is held before testing. The methylene blue reduction
test should not be done if the atmospheric shade temperature exceeds 21°C.
VIVA
FURTHER READING
1 Ananthanarayanan. R, Paniker. C.K, Ananthanarayanan and Paniker’s Textbook Of Microbiology; 7th Edition, Orient Longman, pp 603 –
609, 2005.
2 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. pp. 921, 1996.
254
LESSON
Microbiology of Air
86
LEARNING OBJECTIVES II Reagents and lab wares
These include Petri dishes containing nutrient agar media for
After completing this practical you will be able to identify: the growth of organisms.
1 Bacteria present in the air.
2 Organisms present in the air by settle plate method.
PROCEDURE
INTRODUCTION 1 Prepare nutrient agar media, pour it into plates and dry off
any surface moisture.
Demonstration and estimation of the number of bacteria carrying 2 Remove cover of the Petri dish in its chosen position for the
particles in air may be required in certain situations. For example, measured period of time, and then replace its lid.
in the premises where safe working depends on the bacterial 3 Incubate the plates aerobically for 24 hours at 37°C.
content in air being kept at a very low level and premises where 4 Count the colonies, preferably with the use of a plate microscope.
certain foods or pharmaceutical materials are prepared, the
monitoring of density of microbial pathogens in the air is a priority.
The number of bacteria in the air at any given time is dependent QUALITY CONTROL
on the number of persons present, the amount of their body
movements and the amount of disturbance of their clothing, etc. The Petri dish agar plates should remain open for a specified
The list of bacteria commonly found in the air are summarized and adequate time.
in the table 86-1. The infections that can be transmitted through
air include wound infections, tuberculosis, anthrax,
streptococcal and staphylococcal infections. OBSERVATIONS
Settle plate method and slit sampler method are the two
methods used for routine bacteriological examination of the Observe the colonies grown on the medium after incubation.
air. Settle plate method will be described in this chapter.
KEY FACTS
1 The number of bacteria in the air at any given time is dependent on the number of persons present, the amount of their body
movements and the amount of disturbance of their clothing, etc.
2 Settle plate method and slit sampler method are the two methods used for routine bacteriological examination of the air.
VIVA
FURTHER READINGS
1 Ananthanarayanan. R, Paniker. C.K, Ananthanarayanan and Paniker’s Textbook Of Microbiology; 7th Edition, Orient Longman, pp 603 –
609, 2005.
2 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. pp. 921, 1996.
256
Textbook of Practical Microbiology 257
UNIT
XIII
Animal Experiments
LESSON
Intravenous Inoculation
87 into Mice Tail Vein
After completing this practical you will be able to: Procedure for loading the syringe for injection
1 Inoculate the appropriate culture suspension into the tail 1 Open the culture tube near the flame.
vein of mouse. 2 Fill the syringe with the culture to be injected.
Note: The air is to be expelled by pushing the needle into the
cotton inserted into a sterile tube and the volume to be
INTRODUCTION adjusted
3 The needle should be recapped carefully and placed ready
Mouse is one of the most commonly used lab animal for different for use.
animal experiments. The suckling mouse is used for isolation
and cultivation of viruses like Coxsackie A and B viruses and Animal preparation for injection
arboviruses, and also for testing enterotoxins. The adult mouse
is used for isolation and cultivation of organisms such as 1 Remove the selected animal from the cage.
Mycobacterium leprae, Toxoplasma gondii, Cryptosporidium 2 Keep the animal on a rough surface such as over a mouse
parvum, etc. (Box 87-1). cage or a wire.
3 Lift the mouse by holding the tail halfway.
4 Insert the tail through the mesh of a wire basket and hold the
PRINCIPLE animal in such a way that the animal is inside the wire mesh
and tail outside.
Mouse is a very suitable laboratory animal for different types 5 Wipe clean the tail with spirit or antiseptic soaked cotton.
of pathogens. After inoculation of the animal with appropriate 6 Apply little xylol over the area of the vein, and allow it to act
specimens, the changes can then be observed in the inoculated for a minute so that the vein will be prominent.
mouse and result interpreted.
Injection of material
REQUIREMENTS 1 Hold the syringe and needle in such a way that animal should
be horizontal to the tail and vein.
I Equipments 2 Then insert the needle into the vein midway between base
Mice cage. and tip of tail.
Note: It should be ensured that the needle is inside the vein
II Reagents and lab wares by withdrawing some blood.
Discarding jar, Bunsen flame, 1 ml syringe, 26G needle, culture 3 Inject carefully the material (approx. 0.1 ml and 0.2 ml) into
tube, cotton, colour dye and other standard laboratory glassware. the tail vein.
Anaesthetic agent, and antiseptic. 4 Remove the needle, then place plain sterile cotton over the
prick and hold it firmly until bleeding stops.
III Specimen 5 Keep back the animal in the cage after labeling with colored
Laboratory bred mouse and culture material to be tested. dye.
Textbook of Practical Microbiology 259
Suckling mice
Adult mice
KEY FACTS
1 The mouse to be inoculated should be first checked thoroughly for proper health conditions.
2 The area for inoculation should be disinfected before inoculation.
VIVA
FURTHER READINGS
1 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
2 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. pp. 921, 1996.
Textbook of Practical Microbiology 261
LESSON
Collection of Blood
88 from the Marginal Ear
Vein of Rabbit
After completing this practical you will be able to: 1 Restrain the rabbit by gently wrapping it in a blanket.
2 Shave the hair from the rear margin of an ear. Smear the skin
1 Collect the blood from the ear marginal vein of rabbit. over the marginal ear vein with petroleum jelly to delay clotting.
