Textbook of Practical Microbiology

1 / Running Head1 Textbook of Practical Microbiology Subhash Chandra Parija AHUJA 2 Textbook of Practical Microbiology THE AUTHOR Dr Subhash Chandra Parija MBBS, MD, PhD, FAMS, FICPath, FABMS, FICAI, FISCD and FIMSA is Director-Professor & Head, Department of Microbiology, in the Jawaharlal Institute of Postgraduate Medical Education & Research, Pondicherry. Prof Parija completed his MBBS at SCB Medical College, Cuttack, Utkal Unioversity, Orissa in 1977. He obtained his MD (1978-81) in Medical Microbiology from the Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh. Prof Parija did his PhD from University of Madras in the year 1987 for his work on simple diagnostic methods in amoebiasis. Prof Parija, after completion of the MD, began his carrier at the JIPMER, Pondicherry as senior resident in the year 1981. Subsequently He became Professor of Microbiology in the year 1991, and Director-Professor of Microbiology in the year, 2004. During his tenure at JIPMER, Pondicherry, Prof Parija was sent on deputation by Govt of India to set up and establish the departments of Microbiology & initiate an integrated Clinical Laboratory Services at the B P Koirala Institute of Health Sciences, Dharan, Nepal, of which he was the founder head between 1995-98. In recognition of his excellent contribution to the growth and development of the department of Microbiology, as well for the Institute, the B P Koirala Institute of Health Sciences, Dharan, Nepal, conferred the most prestigious BP Koirala Internal Oration Award. Prof Parija was awarded WHO fellowships for study of DNA probes, PCR and other molecular biological methods in the study of parasitic diseases at the University of Aberdeen, UK. Prof. Parija is member of the ICMR task force on intestinal protozoal infections. He is also the member of the Research Advisory Board of BP Koirala Institute of Health Sciences, Dharan, Nepal; Board of MD Examination in Parasitology, Colombo University, Sri Lanka, and the visiting Professor of the College of Medicine & Health Sciences, Sultan Quaboos University, Muscat, Oman. Author of three books “Text Book of Medical Parasitology”, “Stool Microscopy ” and Sputum Microscopy: a Practical Manual”; editor of a book “Review of Parasitic Zoonoses” and two monographs “Immunizing agents for tropics: success, failure and some practical issues” and “Kala-azar: epidemiology, diagnosis and control in Nepal”; Prof Parija also has contributed several chapters for the books, compendium of lectures and proceedings of scientific meetings. Prof Parija has published more than 137 papers both in the National and International journals of repute. Some of his papers are quoted in text books and serial publications. The development of simple, economical and rapid diagnostic tests in serodiagnosis of parasitic diseases such as amoebiasis and cystic echinococcosis is the main field of his research. Prof Parija was the first to demonstrate excretion of hydatid antigen in urine and its detection for diagnosis of cystic echinococcosis. He Developed for the first time the carbon-immunoassay (CIA) and staphylococci adherence test (SAT) as two simple rapid diagnostic methods using a light microscope, and Co-agglutination (Co-A) and CIEP for the detection of antigen in the serum in the patients with amoebic liver abscess and cystic echinococcosis, and for demonstration of antigen in the hydatid fluid and also in the urine for the diagnosis of cystic echinococcosis. All these tests can be used in the field or in less equipped laboratories in the developing countries like India. Prof Parija was first to report the use of LPCB and KOH in the wet mount preparation of stool for detection of intestinal parasites by light microscopy. Prof Parija is the recipient of the most prestigious Dr BC Roy National Award 2003 of the Medical Council of India in recognition of his immense contribution to the development of Medical Microbiology. He was awarded the coveted Dr B P Pandey Memorial Oration Award of the Indian Society for Parasitology, and BK Aikat Oration Award of the Indian Council of Medical Research for his research in diagnosis and epidemiology of parasitic diseases. Other awards include Dr S.R.Memorial Award 2003 of the Bombay Veterinary College Alumni Association, Dr SC Agarwal Oration Award 2001 of the Indian Association of Medical Microbiologists, Major General Saheb Singh Sokhey Award 1992 of the Indian Council of Medical Research, Third Dr Datta Memorial Award 1999 of the Indian Association for the Advancement of Veterinary Parasitology, IAPM (Orissa chapter) Oration Award 1993 of the Indian Association of Pathologists & Microbiologists (Orissa Chapter), Smt Kuntidevi Malhotra Award 1990 of the Indian Association of Pathologists & Microbiologists, Dr S S Misra Memorial Award 1987 of the National Academy of Medical Sciences, Young Scientist Award 1986 of the Indian Association of Medical Microbiologist and Best Scientific Paper Award 1987 of the JIPMER Scientific Society. Professor Parija is the editor-in chief of the online journal Internet Journal of Parasitic Diseases, and member of the editorial editorial boards of various journals both International (Parasitology International, BMC Infectious Diseases, BMC Clinical Pathology and Health Renaissance) and National (Indian Journal of Medical Microbiology, Journal of Veterinary Parasitology, Journal of Parasitic Diseases and Indian Journal of Pathology & Microbiology) . He has been conferred with various fellowships such as FAMS, FICPath, FABMS, FICAI, FISCD and FIMSA by professional bodies. Prof Parija has visited many countries, delivered invited talks in infectious diseases at universities, institutions conferences, seminars etc, and has chaired scientific sessions in the international as well as national conferences. He has guided both MD and PhD students and has been examiner for MBBS, MD and PhD of various universities of India and abroad. Prof Parija was the former Secretary of the Indian Association of Medical Microbiologists and National Vice President of the Indian Association of Pathologists and Microbiologists. He is currently the National Vice President of the Indian Association for Development of Veterinary Parasitologists . 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 Dr. Subhash Chandra Parija MD, PhD, FAMS,FICAI, FABMS, FISCD, FIMSA, FICPath Director-Professor and Head Department of Microbiology Jawaharlal Institute of Postgraduate Medical Education and Research Pondicherry, India. Ahuja Publishers Bangalore, New Delhi 4 Textbook of Practical Microbiology Textbook of Practical Microbiology Copyright © 2006 Dr. Subhash Chandra Parija All rights reserved. No part of the publication may be reproduced, stored in retrieval system or transmitted by any means, electronic, mechanical, photocopying or otherwise without the prior writtenpermissionof the publisher. First Edition : 2006 Printed in India ISBN Published by Ahuja Publishers 1 / Running Head 5 To my mother Late Smt. Nishamani Parija . 6 Textbook of Practical Microbiology . 1 / Running Head 7 Contents Preface Acknowledgments UNIT I Microscope and Basic Microbiological Techniques Introduction 1 Compound Microscope 2 Darkground Microscopy 3 Measurement of Microorganisms 4 Hanging drop Preparation 5 Isolation of Pure Cultures UNIT II Bacterial Staining 6 Simple Staining 7 Gram’s Staining 8 Acid Fast Staining 9 Albert’s Staining 10 Capsule Staining 11 Spore Staining 12 Negative Staining UNIT III Cultivation of Bacteria 13 14 15 16 17 18 19 Media for Routine Cultivation of Bacteria Temperature Requirement for Growth of Bacteria pH Requirement for Growth of Bacteria Oxygen Requirement for Growth of Bacteria Culture of Anaerobic Bacteria Sterilization of Commonly Used Culture Media Antiseptics and Disinfectants xi xii 1 2 3 7 9 11 14 19 20 23 27 31 34 37 40 43 44 47 49 51 53 56 59 61 62 65 68 71 UNIT IV Enzymatic and Biochemical Activities of Bacteria 20 21 22 23 Catalase Test Oxidase Test Coagulase Test Urease Test . 8 Textbook of Practical Microbiology 24 25 26 27 28 29 30 Indole Test Methyl Red Test Voges-Proskauer Test Citrate Utilization Test Triple Sugar Iron Agar (TSI) Test Hydrogen Sulphide Test Nitrate Reduction Test 74 76 78 80 82 85 88 91 92 95 97 100 102 105 106 108 110 112 114 116 119 121 123 126 128 130 132 135 138 143 144 145 148 152 155 159 160 162 164 166 169 172 175 178 181 184 UNIT V Antimicrobial Sensitivity Tests 31 32 33 34 35 Kirby-Bauer Method Stoke’s Method Agar Dilution Method Broth Dilution Method Epsilometer Test (E-test) UNIT VI Immunology Introduction 36 Bacterial Agglutination Test 37 Blood Grouping 38 Latex Agglutination Test 39 Co-agglutination Test 40 Widal Test 41 Weil Felix Test 42 Anti-Streptolysin O (ASLO) Test 43 VDRL Test 44 Radial Immunodiffusion Test 45 Immunoelectrophoresis Test 46 Counter-current Immunoelectrophoresis Test 47 Indirect Haemagglutination Test 48 Immunofluorescence Test 49 Enzyme-linked Immunosorbent Assay UNIT VII Microbial Genetics and Molecular Techniques Introduction 50 Isolation of Plasmids 51 Polyacrylamide Gel Electrophoresis 52 Isolation of Antibiotic Resistant Mutant 53 Bacterial Conjugation Unit VIII Bacteriology 54 55 56 57 58 59 60 61 62 63 Normal Microbial Flora of the Mouth Normal Microbial Flora of the Throat Normal Microbial Flora of the Skin Identification of Staphylococcus aureus Identification of Streptococcus pneumoniae Identification of b-haemolytic streptococci Identification of Corynebacterium diphtheriae Identification of Lactose Fermenting Enterobacteriacae Identification of Vibrio cholerae Identification of Pseudomonas aeruginosa . 1 / Running Head 9 Unit IX Parasitology Introduction 64 Saline Wet Mount of Stool 65 Iodine Wet Mount of Stool 66 LPCB Wet Mount of Stool 67 Acid-fast Staining of Stool Smears 68 Leishman’s staining of Peripheral Blood Smears 69 Concentration of Stool for Parasites 70 Culture of Stool for Entamoeba histolytica Unit X Mycology Introduction 71 Cultivation of Fungi 72 Gram’s Staining for Fungi 73 Lactophenol Cotton Blue (LPCB) Wet Mount of Fungi 74 Potassium Hydroxide Wet Mount of Fungi 75 Indian Ink Wet Mount Preparation 76 Slide Culture 77 Germ Tube Test 78 Urease Test 79 Carbohydrate Assimilation Test 80 Carbohydrate Fermentation Test 81 Identification of Common Fungi Unit XI Virology 82 Cultivation of Viruses in the Cell lines 83 Cultivation of Viruses in Embryonated Egg Unit XII Microbiology of Water. Milk and Air 84 Microbiology of Water 85 Microbiology of Milk 86 Microbiology of Air Unit XIII Animal Experiments 87 Intravenous Inoculation into Mice Tail Vein 88 Collection of Blood from the Marginal Ear Vein of Rabbit 89 Animals and their uses in the Laboratory Unit XIV Medical Entomology 90 Identification of Common Insects Unit XV Common Viva Spots 91 Identification of Common Viva Spots Index 187 188 189 192 195 198 201 205 208 211 212 213 215 217 219 221 223 225 227 229 231 233 239 240 243 247 248 252 254 257 258 261 263 267 268 273 274 295 . 10 Textbook of Practical Microbiology . Every effort has been made to incorporate all aspects of practical microbiology. and experimental procedure in detail. by laboratory workers and by others who are interested in study of practical microbiology. principle of the test. useful additional informations are provided as box items. Each exercise contains learning objectives. Nishamani Parija and father Shri Mana Govinda Parija without whose encouragement the book would not have been possible. and colour.1 / Running Head 11 Preface Textbook of Practical Microbiology. The book is profusely illustrated with diagrams. medical laboratory technology. Subhash Chandra Parija email: parijasc@vsnl. Important points of the practical experiment are highlighted. I owe a special debt of profound gratitude to my mother late Smt. allied sciences. possible questions with answers are provided and finally. is a performance–based text designed for use by students of medicine. to help students to perform various practicals. I welcome readers views and suggestions for further improvement of the book in future editions. theoretical aspects of the practical. The intent of the book is to provide recent information and explain in detail the routine diagnostic methods performed in a Microbiology laboratory. Textbook of Practical Microbiology consists of 15 learning units. microbiology. Each unit contains many practical exercises. and to learn and apply the knowledge of practical microbiology in clinical medicine.com . and photomicrographs both black and white. A sincere effort is made to provide the essential underlying principles of practical microbiology. Madhuri Parija and Miss Mayuri Parija for their untiring secretarial help towards the preparation of the manuscript. Rajkumar Parija. Subhash Chandra Parija . It is my pleasure to thank my niece Er(Ms) Kukumina Parija. I am very much thankful to Ahuja Publishers. son-in-law Er. daughter-in-law Mrs Smriti Parija. Subhasis Ray. and daughters Dr. New Delhi who have been very supportive of this venture.12 Textbook of Practical Microbiology Acknowledgements I gratefully acknowledge all my colleagues and friends for their valuable advice and assistance in preparation of the manuscript. nephew Er. Textbook of Practical Microbiology UNIT 1 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 . fluorescence microscopy and phase contrast microscopy. dark-field microscopy. Microscopes are of two categories: Light or optical microscopes and Electron microscopes depending upon the principle on which the magnification is based. A microscope may be defined as an optical instrument. (Micro: small. the most important characteristic of microbiology laboratories. for making enlarged or magnified images of minute objects. enables us to see microorganisms and their structures otherwise invisible to the naked eye.400000X. The characteristic morphological studies enabled by the discovery of powerful microscopes. 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. scope: to view).This includes transmission electron microscopy and scanning electron microscopy. which enabled the scientists to study the characteristics more minutely. Later improvements in the compound microscopes were made and Amici discovered oil immersion lens. helped the scientists to classify microorganisms. Light microscopy. The magnitudes attainable by microscopes range 100X. The magnification. . it provides. Microscopes are continuously improved to enable us to have higher magnifications and better resolutions. On the other hand the electron microscopy uses a beam of electrons in place of light waves for visualization of objects . in which the magnification is obtained by a system of optical lenses uses light waves . shape. consisting of a lens or a combination of lenses. He described the size. protozoa and algae.2 UNIT Introduction Microscope is the instrument. movements of bacteria. These findings were later confirmed after the development of compound microscope by Robert Hooke.The light microscopy includes bright field microscopy. The lower end of the tube contains objectives. and e A foot or base upon which the whole instrument rests. 2 Stage. This holds a condenser lens with an inbuilt diaphragm and a holder for light filter and stops. Body and arm: The tube is attached to the microscope by the component of the microscope called the body. body tube and the stage. It consists of : a Main tube. 2 Visualize the cellular morphology from stained slide preparation by using compound microscope. increasing contrast. Foot: The microscope rests firmly on the laboratory bench with the base called foot. A number of objectives of lenses of different magnifications are screwed into the nosepiece of the microscope. Main tube: The main tube primarily holds the objective and eyepiece. . Generally microscopes of this type produce a useful magnification of about 1000x to 2000x. Ordinarily microorganisms do not absorb much light but staining them with a dye greatly increases their light absorbing ability resulting in greater contrast and color differentiation.Textbook of Practical Microbiology 3 LESSON 1 INTRODUCTION Compound Microscope LEARNING OBJECTIVES After completing this practical you will be able to: 1 Become familiar with the principle. Stage: A fixed platform with an opening in the center allows for the passage of light from an illuminating source below to the lens system. Parts of the compound microscope A microscope mainly consists of 1 Microscope stand. d A substage. This may be U-shaped or rectangular. These microscopes are provided by a coiled filament tungsten lamp. This has a standard diameter so that all the eyepieces are interchangeable. Mechanical stage can be moved vertically or horizontally by The commonly used microscope in microbiology laboratory is called compound microscope. c An arm. which may also provide a lifting handle for the microscope. It provides surface for the placement of a slide over the central opening. fungi. At magnifications greater than 2000x. various parts of the compound microscope and its usage in microbiology laboratory. also known as ocular piece. b Body tube. is present at the top of the main tube. The eyepiece is fitted loosely into the upper end of the tube. the microscopic field or area observed is brightly lighted and the objects being studied appear dark because they absorb some of the light. rather than on the object. Closing the aperture of the condenser slightly may aid in detection of certain organisms such as protozoa. In compound microscopy. The body of the microscope and the tube attached to it are supported at the correct height by firm arm. The glowing filaments are prevented from causing glare by focusing their light on the sub-stage condenser. etc. The eyepiece. which supports the main tube. the image becomes hazy. Microscope stand It is the main framework of the microscope. which are screwed into what is known as a revolving nosepiece. Stage can be of fixed or mechanical. because the light will be hitting the edges of the object at a sharper angle. and 3 Microscope optics. Substage: The substage lies immediately below the stage. This is known as Köhler illumination. These objectives can be revolved to increase or decrease the magnification of specimen being examined. the flat side of the mirror should be used. This image is real and can be projected on to a screen. the object is placed between the f and 2f of the objective lens. dark field. The three objectives most commonly used in microbiology laboratory are: (i) A low power objective with focal length 16 mm and magnification 10X. one may find that mirrors are also provided with artificial light. Microscope optics These include objectives. There are four principal types of condensers with respect to correction of optical aberrations. The eyepiece consists of two lenses. Whatever the source of light. The Abbe condenser has two optical lens elements that produce an image of the illuminated field diaphragm that is not sharp and is surrounded by blue and red color at the edges. PRINCIPLE Magnification The purpose of the microscope is to produce an enlarged. The light source A good source of light is needed to examine specimens correctly. a blue filter is placed between the source of illumination and the substage condenser. The condenser adjustment system consists of: a Focusing: It is done by moving the condenser up to down. It should also fill the whole of the back lens of the objective regularly with light. welldefined image of objects too small to be observed with the naked eye. If the object is placed between the f and the lens then an enlarged virtual image which cannot be projected on to a screen is produced. The field lens of the eyepiece . It is carried out by rapid and relatively large movements of the stage. condenser and illuminating source. breadth or diameter but not the area of the object is multiplied. The fine adjustment is carried out when finer focusing is required by using high power (40x) objectives or oil immersion objectives. the concave mirror should be used without the substage condenser. Coarse and fine focusing adjustments The coarse adjustment is required when focusing the specimens with low power (10x) objectives. etc. if not the image will not be clear. (ii) A high power objective with focal length 4 mm and magnification 40X. and a diaphragm between the two lenses. The limit of useful magnification is set by the resolving power. While using electric light. a field lens and an eye lens. With the most powerful lenses. it should fill field of view. In the compound microscope. The degree of enlargement is the magnification or magnifying power and it is expressed as the number of times the length. This may be daylight or electric light. Condenser adjustment Condensers are classified depending on their uses such bright field. Mechanical adjustment of a microscope It is being carried out to focus the specimen examined by the microscope. Stage also contains clips on its surface to hold the slide. The objective produces the primary image. The primary image is real. This adjustment includes coarse and fine focusing adjustments and condenser adjustments. as listed in the table 1-1. (iii) An oil immersion objective with focal length 2 mm and magnification 100X. inverted and magnified. In some other microscopes. which contains the specimen. When daylight is the natural source of light. The next level of condenser correction is split between the aplanatic and achromatic condensers that are corrected exclusively for either spherical (aplanatic) or chromatic (achromatic) optical aberrations. the limit of resolution is about 0. the image is enlarged. eye pieces. phases contrast.2 µm and the greatest useful magnification 1000x or a little less. Table 1-1 Types of condensers Condenser type Abbe Aplanatic Achromatic Aplanatic achromatic Aberrations corrected Spherical Chromatic -----------X ----------X X X Principle involved in the magnification of the object In biconcave lens.4 Compound Microscope means of rack and pinion movement. In such case. Magnification is effected in two stages: the first by the objective lens and the second by the eye-piece lens. including the 2mm oil immersion objective. The total magnification of the microscope can be calculated by multiplying the magnifying power of the objective by that of the eye-piece. b Adjustment of aperture: It is done by opening or closing iris diaphragm. if the object is placed between focal length (f) and 2f. 10 Place a drop of immersion oil on the smear. Resolving power is the ability to reveal two closely adjacent structural details as separate and distinct. 3 Place the slide to be examined on the stage. II Reagents Immersion oil and lens wiping paper.e. until the image comes into view and is sharply focused. As the magnification of the lens increases. The condenser may be raised completely upward for obtaining better illumination. whereas the numerical aperture of the objective lens increases. The greatest possible numerical aperture of a dry lens cannot exceed 1. Actually the highest practical numerical aperture of dry and oil immersion lenses is 0. The condenser contains two lenses that are necessary to produce a maximum numerical aperture. still with the coarse adjustment. Since the intensity of the daylight is an uncontrolled variable. decreases. Illumination Effective illumination is required for efficient magnification and resolving power. The stained smear shows the presence of the bacteria. rack the objective carefully down and using the coarse focusing knob and looking it from the side until the lens is near the slide but not touching it. PROCEDURE 1 Place the microscope on a firm bench so that it does not vibrate. 2 Put on the illumination when using artificial light. It should be preferably away from direct sunlight. the minimum distance between two visible bodies at which they are seen as separate and not in contact with one another. examine for higher magnification. which allows determination of the smallest specimen detail resolvable by the objective and an approximate indication of the depth of field (Box 1-1). Then the eye lens produces the virtual magnified image that is seen by the eye. The greatest resolution in light microscopy is obtained with the shortest wavelength of visible light and an object with maximum numerical aperture. for better visualization of the specimen. BOX 1-1 TERMINOLOGY III Specimen Stained smear on a glass slide. 13 Replace the microscope in its box. rack the objective slowly upward. 6 Focus sharply on the specimen using the fine adjustment. 9 Place the 40x objective in position and examine for higher magnification. moving it by the mechanical stage. expressed quantitatively as microscope's limit of resolution i. artificial light from a tungsten lamp is the most commonly used light source in microscopy. shape. making sure the under side of the slide is completely dry. the distance between the objective lens and slide. Importance of numerical aperture 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. Then while looking through the eyepiece. Then to focus the objective.95 and 1. The condenser may be raised upward for obtaining better illumination. 12 Put off illumination and carefully clean all objective lenses and eyepieces with lens paper. RESULTS AND INTERPRETATION The organisms appear dark against a brightly lighted background. 5 Adjust the illumination in such a way that the illumination of the image is optimum. All modern microscope objectives have the numerical aperture value inscribed on the lens barrel. called working distance. Gram’s reaction arrangement. Use the flat side of the mirror to reflect the light up through the condenser when using artificial light. Focus the 100X objective using the fine adjustment.0. size. 7 Then focus the condenser. 11 Place the oil immersion (100x) objective in position and . REQUIREMENTS I Equipment Compound light microscope. Apochromatic objectives represent high degree of optical perfection used only for critical research and photomicrography due to high expense. Focus the 40x objective using the fine adjustment. 4 Place the low power objective (10x) in position. Begin examination of the slide with 10x objective. The light is passed through the condenser located beneath the stage.Textbook of Practical Microbiology 5 brings the real image to focus at the plane of the diaphragm. 8 Examine the specimen. OBSERVATIONS Carefully observe the morphology and colour (after staining) of bacteria present in the smear and also for uniformity in staining. Sizes of different organisms are summarised in the box 1-1.5 respectively. Numerical aperture The numerical aperture may be defined as the ratio of the diameter of the lens to its focal length. This is within the focal length (f) of the eye lens. Resolution The limit of useful magnification of a microscope is set by its resolving power. ) 1997. Introductory Microbiology. Examination of the slide should always begin with the low power objective (10x). Microscope should be kept away from dust.3 µm to 1µm. 4 Define magnification. After finishing work a cover should be put on the microscope. Evans EGV. London. Taenia saginata. Schreckenbergu PC and Winn Jr.6 Compound Microscope KEY FACTS 1 2 3 4 5 6 One should remember that proper use and care increases the life of a microscope many fold. Medical Microbiology. Eg. Killington RA. USA. BOX 1-2 SIZE OF DIFFERENT ORGANISMS Bacteria Sizes are measured in µm Cocci (spherical shaped bacteria): Size vary from 0. Parvovirus Largest virus measures 300 nm in diameter . numerical aperture and resolution. Smallest virus measures 20 nm in diameter Eg. Color Atlas and Textbook of Diagnostic Microbiology.3 µm x 3.4 µm 8-15 µm 2-30 µm 2-10 µm 3 µm x 5 µm 4-6 µm 4-12 µm Viruses Sizes are measured in nm. rather than on the object. Heritage J. For helminths sizes are variable. Fungus Sizes are measured in µm Yeast like . Show variable sizes .) 2004. moisture and direct sunlight. 3 List the parts of a compound microscope. (McGrow Hill. Janda WM.5 µm to 1 µm. Care must be taken while handling different parts of microscope. Jawetz. (Museum Press. The Microscope. Taenia soluim Nematodes vary in size from 5 mm to even 1 meter in length Eg: Trichinella spiralis. Dracunculus medinesis. Eg: Hymenolepis nana. (Cambridge University Press. Allen SD. Pox virus Parasites For protozoa sizes are measured in µm Most protozoa are around 50 µm in size Balantidium coli is an exception which measures 100 µm or more in size . USA. 5th ed. Koneman EW. In breadth. Butel JS and Morse SA. This is known as Köhler illumination. Staphylococcus aureus Bacilli (rod shaped bacteria): Size vary from 1 µm to 10 µm in length and 0. Eg. London) 1964.) 1996. FURTHER READINGS 1 2 3 4 Brooks GF.appearance 1 2 3 4 5 6 7 Histoplasma capsulatum ___________ Blastomyces dermatitides ___________ Paracoccidioides brasiliensis _______ Sporothrix schenckeii _____________ Candida albicans _________________ Cryptococus neoformans ___________ Chromoblastomycosis _____________ 2. Duddington CL. (Lippincott Williams and Wilkins. 2 What is Köhler illumination? Ans Compound microscopes are provided by a coiled filament tungsten lamp. Attempt should never be made to repair microscope by oneself. 23rd ed. . The glowing filaments are prevented from causing glare by focusing their light on the sub-stage condenser. Melnick and Adelberg. Bacillus anthracis. WC (Eds.).Eg. Sizes are measured ranging from mm to meters Cestodes vary in size from 1mm to several meters in length Mould appearance 1 Aspergillus species ______ 2-5 µm wide hyphae 2 Zygomycetes ____________ 4-5 µm wide hyphae Spherule like appearance 1 Coccidioides immitis ____ 5-60 µm thick walled spherule 2 Rhinosporidium seeberi __ 200-300 µm sporangia VIVA 1 Classify microscopes. slightly raise or lower the condenser to get the bright spot. RESULTS AND INTERPRETATION The wet mount preparation shows motile bacteria. PROCEDURE 1 Take a clean lens wiping paper and carefully clean all the lenses. In dark-ground microscopy. . REQUIREMENTS I Equipments Compound light microscope with dark ground condenser. 11 Record the observations in the note book.Textbook of Practical Microbiology 7 LESSON 2 INTRODUCTION PRINCIPLE Darkground Microscopy LEARNING OBJECTIVES After completing this practical you will be able to: 1 Observe the motility of microorganisms by dark ground microscopy. Hence field appears dark and object is illuminated. hence all rays from the condenser are made to pass outside the objective. OBSERVATIONS Brightly illuminated motile bacteria are observed under dark background. 6 Using the centering screws adjust the light to fall on the center of the microscopic field. focus and examine the slide. and also observe the type of motility of bacteria. 2 Place a drop of oil on the depression present on top lens of the condenser. If a bright spot is not obtained. 10 Observe the entire field for the motile bacteria. II Reagents Cedar wood oil. This can be improved by dark ground microscopy than a compound microscope. lens wiping paper. 8 Remove the high power objective from the light path. the object under examination is illuminated not directly but very obliquely (Box 2-1). 3 Keep the wet mount preparation on the stage. 4 Raise the condenser slowly so that oil touches the bottom of the slide. Visibility of an object by a microscope depends upon contrast between the object and its background. III Specimen Freshly prepared wet mount preparation of bacteria suspension. 5 Place the low power objective in position in the light path and focus. focus and observe the slide. The rays are reflected or diffracted by the object in the specimen. 7 Place the dry high power objective in the light path. Place a drop of oil carefully over the cover slip. 9 Now place the oil immersion objective in the light path. The bright spot of the light must be in the center of the field. This is done by opening the aperture of the condenser completely and inserting a funnel stop below the condenser. London. FURTHER READINGS 1 2 3 4 Brooks GF. the condenser does not allow light to pass directly through the specimen but directs the light to hit the specimen at an oblique angle. USA. Entamoeba histolytica.1 mm thick). such as microorganisms in the specimens will be deflected upward into the objective lens for visualization. BOX 2-1 PRINCIPLE OF DARK GROUND MICROSCOPY Dark ground microscopy involves the alteration of microscopic technique rather than the use of dyes or stains to achieve the contrast. Ans Dark ground microscopy is used commonly for very thin slender bacteria such as spirochetes. All other light that passes through the specimen will miss the objective. Evans EGV. Heritage J. 5th ed. etc. . Introductory Microbiology.) 1996. Koneman EW. Jawetz. Melnick and Adelberg. 23rd ed. 3 The wet mount preparation must be thin and should not be dense. VIVA 1 Describe the principle of the dark ground microscopy. Janda WM.) 1997. The Microscope. Killington RA. thus making the background a dark field. Dark ground microscope also is commonly used for demonstrating motility of the trophozoites of protozoa such as Trichomonas vaginalis. Color Atlas and Textbook of Diagnostic Microbiology. The motility of spirochetes such as treponemes is clearly seen in a dark ground microscope. London) 1964. Allen SD. which are not visible under ordinary illumination. USA.). Schreckenbergu PC and Winn Jr. Medical Microbiology.