3 Grasp the ear near its base so that the venous return is
impeded and the vein raised.
INTRODUCTION 4 With a scalpel blade make a diagonal cut across the vein.
Note: The cut should penetrate the skin and vein but not be
Rabbit is one of the most commonly used lab animal for different so deep as to sever the vein. The ear should not be released
experiments. Infant rabbits are used for the propagation of at this time.
Vibrio cholerae, and for testing of enterotoxins of 5 Blood will then start to flow. When the flow begins to recede,
enteropathogenic pathogens. Adult rabbits are used for the wiping the cut with cotton will restart it.
isolation of Listeria monocytogenes, Streptococcus 6 Collect the blood in a clean sterile test tube.
pneumoniae, etc. 7 After collection release pressure on the vein, cover the cut
Uses of rabbits are summarized in the box 88-1. with a small piece of cotton and press firmly.
8 Clean the ear with a sterile swab and return the rabbit to its
cage.
PRINCIPLE
REQUIREMENTS OBSERVATIONS
I Laboratory wares The inoculated rabbit should be observed and handled with
Jar, Bunsen flame, test tube, cotton, colour dye, and sterile care every day and any change in its health condition should
scalpel. be monitored.
II Reagents
Antiseptic, and petroleum jelly. RESULTS AND INTERPRETATION
Infant rabbit
1. Animal model for Vibrio cholerae.
2. In enteropathogenic organisms, testing of enterotoxins can be done.
Adult rabbit
1. For testing virulence and pathogenicity of
(a) Listeria monocytogenes.
(b) Streptococcus pneumoniae.
(c) Nocardia spp.
(d) Mycobacterium tuberculosis.
(e) Herpes simplex virus.
(f) Candida albicans.
2. Other uses
(a) Differentiating M. tuberculosis and M. bovis.
(b) Sereny’s test.
(c) Rabbit ileal loop test (enterotoxin testing).
(d) Maintenance of Treponema pallidum.
(e) Increasing virulence of rabies virus and vaccinia virus.
(f) For collecting blood for antistreptolysin O test.
(g) To raise antibodies to antigen.
(h) For serum as source of complement for cytotoxic assays.
KEY FACTS
1 After cutting the ear and vein, the ear should not be released immediately.
2 Sudden movements or noise should not be made during the procedure.
3 Blood samples can also be collected from central artery of the ear or directly from the heart by cardiac puncture.
VIVA
1 What are the other routes for the collection of blood from rabbit?
FURTHER READINGS
1 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
2 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. pp. 921, 1996.
Textbook of Practical Microbiology 263
LESSON
Animals and their uses
89 in the Laboratory
LEARNING OBJECTIVES After inoculation following care of the animals should be taken:
After completing this practical you will be able to: 1 Physical properties of the animal should be monitored
regularly.
1 Know the commonly used laboratory animals (mice, rabbit, 2 Health of the animal should be checked regularly.
guinea pig and other animals) for animal experiments. 3 Make sure that the animal is taking normal amount of food
2 Know the uses of these animals in the laboratory. and water as before to inoculation.
4 Take care of animal to prevent it from other infections.
5 Test results should be monitored and recorded regularly.
INTRODUCTION
The animal if dies due to the effect of the specimen inoculated,
Laboratory animals have been used for ages, in laboratories then the animal should be disinfected and incinerated with
for different purposes. These animals include mice, rabbits, proper care.
guinea pigs, hamsters, monkeys, etc. They are used for a wide
range of applications and form an integral part of research (Box
89-1). Animals such as these should be properly taken care of, REQUIREMENTS
and should be in accordance to the governing rule for animal
usage in laboratory. I Equipments
Different animals and their usage are listed in the table 89-1. Wire cage.
QUALITY CONTROL
1 The laboratory animal’s health and physical condition
should be checked before using them.
2 The animal should be labeled properly before and after FIGURE 89-2 Rabbit.
inoculation.
OBSERVATIONS
Depending upon the changes that the animal shows, the results FIGURE 89-3 Guinea pig.
of the test shall be interpreted.
Monkeys
Monkeys are primates and are extensively used in laboratories. Since they are genetically evolved animals, they are considered very suitable
for lab testing.
Uses
1 To raise antiserum.
2 For RBC for serological tests.
3 For testing virulence of Entamoeba histolytica.
4 Testing pathogenicity of polio virus.
5 Testing hemagglutinating property of measles virus.
6 Laboratory cultivation of hepatitis A virus and Plasmodium falciparum.
7 Animal model for influenza virus.
Hamsters
Uses
Infant hamsters
1 To test tumorigenicity of adeno viruses.
Adult hamster
1 For pathogenicity testing of
(a) Leishmania donovani.
(b) Entamoeba histolytica.
(c) Bacillus anthracis.
2 For isolation of
(a) Leptospira species.
(b) Leishmania species.
(c) Toxoplasma gondii.
3 Other uses
(a) Maintenance of virulence of Entamoeba histolytica.
(b) Drug efficiency testing against Leishmania donovani.
Guinea pig
Uses
1 For the isolation of
(a) Mycobacterium tuberculosis.
(b) Mycobacterium bovis.
(c) Yersinia pestis.
(d) Leptospira icterohaemorrhagiae.
(e) Brucella abortus.
(f) Chlamydia species.
(g) Toxoplasma gondii.
3 Pathogenicity testing of
(a) Listeria monocytogenes.
(b) Bacillus anthracis.
(c) Clostridium perfringens.
(d) Entamoeba histolytica.
(e) Cryptococcus neoformans.
4 Other uses
(a) For the preparation of complement.
(b) For the evaluation of bronchodilator compounds.
(c) Pathogenicity testing of M. tuberculosis (spleen enlargement, lesions in spleen and liver).
(d) Pyrogen testing.
(e) Sereny’s test.
266 Animals and their uses in the Laboratory
KEY FACTS
1 Animals should be properly taken care of, and should be in accordance to the governing rule of CPSEA for animal usage in laboratory.
2 The lab animal’s health and physiological condition should be checked before and after inoculation.
VIVA
1 What are the information to be recorded about an animal and inoculation before and after inoculation of test material?
2 What are the different routes of inoculation in lab animals and why are the different routes significant?
3 How should an inoculated animal be looked after and what precautions are to be taken?
FURTHER READINGS
1 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
2 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. pp. 921, 1996.
Textbook of Practical Microbiology 267
UNIT
XIV
Medical Entomology
LESSON
Identification of
90 Common Insects
General features The itch mite or Sarcoptes scabies var hominis belongs to the
class Arachnida.
1 2-4mm in length, smaller than mosquito.
2 Greyish brown having dark conspicuous eye and hairy body. Identifying features
3 Head contains a pair of long, slender, hairy antennae and a
proboscis with 16 segments. 1 It is 0.5mm in size, visible to naked eye.
4 Thorax bears a pair of wings and three pairs of legs. 2 Body is tortoise shaped, rounded above and flattened below.
5 Wings are upright and lanceolate shaped. The second 3 Body is hairy.
longitudinal vein on the wing branches twice, first branching 4 Has 2 pairs of legs in front and 2 pairs behind.
takes place in the middle of the wing. 5 Front legs have suckers at the end, and the hind legs end in
6 Legs are longer and hairy. long bristles.
7 Only females suck blood at night. 6 Males are smaller than the females.
8 Males have three pairs of clappers on the last abdominal 7 In females, the first 2 pairs of legs possess suckers. In males,
segment. the fourth pair of legs also have suckers.
9 They do not fly, they only hop about up to 3 feet. 8 Dorsal surface has backwardly pointed spines.
Diseases transmitted by sand fly are summarized in the
box 90-1. Diseases transmitted
1 Scabies (seven year itch), Norwegian itch of man.
2 Forage mite and house dust mite cause allergic respiratory
HOUSE FLIES distress.
Identifying features
TROMBICULID MITE
1 It is mouse gray in colour.
2 Body is divided in to head, thorax, and abdomen. It is also known as scrub typhus mite.
3 Head bears a pair of antennae, a pair of large compound
eyes and a refractive proboscis, for sucking liquid food. Eyes Identifying features
of the male are close together and those of the female are set 1 Adult mites are 1-2mm in length and are red in colour.
apart widely. 2 Covered dorsally and ventrally with numerous feathered hairs.
4 Thorax is marked with 2 to 4 dark longitudinal stripes, which 3 Has 4 pairs of legs.
is characteristic of the genus Musca. It bears a pair of wing 4 Body is constricted between the 3rd and 4th pairs of legs,
and three pairs of legs. giving a figure of eight appearance.
5 Each leg is provided with a pair of pads which enable the fly
to walk on highly polished surfaces. Diseases transmitted
6 Legs and body are covered with numerous short and stiff Rickettesia orientails (Orientia tsutsugamushi).
hairs called Tenent hairs which secrete a sticky substance. Trombicula dilensis is the vector in India.
7 Abdomen is segmented and shows dark and light markings. Trombicula akamush is the vector in Japan.
270 Identification of Common Insects
6 Abdomen has 5 segments. The last segment has two Diseases transmitted
feathered filaments. In females the first abdominal segment 1 Dracunculiasis. Mesocyclops act as intermediate host for
has got 2 ovisacs on both sides to carry the eggs. Dracunculosis medinensis.
7 Swims with characteristic jerky movements in water. Hence 2 Diphyllobothriasis. First intermediate host for
called as water fleas. They can be seen as small moving Diphyllobothrium latum.
specks in water. 3 They also act as vectors for Gnathostoma spinigerum and
Gnathostoma hispidium.
Diseases Species
Diseases Species
VIVA
FURTHER READINGS
1 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
2 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. pp. 921, 1996.
272
Textbook of Practical Microbiology 273
UNIT
XV
Common Viva Spots
LESSON
Identification of
91 Common Viva Spots
After completing this practical you will be able to identify the Prepared by adding agar in nutrient broth and sterilized by
following common viva spots given in Microbiology autoclaving.
examination:
Modifications
1 Culture media.
2 Culture media with growth. 1 Semi-solid agar if concentration of agar is 0.2–0.5%.
3 Biochemical reactions. 2 Concentrated agar if agar concentration is 2–6%.
4 Specimens of parasites.
5 Glass wares. Uses
6 Instruments, and
7 Microscopic slides. 1 For growing of non fastidious bacteria.
2 Determination of antibiotic sensitivity.
3 Preparation of blood agar.
CULTURE MEDIA 4 Concentrated agar (3% and more) prevents swarming of
bacteria such as Proteus vulgaris, Clostridium tetani, etc.
NUTRIENT AGAR
BLOOD AGAR
Also referred as ‘Agar’ (Fig. 91-1).
The blood agar is an enriched medium as well as an indicator medium.
Composition The colour of the medium is red like that of blood (Fig. 91-2).
Preparation
Uses
FIGURE 91-4 Mac Conkey agar.
1 For growth of most of the pathogenic bacteria.
2 Used in preparation of the potassium tellurite blood agar. Composition
1 Peptone : 20 g.
CHOCOLATE AGAR 2 Sodium taurocholate, commercial : 5 g.