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. Duddington CL. (Cambridge University Press. WC (Eds.) 2004. (Museum Press. By the dark field method. 2 Oil must be used below and above the slide. Butel JS and Morse SA. (McGrow Hill. 2 List the uses of dark ground microscopy for demonstrating motility. Only light that hits objects. (Lippincott Williams and Wilkins. 7 Determine the value of the calibration factor for the oil immersion objective. REQUIREMENTS I Equipments Compound light microscope with ocular micrometer and stage micrometer II Reagents Cedar wood oil and lens wiping paper. namely. first by . slowly rotate the eyepiece to superimpose the ocular micrometer graduations over those of the stage micrometer. This distance is determined by using a stage micrometer.01 mm) and the corresponding number of divisions on ocular micrometer. which also contains graduations that are 0. The calibration procedure for the ocular micrometer requires that the graduations on both micrometers be superimposed on each other. an ocular micrometer and a stage micrometer. This is accomplished by rotating the ocular lens. A determination is then made of the number of ocular divisions per known distance on the stage micrometer.01 mm apart. 6 Determine the distance on the stage micrometer (number of divisions X 0. 4 Add a drop of immersion oil to the stage micrometer. The ocular micrometer is placed on a circular shelf inside the eyepiece. 2 Place the stage micrometer on the microscope stage and center it over the illumination source. 8 Remove the stage micrometer. The distance between these graduations will vary depending on the objective being used. III Specimen Heat fixed smear of Escherichia coli Measurement of size of bacteria in a microscope can be done by micrometry. Then the size of microorganisms can be determined.Textbook of Practical Microbiology 9 LESSON 3 INTRODUCTION PRINCIPLE Measurement of Microorganisms LEARNING OBJECTIVES After completing this practical you will 1 Measure the size of bacteria using a microscope. First the diameter of the microscopic field must be established by means of optic devices. bring the oil immersion objective into position and focus. 3 With the stage micrometer in clear focus under the lowpower objective. Then the calibration factor for one ocular division is calculated by the formula: Known distance between two lines on stage micrometer One division on ocular micrometer = —————————————— Number of divisions on ocular micrometer PROCEDURE 1 Carefully place the ocular micrometer into the eyepiece. 5 Move the mechanical stage so that a line of the stage micrometer coincides with the line of the ocular micrometer at one end. which contains graduations on its surface. Fix another line on the ocular micrometer that coincides with a line on the stage micrometer. 2 Become familiar with the calibration of an ocular micrometer. counting the number of spaces occupied by the organism and second by multiplying this number by the calculated calibration factor for one ocular division. which determines the size of the field. 2 Describe the principle involved in the measurement of the size of microorganisms by microscopy. 4 Determine the size by multiplying the average with calibration factor.) 2004. Killington RA. VIVA 1 Explain the method for the measurement of the size of a microorganism by microscopy. 23rd ed. (McGrow Hill. (Lippincott Williams and Wilkins. Janda WM. 5th ed. Allen SD. 10 Determine the size determined by multiplying the average by calibration factor. (Cambridge University Press.. 11 Record the observations in the note book. OBSERVATIONS 1 Observe the divisions on ocular micrometer. USA. Melnick and Adelberg.) 1996. FURTHER READINGS 1 2 3 4 Brooks GF. Schreckenbergu PC and Winn Jr. Butel JS and Morse SA.6 µm in breadth.). 2 Expert technician help must be taken for inserting ocular micrometer. Evans EGV.10 Measurement of Microorganisms 9 Calculate the number of ocular divisions occupied by each of three separate bacteria and determine the average. WC (Eds.g. 3 Observe the calibration factor. Duddington CL. Color Atlas and Textbook of Diagnostic Microbiology. KEY FACTS 1 Carefully clean the eyepiece and objective lenses before starting experiment. 2 Observe the calibration of ocular micrometer of oil immersion objective. Klebsiella pneumoniae) with known size is examined for comparison. Introductory Microbiology. Heritage J.5 µm in length and 0. coli is 2. Jawetz. London. . London) 1964. RESULTS AND INTERPRETATION The size of E. 3 There must be proper coincidence between the lines of stage micrometer and ocular micrometer. QUALITY CONTROL A stained smear of standard bacterial strain (e. The Microscope. USA. Medical Microbiology.) 1997. Koneman EW. (Museum Press. by dark ground microscopy. iii. 2 Take a clean coverslip. REQUIREMENTS Hanging drop preparation is one of the easiest method to observe motility in a clinical microbiological laboratory. Spirilla. and also observe natural sizes and shapes of the cells by hanging drop preparation.g. and natural sizes and shapes of the bacteria.g. 5 Invert the slide. Examination of living organisms is useful to observe cell activities. shift the focus to high power (40x) and observe. Vibrio cholerae . III Specimen Log phase broth culture of E. The list of motile and non-motile bacteria are summarized in the table 4-1. locate the edge of the drop. Escherichia coli. unstained state. swarming of the bacteria on a non inhibitory medium ( e. do not lend themselves readily to microscopic examination in a living. Spirochaetes are the examples of bacteria which are motile but without presence of any external flagella . blood agar ) and iii. binary fission. 7 Record the observation in a notebook. 4 Place the cavity slide (cavity down) over the coverslip so that the drop is placed in center. I Equipments Compound light microscope. inoculating loop wire. bacteria can be classified as i. . PRINCIPLE Microorganisms such as bacteria. because of their small size and a refractive index that closely approximates that of water.peritrichous ( fagella arranged all around the cell) e. and iv.g.g Pseudomonas aeruginosa . and also to observe natural sizes and shapes of the cells. Craigie ’s tube method . Bacteria are motile due to the presence of flagella .Textbook of Practical Microbiology 11 LESSON 4 INTRODUCTION Hanging Drop Preparation LEARNING OBJECTIVES After completing this practical you will be able to: 1 Observe motility. 3 Place a drop of broth culture on the coverslip with the help of inoculating loop. and observe under microscope.g. This is carried out by putting a loop full of bacterial suspension on the cover slip and placing it over a cavity slide and observing it under a microscope. Advantage of this method is that by this method live bacteria can be observed. amphitrichous (single flagellum at both the ends) e. QUALITY CONTROL The test wet mount preparation is compared with known motile culture of P. lophotrichous (tufts of flagella at one or both ends ) e. ii. apply paraffin to four corners of coverslip. etc . motility. Salmonella sp . viz. Capillary tube method is a useful method for demonstrating the motility of anaerobic bacteria (Box 4-1).Depending on the location of the attachment of the flagella . 6 First observe under low power (10x). coli PROCEDURE 1 Take a clean grease free cavity slide. ii. monotrichous (single polar flagellum at one end) e. II Reagents and glass wares Normal saline. cavity slides and cover slips. Motility of bacteria can also be demonstrated by i. aeruginosa for appropriate details such as motility. staining tray. 12 Hanging drop Preparation OBSERVATIONS The bacteria showing motility are demonstrated in the hanging drop preparation. Pullorum Vibrio cholerae Aeromonas Pleisiomonas Campylobacter Helicobacter Mobiluncus Pseudomonas Burkholderia pseudomallei Burkholderia cepacia Yersinia enterocolitica Ligionella pneumophila Bordetella parapertusis Bordetella bronchiseptica Spirochaetes Treponema Borrelia Leptospira Listeria Spirillum minus Chromobacterium violaceum Eikenella corrodens Alcaligenes faecalis Non motile bacteria 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Staphylococci Micrococci Streptococci Pneumococcus Meningococcus Corynebacterium diphtheriae Bacillus anthracis Clostridium perfringens Lactobacillus Bifidobacterium Propionibacterium Bacteroides Fusobacterium Leptotrichia Klebsiella Shigella Burkholderia mallei Yersinia spp other than Yersinia enterocolitica Pasteurella multocida Francisella tularensis Haemophilus influenzae Moraxella Gardenella Bordetella pertusis Brucella Mycobacterium Mycoplasma Actinomyces Erysipelothrix Streptobacillus Flavobacterium Calymnotobacterium Cardiobacterium Rickettsia Chlamydiae . A broth culture of anaerobic bacteria is prepared and is made to fill into the open capillary tube by capillary action. under the high power objective of the light microscope for the presence of motile anaerobic bacteria. it is sealed at both ends with clay to maintain anaerobiosis. RESULTS AND INTERPRETATION The wet mount preparation shows motile bacteria. A Robertson’s cooked meat medium or thioglycollate broth culture. BOX 4-1 DEMONSTRATING MOTILITY OF ANAEROBIC BACTERIA Capillary tube method is a useful method for demonstrating the motility of anaerobic organisms. Once the tube is filled up.Gallinarum and S. This movement is usually seen around the axis of bacteria. Brownian movement is not true motility. instead it is exibited due to movement of organism as a result of their collision with water molecules. Table 4-1 Motile and non-motile bacteria Motile bacteria 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Other Bacillus species excluding Bacillus anthracis Clostridium tetani Clostridium botulinum Escherichia coli Serratia Proteus Salmonella except S. The tube is then observed. Note: It is important to differentiate active motility from brownian movement. inoculated and incubated with anaerobic bacteria is taken. Heritage J. WC (Eds. USA. VIVA 1 2 3 4 List other methods to confirm motility of the bacteria. London. (McGrow Hill. FURTHER READINGS 1 2 3 4 Brooks GF. 23rd ed. List a non-flagellated motile bacterium. Mention the bacterial appendage responsible for motility of the bacteria. (Cambridge University Press. List motile and non-motile bacteria. (Museum Press. Evans EGV.) 1997. USA. Schreckenbergu PC and Winn Jr.) 1996. 5th ed. (Lippincott Williams and Wilkins. London) 1964. Janda WM. Butel JS and Morse SA. Koneman EW. Medical Microbiology. Killington RA.Textbook of Practical Microbiology 13 KEY FACTS 1 Always make a thin emulsion of bacterial suspension on the glass slide.) 2004. Jawetz. 2 Drop should not touch the surface of cavity. Introductory Microbiology. . Melnick and Adelberg. The Microscope. Color Atlas and Textbook of Diagnostic Microbiology.). 3 Observe the preparation immediately. Duddington CL. Allen SD. individual cells will be sufficiently far apart on the surface of the agar medium. Isolation of pure cultures is very important in a clinical microbiology laboratory.shaped bent gloss rod while the Petri dish is spun on a turntable. air. Environment. sterile Petri dishes and disinfectant. a rapid qualitative isolation method. 70% ethanol (Spread plate method). L. This method involves pouring a specimen of molten agar (at 45°C) mixture in a Petri dish. REQUIREMENTS I Equipment and labwares Bunsen flame. Mixed culture is a culture that contains more than one kind of microorganisms. The resulting diminution of the population size ensures that. 4 Perform the pour plate inoculation procedure PRINCIPLE Obtaining well discreet colonies and isolation of a pure culture initially require the number of organisms in the inoculum be reduced. Molten trypticase soy agar (Pour plate method). Water bath. test tube rack. II Reagents Sterile trypticase soy agar plates (For all methods). and allowing it to solidify and incubating at 37°C for 48-72 hours. Petri plates. skin .14 LESSON 5 INTRODUCTION Isolation of Pure Cultures LEARNING OBJECTIVES After completing this practical you will be able to: 1 Know various procedures for the separation and isolation of the discrete colonies from a mixed culture or a clinical specimen. etc. Most studies and tests of the physiological. mechanical pipetting device. spread plate technique and pour plate technique. Then the plates are incubated at 37°C for 48-72 hours. inoculating loop and marker pen (For all methods). Streak plate method. The method requires a serial dilution of the mixed culture by means of a pipette. The well discrete colonies grown on the medium are carefully transferred in to a fresh medium by any of pure culture method. These microorganisms may be present in extremely large quantities. turntable. The preparation of pure culture involves not only the isolation of a given microorganism from a mixed natural population but also the maintenance of the isolated individual and its progeny in an artificial environment to which the access of other microorganisms is prevented. following inoculation. involves spreading a loop full of culture over the surface of an agar plate. sterile pipettes. Obtaining well discreet colonies and isolation of a pure culture initially require the number of organisms in the inoculum be reduced. whereas pure culture contains only one kind of microorganisms. 3 Perform the spread plate inoculation procedure. intestinal tract. 2 Perform the streak plate inoculation procedure. L-shape glass rod and sterile beaker (Streak plate method). thermometer. immunological and other characters of bacteria are valid only when made on a pure culture ( Box 5-1) . Pour plate method is a rapid quantitative isolation method. . Careful transfer of well discrete colonies in to a fresh medium by any of pure culture method (generally streak plate method) confirms the purity of isolated colony. (Pour plate method). soil and water also consist of mixed populations of bacteria and other organisms. Pure culture isolation methods include streak plate technique. Many different microbial species normally inhabit various parts of our body such as oral cavity. Spread plate method depends on spread of the bacterial colonies over the surface of a solid agar medium with a sterile. This confirms the purity of isolated colony. The microbial population in our environment is large and complex. 5 Remove a tube from the water bath and check the temperature of molten agar medium (45°C). 2 Note the results and colony morphology of different colonies. exudates. with the bent portion of the rod pointing downward to prevent the burning alcohol from running down to arm. 10 Transfer aseptically the single well-isolated colony on to the surface of other agar plate. Allow the agar to solidify. For spread plate method 1 Take 2 trypticase soy agar plates and mark as test and control. 9 Carefully observe the colony morphology of different colonies. 9 Remove the agar plate from turntable. For pour plate method 1 Check the control plates for isolated similar looking colonies only. Note: Flaming of loop can be decided based upon the turbidity of the sample. 48 and 72 hours after first incubation. transfer 0. 8 Stop the turntable. colony to a fresh agar plate with the help of sterile loop. 2 Take an inoculating loop and flame to deep red and cool. 48 and 72 hours after first incubation. 3 Charge the inoculating loop with the specimen to be cultured. QUALITY CONTROL Known mixed culture containing standard strains. 8 Incubate all plates in an inverted position for up to 48-72 hours at 37°C. wipe the outer surface aseptically. 3 With a sterile pipette. incubate the plates in inverted position at 37°C for 48-72 hours. mix the contents well and pour the mixture in to control Petri plate. 7 Repeat the same in area 3 and area 4. 3. . For pour plate method PROCEDURE For streak plate method 1 Take a sterile trypticase soy agar plate and label on the back of the agar surface 1. then spread over a small area at the periphery near the flame. 9 Select a well-discreet colony from each type and aseptically transfer to a fresh separate agar plate and repeat the streak plate method and incubate the plates at 37°C for 48-72 hours. 7 Remove the glass rod. 4 Transfer the loop full of the specimen (control and test) on to the surface of a well-dried agar plate (area 1). 12 Incubate all plates at 37°C for 48. 4 Remove the glass rod from ethanol and pass it through the Bunsen burner flame. Repeat the spread plate procedure. 2. if so discard the plate and repeat the experiment. Allow the agar to solidify. 3 Then add mixed cultures to appropriate tube. For spread plate method 1 Observe the control and test plates after 24. 11 After incubation confirm the purity of isolated colony. 5 Remove the Petri dish cover and spin the turntable. Allow the alcohol to burn off the rod completely. 6 While the turntable is spinning. mix the contents well by rolling the tube between the two hands carefully and pour the mixture in to test Petri plate. dip in alcohol and re flame. stool and other clinical specimens. Replace the cover. Replace the cover on agar plate. 6 Wipe the outside surface dry with a tissue paper. approximately 1 cm away from the edge of the plate. 10 Carefully observe the colonies and transfer a well-discreet OBSERVATIONS For streak plate method 1 Observe all the plates for discrete colonies after 24. 2 Take L shaped 2 glass rods. in an autoclave or by boiling and cool the molten agar tubes and maintain in a water bath at 45°C. 2 Search the entire plate for colonies present outside the streak lines. 10 After incubation confirm the purity of isolated organism. add 1 ml of control culture. 4 at four corners. lightly touch the sterile bent rod to the surface of the agar and move it back and forth. dip the lower bent portion into 70% ethanol. 6 Turn the Petri dish 90° and touch the loop to a corner of the culture in the area 1 and drag it several times across the agar in area 2. Cool the rod for 10-15 seconds. and aseptically add 1 ml of test culture. 4 Take two sterile dry Petri plates and label as test and control. 11 Re flame and cool the loop and repeat the same for all colony types. and incubate in an inverted position at 37°C for 48-72 hours.1 ml of culture from tube 1 to the first agar plate that has been placed on the turntable. 7 Take the second tube. 5 Re flame and cool the loop. 8 After solidifying the agar.72 hours. each containing approximately 20 ml of medium. 2 Take 2 sterile test tubes and label as test and control. 1 Liquify 2 agar deep tubes.Textbook of Practical Microbiology 15 III Specimen Mixed bacterial culture (24-48 hour nutrient broth cultures). discrete colony of all types of colonies (after both incubations) from the test plates. 3 Preserve the colonies for further characterization. 2 Note the colony characters of well. 2 What are the uses of pure cultures in a microbiology laboratory? Ans. 2 Note the colony characters of well. 3 List the selective methods used in the isolation of pure cultures. Write their principles. For spread plate method 1 Check the control plates for isolated same colonies only. Type the isolates. 3 Preserve the colonies for further characterization. Ans. For pour plate method 1 Check the control plates for isolated same colonies only. 3 Preserve the colonies for further characterization. 2 Note the colony characters of well. Determine antibiotic sensitivity pattern. .16 Isolation of Pure Cultures 2 Note the colony characters of well. Selective methods which help in the isolation of pure culture include: Chemical methods Use of a special carbon or nitrogen source Use of dilute media Use of inhibitory or toxic chemicals Physical methods Heat treatment Incubation temperature pH of the medium Biological methods Animal experiments 4 List the pure culture methods. which are present only on streak lines (after both incubations). Estimate viable counts.discrete colony of all types of colonies (after both incubations). 3 Preserve the colonies for further characterization. In the microbiology laboratory pure culture are used to: Isolate bacteria in pure culture. Demonstrate their properties. VIVA 1 Define pure culture. RESULTS AND INTERPRETATION For streak plate method 1 Check the control plates for isolated similar looking colonies only. Obtain sufficient growth for preparation of antigens and for other tests.discrete colony of all types of colonies.discrete colony of all types of colonies (after both incubations). Maintain stock culture. Medical Microbiology. 3 The loop should be properly sterilized and cooled. 5 The exposure time of agar surface to the atmosphere should be minimized.) 2004. London. Duddington CL. 4 Agar plates must be incubated at inverted position. (Lippincott Williams and Wilkins. Allen SD.) 1996. 7 Loop should not touch the wall of the Petri dish any time. 5th ed. Color Atlas and Textbook of Diagnostic Microbiology. London) 1964. 2 Whole procedure must be done near the flame and inside a laminar flow inoculation chamber. USA. Janda WM. (Cambridge University Press.Textbook of Practical Microbiology 17 BOX 5-1 TERMINOLOGY A culture that contains only one type of microorganisms is known as pure culture. Melnick and Adelberg. KEY FACTS 1 Surfaces of agar plates must be dry. Butel JS and Morse SA. Pure culture is also called axenic culture. Koneman EW. it is termed as a clone. Jawetz. (Museum Press. Separation of particular microorganism from a mixed population that exists in nature is called isolation. (McGrow Hill. . 10 Occurrence of air bubbles while pouring the medium-culture mixture into plates should be avoided FURTHER READINGS 1 2 3 4 Brooks GF. Heritage J. Killington RA. The descendants of a single isolation in pure culture comprise a strain. 8 Loop should not enter any other area except the specified in each step. Evans EGV. If a strain is developed from a single parent cell. Introductory Microbiology. WC (Eds. The Microscope. USA.). Schreckenbergu PC and Winn Jr.) 1997. 9 Contamination while transferring material from one tube to another should be avoided. 23rd ed. 18 . Textbook of Practical Microbiology UNIT 19 II Bacterial Staining Lesson 6 Lesson 7 Lesson 8 Lesson 9 Simple Staining Gram’s Staining Acid Fast Staining Albert’s Staining Lesson 10 Capsule Staining Lesson 11 Spore Staining Lesson 12 Negative Staining . The stain maybe ripened quickly by addition of 1% potassium carbonate to the stain. Preparation of Loeffler’s methylene blue stain: This is prepared by dissolving 30 ml of saturated solution of methylene blue in alcohol.01%). 2 Compare various morphological forms and arrangement of bacterial cells. II Reagents and glass wares Loeffler’s methylene blue. The commonly used simple stains are the basic stains such as methylene blue. and imparts same colour to all bacteria. polychrome methylene blue. The slow oxidation of the methylene blue forms a violet compound that gives the stain its polychrome properties. The ripening takes 12 months or more to complete. approximately 0. CSF. PROCEDURE 1 Make a thin exudate smear on a slide. In simple staining. 5 Observe the smear first under low power (10x) objective. OBSERVATIONS 1 Blue coloured spherical cells. 4 Wash the stained smear with water and air dry it. 3 Pour Loeffler’s methylene blue over the smear and allow it to stand for 3 minutes. III Specimen Pus. 6 Record the observations in the note book. carbol fuchsin.20 LESSON 6 INTRODUCTION PRINCIPLE Simple Staining LEARNING OBJECTIVES After completing this practical you will be able to: 1 Stain smears by simple staining method.1 ml of potassium hydroxide (0. Basic stains with chromophore are used in simple staining methods. REQUIREMENTS I Equipments Compound light microscope. . Preparation of polychrome methylene blue: This is prepared by allowing Loeffler’s methylene blue to ‘ripen’ slowly and shaking it at intervals to aerate the contents thoroughly. aspirations. Simple staining employs staining of bacterial smears with a single staining reagent. crystal violet and carbol fuchsin. water. 2 Heat fixes the smear by passing the slide 2–3 times gently over the Bunsen flame with the smear side up. a red-purple hue.5–1 µm size seen arranged in clusters (Fig. in 100 ml of distilled water containing 0. and then under oil immersion (100x) objective. the bacterial smear is stained with a single reagent. QUALITY CONTROL The morphology of the cellular components and bacteria in the known methylene blue stained smear (positive control smears) are compared with that of the blue stained structures in the test smear. They provide good colour contrast and impart the same colour to the stained bacteria (Box 6-1).Bacterial nucleic acids and certain cell wall components carry a negative charge that strongly attracts and binds to the cationic positively charged chromogen. 6-1). microscopic glass slides and Bunsen burner. This stain gives the acidic cell structures of the bacteria. 3 Mention the uses of methylene blue staining in a diagnostic microbiology laboratory. 4 List the uses of polychrome methylene blue staining. Gram’s stain and acid-fast stain are two examples of differential stains. But morphology of leucocytes and other cells are stained less clearly by differential stains. VIVA 1 Describe differences between a simple stain and a differential stain. Polychrome methylene blue: It demonstrates the presence of bacteria in smear or wet mount preparation in addition to the morphology of polymorphs and lymphocytes. KEY FACTS 1. as shown in the Mc Fadyean reaction. b. Smears are required to be stained for 3 minutes and tissues for 5 minutes by simple Loeffler’s methylene blue stain. 5. FIGURE 6-1. Differential stains are those stains that contain two different coloured dyes. blue coloured bacilli and the blue coloured multilobed pus cells. Polychrome methylene blue staining is employed for demonstration of the acidic capsular structure of anthrax bacillus. lymphocytes and other cells more clearly than the Gram stain. It is also employed to demonstrate the acidic capsular material of the anthrax bacilli as seen in the Mc Fadyean reaction. Ans. and these when used impart the same colour to all the bacteria in a stained smear. and c. BOX 6-1 SIMPLE STAINS AND THEIR USES IN MICROBIOLOGY LABORATORY Loeffler’s methylene blue: It demonstrates morphology of polymorphs. lymphocytes and other cells more clearly than stronger stains such as Gram’s stain. . Methylene blue staining is an example of simple staining. approximately 3–5 µm length and 0. Methylene blue is an example of a simple stain. polychrome methylene blue and dilute carbol fuchsin are the examples of other simple stains. 3 Blue coloured round cells with darkly stained multilobed nucleus in pus cells are also seen.Textbook of Practical Microbiology 21 2 Blue coloured elongated rod shaped cells. Ans.5 µm breadth seen. 2 Name other simple stains.2–1. 3. The nature of cellular contents in exudates. Methylene blue staining is used to show a. Methylene blue stained smear of Staphylococcus aureus. It shows the morphology of leucocytes and other cells more clearly. Loeffler’s methylene blue shows the characteristic morphology of polymorphs. Simple staining method uses a single reagent that imparts the same colour to all bacteria and cellular organelles. 4. The presence of bacteria in a smear or wet mount preparation. Ans. RESULTS AND INTERPRETATION The stained smear contains mixture of methylene blue stained cocci in clusters (Fig. Ans. 2. thus differentiating different groups of bacteria. The characteristic morphology of polymorphs. Simple staining method shows the presence of bacteria and cellular contents in exudate smears and also in wet mount preparations. Simple stains are those that contain only a single-coloured dye. x 1000. 6-1). lymphocytes and other cells more clearly. Loeffler’s methylene blue. hence they impart different colours to different bacteria or bacterial structures. Schreckenbergu PC and Winn Jr.) 1997. Sahm DF and Weissfeld AS. Allen SD. (The CV Mosby Company. (Lippincott Williams and Wilkins. Bailey and Scott’s Diagnostic Microbiology. Manual of Basic Techniques for a Health Laboratory. Killington RA. Chapter 4: Staining Techniques.22 Simple Staining FURTHER READINGS 1 Evans EGV.) 1996. 3 Koneman EW. Louis) 2002. (Cambridge University Press. Heritage J. 1980. 11 th ed. Janda WM. Guidelines on Standard Operating Procedures for Microbiology. . 5 th ed. 2 Forbes BA. WC. 5 WHO. Color Atlas and Textbook of Diagnostic Microbiology. Introductory Microbiology. St. 4 WHO. Solution B is made by dissolving 0. . Gram positive bacteria have more acidic protoplasm and hence bind to the basic dye more firmly. PRINCIPLE The Gram reaction is dependent on permeability of the bacterial cell wall and cytoplasmic membrane.Textbook of Practical Microbiology 23 LESSON 7 INTRODUCTION Gram’s Staining LEARNING OBJECTIVES After completing this practical you will be able to: 1 Stain smears by Gram’s staining method. 60%–90% of the Gram positive bacterial cell wall is made up of peptidoglycan and interwoven teichoic acid. lipopolysaccharide. Then 750 ml of distilled water is added and final volume is made up to 1000 ml. Then both solution A and B are mixed well. They stain differently because of the fundamental differences in the structure of their cell walls. Also. Preparation of methyl violet stain: This is prepared by dissolving two solutions: solutions A and B. and 1% safranine or dilute carbol fuchsin (counter stain). marker pen. the dye-iodine complex is not retained within the cell and permeates out of it during the process of decolourisation.8 grams of ammonium oxalate in the above solution A. while only 10%–20% of Gram negative bacterial cell wall is composed of peptidoglycan. Gram’s iodine (mordant). II Reagents and glass wares Bunsen flame and loop wire. Hence when a counter stain is added. 2 Differentiate between Gram positive and Gram negative bacteria. Preparation of 1% safranine: This is prepared by dissolving 1 gram of safranine in 100 ml of distilled water. Solution A is made by dissolving completely 2 grams of crystal violet in 120 ml of ethyl alcohol. Also the alcohol/acetone mixture which act as decolourizing agent. Approximately. cause dehydration of the multi-layered peptidoglycan of the cell wall.This causes decreasing of the space between the molecules causing the cell wall to trap the crystal violet iodine complex within the cell. Preparation of Gram’s iodine: This is made by first dissolving 20 grams of potassium iodide in 250 ml of distilled water and then 10 grams of iodine is further added to it with dissolution. lipoproteins and other proteins. being a lipid solvent. 95% ethanol (decolourising agent). while Gram negative bacteria stain pink when subjected to Gram staining. Different modifications of Gram’s stainings are summarized in the box7-1. they take up the colour of the stain and appear pink. Hence the Gram positive bacteria do not get decolourised and retain primary dye appearing violet. the crystal violet dye-iodine complex combines to form a larger molecule which precipitates within the cell. The Gram negative cell wall is also surrounded by an outer membrane composed of phospholipids. methyl violet (basic dye). forming 2–3 layers. the alcohol. In the case of Gram-negative bacteria. REQUIREMENTS I Equipments Compound light microscope. Gram’s stain was originally devised by histologist Christian Gram (1884) as a method of staining bacteria in tissues. clean grease free slides. In Gram positive bacteria. to the dye-iodine complex. As a result. Gram positive bacteria stain purple. dissolves the outer lipopolysaccharide membrane of the cell wall and also damages the cytoplasmic membrane to which the peptidoglycan is attached. The appearance of purple coloured Gram positive bacteria and pink coloured Gram negative bacteria in the control smears indicate proper staining technique and stained test smear is compared with it. 5 Decolourise the smear with 95% alcohol. 3 Cover the smear with Gram’s iodine (mordant) and allow it to stand for one minute. Various uses of Gram’s staining are summarized in the box 7-2. Streptococcus species Lanceolate shaped in pairs: eg. 5 Heat fix the smear while holding the slide at one end. Escherichia coli Klebsiella pneumoniae Comma shaped. OBSERVATION Presence of 0. PROCEDURE 1 Cover the smear with the methyl violet (basic dye). smears of Staphylococcus aureus (Gram positive bacteria) and Escherichia coli (Gram negative bacteria) are made.. spread the cell suspension into a thin area. Note: List of Gram positive and Gram negative bacteria are summarized in the table 7-1. Bacillus anthracis with spores eg. 2 Take one or two loopfuls of the bacterial cell suspension and place on the clean slide with a bacteriological loop. QUALITY CONTROL On the same slide.85% saline and place it on the centre of the slide. Allow it to stand for one minute. Hold the slide with hand on one corner in a slant position and add drop by drop of absolute alcohol till a faint colour comes out of the smear. 6 Rinse the smear again gently under tap water. 7-2).. 7 Cover the smear with dilute carbol fuchsin (counter stain) for 30 seconds to 1 minute. Staphylococcus species) and Gram negative bacilli (eg.24 Gram’s Staining III Specimen Preparation of bacterial smear: (From liquid culture) 1 Take clean. and by quickly passing the smear over the flame of Bunsen burner two to three times. 4 Allow the smear to air dry. and by quickly passing the smear over the flame of Bunsen burner two to three times. 