3 Water : 1 litre.
The medium is so called because it is chocolate in colour. 4 Agar : 20 g.
Chocolate agar is an enriched medium (Fig. 91-3). 5 Neutral red solution, 2 percent
in 50 percent ethanol : 3.5 ml.
Composition 6 Lactose 10% aqueous solution.
Uses
Preparation
LOEFFLER’S SERUM SLOPE
It is prepared by heating 10% sterile blood in sterile nutrient
agar. The agar is melted and cooled it in water bath at 75°C, Loeffler’s serum slope is an enriched medium. This is used
blood is added and the medium continues to remain at 75°C till for culture and isolation of Corynebacterium diphtheriae
the blood becomes chocolate brown in colour. (Fig. 91-5).
Uses
MACCONKEY AGAR
Composition Preparation
Malachite green solution: Prepare 2% solution of malachite
1 Sterile ox, sheep, horse serum : 300 ml.
green in sterile water with sterile precautions by dissolving the
2 Nutrient broth : 100 ml.
dye in incubator for 1–2 hr.
3 Glucose : 1 g.
Preparation of medium
Preparation Mineral salt solution : 600 ml.
Malachite green solution : 20 ml.
Dissolve glucose in nutrient broth and autoclave at 115°C for
Beaten egg (20 to 22 hen’s
20 min. Add glucose broth to the serum with sterile precautions
eggs depending on size) : 1 litre.
and distribute in test tubes or in 2–5 ml amounts in sterile
conditions to fill up nearly one fourth of the bottles. This medium
Mix the complete medium, distribute it in 5 ml volumess in
is sterilized by inspissation.
sterile McCartney bottles and screw the caps tightly on. Lay
the bottles horizontally in the inspissator and heat at 75°–80°C
Uses for 1 hr. This is to solidify the medium.
FIGURE 91-6 Lowenstein Jenson’s medium. FIGURE 91-7 Robertson’s cooked meat medium. (RCM ) broth.
Textbook of Practical Microbiology 277
broth over meat is 7.5. If test tubes are used the surface of the meat and it is essential that basal medium to which various
medium may be covered with a layer of sterile liquid paraffin carbohydrates added for fermentation tests should be free from
1cm deep but this is not essential. natural sugars (Fig. 91-9).
Uses Composition
Composition
Glucose : 40 g.
Peptone : 10 g.
Agar : 20 g. FIGURE 91-9 Peptone water.
Water : 1 L.
Preparation
Uses
PEPTONE WATER
STREPTOCOCCUS PYOGENES ON
BLOOD AGAR
FIGURE 91-12 Staphylococcus aureus on nutrient agar.
Salient features
1 Blood agar colonies 0.5–1 mm in diameter, after 24 hrs PROTEUS SPP. ON BLOOD AGAR
incubation circular, discrete, semi-transparent, low convex
disks, showing a-hemolysis on fresh blood agar plates (Fig. Salient features
91-11).
2 Virulent strains isolated from lesions give a matt type of 1 Group of cells at the edge of developing micro colony migrate
colony whereas avirulent strains produce glossy colonies. to an uninoculated area of the medium and present as
3 A mucoid colony type is also encountered and corresponds swarming. Swarming is seen in blood agar plates and the
in virulence to matt type. growth may eventually appear either as a uniform growth /
4 Str. pyogenes is the commonest organism causing sore film over the whole plate. Continuous swarming or
throat. discontinuous swarming are a series of concentric circles of
5 Seen as b-hemolytic colonies in blood agar plates. growth around point of inoculation (Fig. 91-13).
6 Gram staining shows Gram positive cocci in chains. 2 Proteus spp. are Gram negative, coccobacilli, 1–3 µm long
7 It is catalase negative. This test differentiates it from and 0.6 µm wide. In young culture they may be filamentous
Staphylococcus. as longs as (80 µm).
3 They are motile by peritrichous flagella. They possess more than
one type of fimbriae. Variants are nonflagellate and non motile.
FIGURE 91-11 Streptococcus pyogenes on blood agar. FIGURE 91-13 Proteus species on blood agar.
Textbook of Practical Microbiology 279
4 The two common species are Proteus vulgaris and Proteus 4 They are indole positive, urease negative.
mirabilis. 5 They cause urinary tract infections and diarrhoea.
5 PPA and urease tests are positive.
KLEBSIELLA SPP ON
PSEUDOMONAS AERUGINOSA ON MACCONKEY AGAR
NUTRIENT AGAR
Salient features
Salient features
1 Klebsiella spp produces lactose fermenting, mucoid colonies
1 On nutrient agar growth of the organism shows green on MacConkey agar (Fig. 91-16).
diffusible pigment (Fig. 91-14). 2 They are Gram negative, non sporing, non motile bacilli, 1–2
2 Ps. aeruginosa is a strict aerobe, slender, Gram negative µm long and 0.5–0.8 µm wide with parallel or bulging sides
bacillus, 1.5–3.0 µm × 0.5 µm arranged singly, in pairs or and slightly pointed or rounded ends.
short chains. 3 Freshly isolated strains possess a well defined
3 It is non sporing, non capsulate, though mucoid strains may polysaccharide capsule.
sometime occur and usually motile by one or two polar flagella. 4 They are urease positive, and indole negative.
4 It is oxidase positive. 5 They cause urinary tract infections, pneumonia, etc.
5 It is one of the common causes of nosocomial infections.
CORYNEBACTERIUM DIPHTHERIAE ON
ESCHERICHIA COLI ON POTASSIUM TELLURITE AGAR
MACCONKEY AGAR
Salient features
Salient features 1 Potassium tellurite agar is used as selective medium for
C. diphtheriae (Fig. 91-17).
1 Escherichia coli shows lactose fermenting, non-mucoid 2 C. diphtheriae produces black coloured colonies in
colonies on MacConkey agar (Fig. 91-15). potassium tellurite agar.
2 It is an aerobe and facultative anaerobe.
3 It is a Gram negative, non capsulated bacillus measuring
1–3 µm × 0.4–0.7 µm. Most of the strains are motile by peritrichate
flagella.
FIGURE 91-15 Escherichia coli on MacConkey agar. FIGURE 91-17 Corynebacterium diphtheriae on potassium tellurite agar.
280 Identification of Common Viva Spots
+ + + –
FIGURE 91-19 Candida albicans on Sabourauds dextrose agar. FIGURE 91-20 Carbohydrate fermentation test.
Textbook of Practical Microbiology 281
INDOLE TEST
Salient features
NEGATIVE POSITIVE
FIGURE 91-24 Citrate utilization test. FIGURE 91-26 Triple sugar iron agar.
PHENYL PYRUVIC ACID TEST (PPA) 4 Yellow colour formation occurs with fermentation of
carbohydrates, while bubbles in butt show the formation of
Salient features gas during fermentation process.
5 When H2S is produced by the bacteria, the medium shows
1 It is used to determine the ability of an organism to deaminate blackish discolouration.
phenyl pyruvic acid (Fig. 91-25). 6 Combinations of TSI reactions:
2 Positive PPA test is indicated by a green colour and negative
PPA test is indicated by no colour change. Examples of PPA K/A (red/yellow) : Glucose only fermented.
positive bacteria are Proteus species, Providencia species, A/A (yellow/yellow) : Glucose, and lactose or sucrose
and Morganella species. fermented or both fermented.
K/K (red / red) : Neither glucose, lactose nor
sucrose fermented.
Note: K – Alkaline, A- Acidic.
SPECIMENS OF PARASITE
Salient features
NEGATIVE POSITIVE 1 Tail end of male worm is curved ventrally in form of a hook,
while in female worm, it is conical and straight (Fig. 91-27).
FIGURE 91-25 Phenyl pyruvic acid test. 2 Route of infection of the organism is by ingestion of the
food or water contaminated with eggs.
3 Embryonated egg is the infective form of the parasite.
Salient features
GLASS WARES Glass syringes (Fig. 91-31) are sterilised by hot air oven.
Uses
UNIVERSAL CONTAINER
1 For collection of blood by venepuncture.
Universal containers (Fig. 91-29) are sterilised by autoclave . 2 To collect body fluids, pus, etc.
3 To collect blood from animals (sheep, rabbit) which may be
used for preparation of blood agar.
4 For injecting medicine to patients.
Uses
1 Specimen collection.
2 For measuring and mixing purposes.
TUBERCULIN SYRINGE
1 Lepromin test. Pasteir pipettes (Fig. 91-35) are sterilised by hot air oven.
2 Tuberculin test.
3 BCG vaccination. Uses
4 Insulin injection.
5 Tetanus toxoid injection. 1 Used for delivering solutions or reagent in test tubes,
6 To give intradermal and other sub cutaneous injections; and containers etc., during various procedures.
to inject small amount of test material into animals. 2 For mixing the constituents for reactions. Example: RBC s
coated with specific antigen to perform IHA test for detection
of antibodies.
PETRI DISH
Uses
SWAB TUBE
GRADUATED PIPETTE
Uses
Uses
FIGURE 91-37 Egg of Enterobius vermicularis, x 400. FIGURE 91-39 Hot air oven.
INSTRUMENTS AUTOCLAVE
Salient features
INCUBATOR
1 Autoclave (Fig. 91-40) uses the principle of moist heat steri-
Salient features lization (121°C at 15 lbs pressure for period of 15 minutes) method.
2 It is used for sterilization of culture media, rubber material,
1 Incubators (Fig. 91-38) are used for incubating culture plates gloves, gowns, dressing, test tubes, etc.
and liquid media at specified temperature for growth of
micoorganisms such as bacteria, fungi, amoebae, etc.
2 Optimum temperature for growing bacteria is 37°C.
3 It contains a thermometer by which periodically temperature
can be monitored.
Salient features
GRAM POSITIVE COCCI
1 In Gram stained smear, Neisseria gonorrhoea appear as pink
Salient features coloured Gram negative diplococci with adjacent sides
concave typically kidney shaped (Fig. 91-43).
1 In Gram stained smear, the Gram positive cocci appear violet 2 Found predominantly within the polymorphonuclear cells,
in colour (Fig. 91-41). but some may be seen outside.
2 Gram positive cocci occur either in pairs, chains or clusters.
Examples
Salient features
HAEMOPHILUS INFLUENZAE
Salient features
CLOSTRIDIUM PERFRINGENS
Salient features
Salient features
TREPONEMA PALLIDUM
Salient features
Salient features
ALBERT STAINING
Salient features
Salient features
FIGURE 91-52 Ziehl Neelsen staining for Mycobacterium leprae,
x 1000.
1 It is a special stain for demonstrating acid fastness of M.
tuberculosis (Fig. 91-51).
2 20% sulphuric acid is used for ZN stain. M. tuberculosis NEGRI BODIES
resist decolourisation with 20% sulphuric acid.
3 M. tuberculosis is both acid and alcohol fast. Salient features
4 M. tuberculosis appear as approx. 1-3 µm size slender pink
coloured acid fast bacilli with curved ends against a blue 1 Negri bodies are 3 - 27 µm size, intra cytoplasmic inclusion
background. body which appear as round , oval, purplish pink structures
5 M. tuberculosis causes tuberculosis. with characteristic basophilic inner granules (Fig. 91-53).
6 Löwenstein Jensen (LJ) medium is the most frequently used 2 Negri bodies are usually found within the nerve cells of the
medium to grow M. tuberculosis. hippocampus and cerebellar region.