4 Allow the smear to air dry. 3 With a straight wire touch the surface of a well isolated colony from the solid media and emulsify in the saline drop forming a thin film. 10 Record the observations in the note book. 7-1) and 1–2 mm sized pink coloured rod shaped bacteria seen ( Fig. The slide with control and test smears is stained by Gram’s staining. coli). Note: Decolourisation is a critical procedure. 3 Then with circular movement of the loop. 9 Observe the smear first under low power (10x) objective. RESULTS AND INTERPRETATION The stained smear contains mixture of Gram positive cocci arranged in grape-like clusters (eg. and then under oil immersion (100x) objective. Vibrio cholerae . Preparation of bacterial smear: (From solid media) 1 Take clean and grease free glass slides for making the smears.5–1 µm sized purple coloured spherical bacteria arranged in clusters ( Fig. Staphylococcus aureus In chains: eg. curved Gram negative bacilli eg. 5 Heat fix the smear while holding the slide at one end. Note: Wipe the slide with a cotton swab dipped in alcohol and then holding its end with forceps roast it free from grease by passing 6 – 12 times through a blue Bunsen flame.Neisseria gonorrhoea Neisseria meningitidis Gram negative bacilli eg. 4 Rinse the smear again gently under tap water. Clostridium species Gram negative bacteria Gram negative cocci In pairs: eg. Streptococcus pneumoniae In pairs and short chains: eg. and grease free glass slides for making the smears. Enterococcus species Gram positive bacilli In chains eg. Table 7-1 List of Gram positive and Gram negative bacteria Gram positive bacteria Gram positive cocci In clusters: eg. 2 Rinse the smear gently under tap water. E. 2 Take a loopful of 0. 8 Rinse the smear again gently under tap water and air dry it. x 1000.Textbook of Practical Microbiology 25 FIGURE 7-1 Gram stained smear of Staphylococcus aureus. BOX: 7-1 MODIFICATIONS OF GRAM’S STAINING 1 Kopeloff and Beerman’s Gram method This method uses acetone as a decolouriser. Clostridium difficile. 3 Preston and Morrell’s Gram method This method uses iodine-acetone as decolouriser giving good results without the need for careful timing of the decolourisation step.000 PMN excreted into urine/hour are likely to be infected. Presence of at least 1 organism/ oil immersion field correlates with significant bacteriuria ( > 105 cfu/ml). 2 Stool specimens Useful to detect Candida. pharyngitis and impetigo. x 1000. 4 Pus specimen Useful to detect Staphylococcus aureus in pus from abscesses. Gram negative rods in pus from cases of chronic otitis media and post operative wound infections. BOX: 7-2 USES OF GRAM’S STAINING 1 Urine specimens Identifies urine specimens that contain bacteria greater than 105 cfu/ml of urine. FIGURE 7-2 Gram stained smear of Gram negative bacilli. . 2 Jensen’s Gram method for smears This method uses alcohol as decolouriser and weak neutral red as counter stain. Presence of polymorphonuclear (PMN’s) in uncentrifuged urine correlates with number of PMN’s excreted per hour. In patients with >400. This modification is recommended for examination of smears for gonococci and meningococci. Streptococcus species in exudates from tonsillitis. 3 Sputum specimens Useful to detect Streptococcus pneumoniae. 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. Other examples of counterstains are basic fuchsin and neutral red. Chapter 4: Staining Techniques. or action of autolytic enzymes. (The CV Mosby Company. “Gram variable’ bacteria are those Gram positive bacteria that have lost their cell wall integrity because of antibiotic treatment. Describe differences between a Gram positive and Gram negative cell wall. Differential stain is defined as those that contain two different coloured dyes. Ans. old age. Guidelines on Standard Operating Procedures for Microbiology. The other differential stain that can be used for staining is acid fast stain and Albert’s stain. Louis) 2002. 5 WHO. Bailey and Scott’s Diagnostic Microbiology. 2 Gram staining differentiates bacteria into two categories: purple coloured Gram positive and pink coloured Gram negative bacteria. 3 Koneman EW. 5th ed. Color Atlas and Textbook of Diagnostic Microbiology. These changes allow crystal violet to come out of the cell wall during process of decolourizing resulting in some cells staining pink and others staining purple.26 Gram’s Staining KEY FACTS 1 Gram staining is a differential stain. ii) Teichoic acid is present in Gram positive cell wall while it is absent in Gram negative cell wall. Manual of Basic Techniques for a Health Laboratory. 5 Give 2 examples each of Gram positive and Gram negative cocci and bacilli. Other examples of decolourizing agents are acetone. 6 What are ‘Gram variable’ bacteria? Ans. VIVA 1 Define differential stain. acetone. 1980. Ans. (Lippincott Williams and Wilkins. 3 Tissue cells. 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. Gram’s stain is said to be a differential stain because it differentiates bacteria as Gram positive bacteria (appears violet) and Gram negative bacteria (appears pink). cultures more than 24 hours old can lose ability to retain dye-iodine complex and appear pink coloured Gram-negative.) 1996. Heritage J. 2 Forbes BA. Janda WM. 95% ethanol. 3. different structures will take up different colours and can be differentiated. iii) Lipopolysaccharide is absent in Gram positive cell wall and present in Gram negative cell wall. WC. leucocytes and the debris of inflammatory exudates all stain pink in Gram’s stained smears.e. Introductory Microbiology. which may result in a Gram-positive bacteria turning as Gram negative. Killington RA. 6 The age of culture can also influence the Gram stain reaction i. penicillin. 4 Gram positive bacteria have a thicker and denser peptidoglycan layer in the cell walls. which make them less permeable to the stain. . (Cambridge University Press. 7 Give other examples of decolourizing agents and counter stains. FURTHER READINGS 1 Evans EGV. most commonly employed for diagnostic identification of bacteria in clinical specimens. What are the other differential stains used for staining? Ans. Schreckenbergu PC and Winn Jr. The conditions that can result in Gram positive bacteria appearing Gram negative are: i) Excess decolourisation by ethanol/ acetone. changes in the pH and aged cultures. 4 WHO.) 1997. Sahm DF and Weissfeld AS. Allen SD. 11 th ed. 4 Describe the conditions. as it can cause in Gram positive bacteria losing the dye-iodine complex and appearing Gram negative. Ans. ii) Loss of cell wall due to action of lysozyme. 2 State why Gram’s stain is said to be a differential stain? Ans. St.alcohol and iodine-acetone. such that when the staining is completed. loop wire. slide rack. The degree of acid fastness varies in different bacteria. about 50 ml at a time. Great care must be taken to avoid spilling the acid on skin. the stain cannot be easily removed. I Equipments Compound light microscope. glass slides. The Ziehl-Neelsen acid-fast staining method has proved to be most useful for staining acid fast bacilli belonging to the genus Mycobacterium especially Mycobacterium tuberculosis and Mycobacterium leprae. II Reagents and glass wares Bunsen flame/ torch soaked in methylated spirit. The ordinary aniline dye solutions do not readily penetrate the substance of the tubercle bacillus and therefore is unsuitable for staining. The non-acid fast bacilli readily absorb the colour of the counter . Once stained. Note: The acid must be added to the water. 2 Become familiar with the chemical basis of the acid-fast stain stain (methylene blue) appearing blue. 50 ml of 95% alcohol or 100% ethanol is added to the solution and mixed thoroughly. REQUIREMENTS The acid fast staining method is a modification of Ehrlich’s (1882) method. The 200ml concentrated sulphuric acid (about 98% or 1. and Loeffler’s methylene blue (counter stain). The mixture becomes hot. The flask containing solution is kept over a boiling water-bath for about 5 minutes. In this staining method. Remainder of acid is added in same manner. These acid fast bacilli have a high concentration of the lipid-mycolic acid in their cell walls. clothing or elsewhere. are considered non-acid fast. The tubercle bacilli resist the decolourizing action of acid-alcohol which confers acid fastness to the bacteria. Preparation of 95% alcohol : This is prepared by adding 95 ml of ethanol and adding water to it to make 100ml. Different modifications of acid-fast staining is summarized in the box 8-1. which are easily decolourised by acid-alcohol. When stained they appear pink against a blue background. strong carbol fuchsin. Preparation of strong carbol fuchsin: This solution is prepared by dissolving 5 grams basic fuchsin powder in 25 grams crystalline phenol by placing them in a 1 litre flask. When the solution is complete. PRINCIPLE Acid fastness of acid-fast bacilli is attributed to the presence of large quantities of unsaponifiable wax fraction called mycolic acid in their cell wall and also the intactness of the cell wall. Ehrlich suggested the method for the differential staining of tubercle bacilli and other acid-fast bacilli with anilinegentian violet followed by strong nitric acid.835g / ml)) is poured slowly down the side of the flask into the water. and also for Nocardia. application of heat helps the dye (a powerful staining solution containing carbol fuchsin and phenol) to penetrate the tubercle bacillus. Preparation of 20% sulphuric acid : 800ml of water is collected in a large flask. Then 500ml of distilled water is added to it and the mixture is filtered before use. acid-alcohol (3 ml HCl + 97 ml ethanol) (decolourising agent). It is dangerous to add the water to the acid. The other microorganisms.Textbook of Practical Microbiology 27 LESSON 8 INTRODUCTION Acid-Fast Staining LEARNING OBJECTIVES After completing this practical you will be able to : 1 Stain smears by acid-fast staining method. shaking the contents from time to time. while the acid-fast cells retain the red colour of primary stain (carbol fuchsin). 5 Cover the smear with 20% sulphuric acid for at least 10 minutes for decolourisation. RESULTS AND INTERPRETATION The stained smear contains pink coloured acid fast bacilli seen among the blue coloured multilobed pus cells. together with grading of the positive smear. Note: Decolourisation with 95% alcohol for 2 minutes is only optional and may be omitted. 6 Wash the slides thoroughly with water to remove all traces of acid. heat the slides from below intermittently by Bunsen flame or torch soaked in methylated spirit without boiling the solution.3 µm sized pink coloured slender rod shaped structures are seen with curved ends. The smear is positive for acid fast bacilli. FIGURE 8-1 Acid fast stained smear of Mycobacterium tuberculosis. Probably.28 Acid Fast Staining Preparation of acid-alcohol decolouriser : This solution contains 75 ml concentrated hydrochloric acid (HCl) and 25 ml of industrial methylated spirit. x 1000 . With appropriate staining. Then hydrochloric acid is added slowly and the top of the flask is covered to stop the fumes from escaping. before declaring the smear negative. PROCEDURE 1 Heat fixes the smears by passing the slide 2–3 times gently over the flame with the smear side up. 9 Observe the smear first under low power (10x) objective. Note: List of other acid–fast structures are provided in the table 8-1. until the steam rises. 7 Cover the smear with Loeffler’s methylene blue for 15–20 seconds. Do not allow the stain to dry on the slide. It is left for 10 minutes. 8-1). 3 During this period. The final concentration of HCl is 3%. Findings are recorded. and if necessary add more carbol fuchsin to cover the smear. Note: Frequently examined specimens for the detection of Mycobacterium tuberculosis are summarized in the box 8-2. and then under oil immersion (100x) objective. Note: The smear should be examined following a zig-zag pattern for at least 10 minutes or 300 fields. Methylated spirit is poured into a large flask. the smear contains Mycobacterium tuberculosis (Fig. The flask is placed in 5–8 cmm of cold water in the sink. FIGURE 8-2 Acid fast stained smear of Mycobacterium leprae. 2 Put the smears on a slide rack and cover the smears with strong carbol fuchsin. and are scattered amidst blue coloured round cells with darkly stained multilobed nucleus. the acid-fast bacillus appears pink against blue background of pus cells. Allow it to stain for 5 minutes. 10 Record the observations in the note book. QUALITY CONTROL A positive control sputum smear from a known case of tuberculosis patient. x 1000. 4 Rinse the smears gently under tap water. OBSERVATION Presence of 3 x 0. stained with Z-N stain is compared with the stained test smear for appropriate morphology and staining appearance. 8 Rinse the smears again under tap water and air dry it. III Specimen Sputum smear positive for tubercle bacilli / culture smear of Mycobacterium species. Allow the smear to be air dried. It is then decanted into a labeled bottle for use. 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+ . Bronchial or laryngeal washings. Liver biopsy. 2 1% sulphuric acid is used as a decolourizing agent for staining Nocardia species. BOX 8-2 FREQUENTLY EXAMINED SPECIMENS FOR THE DETECTION OF MYCOBACTERIUM TUBERCULOSIS Pulmonary tuberculosis Sputum. Gastric lavage (when sputum is swallowed as in children).25% sulphuric acid is used as a decolourizing agent for staining spores. 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. Cryptosporidium and Isospora oocysts (Kinyoun’s modification of acid-fast stain). At the end of the process of decolourisation by sulphuric acid. Miliary tuberculosis Bone marrow. Acid fast bacilli appear pink coloured in stained smears. Renal tuberculosis Urine. 3 0. the smear will appear faintly pink. 8-2) Mycobacterium smegmatis Mycobacterium fortuitum Nocardia asteroids Nocardia brasiliensis Nocardia caviae Parasites Cryptosporidium parvum Cyclospora cayetanensis Isospora belli Fungi Fungal spores Other structures Spermatozoa head BOX 8-1 DIFFERENT MODIFICATIONS OF ACID FAST STAIN AND THEIR USES 1 5% sulphuric acid is used as a decolourizing agent for staining Mycobacterium leprae.Textbook of Practical Microbiology 29 Table 8-1 List of acid –fast structures Bacteria Mycobacterium tuberculosis ( Fig. Tuberculous meningitis Cerebrospinal fluid. 8-1) Mycobacterium leprae ( Fig. KEY FACTS 1 2 3 4 Ziehl-Neelsen stain is intended for the differential staining of tubercle bacilli from other acid fast bacilli. WC. 4 WHO. (Cambridge University Press. Chapter 4: Staining Techniques. Sahm DF and Weissfeld AS. Allen SD. Thus when acid is added as a decolouriser. Heritage J. Some bacteria are acid-fast because their thick waxy cell wall is made up of long chain fatty (mycolic) acids. 3 Koneman EW. Color Atlas and Textbook of Diagnostic Microbiology. 3 How do you grade a positive sputum smear? 4. 11 th ed. St. Schreckenbergu PC and Winn Jr.) 1996. 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. List different modifications of acid fast staining and their uses. . even with acid-alcohol decolourisers. these acid fast bacteria retain the dye because the dye is more soluble in the cytoplasm compared to sulphuric acid. By this method acid fast bacteria fluoresces bright yellow or orange against a greenish background. FURTHER READINGS 1 Evans EGV.) 1997. (The CV Mosby Company. Why are some bacteria acid fast? Ans. 5 WHO. These mycolic acids render the cells resistant to decolourisation.30 Acid Fast Staining VIVA 1. Guidelines on Standard Operating Procedures for Microbiology. 1980. List the acid fast organisms. What are the various specimens obtained in the laboratory for the diagnosis of tuberculosis? 6. List another method that can be used for detecting acid fast bacteria in a smear? Ans. 5th ed. 2 Forbes BA. Introductory Microbiology. Manual of Basic Techniques for a Health Laboratory. 2. Janda WM. Louis) 2002. 5. Bailey and Scott’s Diagnostic Microbiology. Killington RA. (Lippincott Williams and Wilkins. Preparation of Albert’s stain II: Albert’s II (also known as Albert’s iodine) is composed of 6 gram iodine. they stain metachromatically. loop wire. These granules are clearly demonstrated best by special stains such as Albert’s. the bacilli appear green with bluish-black metachromatic granules. Allow it to act for 1 minute. 6 Observe the smear first under low power (10x) objective.5 grams toluidine blue. These granules are known as Volutin granules. and appear reddish purple in colour. and then under oil immersion (100x) objective. refractile bodies within the bacterial cytoplasm.Textbook of Practical Microbiology 31 LESSON 9 INTRODUCTION Albert’s Staining LEARNING OBJECTIVES After completing this practical you will be able to: 1 Stain smears by Albert’s staining to demonstrate the presence of metachromatic granules in the diphtheria bacillus. 9 gram potassium iodide and 900 ml distilled water. Neisser’s or Puch’s stain. Then 290 ml of distilled water is added and final volume is made up to 300 ml. Albert’s stain I and II. diphtheriae. granules tend to stain more strongly than the rest of the bacterium. while the diphtheria bacillus appears green due to malachite green present in Albert I reagent. II Reagents and glass wares Bunsen flame. The stain is then allowed to stand for one day and then filtered. Preparation of Albert’s stain I: This stain is composed of 1. The solution is made by first dissolving 2 gram potassium iodide in 10 ml distilled water and then 1 gram of iodine is further added to it with dissolution. These granules are made up of polymetaphosphate and are seen in unstained wet preparations as round. III Specimen Exudate smear collected directly from pseudomembrane obtained using a throat swab / culture smear of C. polar bodies or metachromatic granules. The diphtheria bacillus. 2 Put the smears on a slide rack and cover the smears with Albert’s stain I. PRINCIPLE The granules present in the diphtheria bacilli exhibit metachromasia property and hence appear bluish-black coloured when stained with the toluidine blue present in Albert I reagent. Allow the smears to be air dried. REQUIREMENTS I Equipments Compound light microscope. With toluidine blue or methylene blue. 10 ml alcohol (95% ethanol) and 1 litre distilled water. glass slides. PROCEDURE 1 Heat fix the smears by passing the slide 2–3 times gently over the flame with the smear side up. With Albert’s staining. Toluidine blue and malachite green are dissolved in the alcohol and then added to the water and acetic acid. . With basic dyes. 4 Then cover the smear with Albert’s stain II. 10 ml glacial acetic acid. 5 Rinse the smears again under tap water and blot those dry. Allow it to stain for 3-5 minutes. 3 Rinse the smears gently under tap water and blot those dry. Corynebacterium diphtheriae has well developed granules within their bacterial cytoplasm. Babes Ernst granules. 2 grams malachite green. Note: Toluidine blue stains the granules bluish black due to metachromatic effect and malachite green stains the bacilli green. plumpy. The significance of metachromatic granules is that they represent storage depots of materials needed to form highenergy phosphate bonds. What is metachromasia? Ans.pathogenic diphtheroids which lack them. diphtheriae? What is the reason for such an arrangement? Ans. thick. OBSERVATION 1–2 µm sized green coloured bacilli showing Chinese letter arrangement at angles to each other. ribonucleic acid and protein. x 1000. diphtheriae from short. When a substance is stained with a particular coloured dye. QUALITY CONTROL A known positive control smear of the diphtheria bacillus stained with Albert’s stain and the stained test smear are compared for appropriate morphology and staining appearance. 9-1). 3 What is the composition of metachromatic granules? Ans. Metachromatic granules are composed of polymetaphosphate. diphtheriae. This method involves the use of specific antibodies raised against C. are seen (Fig. The smear is viewed by immunofluorescent microscopy. 6 Mention the other stains used for the detection of C. together with grading of the positive smear. RESULTS AND INTERPRETATION The stained smear contains malachite green stained bacilli showing bluish-black metachromatic granules. BOX 9-1 RAPID STAINING BY DIRECT FLUORESCENT ANTIBODY METHOD Direct fluorescent antibody testing for Corynebacterium diphtheriae is a rapid diagnostic method like that of Albert’s staining. The typical arrangement of C. The smear is positive for bacilli showing bluish-black metachromatic granules. diphtheriae is Chinese letter pattern or Cuneiform arrangement. the bacillus should appear green with bluish black metachromatic granules. Findings are recorded. diphtheriae conjugated with a fluorescent dye to detect directly the C. 4 What is the significance of metachromatic granules? Ans. non. and if a change in original colour is observed. . Probably. VIVA 1. containing bluish-black metachromatic granules. FIGURE 9-1 Albert’s stained smear of Corynebacterium diphtheriae showing volutin granules. 2 Mention the other names for metachromatic granules. the smear contains Corynebacterium diphtheriae. 5 What is the typical arrangement of C. Their presence in thin slender bacilli helps to distinguish C. diphtheriae. With appropriate staining. 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. This is due to incomplete separation of the daughter cells after longitudinal binary fission. They are not found during active growth period and are depleted under starvation conditions. The method is highly specific as well as sensitive. diphtheriae antigen in the throat swab or smear taken from pseudomembrane. (bacilli are seen arranged at various angles to each other resembling the letters V or L.32 Albert’s Staining 7 Record the observations in the note book. this phenomenon is called metachromasia. This phenomenon is observed with C. WC. Killington RA. St. Neisser’s stain and Puch’s stain are used to demonstrate metachromatic granules. Allen SD.) 1996. 11 th ed. Louis) 2002. 4 WHO. Heritage J. Guidelines on Standard Operating Procedures for Microbiology. 3 Granules exhibit metachromasia and are seen in unstained wet preparations as round refractile bodies within bacterial cytoplasm. Chapter 4: Staining Techniques. (The CV Mosby Company. 5 WHO. Bailey and Scott’s Diagnostic Microbiology. (Lippincott Williams and Wilkins.) 1997. Sahm DF and Weissfeld AS. 3 Koneman EW. diphtheriae are made up of polymetaphosphate. Schreckenbergu PC and Winn Jr. Manual of Basic Techniques for a Health Laboratory. 2 Forbes BA. 1980. (Cambridge University Press. Introductory Microbiology. 5th ed. FURTHER READINGS 1 Evans EGV. Color Atlas and Textbook of Diagnostic Microbiology. Janda WM. . 4 The diphtheria bacillus gives its characteristic volutin staining reactions best in a young culture (18–24 hours) on Loeffler’s serum slope or serum medium.Textbook of Practical Microbiology 33 KEY FACTS 1 Volutin granules of C. 2 Special stains like Albert’s stain. 4 Observe the smear first under low power (10x) objective. the capsule is stained and coloured whereas in the negative staining procedure. The capsule is non-ionic. Note: The smear is not heat fixed.03 grams of nigrosin in 100 ml of distilled water. Allow it to stain for 5-7 minutes. III Specimen Culture of Streptococcus pneumoniae (A capsulated bacterium). Dry-film negative staining methods using India ink. Two types of staining procedures can be employed to demonstrate the capsule. PROCEDURE For positive staining of smears 1 Make a smear from colony of S. Two dyes. so that the dyes commonly used will not bind to it. an artifact that can be mistaken for capsule. pneumoniae on a clean grease free glass slide. 5 Record the observations in the note book. a glycoprotein or a polypeptide. staining tray. the capsular material is a polysaccharide. 2 Differentiate between capsulated and non-capsulated bacteria. by positive staining and negative staining methods. They are the positive and negative staining procedures. blood. and allow it to air dry. are used to stain the background and the cell wall. 2 Put the smear on a slide rack and flood smear with crystal violet. 3 Wash the smear with 20% copper sulphate solution and blot it dry. inoculating loop. respectively. II Reagents and glass wares Bunsen flame. because the resultant cell shrinkage may create a clear zone around the organism. REQUIREMENTS Some bacteria secrete chemical substances that accumulate on the outer surfaces of the cell wall and form capsules. the background is stained and the capsule is seen as unstained hallow around the organism. Capsules may be seen in stained or unstained preparations as a clear zone around the bacteria. 1% crystal violet and 20% copper sulphate (CuSO4 5H2O) for positive staining. the capsular materials are water soluble and may be dislodged and removed with vigorous washing. and then under oil immersion (100x) objective. it is called microcapsule. it is referred to as macrocapsule. PRINCIPLE Chemically. I Equipments Compound light microscope. serous fluids and pus and artificial cultures . since occasionally shrinkage spaces give the appearance of capsules around bacteria that are noncapsulated. The best method for staining capsules on bacteria in either liquid or solid media is the wet-film India ink method.34 LESSON 10 INTRODUCTION Capsule Staining LEARNING OBJECTIVES After completing this practical you will be able to : 1 Demonstrate capsule of the bacteria present in animal tissues. Nigrosin staining is prepared by adding 0. Capsule staining is more difficult than other types of differential staining procedures because . and India ink or Nigrosin stains for negative staining. When the diameter is less than 20 nm and seen under electron microscope. When the capsule has a diameter of 20 nanometer or more and seen under light microscope. one acidic and one basic. glass slides. nigrosin or eosin are somewhat less reliable. Bacterial smears should not be heated. In the positive staining technique. x 1000. OBSERVATION Observation of positive staining method In the culture smear. e. 10-1). and the capsule is seen as an unstained halo around the organism.Textbook of Practical Microbiology 35 For negative staining of smears 1 Take a clean grease free glass slide. x 1000. S. pneumoniae (Fig. S. 3 In the positive staining technique.g. Negative staining method: The wet India-ink film contains capsulated bacteria. 6 Record the observations in the note book. the capsule is seen as a light blue hue in contrast to the deep purple colour of the cell. e. the background is stained. Observation of negative staining method The capsule in negative staining method is seen as clear refractile. 5 Modified India ink preparation using 2% mercurochrome helps clearly demonstrate the internal structure of capsulated budding yeast. RESULTS AND INTERPRETATION Positive staining method: The smear shows capsulated bacteria. 5 Observe the wet film under high power (40x) objective. 2 Capsules of the bacteria can be demonstrated by two methods: positive staining and negative staining procedures. QUALITY CONTROL The test smear subjected to capsular staining (e. In negative staining. the capsule will be obscured by overlying ink and if too thin. so that the film becomes very thin and thus pale in colour. . Bacillus anthracis in the stained blood smears is demonstrated by McFadyean reaction which uses polychrome methylene blue. 4 Take a clean.g. the capsule is stained and coloured. 3 Then add a small loopful of liquid bacterial culture to the India ink and emulsify. pneumoniae. pneumoniae (capsulated bacteria) for appropriate structural details and staining appearance. KEY FACTS 1 Capsule of the bacteria. halo around the organism against a black background. FIGURE 10-1 India ink staining of Streptococcus pneumoniae showing capsule. 6 India ink film should have appropriate thickness if the film is too thick. grease free cover slip and place on the ink drop and press it down. FIGURE 10-2 Gram’s staining showing clear unstained capsulated area around Streptococcus pneumoniae. both positive and negative staining technique) is compared with known stained control smear of S.g. 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. 2 Put a large loopful of undiluted India ink on the slide. the capsules get crushed or flattered and the India ink background becomes too pale to give a good contrast. Guidelines on Standard Operating Procedures for Microbiology. Neisseria memigitidis (fresh isolates capsulated) Bacillus anthracis (polypeptide capsule) Clostridium perfringens Bacteriodes fragilis Escherichia coli (some strains) Klebsiella pneumoniae Vibrio parahaemolyticus Yersinia pestis Francisella tularensis Haemophilus influenzae Bordetella pertusis 3 Describe the functions of the capsule. 3 Koneman EW. 2 Forbes BA.) 1997. ii) They protect the bacteria from antibody. FURTHER READINGS 1 Evans EGV. Bailey and Scott’s Diagnostic Microbiology. Ans: i) Capsules contribute to the virulence of pathogenic bacteria by inhibiting phagocytosis. 5 WHO.36 Capsule Staining VIVA 1 Give examples of bacteria having polysaccharide or polypeptide capsules. and iii) K antigen (acidic capsular polysaccharide antigen) of Escherichia coli protect it from bactericidal effect of complement and phagocytes. Group B streptococci Group D streptococci (polysacchaide capsules) Streptococcus pneumoniae. (The CV Mosby Company. Manual of Basic Techniques for a Health Laboratory. WC. (Lippincott Williams and Wilkins. ii) Capsular polysaccharide antigens of meningococci have been used to classify them into 13 serogroups. 4 Describe the capsular antigens Ans: i) Type specific capsular polysaccharide antigen of pneumococcus is also known as specific soluble substance. Heritage J. and iii) Capsular material is antigenic in nature and may be demonstrated by serological methods such as Quellung reaction widely employed for typing of pneumococci. Color Atlas and Textbook of Diagnostic Microbiology. Janda WM.) 1996. Killington RA. Allen SD. 4 WHO. . (Cambridge University Press. Schreckenbergu PC and Winn Jr. Introductory Microbiology. 5th ed. 1980. Chapter 4: Staining Techniques. 11 th ed. Sahm DF and Weissfeld AS. Louis) 2002. Ans : a) Polysacchride capsule Staphylococcus aureus Group B streptococci Group D streptococci Streptococcus pneumoniae Neisseria meningitidis Haemophilus influenzae Anaerobic Gram negative bacilli b) Polypeptide capsule Bacillus anthracis 2 List various capsulated bacteria Staphylococcus aureus (microscopically visible capsules) Streptococcus pyogenes (some stains) Group C Streptococci ( capsules made of hyaluronic acid). St. radiation.5 gram of safranine in 100 ml distilled water. the body of the bacillus appears deeply stained. Spore production is a very important characteristic of some bacteria such as members of anaerobic genera Clostridium and aerobic genus Bacillus. Once the spore is stained with the malachite green. The morphology of the bacterial endospores is best observed in unstained wet films under the phase-contrast microscope. They occur when environmental conditions become unfavorable for continuing vegetative cellular activities. which will absorb the counterstain and appear red. The spores on staining with modified Ziehl-Neelsen stain appear red and bacilli blue. stain (malachite green) and counter stain (0. whereas the spore is unstained and appears as a clear area in the organism. refractile. where they appear freely or within the bacterial cells as large. as well as to the commonly employed microbiological stains. The spore remains green. also known as Schaeffer-Fulton Method for bacterial endospores uses two different reagents: primary . freezing. both the vegetative cell and spore appear green. glass slides. because the stain does not demonstrate a strong affinity for the vegetative cell components and these vegetable cells therefore become colourless. and chemical agents. The spores retain the green of the primary stain. the spore is resistant to deleterious effects of excessive heat.Ordinary tap water acts as decolourising agent. malachite green and safranine. it cannot be decolourised by tap water. 