7 It takes 3-6 weeks for M. tuberculosis to grow in this medium. 3 These are usually demonstrated in the impression smears of
Textbook of Practical Microbiology 289
Salient features
MALE
FEMALE
FIGURE 91- 57 Plasmodium falciparum ring stage in a Giemsa stained FIGURE 91- 59 Plasmodium falciparum male and female
blood smear, x 1000. gametocytes, in a Giemsa stained blood smear, x 1000.
PLASMODIUM VIVAX
MALE AND FEMALE GAMETOCYTES LD BODIES
1 Male gametocyte (microgametocyte) is spherical and smaller 1 It is a Giemsa stained thin blood smear showing a red
than the female. Cytoplasm stains light blue or pale blue and coloured nucleus and pale blue coloured cytoplasm multiple
nucleus is large (Fig. 91-58). rings in one red blood cell.
2 Female gametocyte (macrogametocyte) is spherical and large 2 Leishmania donovani is the causative agent of visceral
than the male. Cytoplasm is purple and nucleus is small. leishmaniasis or kala-azar (Fig. 91-60).
3 LD bodies, otherwise known as amastigote stage of
L. donovani is found mostly inside the macrophages. Some
LD bodies are found outside macrophages.
4 L. donovani shows frequent drug resistance to pentavalent
antimonials.
PLASMODIUM FALCIPARUM
MALE AND FEMALE GAMETOCYTES TOXOPLASMA GONDII
Salient features Salient features
1 Male gametocyte (microgamete) is sickle shaped, broader 1 The typical active multiplying tachyzoites is an important
and shorter. Cytoplasm stains light blue and nucleus is diagnostic form of Toxoplasma gondii (Fig. 91-61).
diffuse (Fig. 91-59). 2 Tachyzoites appear as 3-7 µm, oval to crescent shaped
2 Female gametocyte are typically crescent (banana) shaped structures with pointed anterior end and rounded posterior
with rounded or pointed ends. Cytoplasm stains deep blue end.
and nucleus is compact. 3 Tachyzoites can be stained with periodic acid schiff (PAS),
Gomori methenamine silver, hematoxylin and eosin, and
Wright-Giemsa stain.
Textbook of Practical Microbiology 291
WUCHERERIA BANCROFTI
MICROFILARIA
FIGURE 91- 63 Cyst of Entamoeba histolytica / dispar, x 400.
Salient features
1 It is a Giemsa stained thin blood smear showing the sheathed CYST OF GIARDIA INTESTINALIS
microfilaria.
2 The body of microfilaria shows few nuclei in the body and
more distinct tail is tapering and pointed.
Salient features
3 No nuclei are present in the tail end.
4 Wuchereria bancrofti is the causative agent of lymphatic 1 Iodine wet mount of stool showing iodine stained brown
filariasis (Fig. 91-62). oval cyst of Giardia intestinalis (Fig. 91-64).
5 Anopheles, Culex and Aedes mosquito transmit the disease 2 Cyst contains four nuclei, and an axostyle which lie
and are the intermediate host. diagonally, forming a dividing line within the cyst wall.The
6 Man is the definitive host. cyst is separated from the cyst wall by a clear space.
3 The cyst is the infective form of the parasite.
4 The infection is transmitted by ingestion of water and food
contaminated with cysts.
5 It is the causative agent of diarrhoea, malabsorption , etc.
1 Bile stained egg of Ascaris lumbricoides in the saline wet 1 Saline wet mount of stool showing plano-convex and non-
mount of stool (Fig. 91-65). bile stained egg of Enterobius vermicularis (Fig. 91-67).
2 Embryonated egg is the infective form of the parasite. 2 Egg is the infective form of the parasite.
3 A. lumbricoides infection is transmitted by faeco -oral 3 E. vermicularis infection is transmitted by faeco-oral
transmission. transmission.
4 It is the causative agent of intestinal ascariasis. 4 It is the causative agent of pruritus ani in children.
5 Auto infection is characteristic of E. vermicularis infection.
Salient features
3 T. trichiura infection is transmitted by faeco-oral 3 They cause opportunistic infection especially in patients
transmission. with HIV.
4 It is the causative agent of gastrointestinal infections. Heavy 4 They cause oral thrush, vaginitis, onychonychia etc., in
infection may complicate as appendicitis. immunocompetent patients.
Salient features
1 It is a yeast-like fungus.
2 In Gram stained smear, C. albicans appear purple in colour
(Fig. 91-70)
FIGURE 91- 70 Candida albicans in a Gram’s stained smear, x 1000. FIGURE 91- 71 Germ tube test, x 400.
FURTHER READINGS
1 Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002.
2 Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. pp. 921, 1996.
3 Parija SC. Textbook of Medical Parasitology. All India Publishers and Distributors. 3nd Edition. 2006.
4 Parija SC. Stool Microscopy. BPKIHS, Dharan, Nepal, 1998.
294
Textbook of Practical Microbiology 295
Index
A Introduction 97
Abbe condenser 4 Principle 97
ABO system 110 Requirements 97
Acetoin 78 Equipments 97
Acetylcholinesterase 140 Reagents and lab wares 97
Acid fast staining method 27 Preparation of stock solutions of antibiotics 97
Acid-alcohol 27 Specimens 97
Acid-fast staining 27 Preparation of suspension of bacteria 97
Learning Objectives 27 Procedure 97
Introduction 27 Test procedure 98
Principle 27 Quality Control 98
Requirements 27 Observations 98
Equipments 27 Results and Interpretation 98
Reagents and glass wares 27 Key Facts 98
Preparation of strong carbol fuchsin 27 Viva 99
Preparation of 20% sulphuric acid 27 Agarose 130
Preparation of 95% alcohol 27 Agglutination 108, 117
Preparation of acid-alcohol decolouriser 28 Albert’s stain 31, 32, 33, 175, 176, 288
Specimen 28 Albert’s staining 31
Procedure 28 Learning Objectives 31
Quality Control 28 Introduction 31
Observation 28 Principle 31
Results and Interpretation 28 Requirements 31
Key Facts 29 Equipments 31
Viva 30 Reagents and glass wares 31
Acid-fast Staining of Stool Smears 198 Preparation of Albert’s stain I 31
Learning Objectives 198 Preparation of Albert’s stain II 31
Introduction 198 Specimen 31
Principle 198 Procedure 31
Requirements 198 Quality Control 32
Equipments 198 Observation 32
Reagents and lab wares 198 Results and Interpretation 32
Specimen 198 Viva 32
Procedure 198 Key Facts 33
Quality Control 198 Alcohol 27, 60
Observations 199 Alkaline peptone water 46, 181
Results and Interpretation 199 Alkaline phosphatase 138, 140
Key Facts 199 Allantoic cavity 243
Viva 200 Allantoic sac 244
Adult hamster 265 Allergic respiratory distress 269
Adult mice 259 Alsever’s solution 121
Adult rabbit 261, 262 Amido black 126, 131
Aedes aegypti 269, 268, 291 Amniotic sac 243, 244
Aerotolerant anaerobes 52 Amoebiasis 269
Aesculin 173 Amoebic antigens 130
Agar 274 Amoebic dysentery 291
Agar dilution method 97, 98 Amoebic liver abscess 291
Learning Objectives 97 Anaerobic bacilli 54
296 Index
Blood group antisera 110 Box 90-2 Diseases Transmitted by Hard Ticks 271
Blood grouping 110 Box 7-1 Modifications of Gram’s staining 25
Learning Objectives 110 Box 7-2 Uses of Gram’s Staining 25
Introduction 110 Brain heart infusion agar 46
Principle 110 Bright field microscopy 2
Requirements 110 Brilliant green bile broth 249
Reagents and lab wares 110 Bromothymol blue 81, 281
Specimen 110 Broth dilution agar method 101
Procedure 110 Broth dilution method 100
Quality Control 110 Learning Objectives 100
Observations 110 Introduction 100
Results and Interpretation 110 Principle 100
Key Facts 111 Requirements 100
Viva 111 Equipments 100
Blood parasites 201 Reagents and lab wares 100
Body louse 270 Preparation of stock solutions of antibiotics 100
Boeck and Drbohlav’s medium 208 Specimens 100
Borrelia duttoni 270 Preparation of suspension of bacteria 100
Borrelia recurrentis 270 Procedure 100
Bound coagulase 68, 69 Quality Control 100
Boutonneuse fever 271 Observations 101
Box 1-1 Terminology 5 Results and Interpretation 101
Box 1-2 Size of Different Organisms 6 Key Facts 101
Box 12-1 List of most common capsulated organisms that can be dem- Viva 101
onstrated by negative staining 41 Browne’s tube 56, 57
Box 17-1 List of Anaerobic Bacilli and Cocci 54 Brownian movement 12
Box 18-1 Pasteurisation 58 Brucellosis 253
Box 19-1 Commonly used disinfectants and their mechanism of actions 60 Buffered distilled water 201
Box 2-1 Principle of Dark Ground Microscopy 8 Buffered methylene blue 191
Box 20-1 Uses of catalase Test 63
Box 20-2 Catalase Test for Mycobacteria 63 C
Box 22-1 Free Coagulase 69 CAMP (Christie, Atkins and Munch-Peterson) test 173
Box 29-1 List of media used for detecting production of hydrogen sul- Candida albicans 6, 215, 293
phide 86 Candida albicans on Sabourauds dextrose agar 280
Box 31-1 Glossary of terms 93 Candida species 225
Box 4-1 Demonstrating Motility of Anaerobic Bacteria 12 Candle jar 51
Box 43-1 VDRL-ELISA 125 Candling 244
Box 43-2 Biological False Positive Reactions of VDRL Test 125 Capillary tube method 11, 12
Box 47-1 Reverse Passive Haemagglutination test 133 Capsular antigens 36
Box 49-1 1st, 2nd and 3rd Generation ELISA 140 Capsulated bacteria 36
Box 49-2 Dot ELISA 140 Capsule 36, 286
Box 5-1 Terminology 17 Capsule Staining 3 4
Box 50-1 Role of Plasmids in drug resistance 146 Learning Objectives 34
Box 51-1 Advantages and Disadvantages of Page 150 Introduction 34
Box 52-1 Point Mutations and Large Scale Mutations 153 Principle 34
Box 52-2 Mechanisms of Mutation 154 Requirements 34
Box 52-3 The Importance of Mutation 154 Equipments 34
Box 53-1 Mechanisms of DNA Transfer 156 Reagents and glass wares 34
Box 54-1 Beneficial Effects of Normal Flora 161 Specimen 34
Box 6-1 Simple stains and their uses in microbiology laboratory 21 Procedure 34