2 Differentiate between bacterial spore and vegetative cell forms in the bacterial smear. REQUIREMENTS I Equipments Compound light microscope. malachite green. Because of the chemical composition of spore layers. oval or spherical bodies. water removes the stain from the vegetable cells. Preparation of malachite green stain: This stain is prepared by dissolving 5 gram of malachite green in 100 ml of distilled water. II Reagents and lab wares Bunsen burner. toluidine blue stain and also Gram’s stain. staining tray. because of its impervious coats. desiccation. Red coloured-safranine as counterstain is used as the second reagent to colour the decolourised vegetative cells. Different staining techniques are available for staining of spores. With the Gram’s stain. beaker of boiling water. which removes only the excess primary stain.5% safranine or 0. The application of heat facilitates penetration of the primary stain. modified Ziehl-Neelsen stain using 0. On the other hand.Textbook of Practical Microbiology 37 LESSON 11 INTRODUCTION Spore Staining LEARNING OBJECTIVES After completing this practical you will be able to: 1 Stain smears for bacterial endospores by malachite green stain. inoculating loop. After the primary stain is applied and the smear is heated. Preparation of safranine stain: This stain is prepared by dissolving 0. These are malachite green stain (Schaeffer and Fulton method). PRINCIPLE Malachite green stain. They are highly resistant and metabolically inactive forms. are not stained by the primary stain easily.05% basic fuchsin). particularly with the exhaustion of nutritional carbon source. the spore.25% sulphuric acid as decolouriser. Unlike most of the vegetative cells that are stained by common procedures. . The spores of Bacillus stearothermophilus are used for testing efficacy of sterilization by moist heat. Clostridium novyi .25% sulphuric acid as decolouriser. vegetative bacilli appear light blue and spores dark blue. VIVA 1 Name spore bearing bacilli causing anthrax and gas gangrene. and its appropriately stained morphology is observed under the microscope and compared with the stained test smear. KEY FACTS 1 Spores are highly resistant. and Clostridium histolyticum. May be sporebearing bacilli (eg.05% basic fuchsin. Spore bearing bacilli causing gas gangrene are Clostridium perfringens . Clostridium septicum . (Fig. while the water continues to boil. resting it on the run with the bacterial film upper most. 5 With toluidine blue staining .bacilli are used to test efficacy of sterilization using i) dry heat and ii) moist heat? Ans: The spores of non toxigenic strain of Clostridium tetani are used for testing efficacy of sterilization by dry heat. 3 The staining methods available for demonstrating spores are Gram’s staining.5% safranine or 0.38 Spore Staining III Specimen Smear collected from 48 hours to 72 hours nutrient agar slant culture of Bacillus cereus/ thioglycollate culture of Clostridium butyricum. and then under oil immersion (100x) objective. metabolically inactive forms occurring when environmental conditions are unfavorable. 4 With malachite green staining vegetative bacilli appear red and spores green. RESULTS AND INTERPRETATION A 2 – 3 µm red coloured rod-shaped structure seen along with an intracellular 0. 2 Spores of which . 3 Wash the smears with cold water. 7. It represents red coloured vegetative bacilli with green coloured spores by the malachite green staining method. flood the smear with 5% acqueous solution of malachite green and allow to act for 1 minute. then air dried and fixed with heat. FIGURE 11-1 Spore staining. Allow it to act for 30 seconds. a smear from the culture is made in saline. 2 Put the slide with the smear over a beaker of boiling water. 2 The morphology of spores is best observed in unstained wet films under the phase-contrast microscope. within several seconds. toluidine blue staining and malachite green staining (Schaeffer-Fulton Method). OBSERVATION Bacterial endospores stain green. On a clean glass slide. Allow the smear to be air dried. x 1000. 11-1) PROCEDURE 1 Heat fix the smears by passing the slide 2–3 times gently over the flame with the smear side up. large droplets have condensed on the underside of the slide. 4 Then cover the smear with 0. 3 Which sterilization method is employed for destruction of spores? Ans: Autoclaving at 120°C for 15 minutes is the sterilization method employed for destruction of spores in clinical specimens. Bacillus species or Clostridium species) QUALITY CONTROL A check smear of a known positive control of bacteria with spores is stained with the malachite green staining method. Record the observations in the note book. modified acid fast staining using 0. and vegetative bacilli stain red. 3 When. 6 Observe the smear first under low power (10x) objective .5 µm sized spherical green coloured structure. 5 Rinse the smears again under tap water and blot those dry. Ans : Spore bearing bacilli causing anthrax is Bacillus anthracis. . Heritage J. 4 WHO.) 1997. Sahm DF and Weissfeld AS. Janda WM. Introductory Microbiology. Schreckenbergu PC and Winn Jr. Color Atlas and Textbook of Diagnostic Microbiology. Chapter 4: Staining Techniques. Bailey and Scott’s Diagnostic Microbiology. Allen SD. 5th ed. 2 Forbes BA. 3 Koneman EW. (Cambridge University Press. WC. Guidelines on Standard Operating Procedures for Microbiology. (Lippincott Williams and Wilkins.Textbook of Practical Microbiology FURTHER READINGS 39 1 Evans EGV. (The CV Mosby Company. 5 WHO. St.) 1996. 11 th ed. Manual of Basic Techniques for a Health Laboratory. Louis) 2002. 1980. Killington RA. 03 gram of nigrosin in 100 ml of distilled water.40 LESSON 12 INTRODUCTION Negative Staining LEARNING OBJECTIVES After completing this practical you will be able to: 1 Perform a negative staining procedure for demonstration of bacteria REQUIREMENTS I Equipment Compound light microscope. II Reagents and glass wares These include Bunsen flame. QUALITY CONTROL The nigrosin-stained preparation of test organism is compared with the preparation of capsulated control organism such as Streptococcus pneumoniae. The slide is then pushed away from the previously spread drop of suspended organism. III Specimen 24 hour broth culture of Klebsiella pneumoniae (A capsulated bacterium). Cryptococcus neoformans . Instead it forms a deposit around the organism. for appropriate staining and capsule demonstration. glass slides and coverslips nigrosin stains. forming a thin smear. 4 With the edge of a second slide. Negative staining finds its utility for the demonstration of capsule and bacteria difficult to stain such as Treponema palladium. PROCEDURE 1 Take a clean grease free glass slide. the unstained cells are easily discernible against the coloured background. In this chapter dry film negative staining by using nigrosin to detect bacteria in dry smears will be described. Therefore. 3 Using a sterile loop. PRINCIPLE The acidic dyes such as India ink. Nigrosin staining solution is prepared by adding 0. leaving the organism itself colourless. A modification of India ink using 2% mercurochrome helps to clearly demonstrate the capsulated budding yeast. It is also known as ‘Indirect staining. 7 Record the observations in the note book. The procedure requires the use of acidic stains such as India ink or Nigrosin. Wet film India-ink method is the best method for staining capsules of bacteria from cultures in either liquid or solid media. staining tray. a loopful of broth culture of the capsulated organism is mixed with the nigrosin drop. and will not readily combine with the negatively charged bacterial cytoplasm. 5 Air dry the preparation without any heat fixation. spread the drop along the edge of the applied slide. held at 30° angle and held in front of the bacterial suspension mixture. nigrosin or eosin have negatively charged chromogen. Negative staining procedure is so called because the background gets stained and the organism remains colourless. . 2 Put a small drop of nigrosin close to one end of a clean slide. 6 Observe the stained smear under oil immersion (100x) objective. Indian ink method for demonstration of capsule is described earlier in the chapter 10. 5th ed. Guidelines on Standard Operating Procedures for Microbiology. 11 th ed.) 1997. Manual of Basic Techniques for a Health Laboratory. (The CV Mosby Company. Ans: In negative staining. . BOX 12-1 LIST OF MOST COMMON CAPSULATED ORGANISMS THAT CAN BE DEMONSTRATED BY NEGATIVE STAINING Bacteria Streptococcus pneumoniae Klebsiella pneumoniae Haemophilus influenzae Group B streptococci Group D streptococci Fungus Cryptococcus neoformans KEY FACTS 1 Negative staining procedure is so called because the background gets stained and the organism remains colourless. WC. (Cambridge University Press. e. RESULTS AND INTERPRETATION The nigrosin-stained smear contains capsulated bacteria. Ans: The different stains employed for negative staining are: India ink. 2 Forbes BA. 12-1). pneumoniae. pneumoniae. Chapter 4: Staining Techniques. Allen SD. Janda WM. modified India ink (using 2% mercurochrome) and 10% nigrosin. 3 Koneman EW. Color Atlas and Textbook of Diagnostic Microbiology. the organism and capsule remained unstained and refractile. 5 WHO.g. St. Louis) 2002. Sahm DF and Weissfeld AS. 2 List the capsulated organisms and bacteria that can be demonstrated by negative staining. FIGURE 12-1 Nigrosin-stained smear showing capsulated K. Bailey and Scott’s Diagnostic Microbiology. Introductory Microbiology. 1980. x 400. Schreckenbergu PC and Winn Jr. the bacteria are mixed with dyes such as India ink or nigrosin that provide a uniformly coloured background against which the unstained bacteria stand out in contrast. (Lippincott Williams and Wilkins.Textbook of Practical Microbiology 41 OBSERVATION The capsule or the bacterial organism is seen as a clear halo against a black or dark background in the wet film or dry film preparation (Fig. 4 WHO. FURTHER READINGS 1 Evans EGV. 2 In negative staining method only background is stained. Heritage J. VIVA 1 List the different stains that can be employed for negative staining. K. 3 What is negative staining? Which bacteria can be demonstrated by India Ink. This is particularly useful in the demonstration of bacterial capsules. Killington RA.) 1996. 42 . Textbook of Practical Microbiology UNIT 43 III Cultivation of Bacteria Lesson 13 Lesson 14 Media for Routine Cultivation of Bacteria Temperature Requirement for Growth of Bacteria Lesson 15 Lesson 16 Lesson 17 Lesson 18 Lesson 19 pH Requirement for Growth of Bacteria Oxygen Requirement for Growth of Bacteria Culture of Anaerobic Bacteria Sterilization of Commonly Used Culture Media Antiseptics and Disinfectants . They favor the growth of some bacteria by extending the lag phase of others eg.g. This medium is used to demonstrate lactose fermenting properties. Media are of different types. d. nutrient agar (Fig. b. The choice of most appropriate media depends on many factors including nutritional and growth requirements of the bacteria. Löwenstein.Jensen medium (Fig. This medium is a selective medium for growth of Salmonella spp. chocolate agar (Fig. Meat extract and peptone are the commonest sources of carbohydrates and amino acids. 13-2). Example: MacConkey (Fig. FIGURE 13-1 Nutrient agar. These are: 1 Basal media: These contain nutrients that support the growth of non-fastidious bacteria. 13-5) agar is a differential medium. e. 13-1). blood agar (Fig. c. They do not confer any selective advantage. Basal medium Differential medium Selective medium Enriched. This medium inhibits Escherichia coli and other Gramnegative bacteria. serum. and Enrichment media Media for Routine Cultivation of Bacteria LEARNING OBJECTIVES After completing this practical you will be able to: 1 Know the following commonly used culture media as well as their uses in a clinical microbiology laboratory: INTRODUCTION Cultivation of bacteria is often the first step in the diagnosis of infectious disease. Since bacteria have varied growth requirements.g. etc. in addition to basal nutrients also contain nutritional supplements like blood. e. while inhibiting others eg. Deoxycholate citrate agar (DCA) medium. which favour the growth of fastidious bacteria. 13-3) . 4 Selective media: These media contain ingredients that selectively enable the growth of some species. Some media may contain additional nutritional supplements. e.. 3 Enrichment media: These are liquid selective media. 13-4). 5 Differential media: These media differentiates between species of bacteria depending on a specific property. nitrogen source and some essential minerals and salts. biochemical properties and many other properties of the bacteria. a wide range of media is available. In addition solid media contain agar as a solidifying agent. Media generally contain a carbon source. Selenite F broth. PRINCIPLE A number of media have been formulated for growing bacteria (Table 13-1).44 LESSON 13 a. 2 Enriched medium: These are solid selective media. . present in stool which contains a mixed bacteria flora. These media. etc. and differentiate between lactose and non-lactose fermenting bacteria. 3 Examine the plate and tubes for growth and record observations. In addition S. into appropriately labeled plates and tubes. aureus. coli. . REQUIREMENTS I Equipments Bacteriological incubator. S. coli and colorless colonies of Proteus spp. FIGURE 13-4 Lowenstein-Jenson’s medium.g. FIGURE 13-2 Blood agar. III Specimen 24 hour broth cultures of Staphylococcus aureus. P. PROCEDURE 1 Inoculate a loopful of the test organism. aureus. 2 Incubate the plates and tubes for 18 hours at 37°C. lactose fermenting colonies of E. and growth negative Proteus spp. using a sterile inoculating loop. Proteus mirabilis and Salmonella spp. 5 Selenite F broth: Growth positive Salmonella spp. OBSERVATIONS All the inoculated bacteria (e. 4 MacConkey agar: Pink. aureus streaked on the plate surrounded by a zone of hemolysis. blood agar. FIGURE 13-3 Chocolate agar.Textbook of Practical Microbiology 45 QUALITY CONTROL 1 One un inoculated set of media as sterility control 2 Nutrient agar: Colonies of non-fastidious bacteria such as S. aureus may or may not produce haemolysis on the blood agar. 3 Blood agar: Haemolytic strain of S. MacConkey agar and Selenite F broth. FIGURE 13-5 Mc Conkey agar. II Reagents and media Different kinds of media such as nutrient agar. E. coli. E. mirabilis and Salmonella spp) produce colonies on the nutrient agar (basal medium) and blood agar (enriched medium). Lyne PM and Grange JM. neutral red (0. FURTHER READINGS 1 Bhattacharya S.aureus. 2 What are the constituents of MacConkey agar? Ans. Guidelines on standard operating procedures for Microbiology. Washington DC. blood agar) is a solid medium where as enrichment medium (e.g. Proteus. Proteus. . Most of the bacteria grow on enriched media. alkaline peptone water) is a liquid medium. S. 2 Collins CH.aureus.g. Microbiological Methods. Klebsiella. 3 Isenberg HD. Blood safety and clinical technology. E. Table 13-1 List of different type of media commonly used for isolation of bacteria Type of medium Basal media Enriched media Enrichment media Selective media Differential media Example Nutrient agar Peptone water Blood agar Brain heart infusion agar Alkaline peptone water Selenite F Broth Pikes medium Cetrimide agar MacConkey agar. Proteus spp. pyogenes. S.5%). 2 S. aureus can grow on MacConkey medium. Parija SC. and for storage of bacterial isolates. 3 Media serve many purposes such as culture and isolation of the bacteria from clinical specimens. 4 PHLS Standard Operating Procedures. CLED medium Bacteria grown/isolated S. Pseudomonas spp. 3 Name the enrichment medium used for Vibrio cholerae. demonstration of growth and biochemical characteristics of bacteria. Selenite F broth inhibits all except S..coli. In SF broth most bacteria are inhibited.aureus. Klebsiella. Klebsiella. Ans Enriched medium (e. Vijayalakshmi N.46 Media for Routine Cultivation of Bacteria On MacConkey agar E. cholerae. Typhi RESULTS AND INTERPRETATION Different colonies of various bacteria on different media show different growth requirement of these bacteria. 1992. KEY FACTS 1 Bacteria have different nutritional requirements. influenzae.SOP 54 Version: 1.coli. American Society for Microbiology. (World Health Organisation. 174-177. 4 Mention an important difference between enriched medium and enrichment medium. coli produces pink. Indian J Med Microbiol. produces colorless non-lactose fermenting colonies. V. sodium taurocholate (0. Pseudomonas S. 5 WHO. Salmonella spp.. Proteus. Enterobacter. Citrobacter. VIVA 1 Give examples of different kinds of media used for culture of bacteria. E. Streptococcus spp. Uncultivable bacteria: Implications and recent trends towards identification.7%). These are the peptone (2%).. 2002: 20. E. Pseudomonas. lactose fermenting colonies where as Proteus spp.coli. etc. (Ed) Clinical Microbiology Procedures Handbook. and agar (2%). Inoculation of Culture Media. Pseudomonas. 1995. No B. Geneva) 1997. Butterworths. lactose (1%). London. 1998. H. Chapter 6: Cultivation of bacteria on laboratory media. However. These factors include availability of proper temperature. 4 Observe the plates for growth of bacteria and pigment production if any. they get denatured. bacteria can survive across a wide range of temperatures from 5 0° C to 80°C. at low temperatures. Enterococcus faecalis and Flavobacterium spp PRINCIPLE Cellular enzymes of the bacteria are optimally active at a certain temperature. II Reagents and media Nutrient agar plates. QUALITY CONTROL 1 Nutrient agar plate inoculated with Proteus mirabilis as quality control for mesophilic bacteria. The ideal temperature for growth may not coincide with that for specific enzyme activities. 2 Using a sterile loop. III Specimen A 24 hour broth culture of Serratia marcescens. aseptically inoculate a drop of each culture. bacteria can be classified into the following groups: 1 Psychrophilic bacteria: These bacteria can grow within a temperature range of 5°C to 20°C. LEARNING OBJECTIVES After completing this practical you will be able to understand: 1 Effect of temperature on growth of the bacteria in culture media. Optimum temperature for growth of these bacteria is 37°C (human body temp). In general. These PROCEDURE 1 Mark the nutrient agar plates into 4 quadrants each. A number of factors influence the growth and culture of bacteria on culture media. 37°C and 60°C for 24-48 hr. Apart from nutritional requirements. On the other hand. . taking care to label each quadrant correctly. 3 Thermophilic bacteria: These bacteria can grow above 35°C. which vary greatly.Textbook of Practical Microbiology 47 LESSON 14 INTRODUCTION Temperature Requirement for Growth of Bacteria bacteria prefer to grow in the bodies of warm blooded hosts and include most bacterial pathogens that cause diseases in humans. 20°C. environmental factors also play an important role. Above this point. Examples of bacteria showing different temperatures for their growth is summarized in the table 14-1. REQUIREMENTS I Equipments Bacteriological incubator. 3 Incubate the plates at 4°C. Based on their temperature requirements. each bacterial species has an optimum growth temperature at which reproduction is most rapid and also a maximum/minimum growth temperature beyond which growth does not occur. Escherichia coli. pH and oxygen in the culture environment. they tend to get inactivated. 2 Mesophilic bacteria: These bacteria can grow within a temperature range of 20°C to 45°C. Either event causes biochemical reaction in bacterial cells to cease. growing only at temperature above 50°C. thereby interfering with the survival of the organism. Optimum temperature for growth of these bacteria varies between 10 °C to 15°C. They are either facultative with an optimum temperature of 45°C -60°C but capable of growth at 37°C or obligate. only E. Campylobacter grows at 42°C. Microbiological Methods. production of flagella. FURTHER READINGS 1 Bhattacharya S. Table 14-1 Examples of bacteria showing different temperatures for their growth Type of bacteria Psychrophilic Mesophilic Thermophilic Example Psychrobacter immobilis.SOP 54 Version: 1. 2 Collins CH. marcescens produces a red/magenta pigment. . (Ed) Clinical Microbiology Procedures Handbook. 2 Give 2 examples each of psychrophilic. 3 Isenberg HD. Geneva) 1997. There is a different temperature requirement for optimal growth and pigment production. mesophilic and thermophilic bacteria. Pigment production will be seen with the cultures of both Flavobacterium and S. 3 Name some other enzymatic activities of the bacteria that are affected by temperature. Different bacteria require different optimal temperature for their growth. Vijayalakshmi N. Uncultivable bacteria: Implications and recent trends towards identification. Escherichia coli. Ans: Pigment production. RESULTS AND INTERPRETATION 1 Growth of all species tested will be absent at 4°C except for Yersinia enterocolitica and Flavobacterium spp. 2002: 20. (World Health Organisation. 5 WHO. they are denatured. Most of the medically important bacteria grow at 35°C -37°C. while at low temperatures. marcescens and Flavobacterium spp. 1995. coli. Pigment production is influenced by temperature with a greater intensity on color at 20°C for both the species tested. Methanosarcina spp. Butterworths. 4 PHLS Standard Operating Procedures. Blood safety and clinical technology. KEY FACTS 1 2 3 4 Temperature requirement for optimal growth and enzyme activity are often different. marcescens. Lyne PM and Grange JM. Enzyme activity in turn is influenced by temperature. No B. VIVA 1 How does temperature affect bacterial growth? Ans: Bacterial growth is dependent on enzyme activity. At high temperatures. and expression of stress proteins. 3 Nutrient agar plate inoculated with Bacillus stearothermophilus as quality control for thermophilic bacteria. Parija SC. 1998. Flavobacterium spp is capable of growth at low temperature as well. Each species has an optimum range of temperature within which its reproductive rate and hence growth is optimum. OBSERVATIONS 1 Reproduction of bacteria is maximal at optimal temperature and will be seen as a luxuriant growth of colonies. Bacillus stearothermophilus. they tend to get inactivated. 174-177. Guidelines on standard operating procedures for Microbiology. 4 At 60°C. 2 At 20°C growth will be minimal/inhibited for E. Inoculation of Culture Media. faecalis will show growth. 2 Pigment production is often optimum at a temperature different from that for growth. 3 At 37°C all the bacterial species tested will produce good growth. S. American Society for Microbiology. DC. 1992. Indian J Med Microbiol. S. London. Washington. Thermus aquaticus.48 Temperature Requirement for Growth of Bacteria 2 Nutrient agar plate inoculated with Pseudomonas aeruginosa as quality control for psychrophilic bacteria. Chapter 6: Cultivation of bacteria on laboratory media. 5. 3 each of pH. 7 and 9.5-7. 5.Textbook of Practical Microbiology 49 LESSON 15 15 INTRODUCTION pH Requirement for Growth of Bacteria LEARNING OBJECTIVES After completing this practical you will be able to understand: 1 The pH requirement of bacteria. 2 Ability to tolerate and grow at a particular pH will be seen as turbidity in culture tubes.2 3 Nutrient broth tube inoculated with A. 5 Only Candida spp shows growth and turbidity at pH 5. faecalis as quality control for growth at alkaline pH. coli. OBSERVATIONS PRINCIPLE Since pH changes can occur during growth of the bacteria due to the accumulation of metabolic byproducts. The ability to grow at different pH varies among bacteria. PROCEDURE 1 Using sterile pipettes. 3.5-8 optimum pH being 6. similar to that of the gut. 3 E. QUALITY CONTROL 1 Nutrient broth tube inoculated with Candida spp as quality control for growth at acidic pH. Alcaligenes faecalis and Candida spp. pH also plays an important role in the growth and reproduction of bacteria. 7 and 9. 2 Incubate the tubes for 24-48 hr. 3. inoculate 0. 3 Check for growth of bacteria in these tubes. REQUIREMENTS I Equipments Bacteriological incubator. coli and A.5 Fungi thrive in an acidic environment of about pH 4-6. . This is a reflection of this natural environment eg. Salts of weak acids and weak bases may also be added. A. Most laboratory media have a neutral pH which suits nearly all organisms.1 ml of each organism into nutrient broth tubes of pH. 4 Only A. II Reagents Nutrient broth tubes. III Specimen A 24 hour broth culture of Escherichia coli . and maximum growth occurs in an optimum pH range. coli as quality control for growth at pH 7. These may include natural buffers like proteins. Like temperature. 2 Nutrient broth tube inoculated with E. peptone and amino acids which retard the shift because of their amphoteric nature. faecalis and Candida spp) show growth and turbidity at pH 7. 2 No microorganism shows any growth at pH 3. faecalis shows growth and turbidity at pH 9. Generally. Each species of bacteria can grow within a particular pH range. 1 Reproduction of bacteria is maximal at optimal pH. bacteria grow best at a pH between 5. buffers are incorporated into the medium to prevent pH change. Most pathogens show an optimum pH close to that of their preferred habitat. RESULTS AND INTERPRETATION 1 All 3 microorganisms (E. enteric bacteria such as Escherichia coli have a broad pH range. faecalis do not show any growth at pH 3 or pH 5. Microbiological Methods. VIVA 1 Name some buffers used in preparing culture media. DC. Chapter 6: Cultivation of bacteria on laboratory media. Washington. bicarbonate buffer and phosphate buffer. London. Vijayalakshmi N. 3 Why does optimum growth of most pathogenic bacteria occur at pH 7. 174-177. Ans: Vibrio cholerae and Enterococcus faecalis. 3 Extremes of pH can be used to selectively grow some microorganisms. 2002: 20. Lyne PM and Grange JM. and hence are best capable of survival at a pH identical to that of the host. 2 Collins CH. 4 PHLS Standard Operating Procedures. Uncultivable bacteria: Implications and recent trends towards identification. Butterworths. 2 Most laboratory media has a pH of 7 and incorporate buffers for maintaining the same. 3 Isenberg HD. 1992. 1995. Inoculation of Culture Media. .SOP 54 Version: 1. Guidelines on standard operating procedures for Microbiology. FURTHER READINGS 1 Bhattacharya S. Ans: Citrate buffer. Blood safety and clinical technology. 1998. (World Health Organisation. 5 WHO.50 pH Requirement of Growth of Bacteria KEY FACTS 1 pH is a critical factor affecting growth and metabolism of bacteria. Parija SC. (Ed) Clinical Microbiology Procedures Handbook.2? Ans: Most body fluids have a pH of 7. 2 Name bacteria that can be selectively grown in alkaline medium. Pathogenic bacteria have adapted to their hosts. Indian J Med Microbiol. No B. American Society for Microbiology. Geneva) 1997.2. Enzyme systems present in these bacteria require oxygen to be the final electron (hydrogen) acceptor especially for the oxidative breakdown of high energy molecule like glucose. Facultative anaerobes are evenly dispersed throughout the medium while microaerophiles grow slightly below the surface (subsurface growth). II Reagents and media Nutrient broth tubes and brain heart infusion (BHI) agar tubes. an anaerobic respiratory I Equipments Bacteriological incubator and water bath. 16-1). sulphates. 4 Aerotolerant anaerobes: They do not use oxygen as a final electron acceptor but possess enzymes like superoxide dismutase and catalase. pathway is followed in absence of oxygen substrate when substances like nitrate. They generally follow an aerobic respiratory pathway. and 48 hour thioglycollate broth culture of Clostridium sporogenes. Pseudomonas aeruginosa. hence other molecule act as the final electron acceptor. Therefore. 5 Facultative anaerobes: These bacteria can grow in the presence or absence of free oxygen. An excess of oxygen is inhibitory to their growth. REQUIREMENTS PRINCIPLE On the basis of oxygen requirement. aerobes grow only on the surface while obligate anaerobes grow only in the depths of the tube. Oxygen is one of the most important growth limiting factor for microorganism. 2 Microaerophiles: These bacteria require small amount of oxygen for their growth and survival. Some bacteria require the presence of 5 to 10% C02 for better growth of the bacteria by using candle jar (Fig. oxygen is not lethal to them. etc. bacteria can be classified into 5 groups as follows: 1 Aerobes: These bacteria can grow only in the presence of free oxygen. which can prevent the accumulation of toxic metabolites produced in the presence of oxygen. Oxygen requirement vary widely among bacteria and this is reflective of the different bio-oxidative enzyme systems present in bacterial cells. It plays a vital role in many biological processes of the microorganisms.. When inoculated into a tube of semisolid or liquid medium. FIGURE 16-1 Candle jar used for incubation and culture of bacteria.Textbook of Practical Microbiology 51 LESSON 16 INTRODUCTION Oxygen Requirement for Growth of Bacteria LEARNING OBJECTIVES After completing this practical you will be able to understand: 1 The oxygen requirement of bacteria. can act as terminal electron acceptor. . III Specimen A 24 hour nutrient broth culture of Escherichia coli . 3 Obligate anaerobes: These bacteria cannot survive in the presence of free oxygen. Blood safety and clinical technology. sporogenes and incubate at 37°C for 48 hours. aeruginosa will grow only on the surface of the medium. sporogenes. 174-177. sporogenes only in the depths at the bottom of the tube. Cl. 6 Innoculate thioglycollate broth with Cl. aeruginosa. coli is a facultative anaerobe. . Ps. 1992. Lyne PM and Grange JM. aerotolerant anaerobes and facultative anaerobes. introduce a loopful of the culture into appropriately labeled tube. However it also results in the accumulation of toxic byproducts. Guidelines on standard operating procedures for Microbiology. etc. and facultative anaerobes throughout the medium in the tube (Table 16-1). The benefits are because of their ability to act as a final electron acceptor in the respiratory pathway with the liberation of much energy. RESULTS AND INTERPRETATION Ps. (World Health Organisation. 2 What are the media commonly used to culture anaerobes? Ans: Robertson’s cooked meat (RCM) medium. thioglycollate broth. obligate anaerobes. QUALITY CONTROL 1 BHI agar tube inoculated with P. Uncultivable bacteria: Implications and recent trends towards identification. (Ed) Clinical Microbiology Procedures Handbook. 3 Anaerobic bacteria can be cultured by the exclusion of air using anaerobic jars. VIVA 1 Give example of obligate aerobes. 3 Isenberg HD. prereduced media. Aerobes will grow on the surface of the medium. Vijayalakshmi N. 3 With a sterile inoculating loop. Indian J Med Microbiol. No B. microaerophiles. the bacteria that does not grow anaerobically 2 Thioglycollate broth culture inoculated with Cl. 4 PHLS Standard Operating Procedures.52 Oxygen Requirement for Growth of Bacteria PROCEDURE 1 Warm the BHI agar tube in a water bath so that the agar melts. Chapter 6: Cultivation of bacteria on laboratory media. Butterworths. 2 Bacteria may possess enzyme system to remove these toxic substances and thus help in their survival in the presence of oxygen. London. Inoculation of Culture Media. blood agar incorporating antibiotics like neomycin or kanamycin. 2 Collins CH. coli will grow throughout the tube and Cl. 1998. E. the bacteria that does not grow aerobically Table 16-1 Examples of bacteria grouped depending on their requirement of oxygen Type Example Vibrio cholerae Clostridium perfringens Clostridium tetani Clostridium histolyticum Escherichia coli OBSERVATIONS 1 Bacteria will grow at different depths in the tube depending on their oxygen requirement. DC. anaerobes in the depth of the medium. 4 Rotate the tubes between palms to evenly dispense the organisms. sporogenes is an obligate anaerobe and E. Microbiological Methods. Parija SC. Obligate aerobes Microaerophiles Obligate anaerobes Aerotolerant anaerobes Facultative anaerobes KEY FACTS 1 Oxygen is both beneficial and toxic to living organisms. 2002: 20. 1995. 5 WHO.SOP 54 Version: 1. 5 Incubate the tubes for 24 hours at 37°C. American Society for Microbiology. 2 Cool the medium to 45°C. 2 Growth will be seen as turbidity in the appropriate section of the tube. Geneva) 1997. aeruginosa is an aerobe. FURTHER READINGS 1 Bhattacharya S. Washington. Deep nutrient agar tubes are the simplest method. II Reagents and media Blood agar plates and thioglycollate broth culture. 3 Stack the plates into the anaerobic jars.5-1% glucose. and obligate aerobes (Pseudomonas aeruginosa). and the number of colonies becomes fewer towards the surface. 4 Incubate the plates at 37ºC for 48 hours. 5 Incubate one plate aerobically. They include the McIntosh and Fildes jar (Fig. A simpler but more expensive technique is the Gaspak system. The tubes are inoculated while still molten. This utilizes a transparent polycarbonate jar with a lid PROCEDURE 1 Divide each plate into 4 quadrants. Anaerobes grow in the depths of the medium. PRINCIPLE There are different ways of creating anaerobic conditions suitable for the growth of obligate anaerobes. Cooked meat particles also act as a good reducing agent Example. combustion involves the combining of oxygen with hydrogen to form water in the presence of a catalyst like palladium or palladinized asbestos. List of anaerobic bacilli and cocci are summarized in the box 17-1. cobalt chloride. Strict anaerobes will not grow within a centimeter of the surface. 0. cooled rapidly and incubated. REQUIREMENTS I Equipments Anaerobic culture systems: McIntosh and Fildes jar and Gaspak system. from which air is excluded. III Specimen A 48 hour thioglycollate broth culture of anaerobic bacteria (Clostridium sporogenes. Bacteroides spp). hence for maintaining anaerobiosis various methods have been devised for anaerobic culture. Anaerobic jars are a constant feature of anaerobic culture. The resulting reaction liberates hydrogen and carbon dioxide. which is then sealed tightly.1% thioglycollate can be added. and a catalyst fitted into the lid. Note: Anaerobic condition should be checked by alkaline methylene blue indicator. For culture of anaerobes. 0. a vacuum pump for evacuating oxygen. introduce the catalyst and quickly seal the lid. These organisms die rapidly on exposure to air. reducing agents like 0. ie. consisting of pellets of sodium borohydride. In many laboratories.1% ascorbic acid. Robertson Cooked meat medium (Fig. Water is added to the sachet and it is immediately placed in the jar. 17-2) . The catalyst. . 0. 17-1). bearing a screened catalyst chamber. Obligate anaerobes can grow in media only in the absence of oxygen. and 24 hour cultures of facultative anaerobes (Escherichia coli ). oxygen must be excluded either by combustion or by replacing it with an inert gas. which has inlets to admit hydrogen and carbon dioxide. 