Box 61-1 Identification of Escherichia Coli 179 For positive staining of smears 34
Box 61-2 Identification of Klebsiella species 179 For negative staining of smears 35
Box 62-1 Identification of Vibrio Cholerae 182 Quality Control 35
Box 63-1 Identification of Pseudomonasaeruginosa 185 Observation 35
Box 67-1 Acid Fast Parasites and Parasitic Components 199 Observation of positive staining method 35
Box 68-1 The Parasites found in the Peripheral Blood Smear 203 Observation of negative staining method 35
Box 69-1 Advantages and Disadvantages of the Concentration Results and Interpretation 35
Methods 207 Positive staining method 35
Box 71-1 Sabouraud’s Dextrose Agar 214 Negative staining method 35
Box 77-1 Predisposing Factors for Candidiasis 226 Key Facts 35
Box 8-1 Different Modifications of Acid Fast Stain and their uses 29 Viva 36
Box 8-2 Frequently examined specimens for the Detection of Carbohydrate assimilation 229
Mycobacterium Tuberculosis 29 Carbohydrate Assimilation Test 229, 230
Box 87-1 Uses of Mice in Laboratory 259 Learning Objectives 229
Box 88-1 Use of Rabbits in Laboratory 262 Introduction 229
Box 89-1 Use of Laboratory Animals 264 Principle 229
Box 9-1 Rapid staining by direct fluorescent antibody method 32 Requirements 229
Box 90-1 Diseases Transmitted by Sand Fly 271 Equipments 229
298 Index
Uses 274, 275, 276, 277, 278, 283, 284, 285 Universal Container 283
Blood Agar 274 Bijou Bottle 283
Composition 274 Tuberculin Syringe 283
Preparation 275 Graduated Pipette 284
Chocolate Agar 275 Pasteur Pipette 284
Composition 275 NIH Swab 285
Preparation 275 Incubator 285
MacConkey Agar 275 Salient features 285
Composition 275 Hot Air Oven 285
Preparation 275 Salient features 285
Loeffler’s Serum Slope 275 Autoclave 285
Composition 276 Salient features 285
Preparation 276 Microscopy Slides 286
Lowenstein-Jensen (LJ) Medium 276 Gram Positive Cocci 286
Composition 276 Salient features 286
Preparation 276 Streptococcus pneumoniae 286
Robertson Cooked Meat (RCM) Broth 276 Salient features 286
Composition 276 Neisseria gonorrhoeae 286
Preparation 276 Salient features 286
Sabouraud’s Dextrose Agar (SDA) 277 Gram Negative Bacilli 286
Composition 277 Salient features 286
Preparation 277 Haemophilus influenzae 287
Peptone water 277 Salient features 287
Composition 277 Vibrio cholerae 287
Preparation 277 Salient features 287
Glucose Broth 277 Bacillus anthracis 287
Composition 278 Salient features 287
Preparation 278 Clostridium perfringens 287
Culture Media with Growth 278 Salient features 287
Streptococcus pyogenes on Blood Agar 278 Treponema pallidum 287
Salient features 278 Salient features 287
Staphylococcus aureus on Nutrient Agar 278 Albert staining 288
Salient features 278 Salient features 288
Proteus spp. on Blood Agar 278 Ziehl-Neelsen Staining for Mycobacterium tuberculosis 288
Salient features 278 Salient features 288
Pseudomonas aeruginosa on Nutrient Agar 279 Ziehl-Neelsen Staining for Mycobacterium leprae 288
Salient features 279 Salient features 288
Escherichia coli on MacConkey Agar 279 Negri Bodies 288
Salient features 279 Salient features 288
Klebsiella spp. on MacConkey Agar 279 Multinucleate Giant Cells Measles 289
Salient features 279 Salient features 289
Corynebacterium diphtheriae on Potassium Tellurite Agar 279 Molluscum Bodies 289
Salient features 279 Salient features 289
Mycobacterium tuberculosis on Lowenstein Jensen (LJ) Medium 280 Plasmodium vivax Ring Stage 289
Salient features 280 Salient features 289
Candida albicans on Sabouraud’s Dextrose Agar (SDA) 280 Plasmodium falciparum Ring Stage 289
Salient features 280 Salient features 289
Biochemical Reactions 280 Plasmodium vivax Male and Female Gametocytes 290
Carbohydrate Fermentation Tests 280 Salient features 290
Salient features 280 Plasmodium falciparum Male and Female Gametocytes 290
Glucose with Durham’s Tube 281 Salient features 290
Salient features 281 LD Bodies 290
Indole Test 281 Salient features 290
Salient features 281 Toxoplasma gondii 290
Urease Test 281 Salient features 290
Salient features 281 Wuchereria bancrofti Microfilaria 291
Citrate Utilization Test 281 Salient features 291
Salient features 281 Cyst of Entamoeba histolytica/dispar 291
Phenyl Pyruvic Acid Test (PPA) 282 Salient features 291
Salient features 282 Cyst of Giardia intestinalis 291
Triple Sugar Iron (TSI) Agar 282 Salient features 291
Salient features 282 Egg of Round Worm 292
Specimens of Parasite 282 Salient features 292
Ascaris lumbricoides Adult Worm 282 Egg of Hook Worm 292
Salient features 282 Salient features 292
Hydatid Cyst 283 Egg of Enterobius vermicularis 292
Salient features 283 Salient features 292
Glass Wares 283 Egg of Hymenolepis nana 292
304 Index
Z Zinc dusts 89
Zephiran 60 Zinc sulphate floatation method 206
Ziehl Neelsen staining 288 Zone reactions 124, 125
Ziehl-Neelsen acid-fast staining 27 Zygomycetes 6
Ziehl-Neelsen stain 29, 37
1 / Running Head 13
TEXTBOOK OF
PRACTICAL MICROBIOLOGY
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