6 Remove the plates from the jars and examine for growth. citric acid and sodium bicarbonate is contained in sachets.Textbook of Practical Microbiology 53 LESSON 17 17 INTRODUCTION Culture of Anaerobic Bacteria LEARNING OBJECTIVES After completing this practical you will be able to understand: 1 The methods of culture of anaerobic bacteria. Alternatively.1% cysteine. 2 Inoculate a loopful of each organism into a quadrant. An indicator is also added to demonstrate anaerobiosis. the bacteria that does not grow aerobically Cl. coli will grow on all the plates. E. 2. aeruginosa. FIGURE 17-2 Macintosh Filde’s jar. either incubated aerobically or anaerobically. The Hungate procedure is a method for the anaerobic culture of bacteria. P. usually nitrogen. obligate anaerobes like Cl. sporogenes and Bacteroides are strict anaerobes that cannot grow if oxygen is present. The organisms are then inoculated. the bacteria that does not grow anaerobically 2 Thioglycollate broth culture inoculated with Cl. . OBSERVATIONS If anaerobiosis is complete. E. BOX 17-1 LIST OF ANAEROBIC BACILLI AND COCCI Anaerobic bacilli Gram positive bacilli Bifidobacterium Propionobacterium Eubacterium Lactobacillus Actonomyces Anaerobic cocci Gram positive cocci Peptosteptococcus Coprococcus Ruminococcus Gram negative Bacilli Bacteroides Fusobacterium RESULTS AND INTERPRETATION P. while Cl. coli is a facultative anaerobe and can grow either in the presence or absence of oxygen. P. Gram Negative cocci Veillonella Acidaminococcus Megasphaera VIVA 1 List some methods of anaerobic culture not utilizing anaerobic jars. aeruginosa is a strict aerobe that cannot grow in the absence of oxygen. This ensures anaerobic conditions inside the tube.54 Culture of Anaerobic Bacteria FIGURE 17-1 Robertson’s cooked meat medium. sporogenes will grow. sporogenes or Bacteroides spp will not grow. The tubes are then sealed. sporogenes and Bacteroides spp will grow on the blood gar plates. also in the presence of the same gas. sporogenes. aeruginosa will show growth on the aerobically incubated plate while Cl. while obligate aerobes like P. QUALITY CONTROL 1 Blood agar inoculated with P. aeruginosa will not show growth on the plates incubated anaerobically. a thin layer of agar is coated on the inside of the culture tube. aeruginosa will not grow. In the presence of an oxygen free gas. What is the Hungate procedure of anaerobiosis? Ans. (Ed) Clinical Microbiology Procedures Handbook. 2 The addition of indicators to an anaerobic system is a useful way of ascertaining absence or presence of oxygen. No B. Indian J Med Microbiol. . Washington. Blood safety and clinical technology. Inoculation of Culture Media. London. 1992. Microbiological Methods. Geneva) 1997. 4 PHLS Standard Operating Procedures. 5 WHO. 3 Isenberg HD.Textbook of Practical Microbiology 55 KEY FACTS 1 If anaerobiosis is inadequate. Chapter 6: Cultivation of bacteria on laboratory media. 2 Collins CH. Guidelines on standard operating procedures for Microbiology. Lyne PM and Grange JM. 1998. DC. obligate anaerobes may fail to grow. 1995. FURTHER READINGS 1 Bhattacharya S. 2002: 20. Butterworths. American Society for Microbiology. Uncultivable bacteria: Implications and recent trends towards identification. (World Health Organisation.SOP 54 Version: 1. 174-177. Parija SC. Vijayalakshmi N. 3. 4 Autoclave the 4th. 5 After cooling. There are different types of filters. PROCEDURE 1 Soak strips of filter paper in the spore suspensions of Bacillus spp and dry them in a Petri dish in the incubator. and 30 minutes respectively. Moist heat has greater penetrating power than the dry heat and so relatively lower temperature is required for sterilization by this method. II Reagents Sterile nutrient broth. hot air oven (Fig. including spores. Berkefield. Seitz filter (Fig. In a hot air oven. 9 Aseptically inoculate the filtrate onto a nutrient agar plate and incubate both plates at 37°C for 24 hours. for an hour is effective for sterilization. This is vital for isolation and maintenance of microbes. III Specimen Spore suspensions of Bacillus spp and suspension of Escherichia coli PRINCIPLE Sterilization by heat This can be performed by two methods as mentioned below: Dry heat: Sterilisation by dry heat kills the bacteria by oxidizing essential cell components of the cell.coli suspension onto a nutrient agar plate.56 LESSON 18 INTRODUCTION Sterilization of Commonly Used Culture Media Chamber land). powders etc.. Of these cellulose membrane filter is most extensively used now a days. oils. 8 Filter the suspension through a Seitz filter. 5th and 6th tubes for 10.b) asbestos filters (e. Pasteurisation is an example of sterilization by moist heat (Box 18-1).g. 7 Inoculate a drop of the E.g. 2 Take 7 test tubes and place a dried strip in each of them. I Equipments Autoclave (Fig. REQUIREMENTS Sterilization is the process by which an article is freed of all living organisms. antibiotics. temperatures maintained at 160°C. 3 Place the first three tubes in a hot air oven at 160°C for 20. Different methods of sterilization of various substances are summarized in the table 18-1. 6 The 7th tube acts as control. c) sintered glass filter (Fig. 18-1). 18-2). 18-3). The hot air oven is commonly used in a microbiology laboratory for sterilization of laboratory glassware. aseptically add 5 ml of nutrient broth to each tube and incubate for 24 hours at 37°C. Sterilization by filtration Heat labile fluids such as serum. 30 and 60 minutes respectively. The common methods used for sterilization of media is heat and filtration. LEARNING OBJECTIVES After completing this practical you will be able to become familiar with: 1 The commonly used techniques for the sterilization of culture media. and d) cellulose membrane filters. Moist heat: Moist heat kills the microorganisms by coagulating their proteins and denaturing their enzymes. . Browne’s tube no. and filter paper strips impregnated with spore of Clostridium tetani and filter paper strips impregnated with spores of Bacillus stearothermophilus. are sterilized by passing them through special filters. 20.. etc. 18-4) and Seitz filter. These include a) earthenware candles (e. 2 Only the tube autoclaved for 10 minutes will show growth. FIGURE 18-4 Hot air oven. 3 There will be no growth in the culture filtrates. tetani heated along with the load and incubated in appropriate media. No growth should occur. if the bacteria have been held back by the filter there should be no growth in the sample of filtrate. The other two tubes will remain free of growth. Filtration is also a suitable method of sterilization. FIGURE 18-2 Sintered glass filter. 3: Color change from red to green is a satisfactory result. Similarly. kept in the hot air oven for 20 minutes and 30 minutes but not for 60 minutes. 3 Filters: Efficient filters should be able to retain Serratia marcescens. Both dry and moist heats are effective sterilization methods. FIGURE 18-3 Laboratory autoclave. RESULTS AND INTERPRETATION 1 There will be growth in tubes.Textbook of Practical Microbiology 57 FIGURE 18-1 Seitz filter. However moist heat is more effective requiring less time and temperature. OBSERVATIONS If the spores have been killed by the heat process there will be no growth as demonstrated by a lack of turbidity. QUALITY CONTROL 1 Autoclave: Filter paper strips impregnated with spores of B. b) Filter paper strips impregnated with spores of non toxigenic strain of C. . If the correct sterilization conditions have been achieved no growth should occur. 2 Hot air oven: This can be tested by using a) Browne’s tube no.stearothermophilus are autoclaved along with the normal load and then transferred to nutrient broth and incubated. Heat labile media. Glassware. but not spores. Guidelines on standard operating procedures for Microbiology.used gloves. Chapter 6: Cultivation of bacteria on laboratory media.58 Sterilization of Commonly Used Culture Media BOX 18-1 PASTEURISATION Pasteurization is a method of sterilisation by moist heat. KEY FACTS 1 Various factors influence sterilization by heat. temperature. hot air oven and incineration. Löwenstein Jensen media. Sugar solution. . 3 How would you sterilize a) sera. 5 Filters should be assembled and autoclaved prior to use. b) Löwenstein Jensen medium. 2 Collins CH. It is sufficient to kill heat labile bacteria. so this is not a very efficient method of sterilization. Vijayalakshmi N. loops. 1998. Microbiological Methods. etc. and thus sterilization cannot be guaranteed. These include time. 3 The load should be properly plugged or wrapped to ensure sterility. etc. 3 Isenberg HD. (Ed) Clinical Microbiology Procedures Handbook. powders. No B. Butterworths. Biohazardous material. VIVA 1 Why should media not be sterilized in large quantities? Ans: Large quantities of media are difficult to sterilize by heat. 2 It is important not to overfill the media container. Parija SC. If methods like filtration are used. large volumes cannot be handled by the filtration apparatus. oils. Indian J Med Microbiol. Needles. Table18-1 Different methods of sterilization of various substances Methods of sterilization Dry heat Flaming Hot air oven Incineration Moist heat Pasteurization Inspissation Boiling Tyndallization Autoclaving Filtration Substances sterilized Inoculating wires. etc. 174-177. antibiotics. Washington. 2002: 20. 2 What are the different methods of sterilizing by dry heat? Ans: These are sterilization by red heat.SOP 54 Version: 1. Heat stable media such as nutrient agar. Inoculation of Culture Media. needles. Uncultivable bacteria: Implications and recent trends towards identification. 5 WHO. glass syringes. 1995. flaming. Some pathogens such as Coxiella burnetii is not destroyed. Two methods are available: holder method (60°C for 30 minutes) and flash method (72° C for 15 seconds). Lyne PM and Grange JM. 4 PHLS Standard Operating Procedures. (World Health Organisation. c) nutrient agar and d) paraffin? FURTHER READINGS 1 Bhattacharya S. Loeffler’s serum slope. American Society for Microbiology. Blood safety and clinical technology. It will not be possible to ensure that all parts of the media have attained sterilization temperature. Serum. nature of material. paraffin . 4 The load should be cooled before being removed from the autoclave/hot air oven. serum. 1992. milk. DC. Geneva) 1997. London. number of microorganisms and spores and their type. 4 Incubate one plate for 3 days at 37°C and the other for 7 days at room temperature. OBSERVATION If the disinfectant has lost activity it will fail to kill microorganism on objects immersed in it. the activity of a disinfectant decreases. 1 With a sterile pipette. The fresh disinfectant will be able to bring about disinfection more effectively than the used one. The concentration at which they are used should be accurate for optimal activity.Textbook of Practical Microbiology 59 LESSON 19 17 INTRODUCTION Antiseptics and Disinfectants LEARNING OBJECTIVES After completing this practical you will be able to become familiar with: 1 The commonly used antiseptics and disinfectants and methods of testing them. A chemical agent that disinfects a substance is called a disinfectant. 1 Fresh disinfectant should be a) sterile. This will show a growth on the nutrient agar plate. With time and use. 3 Inoculate this mixture onto 10 different areas of two well dried nutrient agar plates each. A disinfectant that can be safely applied to living tissue is called an antiseptic. Commonly used disinfectants and their mechanism of actions are summarized in the box 19-1. PROCEDURE Disinfection is defined as the destruction of microorganisms not including bacterial spores. II Reagents and media Sterile nutrient broth. They have markedly different activity against different microorganisms and are generally most effective against Gram positive bacteria. used as well as fresh disinfectant (e. 2 Also mix 1 ml of fresh disinfectant with 9 ml of E coli culture. III Specimen A 24 hour broth culture of Escherichia coli. . QUALITY CONTROL PRINCIPLE Disinfectants are used for medical devices where sterility is not required. transfer 1 ml of the used disinfectant into 9 ml sterile nutrient broth.g. and b) should kill a test inoculum of E coli within 10 minutes. Table 19-1 summarises the list of commonly used disinfectants and antiseptics. RESULTS AND INTERPRETATION If growth occurs on 5 or more spots on either plate. nutrient agar plates. The article to be disinfected should be cleaned thoroughly (to reduce organic matter which may inactivate the agent) or immersed in the disinfection for the required amount of time. The process reduces microorganism to a level acceptable for a defined purpose. lysol). the used disinfectant is said to have failed the test. REQUIREMENTS I Equipments Sterile pipette. Antiseptics can be used for decontamination of living tissue. Uncultivable bacteria: Implications and recent trends towards identification. chlorine and sodium hypochlorite): Iodine is commonly used as an antiseptic. American Society for Microbiology. DC. Chapter 6: Cultivation of bacteria on laboratory media. . Microbiological Methods. Lyne PM and Grange JM. zephiran): They act on the cell membrane of bacteria. Indian J Med Microbiol.g. London. 3 Isenberg HD. FURTHER READINGS 1 Bhattacharya S.g. chloroxylenol): They act by a number of mechanisms such as disruption of cells. Washington. vegetative cells. Vijayalakshmi N. 2 Alcohols (e. 4 Heavy metals and their compounds (e. Phenol is the standard disinfectant against which other disinfectants are compared. copper sulphate): They act by inactivating cellular protein. 4 PHLS Standard Operating Procedures. etc. They act by denaturing proteins damaging lipid complexes and dehydration. sodium lauryl sulfate): They help in mechanical removal of microorganism. Geneva) 1997.g. Blood safety and clinical technology. ethyl alcohol. VIVA 1 What are the commonly used disinfectants and antiseptics? 2 What is the difference between sterilization and disinfection? Ans: Sterilization frees an article of all infectious materials including spores. b) Chick Martin test and c) In-use test. Disinfectants are used for decontamination of non-living tissue.g. 3 Halogens (eg. 1998. iodine. 174-177. It acts by oxidation. Guidelines on standard operating procedures for Microbiology. (Ed) Clinical Microbiology Procedures Handbook. (World Health Organisation. In general. Parija SC. lysol. Butterworths. 2 Collins CH. KEY FACTS 1 2 3 4 Disinfectant does not guarantee sterility. the temperatures used in sterilization are higher than those used for disinfection. and the concentration of chemical agents is higher when they are used for sterilization than disinfection. 5 WHO. 5 Synthetic detergents (e. It doesn’t guarantee the removal or inactivation of spores. 1995. Inoculation of Culture Media.g. precipitation of protein inactivation of enzyme.60 Antiseptics and Disinfectants Table 19-1 List of commonly used disinfectants and antiseptics Disinfectants Lysol Zephiran Glutaraldehyde Sodium hypochlorite Antiseptics 70% ethyl alcohol Chlorhexidine Iodine Dettol BOX 19-1 COMMONLY USED DISINFECTANTS AND THEIR MECHANISM OF ACTIONS 1 Phenols (e. usually at a concentration of 70%): They can also be used as antiseptics and are active against viruses as well. No B. Chlorine is a widely used disinfectant. 6 Quaternary ammonium compound (e. whereas disinfection merely frees an article of infectious.SOP 54 Version: 1. They merely ensure that an object is relatively free of microbial contamination. 2002: 20. 3 What are different methods used for testing disinfectant? Ans: These are : a) Rideal Walker test. 1992. The compounds act by the liberation of nascent oxygen and are effective against most bacteria and viruses. It is essential to test disinfectant from time to time to detect loss of activity. Textbook of Practical Microbiology UNIT 61 IV ENZYMATIC AND BIOCHEMICAL ACTIVITIES OF BACTERIA Lesson 20 Catalase Test Lesson 21 Oxidase Test Lesson 22 Coagulase Test Lesson 23 Urease Test Lesson 24 Indole Test Lesson 25 Methyl Red Test Lesson 26 Voges-Proskauer Test Lesson 27 Citrate Utilization Test Lesson 28 Triple Sugar Iron (TSI) Agar Test Lesson 29 Hydrogen Sulphide Test Lesson 30 Nitrate Reduction Test . Muller-Hinton agar) is tested. Hydrogen peroxide is a by product of aerobic respiration and is lethal if it accumulates in the bacterial cell. Catalase degrades the hydrogen peroxide in the bacterial cell before it can do any damage to the bacterial cell. catalase is a haemoprotein. an intracellular enzyme in the bacteria. the hydrogen peroxide on exposure to light will be broken down into oxygen and water.62 LESSON 20 INTRODUCTION Catalase Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Demonstrate the presence of catalase. Catalase is an enzyme produced by many bacteria. 1 Take 1 ml of 3% hydrogen peroxide in 12 x 100 mm test tube. similar in structure to haemoglobin. test tubes. Note: Hydrogen peroxide must be stored in amber coloured bottles. The enzyme converts hydrogen peroxide into water and oxygen. PROCEDURE Test can be done by 2 methods as follows: 1 Slide method 2 Tube method PRINCIPLE Slide method Chemically. II Specimen Pure growth of bacteria from solid media preferably from nonblood agar plates (Examples: nutrient agar. glass rod / platinum loop / plastic loop and other standard lab wares. 3 Observe the release of bubbles. REQUIREMENTS I Reagents and glass wares 3% hydrogen peroxide. List of catalase positive and negative bacteria are summarized in the table 20-1. 3 Observe for bubble formation. If catalase is present it is indicated by the presence of free gas bubbles. The enzyme splits hydrogen peroxide into water and oxygen. The catalase test is most commonly used to differentiate members of the family Micrococcaceae from members of the family Streptococcaceae (Box 20-1). Use of this solution will give false negative results. 2 Distinguish the bacteria based on the catalase activity. no bubbles will be seen. . Tube method Production of the enzyme catalase can be demonstrated by adding hydrogen peroxide to colonies of the bacteria. Catalase test is also carried out for Mycobacteria to differentiate tubercle bacilli from atypical mycobacteria (Box 20-2). If catalase is absent. except that the four iron atoms in the molecule are in the oxidized (Fe3+) rather than the reduced (Fe2+) state. 2 Immediately add a drop of 3% hydrogen peroxide to the growth. 2 Introduce small quantity of bacterial growth into the fluid with the help of a glass rod or plastic loop and touch the side of the tube. glass slides. If stored in colourless penicillin vials. 2H202 —— 2H2 0+02 (gas bubbles) 1 Transfer pure growth of the organism from the agar to a clean slide with a loop or glass rod. The test is performed by mixing equal volumes of 30% hydrogen peroxide and 0. produce the intracellular enzyme catalase. M. while tubercle bacilli are weakly positive. 1 Staphylococci (catalase +ve) from streptococci and enterococci (catalase . It means bacteria possesses the enzyme catalase. tuberculosis. gastri.ve).ve). b) By the ability of the enzyme catalase to remain active after heating. . forming a few tiny bubbles after 20-30 seconds is not considered a positive test. bovis. On the basis of the semi quantitative catalase test. except certain strains of Mycobacterium tuberculosis complex (some isoniazid resistant strains) and M. It is allowed to stand for a few minutes. gastri and M. Most strains of mycobacteria. Negative control: Streptococcus species (catalase negative bacteria).Textbook of Practical Microbiology 63 QUALITY CONTROL Positive control: Staphylococcus aureus (catalase positive bacteria). M. BOX 20-2 CATALASE TEST FOR MYCOBACTERIA It helps to differentiate tubercle bacilli from atypical mycobacteria.those producing >45mm column height of bubbles. 2 Some bacteria possess enzymes other than catalase that can decompose hydrogen peroxide. haemophilum becomes inactivated. BOX 20-1 USES OF CATALASE TEST The catalase test is useful to differentiate: RESULTS AND INTERPRETATION 1 The rapid and sustained appearance of bubbles or effervescence constitutes a positive test. Most atypical mycobacteria are strongly catalase positive.ve). Tube method Gas bubbles are released when colonies are introduced into the hydrogen peroxide in the test tube.2% catechol in distilled water and then adding it to 5ml of a mycobacterial test culture. mycobacteria are classified in to 2 groups: i. non-spore forming bacilli (catalase . When heated to 68°C for 20 minutes. hence is catalase positive. Effervescence indicates catalase production. 2 Listeria monocytogenes and Corynebacteria (catalase +ve) from other Gram positive. 4 Aerobic bacteria (catalase +ve) from anaerobic bacteria (catalase . Hence. and ii. Table 20-1 Catalase positive and negative bacteria Catalase positive bacteria Staphylococci Micrococci Corynebacterium diphtheriae Enterobacteriaceae Catalase negative bacteria Streptococcus pyogenes Gardnerella vaginalis Fusobacterium species Eikenella corrodens Kingella kinge Shigella dysenteriae type 1 Fatumella ptysees OBSERVATIONS Slide method Gas bubbles are formed immediately when 3% H2 02 is added to the colony. those producing <45mm column height of bubbles. 3 Members of family Enterobacteriacae. Catalase production is assessed by a) Relative activity of the enzyme catalase determined by the height of the column of oxygen bubbles formed by the action of untreated enzyme produced by the organism (Semi quantitative catalase test). the catalase of M. a measure of the heat stability of the enzyme (Heat stable catalase test). 5th ed. 2 Forbes BA. Name some catalase positive and negative organisms. Butterworths. University of Texas. . Allen SD.Houston Medical School. Louis) 2002. DPALM Medic. FURTHER READINGS 1 Collins CH. Most of the aerobes are catalase positive. Schreckenbergu PC and Winn Jr. 1997. catalase. (The CV Mosby Company. Bailey and Scott’s Diagnostic Microbiology. Sahm DF and Weissfeld AS (Eds). Care must be taken while performing catalase test in growth from blood agar plate. WC (Eds). which gives false positive reactions. Most of the anaerobes do not possess the enzyme. London. USA). St. Lyne PM and Grange JM. (Lippincott Williams and Wilkins. Blood (RBC) contains catalase. High concentration of H2 02 is toxic to the cell. In aerobic organisms. VIVA 1 2 3 4 5 What is catalase test? What are the positive and negative controls used in the catalase test? What is the importance of catalase test? Can you take colonies from blood agar plate for testing catalase reaction? Give explanations. Microbiological Methods. Janda WM. 11 th ed. 1995. 1995.64 Catalase Test KEY FACTS 1 2 3 4 5 6 Catalase enzyme splits hydrogen peroxide into oxygen and water. 02 serves as H2 acceptor and hydrogen peroxide (H2 02) is formed in the cell. Color Atlas and Textbook of Diagnostic Microbiology. 3 Introduction to Clinical Microbiology. aerobic bacteria possess catalase to split toxic hydrogen peroxide. Therefore. 4 Koneman EW. an intracellular enzyme in the oxidase-positive bacteria.Aerobic bacteria. 2 Distinguish the bacteria based on the cytochrome oxidase activity. REQUIREMENTS I Reagents and glass wares Fresh reagent: Tetramethyl – p-phenylene diamine . preferably less than 24 hours old. and filter paper. as well as some facultative anaerobes and microaerophiles exhibit oxidase activity. growing on an agar plate or agar slant. The cytochrome oxidase test uses certain reagent dyes such as p-phenylene diamine dihydrochloride which acts as a substitute for oxygen as artificial electron acceptors. List of oxidase positive and negative bacteria is presented in the table 21-1. PROCEDURE The test is performed by following two methods: 1 Direct plate technique. 3 Pick up the colonies to be tested with the help of a glass rod or plastic loop or platinum wire.phenylene diamine dihydrochloride) directly to the bacterial colonies growing on medium in the plate. a purplish blue coloured product. 4 Smear the colonies into the reagent zone of the filter paper. Note: Oxidase reagent is freshly prepared in distilled water every day. 2 Add 2 to 3 drops of reagent (tetramethyl p-phenylene diamine hydrochloride or dimethyl-p. 3 Note the change of colour of the colonies. and 2 Indirect paper strip procedure. and dimethyl – p – phenylene diamine dihydrochloride (1%). Indirect filter paper strip procedure 1 Take a filter paper strip. Cytochrome oxidase catalyzes the oxidation of a reduced cytochrome by molecular oxygen. PRINCIPLE The cytochromes are iron containing haemoproteins that act as the last link in the chains of aerobic respiration by transferring electrons (hydrogen) to oxygen. II Specimen Young culture of bacteria to be tested. dihydrochloride (1%).Textbook of Practical Microbiology 65 LESSON 21 17 INTRODUCTION Oxidase Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Demonstrate the presence of oxidase. The enzyme oxidase plays a vital role in the operation of the electron transport system during aerobic respiration. 5 Note the change in colour if any within 10 seconds. Wood stick/platinum loop/glass rod. 2 Moisten the filter paper strip with freshly prepared 1% oxidase reagent. Direct plate technique 1 Take a nutrient agar plate with colonies of bacteria to be tested. This enzyme oxidises the reagent N-N tetramethyl para-phenylene diamine hydrochloride (a colour less reagent in reduced form) to indophenol blue. resulting in the formation of water or hydrogen peroxide. with the formation of water. Camphylobacter spp. deep blue colour develops at the site of smear within 10 seconds. It means bacteria possesses the enzyme oxidase. Colonies should not be picked up from selective media.parapertusis) 12. Plesiomonas spp. 2. 4. a deep blue colour develops at the site of smear in the filter paper. 7. Branhamella spp. e. Negative control: Escherichia coli (oxidase negative bacteria). Chromobacterium spp. bacterial colonies on the plate develop a deep blue colour at the site of inoculation within 10 seconds. Nichrome wire loops should not be used. Aeromonas spp.Acinetobacter calcoaceticus 3.Brucella canis 5. Cardiobacterium spp. 14. within 10 seconds (Fig. In a negative test the colour of the smear in the zone of the filter paper remain unchanged. hence is oxidase positive. All genera in family Enterobacteriaceae 2. 5. 6 What is chemical name of the oxidase reagent? . Haemophilus spp. Vibrio spp. In a negative test the colour of the colonies remain unchanged. Neisseria spp. Pasteurella multocida Gram negative cocci 1. Bordetella parapertusis 4. Achromobacter spp. Brucella spp (except B. 2. 8.canis) 13. 9. RESULTS AND INTERPRETATION Bacterial colonies having cytochrome oxidase activity develop a deep blue colour at the inoculation site within 10 seconds.Francisella tularensis 6. 15. b. 4 Name oxidase positive bacteria. Results must be observed within 10 seconds.Fresh reagent must be used. cepacia). Pseudomonas spp(except Ps. 16. 21-1). c. 10. Alcaligenes spp. Oxidative negative bacteria 1. Table 21-1 List of oxidase positive and negative bacteria Oxidase positive bacteria Gram negative rods 1. 11. 6. FIGURE 21-1 Slide oxidase test VIVA 1 What is the principle of oxidase test? 2 How is oxidase test done? 3 What are the precautions to be observed while doing oxidase test? Ans: a. Eikinella spp. 3. In filter paper test.Gardnerella vaginalis OBSERVATIONS Direct plate technique In a positive test. Indirect filter paper strip procedure In a positive test. Moraxella spp. Bordetella spp(except B.66 Oxidase Test QUALITY CONTROL Positive control: Pseudomonas aeruginosa (oxidase positive bacteria). 5 Name oxidase negative bacteria. Iron wire loops should not be used. d. Flavobacterium spp. ii more sensitive to the detection of cytochrome oxidase. . 11 th ed. USA). 7 Colonies for testing should not be taken from blood agar. 1995. Schreckenbergu PC and Winn Jr. Sahm DF and Weissfeld AS (Eds). University of Texas. less toxic than dimethyl derivative. (The CV Mosby Company. Lyne PM and Grange JM.Houston Medical School. Colour change must be noted within 10 seconds. 1997.Textbook of Practical Microbiology 67 KEY FACTS 1 2 3 4 The dye. In reduced state the dye is colourless and in oxidized state deep purple in colour. 1995. Microbiological Methods. Tetramethyl derivative of p-phenylene diamine is recommended because the reagent is i more stable in storage. Butterworths. WC (Eds). and iii. Color Atlas and Textbook of Diagnostic Microbiology. p-phenylene diamine dihydrochloride is the substitute for oxygen as artificial election acceptors. Louis) 2002. FURTHER READINGS 1 Collins CH. 2 Forbes BA. 4 Koneman EW. St. London. 3 Introduction to Clinical Microbiology. (Lippincott Williams and Wilkins. DPALM Medic. 5th ed. 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. Janda WM. Allen SD. 6 Always freshly prepared reagent should be used. Bailey and Scott’s Diagnostic Microbiology. an intracellular enzyme in the bacteria. bound coagulase and b. In a positive test. aureus culture filtrates (Box 22 -1). Bound coagulase Bound coagulase is also known as clumping factor. saline. aureus produces the enzyme coagulase in 2 forms: a. aureus from solid media preferably from nonblood agar plates (Examples: nutrient agar. 4 Pick up the colonies of S. which reacts with fibrinogen to produce a visible fibrin clot. by tube coagulase test. PROCEDURE PRINCIPLE Slide test S. In this method. which results in the formation of visible clot. especially Staphylococcus aureus. produced by a few Staphylococcus species. is a key feature of pathogenic Staphylococcus. glass slides. The enzyme causes coagulation of blood. and observing for the prompt clumping of the bacterial suspension within 10-15 seconds. back and froth. the coagulase test is used to identify S. 6 Place another drop of saline in other half of the slide as a control. aureus is liberated to the medium. Presence of free coagulase is tested 1 Take 0. aureus to be tested from agar culture and gently emulsify with drops of saline. . glass rod/platinum loop/plastic loop and other standard lab wares. II Specimen Pure growth of S. a suspension of coagulase producing staphylococci is prepared in plasma in a test tube. Free coagulase Tube test Free coagulase is a thrombin-like substance present in S. I Reagents and lab wares Rabbit plasma with EDTA anticoagulant. allowing the organism to “wall” its infection off from the host’s protective mechanisms rather effectively. coagulase. Fibrin strands are formed between the bacterial cells when suspended in plasma (fibrinogen). 2 Mark it into two halves by a glass marking pencil. 5 Add a drop of undiluted plasma to the bacterial suspension and mix with a wooden applicator sticks. test tubes. Muller-Hinton agar). the enzyme coagulase secreted by S. free coagulase. Presence of this enzyme is tested by slide coagulase test. 1 Take a clean glass slide. 3 Add two drops of sterile saline on two halves of the glass slides. REQUIREMENTS The enzyme.68 LESSON 22 INTRODUCTION Coagulase Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Demonstrate the presence of coagulase. It is bound to the bacterial cell wall and is not present in culture filtrates.5 ml of rabbit plasma (diluted 1 in 5 with saline) in a test tube. In the laboratory. and incubated at 37°C for 3-6 hours. aureus and differentiate it from the other species of coagulase-negative Staphylococcus. 2 Distinguish the bacteria based on the coagulase activity. causing them to clump into visible aggregates. Coagulase is a protein having a prothrombin-like activity capable of converting fibrinogen into fibrin. 7 Rock the slide. List of coagulase positive bacteria is presented in the table 22-1. aureus. A clot should form within 10 to 15 seconds. All strains producing negative slide tests must be tested with the tube coagulase test. 22-1). Table 22-1 List of coagulase positive bacteria 1 2 4 5 6 7 8 Staphylococcus aureus. Bound coagulase is tested by slide coagulase test.Most human strains of S. POSITIVE QUALITY CONTROL Positive control: S. reincubate the tube at room temperature and read again after 18 hours. NEGATIVE FIGURE 22-1 Tube coagulase test. aureus. the plasma in the tube clots and does not flow when the tube is inverted (Fig. These are bound coagulase. Positive reaction will be detected within 10–15 seconds of mixing the plasma with the suspension by the formation of a white precipitate and agglutination of the BOX 22-1 FREE COAGULASE Free coagulase is an extracellular enzyme produced by Staphylococcus aureus. Staphylococcus schleiferi Staphylococcus felis Staphylococcus lutrae Staphylococcus intermedius Staphylococcus hyicus Peptostreptococcus hydrogenalis RESULTS AND INTERPRETATION In slide test.Textbook of Practical Microbiology 69 2 Add approximately 5 drops (250 µl) of overnight broth culture or small amount of the colony growth of S. All coagulase producing staphylococci are S. aureus. Pooled human plasma can be used after checking with a standard strain of S. There are 8 antigenic types of coagulase (designated as A to H). 4 Observe for clot formation by gently tilting the tube. Coagulase may act to coat the bacterial cells with fibrin rendering them resistant to opsonization and phagocytosis. aureus to the diluted plasma in the test tube. Free coagulase is tested by tube coagulase test. Note: On continued incubation. 5 If no clot is observed at that time.5 ml of the reconstituted plasma. and tube coagulase. organisms. aureus produce coagulase A. Coagulability of plasma may be tested by adding one drop of 5% calcium chloride to 0. KEY FACTS 1 2 3 4 5 6 Two types of coagulase are produced by S. the clot may be lysed by fibrinolysin secreted by some strains. The tube coagulase test is considered positive if any degree of clotting is noted. Coagulase does not clot plasma of guinea pigs because they lack CRF. 3 Incubate the tube at 37°C for 4 hours. aureus and as a result coagulase production is considered the best laboratory evidence for the potential pathogenicity of Staphylococcus. All strains that are coagulase positive can be reported as S. aureus (Coagulase positive bacteria). OBSERVATION In a positive slide test. In a positive tube test. the clot already formed may be lysed by fibrinolysin secreted by some strains. Negative control: S. In the tube coagulase test. Rabbit plasma with EDTA is used in both tests. The test is considered negative if no agglutination is observed after 2 minutes. prompt clumping of the organism shows the presence of the bound coagulase. on continued incubation. epidermidis (Coagulase negative bacteria). . It activates a coagulase reacting factor (CRF) normally present in the plasma to clot by the conversion of fibrinogen to fibrin. University of Texas. St. 4 Koneman EW. 1995. Microbiological Methods. Lyne PM and Grange JM. 5th ed. 2 Forbes BA. Janda WM. Sahm DF and Weissfeld AS (Eds). 11 th ed. London. 3 Introduction to Clinical Microbiology. USA). . Color Atlas and Textbook of Diagnostic Microbiology. FURTHER READINGS 1 Collins CH. 1995. WC (Eds). 1997.70 Coagulase Test VIVA 1 2 3 4 5 6 7 What is the principle of slide coagulase test? What is the principle of tube coagulase test? What is the positive control in the test? What is the negative control in the test? How will you interpret the slide coagulase test? How will you interpret the tube coagulase test? Give examples of coagulase positive bacteria. (Lippincott Williams and Wilkins. Allen SD.Houston Medical School. Bailey and Scott’s Diagnostic Microbiology. Louis) 2002. Schreckenbergu PC and Winn Jr. Butterworths. (The CV Mosby Company. DPALM Medic. PRINCIPLE Urea is a diamide of carbonic acid. 4 Observe any change of colour in the inoculated medium.1 gm. Christensen’s urea agar. The composition and preparation of Christensen’s urea agar is described in the box 23-1. Preparation Prepare the base. thus indicating the presence of urease activity. Urease. 3 Incubate the tube at 37°C for 18 hours. List of urease producing microorganisms is summarized in the Table 23-1.2 gm. Ammonia reacts in solution to form ammonium carbonate. NaCl – 0. Allow the medium to solidify in a slanting position in such a way to get half inch butt and one inch slant. Phenol red (1. Monopotassium phosphate – 0. Distilled water – 100 ml. REQUIREMENTS I Equipments Incubator. . Phenol red that is incorporated in the medium changes its color from yellow to red in alkaline pH. hydrolyses urea and releases ammonia and carbon dioxide.Textbook of Practical Microbiology 71 LESSON 23 INTRODUCTION Urease Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Demonstrate the presence of urease. This produces a change in the pH of the medium that can be detected by the color change in the indicator dye. III Specimen Pure growth of Proteus mirabilis from solid media preferably from non-blood agar plates (Examples: nutrient agar. MullerHinton agar) is tested. 2 Distinguish the bacteria based on the urease activity. II Reagents and glass wares Inoculating wire.0 ml. 1 Pick up the colonies of P. Agar – 2 gm.8. mirabilis from the culture on nutrient agar. which is alkaline leading to an increase in pH of the medium. sterilize by autoclaving at 121°C for 15 min. pH – 6. BOX 23-1 CHRISTENSEN’S UREA AGAR Composition Peptone – 0. Cool to 50°C in a waterbath and then add 5 ml of filter sterilized 40% urea solution. the enzyme produced by the bacteria and fungi. This test can be used to differentiate different groups of bacteria and fungi. and 12 × 100 mm test tubes. an intracellular enzyme in the bacteria. PROCEDURE Certain bacteria and fungi possess the enzyme urease that hydrolyzes urea releasing ammonia into the medium. Glucose – 0. 2 Inoculate Christensen’s urea agar slope with these bacterial colonies. Mix. distribute in 2–4 ml amounts in 12×100 mm test tubes.5 gm.1 gm.2%) – 1. The test is considered negative if no colour change of the medium is observed (E. thus indicating the presence of urease activity. 23-1). mirabilis). When positive. the color of the medium changes to purple pink (P. the colour of the medium changes to purple pink. mirabilis (urease positive bacteria). Name the slow. purple pink colour is seen throughout the medium which indicates alkalinization and urea hydrolysis. purple pink colour throughout the medium indicates alkalinization and urea hydrolysis.The test should not be considered negative till after four days of incubation. mirabilis tested is a urease producing bacteria. 4. rapid. after incubation. Ans: Urease test is used in mycology for identification of urease positive Cryptococcus neoformans.72 Urease Test Table 23-1 Urease producing bacteria and fungi Urease producing bacteria Strong (or) most rapid urease producers Brucella species Helicobacter pylori Rapid urease producers Proteus species Morganella species Slow urease producers Klebsiella species Enterobacter species Urease producing fungi Cryptococcus neoformans Trichophyton mentagrophytes FIGURE 23-1 Urease negative and positive test. KEY FACTS 1 Certain bacteria and fungi possess the enzyme urease that hydrolyzes urea releasing ammonia into the medium. and most rapid urease producing bacteria. coli) (Fig. P. 5. Describe principle of the urease test. The uninoculated medium is colour less. Christensen’s urea agar is a solid medium whereas Stuart’s broth is a liquid medium used for testing the urease activity of the bacteria. List urease producing microorganisms. List different media used for testing urease activity of the microorganism and how do you interpret the results? Ans. 3. List an important use of urease test in mycology. coli does not produce the enzyme urease. RESULTS AND INTERPRETATION Positive reaction is detected after 18 hours of incubation. List the compositions of the Christensen’s urea agar medium. VIVA 1. OBSERVATION Examine the medium after four hours and after overnight incubation. In Christensen’s agar. In Stuart’s broth. Negative control: Escherichia coli (urease negative bacteria). 2 Phenol red that is incorporated in the medium changes its color from yellow to red in alkaline pH. NEGATIVE POSITIVE QUALITY CONTROL Positive control: P. 3 Control strains should be used for interpretation of results. 6. 2. In a positive test. E. . An uninoculated medium is incubated along with the test to compare the colour change. WC (Eds). Color Atlas and Textbook of Diagnostic Microbiology. 2 Forbes BA.Houston Medical School. 3 Introduction to Clinical Microbiology. Butterworths. Schreckenbergu PC and Winn Jr. 11 th ed.Textbook of Practical Microbiology 73 FURTHER READINGS 1 Collins CH. Bailey and Scott’s Diagnostic Microbiology. Louis) 2002. 1995. 4 Koneman EW. Microbiological Methods. . Janda WM. Sahm DF and Weissfeld AS (Eds). Lyne PM and Grange JM. St. Allen SD. DPALM Medic. 5th ed. University of Texas. London. (The CV Mosby Company. 1997. USA). (Lippincott Williams and Wilkins. 1995. 0 gm. Positive indole test is indicated by the appearance of red-violet ring on adding the reagent. absolute ethyl alcohol. Kovac’s reagent consists of para-dimethyl amino benzaldehyde.Only some bacteria produce indole.0 ml. 2 Distinguish the bacteria based on the indole activity. and when acted upon by the enzyme tryptophanase.74 LESSON 24 INTRODUCTION Indole Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Determine the ability of bacteria to degrade the amino acid tryptophan.0 gm. E. Tryptophan is an essential amino acid that can undergo oxidation by enzymatic activities of some bacteria. a red-violet ring develops within minutes on addition of Kovac’s reagent. coli colonies tested are an indole producing bacteria. skatole and indole acetic acid. isoamyl alcohol. 75. In a negative test a yellow ring appears. and concentrated hydrochloric acid. 380. coli in a small test tube. and concentrated hydrochloric acid. 24-1). Kovac’s reagent.5 ml of 24 hours to 48 hours peptone water cultures of E. REQUIREMENTS RESULTS AND INTERPRETATION I Equipments Incubator. or Ehrlich’s reagent. 80. Conversion of tryptophan into metabolic products is mediated by the enzyme tryptophanase. OBSERVATION In a positive test. Ehrlich’s reagent consists of p-dimethyl amino benzaldehyde. The indole reacts with aldehydes to produce a red coloured product. coli (indole positive bacteria). 4. Negative reaction is indicated by developing a yellow ring (Fig. List of indole positive and negative bacteria are presented in the Table 24-1 QUALITY CONTROL Positive control: E. 3 Allow it to stand for few minutes and read the result. it is converted into indole. II Reagents and lab wares Peptone water / tryptone broth.0 ml. K.0 ml. The aldehyde used in the test is para dimethyl amino benzaldehyde.2 ml of Kovac’s reagent to the peptone water and shake. 2 Add 0. 25. glass tubes and inoculating wire. PROCEDURE 1 Take 0. PRINCIPLE Tryptophan is an amino acid. 5.0 ml. Negative control: Klebsiella pneumoniae (indole negative bacteria).pneumoniae does not produce the indole . The ability to hydrolyse tryptophan with the production of indole is not a characteristic of all bacteria. The metabolic end products are indole. III Specimen 24 hours to 48 hours peptone water culture of Escherichia coli incubated at 37°C. skatole and indole acetic acid.This is present in peptone water of the culture medium. London. Aeromonas hydrophila 7.Take 0. Louis) 2002. 1995. Ans. Color Atlas and Textbook of Diagnostic Microbiology. WC (Eds). This can be also tested in another method by adding 0. Klebsiella oxytoca 3. 1995. USA). Proteus mirabilis 4. Schreckenbergu PC and Winn Jr. Then 0. Sahm DF and Weissfeld AS (Eds). Plesiomonas shigelloides Indole negative bacteria 1. Morganella morganii 5. Janda WM. Microbiological Methods. Klebsiella pneumoniae 3. University of Texas.5 ml of Ehrlich’s reagent is added and the result read. 4 Koneman EW. Salmonella Typhi 5. (Lippincott Williams and Wilkins. Providencia rettgeri 6. Lyne PM and Grange JM. 11 th ed. .5-1 ml of xylene or ether to the culture broth and shaking it well.Shake and allow it to stand for few minutes then observe the result. VIVA 1 2 3 4 5 6 What are the end products of tryptophan metabolism? What is the principle behind the indole production test? What are the reagents used in this test? What are the constituents of Kovac’s reagent? What are the constituents of Ehrlich’s reagent? How to test indole production by adding Ehrlich reagent. FURTHER READINGS 1 Collins CH. Proteus vulgaris 4. Bailey and Scott’s Diagnostic Microbiology. Vibrio cholerae 9.5 ml of culture broth and mix with equal volumes of Ehrlich’s reagent. Positive reaction is indicated by development of red-violet ring and negative by yellow ring. Pasteurella multocida 8. (The CV Mosby Company. 1997. Escherichia vulnaris 2. DPALM Medic. Shigella sonnei NEGATIVE POSITIVE FIGURE 24-1 Indole negative and positive test. 5th ed. KEY FACTS 1 End product of tryptophan metabolism by tryptophanase is indole. and indole acetic acid. St. Butterworths. Allen SD. Escherichia coli 2. skatole. 3 Introduction to Clinical Microbiology.Houston Medical School. Falvobacterium 10. 2 Forbes BA.Textbook of Practical Microbiology 75 Table 24-1 List of indole positive and negative bacteria Indole positive bacteria 1. 2 Indole reacts with p-dimethyl amino benzaldehyde to form Quinoidal red-violet compound. The pH at which methyl red detects acid is considerably lower than the pH of other indicators used in bacteriologic culture media. only organisms that can maintain this low pH after prolonged incubation (48–72 hours) overcoming the pH buffering system of the medium can be called methyl red positive. PRINCIPLE PROCEDURE Methyl red is a pH indicator with a range between 6.This test is of value to differentiate between E. The low acidic pH (4) is stabilized and maintained by E. the test organism must produce large quantities of acid from the carbohydrate substrate being used. dipotassium phosphate. 5 gm.4 (red).04% solution of methyl red directly to the broth culture and mix well. coli in a small test tube. 2 Add five drops of 0.9. 5 gm.76 LESSON 25 INTRODUCTION Methyl Red Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Determine the ability of bacteria to oxidise glucose with the production of high concentrations of acidic end products by methyl red test. 0. 3 – butanediol and acetoin (acetyl methyl carbinol) resulting in an elevated pH of 6.0 (yellow) and 4. coli. formic acid) from glucose through the mixed acid fermentation pathway. It consists of methyl red. During the later period of incubation. 3 Note any change in the colour of medium at once. acetic acid. 1 Take 0. Because many species of the Enterobacteriaceae may produce sufficient quantities of strong acids that can be detected by methyl red indicator during the initial phase of incubation. aerogenes and Klebsiella pneumoniae in glucose phosphate medium incubated at 30°C for five days. 7 gm.1 g in 300 ml of 95% ethyl alcohol. and distilled water. The methyl red test is a quantitative test for acid production. requiring positive organisms to produce strong acids (lactic acid. Methyl red test broth. aerogenes enzymatically converts these acids to non-acidic end products such as 2. Thus to produce a colour change. III Specimen Culture of E. REQUIREMENTS I Equipments Incubator. It consists of poly peptone. In MR test the methyl red is the pH indicator.5 ml of broth cultures of E. Methyl red indicator. Both these bacteria initially produce organic acidic products during the early period of incubation. glucose. 2 Differentiate between all glucose oxidizing enteric bacteria particularly Escherichia coli and Enterobacter aerogenes. E. II Reagents and lab wares Inoculating wire. and other members of the family Enterobacteriacae. The methyl red detects the presence of large concentrations of acidic end products. coli at the end of incubation. . List of MR positive and negative bacteria is presented in the table 25-1. The end products of this process vary depending on the specific enzymatic pathways present in the bacteria. coli and E. aerogenes. E.The hexose monosaccharide glucose is the major substrate oxidized by the enteric bacteria. 1l at a pH of 6. All enteric bacteria ferment glucose with the production of organic acids and energy. Because other organisms may produce smaller quantities of acid from the test substrate. 5th ed. Janda WM. 1997. . Development of red colour on addition of methyl red to broth culture of bacteria is considered positive. Enterobacter spp RESULTS AND INTERPRETATION The development of a stable red colour in the surface of the medium indicates sufficient acid production to lower the pH to 4.4 and constitutes a positive test. 4 Koneman EW. St. Microbiological Methods. because the organisms have to produce sufficient quantities of acid and it has to be maintained in the same acidic pH which turns the methyl red indicator to red colour. E. aerogenes. 1995. The test differentiates between E. INDOLE MR VP CITRATE FIGURE 25-1 IMViC Test. coli and E. Salmonellae 7. WC (Eds). Allen SD. 25-1). University of Texas. KEY FACTS 1 2 3 4 5 MR test detects the products of stable high concentration of acidic end products. Butterworths. This does not indicate a positive test. USA). rhinoscleromatis 4. Proteae 9. K. aerogenes OBSERVATION Look for the development of stable red colour on adding methyl red indicator. Bailey and Scott’s Diagnostic Microbiology. Schreckenbergu PC and Winn Jr. Citrobacter 8. 1995. coli Negative control: E. Yellow colour indicates a negative test (Fig. an intermediate orange colour between yellow and red may develop. ornitholytica 5. 2 Forbes BA. 5 What is the indicator used in this test? FURTHER READINGS 1 Collins CH. 11 th ed. pneumoniae 2. QUALITY CONTROL Positive control: E. coli 2. VIVA 1 2 3 4 What are the group of organisms that can be differentiated by MR test? What is the principle of MR Test? What are positive and negative controls used in the test? How long you have to incubate the test and why? Ans: The broth is incubated for 48-72 hr. (Lippincott Williams and Wilkins. Lyne PM and Grange JM.Textbook of Practical Microbiology 77 Table25-1 List of MR positive and negative bacteria MR positive bacteria 1. London. DPALM Medic. Yersinia MR negative bacteria 1. K. Methyl red is yellow in alkaline pH and red in acidic pH. Sahm DF and Weissfeld AS (Eds). K. Edwardsielleae 6. ozaenae 3.Houston Medical School. K. (The CV Mosby Company. 3 Introduction to Clinical Microbiology. Methyl red is the indicator used in the test. Louis) 2002. Color Atlas and Textbook of Diagnostic Microbiology. .78 LESSON 26 INTRODUCTION Voges-Proskauer Test LEARNING OBJECTIVES After completing this practical you will be able to: small amount of acetyl methyl carbinol present in the medium is converted to diacetyl. glucose. REQUIREMENTS Voges-Proskauer is a double eponym. In the presence of atmospheric oxygen and alkali (40% potassium hydroxide). It was later discovered that the active product in the medium formed by bacterial metabolism is acetyl methyl carbinol. List of VP positive and negative bacteria is presented in the table 26-1. II Reagents and lab wares Inoculating loop. It consists of polypeptone. It serves as the colour intensifier.6 ml of a 5% solution of a naphthol in ethanol to the broth culture and shake gently. PRINCIPLE The Voges-Proskauer test determines the capability of some bacteria to produce non-acidic or neutral end products such as acetyl methyl carbinol from the organic acids produced as a result of glucose metabolism. 7 gm. It consists of 40 gm potassium hydroxide in 100 ml distilled water. VP broth. a product of the butylene glycol pathway. They first observed the red colour reaction produced by appropriate culture media after treatment with potassium hydroxide. 5 gm and distilled water. the pivotal compound formed in the fermentative degradation of glucose is further metabolised through various metabolic pathways. I Equipments Incubator. 2 First add 40% KOH and then add 0. 3 Note any change in the colour of medium within 2-5 minutes. 40% potassium hydroxide. and absolute ethyl alcohol.5 gm. a neutral-reacting end product. dipotassium phosphate. depending on the enzyme systems possessed by different bacteria. It consists of a naphthol. The test depends on the production of acetyl methyl carbinol from pyruvic acid. which reacts with the peptone of the broth to produce a red colour. 5% a naphthol. 100 ml. named after two microbiologists working at the beginning of the 20th century. the PROCEDURE 1 Take 1 ml of broth cultures of E. III Specimen Culture of Escherichia coli. 1 Perform Voges-Proskauer test. 5 gm. Enterobacter aerogenes and Klebsiella pneumoniae in glucose phosphate medium incubated at 30°C for five days or 37°C for 48 hours. One such pathway results in the production of acetoin (acetyl methyl carbinol). It serves as the oxidising agent. coli in a small test tube. Enteric bacteria such as members of the Klebsiella-EnterobacterHafnia-Serratia group produce acetoin as the chief end products of glucose metabolism and form smaller quantities of mixed acids.9. Pyruvic acid. as an intermediate product in its conversion to 2: 3 butylene glycol. It is essential that the reagents are added in this order. 1 litre at a pH of 6. coli. 3 Introduction to Clinical Microbiology. Proteae 5. Sphingomonas paucinobilix VP negative bacteria 1. Schreckenbergu PC and Winn Jr. University of Texas. DPALM Medic. A negative test is indicated by colour less reaction for half an KEY FACTS 1 The VP test shows the presence of non acidic neutral end product acetyl methyl carbinol. 2 Forbes BA. Table 26-1 VP positive and negative bacteria OBSERVATIONS Look for the development of pink colour 15 minutes or more after addition of the reagents. 4 Some times prolonged incubation of cultures is required before doing the test. 11 th ed. Color Atlas and Textbook of Diagnostic Microbiology. FURTHER READINGS 1 Collins CH. 1997. Serratia marcescens 6. deepening to magenta or crimson in half an hour. VP positive bacteria 1. Butterworths. Lyne PM and Grange JM. Sahm DF and Weissfeld AS (Eds). Chryseomonas luteola 9. Salmonellae 4.The test should not be read after standing for over 1 hour because negative VP test may produce a copper-like colour. Aeromonas sobria 7. Klebsiella pneumoniae 2. Bailey and Scott’s Diagnostic Microbiology. 2 A positive test is represented by the development of a pink colour 15 minutes or more after addition of the reagents. . Allen SD. 3 A negative test is indicated by colour less reaction for half an hour.Textbook of Practical Microbiology 79 QUALITY CONTROL Positive control: E. WC (Eds). Flavimonas oryzihabitans 10. USA). 1995. Vibrio cholerae 8. (The CV Mosby Company. 4 Koneman EW. Louis) 2002. Cedicia netri 4.This indicates the presence of diacetyl. Escherichia coli 2. 5th ed. Microbiological Methods. Enterobacter cloacae 3. (Lippincott Williams and Wilkins. leading to a false positive interpretation. London. St. Yersinieae RESULTS AND INTERPRETATION A positive test is represented by the development of a pink colour 15 minutes or more after addition of the reagents. Janda WM. hour. Negative control: E. 1995. the oxidation product of acetoin. Ewingella americana 5. Edwardsiella tarda 3. deepening to magenta or crimson in half an hour. VIVA 1 2 3 4 What is the principle of VP test? What are the positive and negative controls used in the VP test? Name some VP positive bacteria? Describe the biochemical reaction in the VP test. aerogenes.Houston Medical School. List of citrate positive and negative bacteria are presented in the table 27-2. any medium used to demonstrate citrate utilisation by test bacteria must be devoid of protein and carbohydrates as sources of carbon. PRINCIPLE In the absence of fermentable glucose or lactose. magnesium sulfate. a citrate permease that facilitates the transport of citrate in the cell. 0. Enterobacter aerogenes and Klebsiella pneumoniae in glucose phosphate medium incubated at 37°C for 48 hours. Citrate is the first major intermediate in the Krebs cycle and is produced by the condensation of active acetyl with oxaloacetic acid. III Specimen Culture of Escherichia coli.9 to deep Prussian blue at pH 7. PROCEDURE 1 Using sterile technique. This ability depends on the presence of the enzyme. agar. Koser’s citrate It is a broth (liquid medium) It contains no agar It does not contain any indicator Positive test is by observing the turbidity in the medium . Some bacteria can also obtain energy by using citrate as the sole source of carbon. an alkaline product. 5 gm. dipotassium phosphate. These products are then enzymatically converted to pyruvic acid and carbon dioxide (CO2). 2 gm. During this reaction the medium becomes alkaline because the CO2 that is generated combines with sodium and water to form sodium carbonate. Sodium citrate is a salt of citric acid. some bacteria are capable of using citrate as a sole source of carbon for their energy. Simmon’s citrate and Koser’s citrate are two examples of different types of citrate media used in the test. 1 gm. inoculate each bacteria into its appropriately labeled tube by means of a stab and streak inoculation. The medium is poured into a tube on a slant. Hence. 2 Incubate all cultures for 24 hours to 48 hours at 37°C.6. The presence of sodium carbonate changes the indicator.9. Citrate is acted on by the enzyme citrase which produces oxaloacetic acid and acetate. a simple organic compound produced as one of the metabolites in the tricarboxylic acid cycle of the bacteria. bromo thymol blue present in the medium from green at pH 6.08 gm and distilled water 1 litre) pH adjusted to 6. sodium chloride. sodium citrate. Table 27-1 Differences between Simmon’s citrate and Koser’s citrate Simmon’s citrate It is a slant (solid medium) It contains agar This medium contains bromothymol blue as indicator Positive test is indicated by growth on the medium and change in the colour of the medium. Simmon’s citrate medium (It consists of ammonium dihydrogen phosphate. 1 gm. 0. Differences between the two media are summarized in the table 27-1. bromo thymol blue. II Reagents and lab wares Inoculating loop. REQUIREMENTS I Equipments Incubator.20 gm. 15 g.80 LESSON 27 INTRODUCTION Citrate Utilisation Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Differentiate certain enteric organisms on the basis of their ability to utilize citrate as a sole source of carbon. Lyne PM and Grange JM. with the production of alkaline products. Butterworths. VIVA 1 2 3 4 5 What are the constituents of Simmon’s citrate medium? What is the principle behind citrate utilisation test? What is the positive control and negative controls used in the citrate utilisation test? What are the organisms that are citrate utilisation positive and negative? 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. Sahm DF and Weissfeld AS (Eds). Development of deep blue colour is taken as positive. . Ans: Simmon’s citrate utilization test and Koser’s citrate utilization test. Salmonella Paratyphi A 4. Escherichia coli 2. Bailey and Scott’s Diagnostic Microbiology. Citrobacter diversus 2. Acroncobacter oxylosoxidans 7.Textbook of Practical Microbiology 81 QUALITY CONTROL Positive control: E. 27-1).Houston Medical School. KEY FACTS 1 2 3 4 Simmon’s citrate medium contains sodium citrate which acts as a sole source of carbon. Yersinia enterocolitica 6. 5th ed. Louis) 2002. 1995. OBSERVATIONS Look for the development of deep blue colour within 24-48 hours of incubation of the inoculated tube. Providencia alcalifaecians 5. A negative test is indicated by no change of colour of the citrate medium (Fig. Vibrio holisae 8. St. Euringella americana 6. coli. Color Atlas and Textbook of Diagnostic Microbiology. DPALM Medic. FURTHER READINGS 1 Collins CH. 1995. (Lippincott Williams and Wilkins. Serratia marcescens 4. 11 th ed. 2 Forbes BA. WC (Eds). with the production of alkaline products. University of Texas. 3 Introduction to Clinical Microbiology. Enterobacter cloacae 3. Vibrio vulnificus NEGATIVE POSITIVE FIGURE 27-1 Citrate negative and positive test. 6 Mention different types of citrate utilization tests. 4 Koneman EW. A negative test is indicated by no change of colour of the citrate medium. USA). (The CV Mosby Company. Edwardsiella tarda 7. aerogenes. Janda WM. Microbiological Methods. Salmonella Typhi 3. 7 List differences between Koser’s citrate and Simmon’s citrate media. Table 27-2 List of citrate positive and negative bacteria Citrate positive bacteria Citrate negative bacteria 1. Shigella species 5. Schreckenbergu PC and Winn Jr. London. Allen SD. Bromothymol blue is the indicator used in Simmon’s citrate medium. Negative control: E. 1997. Klebsiella pneumoniae 1. RESULTS AND INTERPRETATION A positive test is represented by the development of a deep blue colour within 24 hours to 48 hours. indicating that the test organism has been able to utilize the citrate contained in the medium. Phenol red is the acid base indicator incorporated in the medium. sucrose. phenol red. lactose. So the acid production continues to occur even after 18–24 hours and both the slant and the butt appear yellow.This indicator helps to detect carbohydrate fermentation that is indicated by a change in colour of the medium from orange red to yellow in the presence of acid. fermentation continues as the organism is able to use lactose which is present in concentration 10 times that of glucose. Sodium thiosulfate is the source of sulfur atoms. The triple sugar iron (TSI) agar is an example of a composite medium used widely for the identification of bacterial isolates. then even after the glucose is completely used up in first 8–12 hours. II Reagents and lab wares Inoculating loop and triple sugar iron agar (It contains beef extract. REQUIREMENTS I Equipments Incubator. 15g. glucose.82 LESSON 28 INTRODUCTION Triple Sugar Iron (TSI) Agar Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Differentiate among the members of the family Enterobacteriaceae by the TSI test. In the deeper part of the tube. agar. 12 gm. sodium chloride.TSI medium indicates whether the bacteria ferments glucose only. peptone. 5g. to yellow and the whole medium appears yellow in colour. 0.3g. and distilled water to equal 1 litre) at pH 7. The TSI agar has glucose. 1 gm. medium must include an indicator to detect the H2 S. The TSI agar is designed to differentiate among different groups or genera of the family Enterobacteriaceae. After further incubation as the glucose is fully exhausted. the bacteria begin to oxidatively degrade the amino acid present in the medium. or lactose and sucrose also with or without production of gas. ferrous sulfate. lactose. so medium in the butt part remains yellow in colour.This medium is convenient and economical. The medium can detect production of hydrogen sulphide (H2S) as well as other bacteria which utilizes only glucose (but not lactose or sucrose). 10 gm.4.024 g. The differentiation can be made on the basis of differences in carbohydrate fermentation and hydrogen sulfide production by various intestinal bacteria. proteose peptone. Proteus mirabilis and Klebsiella pneumoniae in glucose phosphate medium incubated at 37°C for 48 hours.2 gm. Ferrous sulfate is the indicator used for the detection of the H2S which is indicated by the production of insoluble black precipitate. For the detection of H2S which is a colourless gas. PRINCIPLE The TSI agar is distributed in the tube which contains a slant and a butt. If the TSI medium is inoculated with lactose fermenting organism. The latter are the Gram Negative bacilli capable of fermenting glucose with the production of acid. This oxidative degradation results in production of alkali products. amino acid degradation is insufficient to overcome the acid formed. 5g. oxidative degradation occurs only in the slant portion. III Specimen Culture of test organisms such as Escherichia coli. Since oxygen is exposed only to the slant portion. and sucrose as the sources of carbohydrates.1%. 3g. sodium thiosulfate. yeast extract. which reverts the colour of the slant to red colour. 10g. 0. because as a single composite medium different properties of the bacteria which otherwise would have required the use of many separate media could be used. Due to the acid production. 0. the colour of the phenol red (indicator) is changed . The slant contains lactose and sucrose in the concentration of 1% and glucose in the concentration of 0. 3g. glucose and peptone 4. This is characteristic of non -lactose fermenting. OBSERVATIONS Look for the colour change in the slant and butt after 18–24 hours incubation and also look for the development of black precipitate to indicate H2S production (Fig. Example: P. Proteus spp. 5 Acid butt/acid slant. What is the H2S indicator? 7. RESULTS AND INTERPRETATION 1 Alkaline slant /alkaline butt (K/K reaction): This shows no carbohydrate fermentation. Enterobacter spp. 3 Alkaline slant /acidic butt /production of H2 S (K/A reaction and positive for H2 S): Salmonella spp. . Glucose fermenters break down glucose first then lactose. So the pH of the slant is alkaline after 8 hours. and sucrose) are fermented. QUALITY CONTROL 1 Alkaline slant /alkaline butt (K/K reaction): Pseudomonas aeruginosa. When the reading of TSI test is taken and why? Ans: The readings of TSI test is taken only after 18-24 hrs. If the organism is non lactose or sucrose fermenter. Lactose and sucrose are not fermented. 2 Incubate all cultures for 18 hours to 24 hours at 37°C. coli. Enterobacter spp. This is characteristic of lactose fermenting coliforms. inoculate each bacterial colony into its appropriately labeled tube by means of a stab and streak inoculation. Klebsiella spp. What is the source of nutrition? Ans: Sucrose. A+/A- Un inoculated K-/K- K+/A+ FIGURE 28-1 TSI reactions. Citrobacter spp. Example: Salmonella spp. Example E. 3 Alkaline slant /acidic butt /black precipitate of H2S (K/A reaction and positive for H2S): Glucose is fermented. What is the source of sulfur? 3. producing alkaline end products. VIVA 1. H2 S positive: Citrobacter. lactose.Textbook of Practical Microbiology 83 PROCEDURE 1 Using sterile technique. Example: Shigella. It indicates that the organism is a non -lactose fermenter. coli. hydrogen sulfide producing bacteria. The reason behind this is that the glucose concentration is 10 times less than that of lactose and sucrose.28-1). 4 Acidic slant /acidic butt (A/A reaction): E. In the first 6-8 hours whole of the tube will be acidic and will be yellow in colour. What is the principle behind this test? 6. Vibrio. Production of alkaline end products results in the change of pH alkaline side and pink colour develops in the slant. So the results are read after 24 hours. Citrobacter spp. 2 Alkaline slant /acidic butt (K/A reaction): Glucose is fermented. Vibrio. What are the sugars used and at what concentration they are used in the TSI agar? 5. Proteus spp. aeruginosa. lactose. Klebsiella spp. This indicates that the bacteria are non-fermented. Lactose and sucrose is not fermented. the organisms metabolise peptones aerobically for their energy. 2 Alkaline slant /acidic butt (K/A reaction): Shigella. 4 Acidic slant /acidic butt (A/A reaction): All the sugars (glucose. What are the constituents of TSI medium? 2. Butterworths. 5th ed.Houston Medical School. Louis) 2002. 4 Koneman EW. Lyne PM and Grange JM. DPALM Medic.84 Triple Sugar Iron (TSI) Agar Test KEY FACTS 1 2 3 4 5 6 Beef extract. Ferrous sulfate is the indicator for the production of the hydrogen sulfide. Allen SD. Sahm DF and Weissfeld AS (Eds). Color Atlas and Textbook of Diagnostic Microbiology. FURTHER READINGS 1 Collins CH. Microbiological Methods. USA). Phenol red is the indicator used to detect acid /alkaline changes in the medium. Oxidative degradation of amino acids in the medium occurs only in the slant. and sucrose are added in the ratio of 1: 10 in the TSI agar. (The CV Mosby Company. Bailey and Scott’s Diagnostic Microbiology. and proteose peptone makes the medium nutritionally rich. Sodium thiosulfate is the source of sulfur. . 11 th ed. 2 Forbes BA. Janda WM. St. 1995. 3 Introduction to Clinical Microbiology. yeast extract. WC (Eds). (Lippincott Williams and Wilkins. 1997. peptone. University of Texas. Schreckenbergu PC and Winn Jr. 1995. Glucose and lactose. London. the indicators such as heavy metal ions are added to the medium. Bacteria which are tested for the production of H2 S must contain enzyme system which release sulfide from the sulfur source. which support the growth of the organism. cystine. which is the source of sulfur in the medium. Pathway 1: Gaseous H2S may be produced by the reduction (hydrogenation) of organic sulfur present in the amino acid cysteine. The medium contains sodium thiosulfate. This amino acid in the presence of the enzyme. 3 Incubate all cultures for 18 hours to 24 hours at 37°C. cysteine or inorganic compounds such as this sulphates. REQUIREMENTS I Equipments Incubator. 2 Inoculate by stabbing down the center of agar butt carefully and then streak the surface of the slant. III Specimen Soy broth cultures of test bacteria such as Enterobacter aerogenes. Proteus vulgaris and Salmonella Typhimurium incubated at 37°C for 24. etc. should be present in the medium to detect the presence of the H2 S. H2S being a gas. pick up the organisms from the top of a single colony from primary isolation plate or from pure growth with a straight wire. Sulfides combine with hydrogen ion to form H2 S. The sulfur atoms act as hydrogen acceptors during oxidation of the inorganic compound. Sulfur containing amino acids such as methionine. H2 S combines with heavy metals to form insoluble black precipitate. PRINCIPLE There are two major fermentative pathways by which hydrogen sulfide is produced by bacteria. will escape from the medium. and inoculating loop. Some bacteria liberate sulfur from sulfur containing amino acids or other sulfur containing compounds.Textbook of Practical Microbiology 85 LESSON 29 INTRODUCTION Hydrogen Sulfide Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Determine the ability of certain bacteria to produce hydrogen sulfide from substrates such as the sulfur containing amino acids or inorganic sulfur compounds.48 hours. Pathway 2: Gaseous H2S may also be produced by the PROCEDURE 1 Using sterile technique. sulfates (SO4) or sulfites (SO3). List of bacteria producing hydrogen sulphide is presented in the table 29-1. a cysteine desulfurase. TSI agar or lead acetate agar. List of media used for detecting production of hydrogen sulphide is presented in the box 29-1. which are reduced to sulfite by certain microorganisms with the liberation of hydrogen sulfide. So. II Reagents and lab wares Sulphur containing medium such as Kligler iron agar. etc. Liberated H2S combines with indicator like ferrous sulfate (Fe SO4) to produce black insoluble precipitate (ferrous sulfide). . The sulfur is used as final hydrogen acceptor leading to the formation of hydrogen sulphide (H2S). loses the sulfur atom and is then reduced by the addition of hydrogen from water to form hydrogen gas. reduction of inorganic sulfur compounds such as the thiosulfates (S2O3). Deoxycholate citrate agar 4. Lead acetate agar 8. Peptone 3. Citrate sulfide agar 3. Kligler iron agar 6.86 Hydrogen Sulphide Test BOX 29-1 LIST OF MEDIA USED FOR DETECTING PRODUCTION OF HYDROGEN SULFIDE List of media 1. aerogenes. Hektoen enteric agar 12. Xylose lysine deoxycholate agar 11. Lead acetate 4. Sulphur indole motility medium 10. Typhimurium. Peptone water. Sulfite 2. vulgaris. NEGATIVE POSITIVE FIGURE 29-1 TSI agar showing absence and presence of H2S. 29-1). If black colour is not produced then H2S is not produced (Fig. Negative control: E. QUALITY CONTROL Positive control: P. Sources of sulfur 1. Note: Source of sulfur and H2S indicators are different for each medium. Ferric ammonium citrate 3. Bismuth sulfite agar 2. S. 5. lead acetate paper inserts. Paratyphi A) 4 Proteus mirabilis 5 Proteus vulgaris 6 Edwardsiella tarda 7 Edwardsiella hoshinae 8 Shewanella putrefaciens 9 Campylobacter sputorum 10 Brucella abortus 11 Brucella suis. 12 Erysiphilothrix rhusiopathiae RESULTS AND INTERPRETATION Black coloration along the streak line or throughout the medium indicates H2S production. TSI agar 7. Salmonella-Shigella agar 9. Table 29-1 List of H2S positive bacteria 1 Citrobacter freundii 2 Salmonella Arizona 3 Salmonellae spp (except S. Peptonised iron. . Lysine iron agar. OBSERVATIONS Look for the development of black coloured streak line after 18–24 hours incubation. Sodium thiosulfate H2S indicator 1. VIVA 1 2 3 4 5 6 7 8 What are the conditions required for the production and demonstration of H2S by bacteria? What are the sequence of events occurring in the production and detection of H2S? What is the source of sulfur in the media? What are the indicators of H2S production? What are the sulfur containing amino acids? How will you pick up the colonies for inoculation? What are the media that can be used in H2S production detection? Name some H2S producing bacteria. Ferrous sulfate 2. 1995. Bailey and Scott’s Diagnostic Microbiology. • Medium must support the growth of bacteria being tested. 5th ed. These may interfere with the reaction. 3 Introduction to Clinical Microbiology. lead to produce insoluble heavy metal sulfides that appear black precipitate. Janda WM. 3 Second step is the coupling of sulfide to H+ ions to form H2S. • A H2S indicator in the medium. 11 th ed.Textbook of Practical Microbiology 87 KEY FACTS 1 Requirements to demonstrate H2S production. since inhibited flora may still be present and viable. lead acetate and peptonised iron. 2 Forbes BA. Butterworths. bismuth. Allen SD. 1997. Sahm DF and Weissfeld AS (Eds). Lyne PM and Grange JM. Color Atlas and Textbook of Diagnostic Microbiology. 6 Only tops of the colonies growing on selective media must be touched. sodium thiosulfate. Microbiological Methods. 7 Sources of sulfur in the medium may be sulfite. (Lippincott Williams and Wilkins. University of Texas. ferric ammonium citrate. Schreckenbergu PC and Winn Jr. DPALM Medic. 4 Third step is H2S reacting with heavy metals like iron. 8 Different H2S indicators used in various media are ferrous sulfate. . FURTHER READINGS 1 Collins CH. London. WC (Eds). Louis) 2002. 2 First step in the production of H2S is release of sulfide ions from sulfur containing amino acids or thiosulfates by bacterial enzymes. or peptone.Houston Medical School. • A source of sulfur in the medium. (The CV Mosby Company. 4 Koneman EW. cystine. 1995. USA). 5 Cysteine. St. Otherwise single colony of purified culture must be used. methionine are some sulfur containing amino acids. • The bacteria must possess the enzyme system to produce H2S. Acinetobacter baumannii. p-sulfobenzena-azo-anaphthylamine. Some Pseudomonas and nonfermenters reduce nitrate to nitrite and further down to N2 and molecular nitrogen. Negative control: A. PROCEDURE 1 Mix an equal volume (0.5 gm.88 LESSON 30 INTRODUCTION Nitrate Reduction Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Determine the ability of certain bacteria to reduce nitrates (NO3–) to nitrites (NO2–) or beyond the nitrite stage. Haemophilus and Branhamella. acetic acid (5N) 30%. This character is also useful for the identification of species in the genera Neisseria. REQUIREMENTS I Equipments Incubator. Reagent A (a naphthylamine. PRINCIPLE Bacteria demonstrating nitrate reduction have the capability of extracting oxygen from nitrates to form nitrite and other reduction products. Most of the bacteria belonging to the family Enterobacteriaceae reduce nitrates. II Reagents and lab wares 1% potassium nitrate broth (KNO3) or nitrate agar slant (beef RESULTS AND INTERPRETATION The development of a red colour within 30 seconds. Many bacteria can be differentiated and are identified by their capacity to reduce nitrates to nitrites. etc. Inoculating loop. baumannii. 2 Add 0. 3 gm. 8 g.12 gm. This is called denitrification. after adding the test reagents indicates the presence of nitrites and represents positive reaction (Fig. agar. 3 Observe for any change of colour immediately within few minutes. The presence of nitrites in the test medium is detected by the addition of a-naphthylamine and sulphanilic acid. Nitrates serve as a source of nitrogen for many bacteria. peptone. Reagent B (sulphanilic acid. coli. extract. potassium nitrate. acetic acid (5N) 30%. with the formation of a red diagonium dye. The chemical reaction is NO3–+ 2e– + 2H+®NO2– + H2O. 30-1). 1 L).1 gm.5 ml) of reagent A with 0. 1 L). and distilled water.5 ml of reagent B just before use. 5 g.1 ml of the test reagent to 1 ml of the culture broth of the bacteria to be tested. They can also act as final electron acceptor. 1 L). . in a broth containing 1% potassium nitrate broth (KNO3) incubated at 37°C for 5 days. QUALITY CONTROL Positive control: E. III Specimen Cultures of test bacteria such as Escherichia coli. OBSERVATION Observe for the development of red colour immediately within few minutes of adding the reagents. Butterworths. 4 Koneman EW. 1995. Janda WM. Thus it is necessary to add a small quantity of zinc dust to all negative reactions. and the development of a red colour after adding zinc dust indicates the presence of residual nitrates and confirms a true negative reaction. 3 Introduction to Clinical Microbiology. University of Texas. 3 Zinc dust is added to detect all negative reactions. FURTHER READINGS 1 Collins CH. the reaction may show a false negative reading. 1995. Microbiological Methods. 11 th ed. 2 Two reagents used in the test are: a naphthylamine and sulphanilic acid. Sahm DF and Weissfeld AS (Eds). In this case. 5th ed. WC (Eds). Louis) 2002.Textbook of Practical Microbiology 89 If no colour develops after adding the test reagents. It might have been reduced to products other than nitrites such as molecular nitrogen. VIVA 1 2 3 4 5 What is the principle behind nitrate reduction test? What are the ingredients present in nitrate broth? What are reagents added in the reactions? What is the positive and negative control for nitrate reduction test? What is the purpose of adding zinc dust to all negative reaction? Ans: Negative reaction in nitrate reduction test may be of 2 types. USA). indicates the presence of residual nitrates and confirms a true negative reaction. Allen SD. (Lippincott Williams and Wilkins. The reagents can detect only the presence of nitrites. Color Atlas and Textbook of Diagnostic Microbiology. 2 Forbes BA. it is taken as negative test. Bailey and Scott’s Diagnostic Microbiology. NO or N02. Schreckenbergu PC and Winn Jr. DPALM Medic. 6 How will you avoid false negative reaction? Explain. . St. London. NEGATIVE POSITIVE FIGURE 30-1 Nitrate reduction test. nitric oxide or nitrous oxide. One is the true negative reaction in which nitrates are not reduced to nitrites or in false negative reactions the nitrates are reduced to products other than nitrites such as N2. (The CV Mosby Company. Lyne PM and Grange JM. Zinc ions reduces nitrates to nitrites and the development of a red colour after adding zinc dust. Zinc dusts reduce nitrates to nitrites.Houston Medical School. KEY FACTS 1 Nitrates are reduced to nitrites by some organisms. 1997. 90 . Textbook of Practical Microbiology UNIT 91 V Antimicrobial Sensitivity Tests Lesson 31 Kirby-Bauer Method Lesson 32 Stoke’s Method Lesson 33 Agar Dilution Method Lesson 34 Broth Dilution Method Lesson 35 Epsilometer Test (E-test) . PROCEDURE 1 After standardisation of bacterial suspension. Mueller Hinton agar plates (pH 7. Some have a limited spectrum while others have a wide spectrum of activities against bacteria. millimeter ruler.). 2H2O) to 100ml distilled water.4) with a depth of 4 mm. a standard suspension of bacteria to be tested are inoculated on the surface of Mueller Hinton agar plates. Due to emergence of many antibiotic resistant strains of bacteria. Then 0. In this method. Escherichia coli ATCC 25922. forceps and inoculating wire. sterile cotton swabs.5 McFarland standard.4). III Specimens Staphylococcus aureus ATCC 25923. Preparation of suspension of bacteria: Approximately.92 LESSON 31 INTRODUCTION Kirby–Bauer Method LEARNING OBJECTIVES After completing this practical you will be able to: 1 Determine antibacterial sensitivity of bacterial isolates by Kirby –Bauer disc diffusion method. and Pseudomonas aeruginosa ATCC 27853. the zone of inhibition of growth of bacteria around each disc is measured and the susceptibility is determined. It is streaked in three directions by rotating the plate 60° after each streak. antimicrobial susceptibility testing is done in order to determine which antimicrobial agent to use against a specific strain of bacteria. and is incubated at 37 °C for 3-4 hours. Solution B is prepared by adding 1ml of sulphuric acid (H2S04 (0.5 ml of solution B. immerse a sterile cotton swab in it and rotate the swab several times with firm pressure on the inside wall of the tube to remove excess fluid. The mixture is stored in the dark.5 McFarland standards. Filter paper discs containing specific concentration of antimicrobial agents are pressed on to the surface and incubated at 35°C overnight (18-24 hr. peptone water .27.5 ml of solution A is added to 99. After incubation. The turbidity of the suspension is adjusted to match 0. 3 Inoculate the dried surface of the MHA agar plate by streaking the swab three times over the entire agar surface. REQUIREMENTS I Equipments Incubator. . The cap is closed tightly to avoid evaporation. Enterococcus faecalis ATCC 29212. Preparation of 0.36N) to 100 ml of distilled water. II Reagents and lab wares 0. PRINCIPLE Kirby – Bauer method is a method of determination of antibiotic sensitivity of the bacteria by disc diffusion method. mixed well and distributed in test tubes with a screw cap. The comparison is made against a white back ground with a contrasting black line. 4-5 well isolated colonies of the bacterial strain to be tested are inoculated into 5 ml of peptone water. The solution is agitated vigorously before using it.5 McFarland standard: Solution A is prepared by adding barium chloride (BaCl 2. The available chemotherapeutic agents vary in their scope of antimicrobial activity.2-7. If the density is more it is diluted with sterile saline. filter paper discs impregnated with appropriate concentration of antibiotics. 2 Prepare a Mueller Hinton agar (MHA) plate (pH 7. The bacterial strains isolated from clinical samples should be tested for antimicrobial sensitivity because it gives the clinician an idea as to what antimicrobial therapy should be started to the patients (Box 31-1). 31-1): Sensitive (S): Infection treatable with normal dosage of the antibiotic. RESULTS AND INTERPRETATION Each antibiotics produces a specific zone size for each bacteria tested. 4 Measure the inhibitory zones for each antimicrobial agent. 2 The diameter of the inhibitory zone including the diameter of the disc is measured by using a millimeter scale upto the nearest millimeter. BOX 31-1 GLOSSARY OF TERMS Antibiotics: It is a substance produced by microorganisms or a similar substance produced wholly or partially by chemical synthesis that inhibits the growth or causes death of other organisms in low concentrations. Note: 6 antibiotic discs may be put in an 85 mm plate. Do not move the disc once it has contacted the agar because some of the antibiotics diffuse almost immediately Discs must be placed in such a way that they are at least 20 mm from one another. e. so that they retain their potency. tetracycline. Antimicrobial agent: It is a chemical substance inhibiting the growth or causing death of microorganism. as growth denser than this or lighter than this give problem while reading zone size. E. the bacteria are classified as follows (Fig. They do not cause death. 2 Standardization of the bacterial inoculum is important. It should be such that it gives rise to a semi confluent growth. These are called bacteriostatic agents. 3 All measurements are made with unaided eye while viewing the back of the petri dish with reflected light against a black non-reflecting background. and sulphonamides. penicillin and quinolones. faecalis ATCC 29212. 6 Invert the plates and incubate at 35 °C -37°C for 16-18 hr. 3 Proper storage of the antimicrobial discs. compare with the standard Kirby-Bauer’s chart and interpret the zone of inhibition as sensitive. eg. aeruginosa ATCC 27853 should be tested periodically. and P. coli ATCC 25922. . aureus ATCC 25923. QUALITY CONTROL S. OBSERVATIONS 1 Examine the plates for the presence and size of inhibitory zones. E.g. Depending on the zone size. Intermediate (I): Infection may respond to therapy with higher dosage Resistant (R): Unlikely to respond to the antibiotic at the usual dosage. Bactericidal drug: The drugs that cause irreversible damage to bacteria resulting in death are called bactericidal drugs.Textbook of Practical Microbiology 93 4 Place the appropriate antimicrobial impregnated discs on the surface of the agar using sterile forceps. intermediate or resistant. FIGURE 31-1 Kirby-Baur method of antibiotic susceptibility testing. 5 Gently press each disc onto the agar to provide uniform contact. erythromycin. aminoglycosides. Bacteriostatic drug: Certain antimicrobial agents inhibit the growth by preventing the multiplication of organisms. 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. Bailey and Scott’s Diagnostic Microbiology. 6 Any disc that is not to be used on the day of preparation is stored at -20 °C to 8 °C. 1395. Lyne PM and Grange JM.K. Guidelines on Standard Operating Procedures for Microbiology. c) bacteriostatic drug and d) bactericidal drug. 5 Each disc is impregnated with a drop of the solution and allowed to dry in the incubator at 37°C for 30 mins. and novobiocin. Antibiotics stimulated by fall in pH i. Sahm DF and Weissfeld AS. Antibiotics stimulated by an alkaline pH – Aminoglycosides and macrolides such as erythromycin. Vellore.e. Blood safety and Clinical Technology. Butterworths. Allen SD. 3 A 27 SWG needle. 5 WHO. 5th Edition. FURTHER READINGS 1 Collins CH. 3 Koneman EW. St. London. 4 A 6 mm disc is able to completely absorb the fluid volume (7 µl). larger zone sizes – Tetracycline. Microbiological Methods. Christian Medical College. (The CV Mosby Company. Louis) 2002. 1 using standard stationary paper punch. Schreckenbergu PC and Winn Jr. methicillin. Janda WM. 94-96. 4 How are antimicrobial discs prepared? Ans: 1 Discs are made from Whatman filter paper No. 4 Lalitha M. . pp. 2 What is the importance of antimicrobial susceptibility testing? 3 Why is the standardization of pH of medium important in disc diffusion test? Ans: Standardization of pH of the medium is important because some antibiotics show increased or decreased zone sizes depending on pH. 1997. 1995. bevel cut off is used to drop the antibiotics. This delivers 140 drops/ ml. 2 Forbes BA. Chapter 7: Antimicrobial Susceptibility Testing. WC. b) antimicrobial agent. 2 Discs are arranged separately in a Petri dish and sterilized in hot air oven at 160°C for 1 hour. 11 th ed.94 Kirby-Bauer Method VIVA 1 Define a) antibiotics. 2004. Color Atlas and Textbook of Diagnostic Microbiology. It is attached to a tuberculin syringe fitted with a teat. Manual on Antimicrobial Susceptibility Testing. Lippincott Williams and Wilkins. pp 43. II Reagents and lab wares 0. 45 well isolated colonies of the bacterial strain to be tested are transferred to Tryptic soy broth or BHI broth. Millimeter ruler and sterile forceps. There should be a minimum distance of 2 cm between two discs. Pseutomonas aeruginosa NCTC 10662 against aminoglycosides. 2 The control culture is applied in two bands on either side of the plate leaving a central band uninoculated with the help of sterile swab. In such a method 6 discs can be put on an 85 mm circular plate. REQUIREMENTS I Equipments Incubator. coli NCTC 10414 and P. sterile cotton swabs. The turbidity of the suspension is adjusted to match 0. QUALITY CONTROL S. PROCEDURE 1 The inoculation plates are dried with lids open so that there are no droplets of moisture on the surface. Kirby-Bauer and Stokes methods are compared in the table 32-1. Mueller-Hinton agar plates (pH 7. antibiotics discs. aureus NCTC 6571. A set of standard strains are used as control strains depending on the bacterium to be tested.5 McFarland standards.4). PRINCIPLE In this test antibiotic discs are applied between the standard and test inocula. 5 For inoculation. Escherichia coli NCTC 10414 and Pseudomonas aeruginosa NCTC 10662. a rotatory plating method can also be used wherein the control strain is applied on the outer periphery and the test strain is applied in the central portion.27. 6 The plates are then incubated overnight at 35°C –37°C. 3 The test organism is applied in the central portion without touching the either sides. E.5 McFarland standards. 4 Antibiotic discs are applied with forceps on the line between the test and control organisms and pressed gently to ensure even contact with the medium. The method makes use of in-built controls against many variables and therefore provides dependable results. so that zones of inhibition formed around each disc are composed of standard and test bacteria. III Specimens Control strains: Staphylococcus aureus NCTC 6571. Preparation of suspension of bacteria: Approximately. aeruginosa NCTC 10662.Textbook of Practical Microbiology 95 LESSON 32 INTRODUCTION The Stokes Method LEARNING OBJECTIVES After completing this practical you will be able to: 1 Determine antibacterial sensitivity of bacterial isolates by Stokes method. The diffusion of antibiotic takes place and thus the susceptibility of those organisms to the antibiotic are known by measuring zone size. The control strains are Escherechia coli NCTC 10414 for testing coliform bacilli from urinary tract. Stokes method is an example of the disc diffusion test and is another method used for routine antibiotic sensitivity testing of bacterial strains. Four discs can be accommodated on an 85 mm circular plate. . RESULTS AND INTERPRETATION Each zone size is interpreted as follows (Fig. Vellore. 1995. VIVA 1 How is the zone size measured in Stokes method? 2 How many discs can be applied on a plate? Ans: A total of 4 discs can be accommodated on an 85 mm circular plate. pp 43. 1395. Resistant: Zone size 2 mm or less. 5th Edition. Microbiological Methods. Lyne PM and Grange JM. Test and control strains tested on the same 2. but smaller than the control to more than 3 mm. 3 Koneman EW. Comparison of the zones of inhibition between the standard and test bacteria indicates the sensitivity of the test bacteria. There should be a minimum distance of 2 cm between 2 discs. Intermediate: Zone size greater than 2 mm. 2 Forbes BA. 3 The advantage of Stoke’s method is that both control and test organisms can be tested on the same plate under similar conditions and difference between zone sizes can be directly measured. Louis) 2002. 2004. Stokes Method 1. 2 Penicillinase producing Staphylococci. Butterworths. there is no need to perform any measurement with calipers or a millimeter scale. Christian Medical College. FIGURE 32-1 Stokes method. Schreckenbergu PC and Winn Jr. 4 Lalitha M. (The CV Mosby Company. showing heaped up. 5 WHO.K. Lippincott Williams and Wilkins. 11 th ed. 3. Sahm DF and Weissfeld AS. Test and control strains have to be tested on separate plates. Color Atlas and Textbook of Diagnostic Microbiology. 94-96. Table32-1 Comparison of Kirby-Bauer and Stokes methods Kirby-Bauer Method 1. Blood safety and Clinical Technology. If the test zones are obviously larger than the control or give no zone of inhibition at all.96 The Stoke’s Method OBSERVATIONS Zone sizes are measured from the edge of the discs to the edge of the zone. 4 discs on 85mm plate and 6 discs by rotatory method. 3. KEY FACTS 1 It is better to apply the control organisms followed by test organisms. Manual on Antimicrobial Susceptibility Testing. St. Bailey and Scott’s Diagnostic Microbiology. London. 32-1): Sensitive: Zone size equal to wider than or not more than 3 mm smaller than control. WC. Guidelines on Standard Operating Procedures for Microbiology. 1997. Zone diameter measured from edge of disc to edge of zone of inhibitation the disc. 6 discs applied on 85mm plate. Zone diameter is measured including disc diameter. If rotatory plating method is used 6 discs can be accommodated on an 85mm circular plate. . Chapter 7: Antimicrobial Susceptibility Testing. 2. Allen SD. Janda WM. 3 Compare and contrast Kirby-Bauer and Stokes methods. clearly defined zone edges should be reported resistant irrespective of zone size. FURTHER READINGS 1 Collins CH. pp. shake well and pour into sterile petri dish. the quantitation of antibiotic against pathogen needs to be more precise So when in doubt about the sensitivity of pathogen the way to a precise assessment is to determine the minimum inhibitory concentration (MIC) of the antibiotic to the organisms concerned.85 %) and stock solution of antibiotic.g.4). II Reagents and lab wares 0. sterile Mueller Hinton agar (pH 7. In these cases. Agar dilutions methods have the advantage that it is possible to test a number of organisms on each plate containing an antibiotics solution. It is mainly useful in testing isolates from serious infections like bacterial endocarditis or to verify equivocal results (e. REQUIREMENTS I Equipments Water bath. Note: By this method.27.5 ml distilled water (working solution contains antibiotics at a strength of 200 µg / ml-solution A) III Specimens Preparation of suspension of bacteria: Approximately. PROCEDURE PRINCIPLE In agar dilution method.Textbook of Practical Microbiology 97 LESSON 33 INTRODUCTION Agar Dilution Method LEARNING OBJECTIVES After completing this practical you will be able to: 1 Determine antibacterial sensitivity of bacterial isolates by agar dilution method. bacterial endocarditis. Prepare a stock solution containing 2000 µg / ml of the antibiotic to be tested. or in prolonged serious infections eg. Note: It is advisable to start wilh the highest dilution so that single pipette can be used to dispense all the dilutions prepared. serial dilutions of the antibiotics are prepared in agar and poured into petri dishes. Diffusion tests used to determine the susceptibility of organisms isolated from clinical specimens have their own limitations. exact volume of medium (22.5 McFarland standards (106 organisms/ml).5 ml of this solution with 9. The turbidity of the suspension is adjusted to match 0. sterile test tubes.6 ml) is delivered into the screw capped bottles without the danger of the molten agar jellifying during transfer into dilution of the antibiotic (Table 33-1). . sterile Mueller Hinton broth. Preparation of the agar plate with different concentration of the antibiotics 1 Dispense 2 ml of the diluted antibiotic solution into each of the marked sterile screw capped bottle. Mix 0. The dilutions are made in a small volume of water and added to agar which has been melted and cooled to not more than 60°C. intermediate susceptibility of ciprofloxacin against Salmonella Typhi). 4-5 wellisolated colonies of the bacterial strain to be tested are transferred to Tryptic soy broth or BHI broth. Agar dilution is a quantitative method for determining the minimum inhibitory concentration of the antibiotics against bacteria to be tested. These also include sterile saline (0. For example weigh 200 mg of the antibiotic powder and dissolve in 5 ml of distilled water / appropriate solvent. Preparation of stock solutions of antibiotics: The required dilutions of the antibiotics are made as per the table 33-1. 2 Sterile Muller-Hinton agar is cooled and maintained at 50°C – 55°C in a water bath (Table 33-1). when equivocal results are obtained. 3 Pour this medium (18 ml) into the screw capped bottle containing the different concentration of antibiotic. screw capped flat bottomed bottles (25 ml capacity) and Petri dishes (90 mm diameter).5 McFarland standard. pipettes. antibiotic powder. B 640 . 5 Allow the drops to dry and incubate the plates without inverting at 37°C for 16 –18 hours.F 8 40 .4 2 1 1 2 1 1 2 1 1 + Sterile Water (Vol) µg/ml 2000 .25 128 = Intermediate conc (µg / ml) in tubes Final conc at 1:10 in agar plates (µg / ml) 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.J 0.E 20 . 4 Inoculate a control plate without antibiotics simultaneously as control.98 Agar Dilution Method 4 Keep the poured plates at 4°C for setting. Check the control plate for growth. Escherichia coli ATCC 25922.6 2 3 7 2 3 7 2 3 7 1280 . Note: Inoculation is done starting with the plates containing highest dilution of the antibiotic. and Pseudomonas aeruginosa ATCC 27853 are used as control strains.H 20 . The plates must be dry before performing the test.H 20 .001 – 0.H 3.C 64 320 .D32 160 .K 0.5 .E 160 .B 1280 .E 16 80 . 3 Inoculate the culture on the surface of the medium.5 2. 5 After the plates have set. 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. Enterococcus faecalis ATCC 29212. In each case 104 bacteria is delivered to a spot 5 – 8 mm in diameter. 3 It is mainly useful in testing isolates from serious infections like bacterial endocarditis or to verify equivocal results (e. indicated by the square marked below.g. QUALITY CONTROL Staphylococcus aureus ATCC 25923. OBSERVATIONS Read the plates for presence or absence of growth. 4 Selective media should not be used for performing agar dilution method. Test procedure 1 A grid is marked on the bottom of the plates containing antibiotics. 2 A loopful of inoculating loop is calibrated to deliver 0. . 2 The method is carried out on Muller Hinton agar. Control plate must show confluent or near confluent growth.I 1 5 . The concentration at which growth is completely inhibited is considered as the minimum inhibitory concentration (MIC).B 160 .G 4 20 .A (Stock) 1280 . RESULTS AND INTERPRETATION The highest dilution of antibiotic showing more than 99% inhibition of growth of bacteria is considered as the minimum inhibitory concentration (MIC) of the bacteria. The organisms are reported sensitive. dry the plates well in an incubator at 37°C for 30-60 mins. intermediate susceptibility of ciprofloxacin against Salmonella Typhi). Read the test plate.E 160 . Note: 20 – 25 strains can be tested in plate including the control.1 System for preparing dilutions for agar dilution method Antibiotic Solution Volume 6. Table 33.B 1280 . intermediate or resistant by comparing the test MIC values with that given in the NCCLS table.H 2 10 .002 ml (1-2 µl) of the culture. WC. Christian Medical College. 1995. 4 Lalitha M. 5 WHO. . 2 What is the automated method used to inoculate the organisms? Ans: Steer’s Replicator is used as the automated method to inoculate the organisms. 94-96. Bailey and Scott’s Diagnostic Microbiology. Sahm DF and Weissfeld AS. London. Lyne PM and Grange JM. pp 43. 5th Edition. 3 Mention the merits and demerits of broth dilution method. 2004. Lippincott Williams and Wilkins. 2 Forbes BA. Color Atlas and Textbook of Diagnostic Microbiology. Demerit is that it is a cumbersome procedure. 3 Koneman EW. St. 1997. Allen SD. (The CV Mosby Company.Textbook of Practical Microbiology 99 VIVA 1 What is the break point of an antimicrobial agent? Ans: Break point of an antimicrobial agent is defined as the concentration of that agent that can be achieved in the serum with optimal therapy. FURTHER READINGS 1 Collins CH. Chapter 7: Antimicrobial Susceptibility Testing. Butterworths. Guidelines on Standard Operating Procedures for Microbiology. Microbiological Methods. Schreckenbergu PC and Winn Jr. Janda WM. Ans: Merit of the test is that it is possible to test many organisms on each plate. Manual on Antimicrobial Susceptibility Testing. Louis) 2002.K. Blood safety and Clinical Technology. 1395. pp. Vellore. 11 th ed. 4-5 well isolated colonies of the bacterial strain to be tested are transferred to Tryptic are soy broth or BHI broth. 4 Standardised suspension of the microorganisms to be tested is inoculated into the tubes.5 ml of this solution with 9. 2 The last tube is kept free of antibiotic and serves as a growth control. It is another quantitative method for determining the minimum inhibitory concentration (MIC) of the antibiotic against a bacteria to be tested. . Enterococcus faecalis ATCC 29212. antibiotic powder. II Reagents and lab wares 0. In this method. A standard suspension of the organism is inoculated into the medium with one antimicrobial agent -free medium as control. sterile Mueller Hinton broth. and examined for growth. Prepare a stock solution containing 2000 µg / ml of the antibiotic to be tested. 5 Tubes are incubated at 35-37°C for 18 hours. Preparation of stock solutions of antibiotics: The required dilutions of the antibiotics are made as per the table 34-1. Escherichia coli ATCC 25922. Broth dilution is also known as tube dilution method. sterile capped tubes and test tube rack. the MIC of the antibiotics is determined by observing the lowest concentration of antibiotics that inhibits growth of the bacteria.5 McFarland standards.5 ml distilled water (stock solution contains antibiotics at a strength of 200 µg / ml-solution A) III Specimens Preparation of suspension of bacteria: Approximately. REQUIREMENTS I Equipments Water bath. 3 Arrange the test tubes in a rack. and Pseudomonas aeruginosa ATCC 27853 are used as control strains. 2 ml and 1 ml. Two fold dilutions of this solution is prepared in suitable broth. PRINCIPLE A stock solution of antimicrobial agent to be tested is prepared. These also include stock solution of antibiotic. MIC is taken as the lowest concentration of antimicrobial agent which completely inhibits the growth. QUALITY CONTROL Staphylococcus aureus ATCC 25923. The turbidity of the suspension is adjusted to match 0. sterile pipettes of 10ml. PROCEDURE 1 Serial dilutions of the antimicrobial agent are made in broth and are kept in test tubes. For example weigh 200 mg of the antibiotic powder and dissolve in 5 ml of distilled water / appropriate solvent. sterile test tubes. 5 ml.100 LESSON 34 INTRODUCTION Broth Dilution Method LEARNING OBJECTIVES After completing this practical you will be able to: 1 Determine antibacterial sensitivity of bacterial isolates by broth dilution method.5 McFarland standard. After incubating overnight . The inoculated media are inoculated at 35-37°C for 18-24 hr. serial dilutions of the antibiotics are taken in test tubes and a standardise suspension of the bacterium is inoculated. Mix 0. The minimum bactericidal concentration (MBC) can also be estimated by this method by subculturing from the lowest concentration of drug that kills the bacteria. 2 Because very faint turbidity may be given by the inoculum itself. 3. 2. e.K. Guidelines on Standard Operating Procedures for Microbiology. penicillin and quinolones. Louis) 2002. The main advantage of the broth dilution method for MIC determination is that it can readily be converted to determine the minimum bactericidal concentration (MBC) also. The tubes not showing visible growth are subcultured on solid medium and incubated at 37°C overnight. Mention merits and demerits of broth dilution agar method. KEY FACTS 1 Minimum inhibitory concentration (MIC) is defined as the highest dilution which inhibits growth of the bacteria. 5. 5th Edition. Sahm DF and Weissfeld AS. 1395.Textbook of Practical Microbiology 101 OBSERVATIONS At the end of the incubation period the tubes are examined for turbidity. 4 Lalitha M. 3 Standard strain of known MIC should be tested as control to check the reagents and conditions. They do not cause death. Lyne PM and Grange JM. Manual on Antimicrobial Susceptibility Testing. e. Butterworths. (The CV Mosby Company. These are called bacteriostatic agents. Janda WM. Lippincott Williams and Wilkins. 2 Forbes BA. pp 43. Bailey and Scott’s Diagnostic Microbiology. Schreckenbergu PC and Winn Jr. It is noted by lack of turbidity in the tube. the inoculated tube kept in the refrigerator overnight may be used as the standard for the determination of complete inhibition. Table 34-1 Preparation of stock dilutions of the antibiotic stock solutions Stock dilutions of the antibiotic stock solutions can be prepared using the formula: 1000 x V X C =W P Where: P =Potency given by manufacturer V= Volume (ml) require C= Final concentration of solution (per ml) W= Weight of antimicrobial to be dissolved in volume V RESULTS AND INTERPRETATION Minimum inhibitory concentration (MIC) is defined as the highest dilution which inhibits growth judged by lack of turbidity in the tube. What is a bacteriostatic agent? Give examples. 1995. 1997. Allen SD. Cloudiness indicates that bacterial growth has not been inhibited by the concentration of antibiotic present in the medium. 11 th ed. St. Blood safety and Clinical Technology. 5 WHO. Microbiological Methods. Color Atlas and Textbook of Diagnostic Microbiology. VIVA 1. Demerits There may be non specific turbidity due to the inoculum itself. 94-96. London. FURTHER READINGS 1 Collins CH. Vellore. What is minimum inhibitory concentration (MIC)? 4.g. Ans: Certain antimicrobial agents inhibit the growth by preventing the multiplication of organisms. pp.g. WC. Ans: The drugs that cause irreversible damage to bacteria resulting in death are called bactericidal drugs. tetracycline. 2 Same tubes can be used for MBC tests also. aminoglycosides. 2004. What is a bactericidal agent? Give examples. sulphonamides. even single isolates. What is minimum bactericidal concentration (MBC)? Ans: The lowest concentration of the antimicrobial agent that allows < 0. The highest dilution showing at least 99% inhibition is taken as MBC. etc. Ans: Merits 1 Simple procedure for testing a small number of isolates. Christian Medical College. .1 % of the original inoculum to survive is called the minimum bactericidal concentration (MBC). 3 Koneman EW. erythromycin. Chapter 7: Antimicrobial Susceptibility Testing. e. the area labeled E. at least 4 antibiotic strips can be tested. 3 Inspect the package for holes or cracks.102 LESSON 35 INTRODUCTION Epsilometer Test (E-test) LEARNING OBJECTIVES After completing this practical you will be able to: 1 Determine antibacterial sensitivity of bacterial isolates by Epsilometer test (E-test).5 McFarland standards. The turbidity of the suspension is adjusted to match 0. and sterile swabs II Specimens Preparation of suspension of bacteria: Inoculate peptone water with test organism and incubate at 37 °C for 3-4 hours. In a 90 mm plate. twist them apart with your fingers. This method produces a means for producing MIC data in those situations in which the level of resistance can be clinically important. It is easy to interpret the result of MIC. An elliptical zone of growth inhibition is seen around the strip after incubation. antibiotic carrier) with a continuous gradient of antibiotic immobilized on one side and MIC interpretative scale corresponding to 15 two fold MIC dilutions on the other side. Ensure the MIC scale is facing upwards i.e.g. REQUIREMENTS I Reagents and lab wares Commercially available E-test strips. The E-test is an automated system for measuring the MIC of the bacterial isolate. 7 Touch only the handle. In a 150 mm plate. When transferred to the agar.. 4 Cut along the broken line at the top of a blister. towards the opening of the plate. e. Do not cut in between the blisters. Application of strips 1 Apply E-test strips with forceps. 1 Remove the package stored at – 20°C or .85% saline for inoculum preparation. PROCEDURE Opening an E-test package PRINCIPLE The E-test is based on the principle of disc diffusion where the antibiotic diffuses into the medium when the strip is placed on the medium. an automated system for measuring the minimum inhibitory concentration (MIC) of the bacteria. Ensure all the moisture has evaporated before opening. 5 Tip the strips out of the opening slightly and take them out with forceps. A predefined antibiotic gradient is immobilized on the surface opposite the MIC scale. The MIC is read from the scale at the intersection of the zone with the strip. Do not use if damaged. 8 Place the strips to be used into a dry clean petri dish. These also include foreceps.70°C. sterile Mueller Hinton agar plates (150 or 90 mm with a depth of 4 mm). The E-test is a plastic strip (5 x 50 mm. 0. i.5 McFarland standards. This takes approximately 30 minutes if stored at – 20° C and approximately one hour if stored at – 70° C. a single antibiotic strip can be tested. 6 If strips stick together. penicillin or cephalosporins against Streptococcus pneumoniae. the continuous antibiotic gradient established under the strip remains stable over a period covering the critical times of most microorganisms subjected to susceptibility testing. . 0. 2 Equilibrate at room temperature. It is a very simple test to perform MIC of the bacterial isolate as compared to the other techniques like broth and agar dilution methods which are technically cumbersome. . 2 Forbes BA. Dip a swab in the inoculum. Always round up these values to the next two-fold dilution before interpretation. Chapter 7: Antimicrobial Susceptibility Testing. London. The MIC is read from the scale at the intersection of the zone with the strip (Fig. Lyne PM and Grange JM. RESULTS AND INTERPRETATION Read plates after the recommended incubation period only if sufficient growth is seen and the inhibition eclipse is clearly visible. I = 2. 1997. Always read the end point at complete inhibition of all growth including hazes and isolated colonies. Color Atlas and Textbook of Diagnostic Microbiology. 6 Use templates to position 4 to 6 strips on a 150 mm plate or one to two strips on a 90 mm plate. Lippincott Williams and Wilkins. Schreckenbergu PC and Winn Jr. Blood safety and Clinical Technology. VIVA 1 What is the principle of the E-test? 2 What is the advantage of E-test? 3 How is the result for MIC interpreted? FURTHER READINGS 1 Collins CH. KEY FACTS 1 The E-test strips have to be placed in proper orientation. Guidelines on Standard Operating Procedures for Microbiology. Microbiological Methods. 94-96. 7 Place the handle of the strip closest to the rim of the plate. 4 Open the E-test package and place the strips in a dry petri dish. remove excess fluid and swab the entire agar surface evenly in 3 directions. Louis) 2002. 5 WHO. 3 Koneman EW. St. 2 Diffusion of antibiotics begins immediately after placement of the strips. Note: Be firm when applying the strip. then an E-test MIC of 1. MIC values in between two – fold dilutions can be obtained. For example: If ampicillin breakpoints are given as S=1. 8 Incubate at 37°C for 18-24hours.Textbook of Practical Microbiology 103 2 Ensure that the agar surface is dry before swabbing it. (The CV Mosby Company. 1395. Since E. pp 43. Christian Medical College. 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. NCCLS QC ranges and interpretive guidelines. Once applied. FIGURE 35-1 E-Test. WC. Vellore. Bailey and Scott’s Diagnostic Microbiology. Butterworths. Read the MIC where the ellipse intersects the scale. Allen SD. 5 Always store unused strips in airtight containers at -20°C or -70°C. Note: Always store unused strips in airtight containers at 20°C or -70°C. 35-1).5 µg/ml is rounded up to 2 µg/ml and the category reported as Intermediate (I) QUALITY CONTROL Package labels for each antibiotic will carry performance and reproducibility data. 4 Lalitha M. 5 Apply the strips to the agar surface with a forceps. OBSERVATIONS After incubation an elliptical zone of growth inhibition is seen around the strip. do not move the strip.test comprises a continuous gradient. 5th Edition. which cannot be moved once it has touched the agar. 11 th ed. Janda WM. pp. 1995. 2004. 3 Allow the agar surface to dry for 10 minutes to 15 minutes on the bench or in the incubator. R=4 µg/ml. Manual on Antimicrobial Susceptibility Testing.K. Placing strips upside down on the agar will alter the results. Always apply the strip with the MIC scale facing the opening of the plate. Do not apply it upside down. Sahm DF and Weissfeld AS. 104 . Textbook of Practical Microbiology UNIT 105 VI Immunology Introduction Bacterial Agglutination Test Blood Grouping Latex Agglutination Test Co-agglutination Test Widal Test Weil Felix Test Anti-Streptolysin O (ASLO) Test VDRL Test Radial Immunodiffusion Test Immunoelectrophoresis Test Counter-current Immunoelectrophoresis Test Indirect Haemagglutination Test Immunofluorescence Test Enzyme-Linked Immunosorbent Assay Lesson Lesson Lesson Lesson Lesson Lesson Lesson Lesson Lesson Lesson Lesson Lesson Lesson Lesson 36 37 38 39 40 41 42 43 44 45 46 47 48 49 . Standard agglutination test. . For blood group antigen detection A.106 Introduction Serological tests are widely used for diagnosis of many infectious diseases including bacterial. NAME OF THE TEST Bacterial agglutination test. viral. Salmonella agglutination. Paratyphi B S. Tube agglutination tests. Paratyphi A S. Latex agglutination test. For antibody detection in: Amoebiasis Lymphatic filariasis Echinococcosis Toxoplasmosis Rickettsial infection. etc. Indirect haemagglutination test. List of few common serological tests are mentioned in the table. For antigen detection in infections caused by: Streptococcus pneumoniae Haemophilus influenzae Cryptococcus neoformans Echinococcus granulosus. These tests may be agglutination. For antigen detection in: Cryptococcosis Echinococcosis Lymphatic filariasis Blood grouping. For antibody detection against Mycoplasma pneumoniae. Paratyphi C. Table Applications of various tests used in a microbiology laboratory TYPE OF THE TEST Slide agglutination tests. AB and O. Cold agglutination test. Co-agglutination test. Shigella agglutination. Widal test. fungal and parasitic. with a variation in their sensitivity and specificity. For antibody detection against Brucella species. Streptococcus Lance field’s grouping. APPLICATION For confirmation of identification of the bacterial isolates by using specific antisera. For antibody detection against Salmonella Typhi S. precipitation. Passive agglutination tests. neutralization. Vibrio agglutination. B. For antigen detection in infections caused by: Brucella (Milk ring test) B. Immunofluorescence test For antigen detection in infections caused by: Respiratory syncytial virus Measles Mumps Rabies Influenza Indirect immunofluorescence test. Weil-Felix test. Precipitation tests Ring test. For detection of: a-Fetoprotein N. Slide flocculation test for demonstration of reaginic antibodies in syphilis. For heterophile antibody detection in Scrub typhus Endemic typhus. For detection of antibodies (Anti-streptolysin) in acute rheumatic fever. Gel diffusion test. .anthracis (Ascoli’s test) Toxoid precipitation for diphtheria. Paul-Bunnel test.Textbook of Practical Microbiology 107 Heterophile agglutination tests. Epidemic typhus. meningitidis antigen HBs antigen. VDRL test. Tube test. Neutralization test ASLO test. For antibody detection in: Toxoplasmosis Amoebiasis. Direct immunofluorescence test. For heterophile antibody detection in Infectious mononucleosis (EBV). In this case.g. REQUIREMENTS I Reagents and glass wares Glass slides. hence called slide agglutination test. specific antiserum against the bacterium(e. As in the case with precipitation reaction. they help to neutralize the slight negative charge that particles in solution normally carry (the zeta potential). When a particulate antigen is mixed with its antibody in the presence of electrolytes at a suitable temperature and pH. This phenomenon is known as prozone reaction. The soluble antigens can be tested in agglutination reaction by coating them with carrier particles such as red blood cells.. Antibodies cause agglutination by binding to antigens on the particles. with one of the five subunits binding to one particle. This is probably because of restricted movement in the hinge region of the immunoglobulin. The slide agglutination tests have many uses. The slide agglutination test is rapid and convenient.85%). Muller-Hinton agar). glass marking pencil. They are used for confirmatory identification of Salmonella. The reaction is facilitated by mixing of the bacterial colony and the antiserum with a loop. Shigella flexneri or Vibrio cholerae) to be tested. IgG4 antibodies are an example of incomplete antibodies. identification of Bordetella pertussis and typing of streptococci (e. IgM immunoglobulins actually function as a bridge between two particles.g. uniform suspension of a bacterial isolate from the clinical specimens resulting in agglutination. and they will not clump together. This allows the particles to approach each other.. and another subunit binding to another particle. . PRINCIPLE Agglutination is an example of antigen-antibody reaction in which antigen is particulate or insoluble in nature.g. The test is performed on a glass slide. PROCEDURE 1 Take a clean glass slide. are called “incomplete” antibodies. This method is useful where In the slide agglutination test a drop of the appropriate specific antiserum is added to a smooth. The slide agglutination is a frequently used procedure for the identification of many bacterial isolates from clinical specimens. A positive reaction is identified by the clumping together of bacteria and clearing of the drop. the particles are clumped or agglutinated. more quantity of antibody can actually inhibit agglutination. Not all antibodies can agglutinate particles. which cannot cause agglutination. Salmonella Typhi. saline (0. only small quantities of culture are available. bacteria or inorganic particles such as the latex. II Specimen Pure 24 hour growth of bacteria (e. antiserum against Salmonella Typhi. each particle will be completely covered with antibodies. Antibodies. bacteriological loop. Streptococcus Lance field’s grouping) and pneumococci. Shigella flexneri or Vibrio cholerae) from solid media preferably from non-blood agar plates (Examples : nutrient agar. The agglutination reaction occurs instantly or within seconds. This method is also used for blood grouping and cross matching. Shigella and Vibrio isolates.108 LESSON 36 INTRODUCTION Bacterial Agglutination Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Demonstrate the application of slide agglutination reaction for the identification of bacteria. 3 Stites DP. Allen SD. 2 Koneman EW. VIVA 1 Discuss application of slide bacterial agglutination tests. 4 Pick up the colonies of Salmonella to be tested from agar culture and gently emulsify with drops of saline in both the halves by loop. Schreckenbergu PC and Winn Jr. 3 Place a drop of saline in both the halves. 10th Edition. Janda WM. 8 Observe the clumping of the bacterial suspension in the test. Butterworth. 1995. 2001. WC. Test is fallacious. Auto agglutination: Clumping in both test and control halves. A markedly stronger agglutination reaction in POSITIVE NEGATIVE FIGURE 36-1 Slide agglutination test. Microbiological Methods. hence discard the result. 6 Place another drop of saline in the half of the slide labeled as control. pp. Negative agglutination: No clumping either in the test half or control half. Lyne PM and Grange JM. 5 Add a drop of specific antisera to the bacterial suspension in the half labeled as test and mix. Terr AI and Parslow TG. the test reagent as compared to control reagent is indicative of a positive reaction. RESULTS AND INTERPRETATION Positive agglutination: Clumping in the test half and no clumping in the control half. London. Color Atlas and Textbook of Diagnostic Microbiology.Textbook of Practical Microbiology 109 2 Mark it into two halves by a glass marking pencil and label them as test and control. KEY FACTS 1 Fresh young cultures are always used for the test. Absence of reaction with a test reagent indicates a negative reaction irrespective of any reaction with control organism. pp. 2 Check for the auto agglutination of the test organism. 902. Medical Immunology. OBSERVATIONS Observe the test mixture for agglutination by naked eye observation. 2 How do you check for the auto agglutination of the test organism? FURTHER READINGS 1 Collins CH. It identifies specific bacteria (Fig. 1395. 5th Edition. It shows either antiserum is not good or bacteria and the antisera are not specific to each other. QUALITY CONTROL On the same slide a control consisting of the bacterial suspension in saline without the antisera is used to ensure that bacteria is not auto agglutinable. . 7 Gently rock the slide. back and froth for 2 minutes. Lippincott Williams and Wilkins. 1997. 36-1). and mix. 3 On the same slide a control consisting of the bacterial suspension in saline without the antisera is used to ensure that bacteria are not auto agglutinable. but sometimes it may require confirmation under the microscope. 94-96. 110 LESSON 37 INTRODUCTION Blood Grouping LEARNING OBJECTIVES After completing this practical you will be able to: 1 Demonstrate blood grouping by agglutination reaction. PROCEDURE 1 Take three different slides and label the slides as A, B, and D. 2 Clean middle finger of the left hand with the spirit and allow it to dry. 3 Prick the finger with sterile lancet. 4 Collect 3 drops of blood on three different slides labeled as A, B, and D. 5 Add a drop of antiserum A to A and anti B to B anti D to D. 6 Mix with an applicator stick. Mix the samples on the slide by gentle rocking for about two minutes. 7 Examine each zone for agglutination of RBCs. 8 Record result of the test immediately before the drop dries out. The ABO system contains four blood groups and is determined by the presence or absence of two distinct antigens, A and B, on the surface of erythrocytes. Red cells of group A carry antigen A, cells of group B antigen B and cells of group AB have both A and B antigens, while group O cells have neither A nor B antigen. The 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. Blood group antigens are inherited according to Mendelian laws. Their synthesis is determined by allelomorphic genes A, B and O. Genes A and B give rise to the corresponding antigens, but O is an amorph and does not produce any antigen. Group O is the commonest group and group AB is the rarest. In India the distribution is O – 40%, A – 22%, B – 33%, and AB – 5%. O group population is called universal donors and AB is called universal recipients. QUALITY CONTROL Blood group antisera (Anti-A, anti-B, anti-D or Rh antiserum) must be checked with known blood before to test. OBSERVATIONS 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 a drop of blood, the antibody binds with its specific antigen present on the RBCs and causes agglutination of the RBCs. RESULTS AND INTERPRETATION A clear agglutination indicates positive result. If the test sample is agglutinated with anti A antibody then the blood is Group A. If the test sample is agglutinated with anti B antibody then the blood is Group B. If the test sample is agglutinated with anti A and anti B antibodies both, then the blood is Group AB. If the test sample is not agglutinated with anti A and anti B antibodies, then the blood is Group O. REQUIREMENTS I Reagents and lab wares Blood group antisera (Anti-A, anti-B, anti-D or Rh antiserum), glazed white ceramic slide, applicator stick and lancet. II Specimen Blood with anticoagulant or finger prick blood. Textbook of Practical Microbiology 111 KEY FACTS 1 Autoagglutination of RBCs during the test procedure should be checked. 2 Record result of the test immediately before the drop dries out. VIVA 1 What exactly does it tell you about a person’s blood cells if you know that they are of blood type A, B, AB, or O? Ans: It shows the presence or absence of two distinct antigens, A and B, on the surface of erythrocytes. 2 Explain what blood types a person of type A, B, AB, or O can receive and why? Ans :Red cells of group A carry antigen A, cells of group B carry antigen B and cells of group AB have both A and B antigens, while group O cells have neither A nor B antigen. The serum contains the isoantibodies specific for the antigen that is absent on the red cell. Immune isoantibodies may develop following ABO incompatible transfusion. Red cells also carry another antigen that reacts with rabbit serum to Rhesus monkey erythrocytes called Rhesus or Rh factor. Rh-negative population contains anti Rh antibodies in their blood. Hence a person should not receive blood, which contains isoantibodies to own RBC. O group population is called universal donors since their blood does not contain any blood group antigens (A, B and AB). AB group is called universal recipients due to absence of isoantibodies. FURTHER READINGS 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 38 INTRODUCTION Latex Agglutination Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Demonstrate the application of latex agglutination test for detection of soluble antigen. of the latex particles and form visible agglutination or clumping of the particles. REQUIREMENTS I Reagents and lab wares Hydatid antibody-coated latex bead, glass slides, and applicator stick. II Specimen Serum sample to be tested. Latex agglutination test is an example of slide agglutination test in which polystyrene spheres such as latex particles have been used as carrier particles for coating with antigens. In this test both soluble proteins and non-protein antigens can be bound on the surface of latex beads, which then can be used to detect antibodies by demonstrating the agglutination of the antigen-carrying latex particles. The latex agglutination test is used for demonstration of antibodies on a variety of bacterial, parasitic and fungal infections. The LAT are widely used for diagnosis of typhoid fever, syphilis, infectious mononucleosis, amoebiasis and hydatid diseases. The LAT can also be used for detection of soluble antigen by using latex particles coated with the specific antibodies. The LAT is used to demonstrate rheumatoid factor (RF) in the serum, Streptococcus pneumoniae and Haemophilus influenzae and cryptococcal antigen in the cerebrospinal fluid for diagnosis of meningitis, Clostridium difficile antigens in the stool for diarrhoea and hydatid antigen in the serum for diagnosis of hydatid disease. In this exercise you will perform the LAT for detection of antigen in the serum for diagnosis of hydatid disease. PROCEDURE 1 Take a clean glass slide. 2 Mark it into two halves by a glass marking pencil and label them as test and control. 3 Add a drop of test serum in the test half, and a drop of saline in the control half. 4 Add a drop of hydatid antibody-coated latex reagent to the serum in the half labeled as test and mix. 5 Place another drop of hydatid antibody-coated latex reagent in half of the slide labeled as control, and mix. 6 Gently rock the slide, back and froth for 2 minutes. 7 Observe the clumping of the latex reagent in the test. QUALITY CONTROL The LAT is performed with a known positive and negative hydatid sera, every time the test is performed with the test sera. PRINCIPLE In the LAT for detection of hydatid antigen in the serum, the latex particles are coated with specific polyclonal hydatid antibodies. Then hydatid antibody coated latex particles are mixed with serum to be tested for the antigen, If serum contain antigen, then the latter combines with the antibodies on surface OBSERVATIONS Observe the test half for agglutination of latex reagent. Observe the control half for autoagglutination. Textbook of Practical Microbiology 113 RESULTS AND INTERPRETATION Agglutination of latex reagents with the sera, and absence of any agglutination in the control half indicates the LAT to be positive and shows the presence of hydatid antigen in the serum (Fig. 38-1). Absence of agglutination either in the test half or in the control half indicates the test to be negative and shows the absence of hydatid antigen in the serum. NEGATIVE POSITIVE FIGURE 38-1 Latex agglutination test. KEY FACTS 1 Latex agglutination test is an example of slide agglutination test. 2 The latex agglutination test is used for demonstration of antibodies on a variety of bacterial, parasitic and fungal infections. 3 The LAT can also be used for detection of soluble antigen by latex particles coated with the specific antibodies. VIVA 1 List application of the LAT for diagnosis of infectious diseases by demonstration of antibodies in the serum. 2 List application of the LAT for diagnosis of infectious diseases by demonstration of antigens in the serum. FURTHER READINGS 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 39 INTRODUCTION Co-Agglutination Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Demonstrate the application of Co-agglutination test for detection of soluble antigen. PROCEDURE 1 Take a clean glass slide. 2 Mark it into two halves by a glass marking pencil and label 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. Note: The CSF specimen has to be absorbed with stabilized SAPA cells (not coated with specific pneumococcal antibody) to prevent nonspecific agglutination with human IgG. 4 Add a drop of pneumococcal antibody-coated Co-A reagent to the serum in the half labeled as test and mix. 5 Place another drop of pneumococcal antibody-coated Co-A reagent in the other half of the slide labeled as control, and mix. 6 Gently rock the slide, back and froth for 2 minutes. 7 Observe the agglutination of the bacteria in the test. The Co-agglutination (Co-A) test is a simple slide agglutination test. The test is being used for detection of specific antigen in the serum, cerebrospinal fluid (CSF), urine and other body fluids. Commercial Co-A kits are now available for detection of haemophilus, meningococcal and pneumococcal antigens in the CSF, and salmonella antigen in the serum. The kits are also available for identification of Neisseria gonorrhoeae and sero 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 The Co-A is performed with a known pneumococcal antigen positive and negative CSF, every time the test is performed with the test sera. PRINCIPLE Co- agglutination test means cocci- mediated agglutination. In this test certain strains of S. aureus organisms (Cowan’s I strain) containing a large amount of an antibody binding Protein A (SAPA) in their cell walls is utilized as a carrier particle. These cocci on mixing with the specific antibodies (raised against Streptococcus pneumoniae to be detected in the CSF) bind IgG non-specifically through the Fc region leaving specific Fab sites free. The subsequent reaction of Fab with pneumococcal antigen in the test CSF is visualized by clumping. OBSERVATIONS Observe the test half for agglutination of Co-A reagent. Observe the control half for auto agglutination. RESULTS AND INTERPRETATION Agglutination of the Co-A reagents with the CSF , and absence of any agglutination in the control half indicates the Co-A to be positive and shows the presence of pneumococcal antigen in the CSF. Absence of agglutination either in the test half or in the control half indicates the test to be negative and shows the absence of pneumococcal antigen in the CSF. REQUIREMENTS I Reagents and lab wares Co-agglutination reagent, glass slides, and applicator stick. II Specimen CSF sample to be tested. Textbook of Practical Microbiology 115 KEY FACTS 1 The Co-agglutination test is an example of slide agglutination test. 2 S. aureus organisms (Cowan’s I strain) containing a large amount of an antibody binding Protein A (SAPA) in their cell walls is utilized as a carrier particle. 3 The Co-A test is used for demonstration of antigens in the serum, urine and CSF in a variety of infections. 4 The commercial Co-A reagents are available for identification of N. gonorrhoeae and serogrouping of S. pyogenes A, B, C, D or G. 5 The reagents are also available commercially for identification of meningococcal, pneumococcal and haemophilus antigen in the CSF for diagnosis of meningitis. VIVA 1 Explain the principle of the Co-agglutination test. 2 What is the strain used in Co-agglutination test? 3 List the uses of Co-agglutination test. FURTHER READINGS 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. REQUIREMENTS I Equipments Water bath (Fig. 3 Add 0. Typhi and against H suspension of S.2 ml of serum to the test tube. Performance of the test 1 Arrange 4 rows of test tubes. AH and BH. They may be stored at –20 °C for the period beyond 2 weeks. rarely by S. Enteric fever is caused by Salmonella Typhi. which makes the serum dilution as 1: 12. the O antigen of S. TO. . Paratyphi B are tested separately. and pipettes. In Widal test serum of the patients is tested for antibodies against H and O antigens of S. Paratyphi B are added. The test is then incubated in water bath at 37°C and is checked for agglutination by examining the bottom of the tubes for sediment. Widal test is an example of tube agglutination test used widely for diagnosis of enteric fever. Saline (0. Kasauli. 2 Add 0. Four rows of such dilutions are prepared and to that antigen suspensions of O antigen of S. (Himachal Pradesh). Paratyphi A and S. Paratyphi B. Typhi and H antigens of S. Paratyphi A and S. PROCEDURE Preparation of master dilution of the serum 1 Add 2.2 ml of saline to all the tubes from 2nd to 7th in all the rows including the 7th control tube. Paratyphi C infections are not found in India. PRINCIPLE Widal test detects antibodies in serum against H and O suspensions of S. Since “O” antigens are related to each other. 3 Patient’s serum is tested in a series of dilutions against each of the different antigen suspensions.116 LESSON 40 INTRODUCTION Widal Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Perform Widal test for diagnosis of enteric fever. Typhi is only tested. These antigens can be prepared in the laboratory or commercially prepared suspensions are available from the Central Research Institute. and by S. and salmonella antigen suspensions. 2 Label the row 1 as TO. serum antibodies against this species are not tested. Since S.85 % NaCl). II Reagents and glass wares Small test tubes. Typhi. Typhi. while H antigens of S. In India enteric fever is caused commonly by S. The titre of patient serum is calculated for each salmonella suspension as the highest dilution producing visible agglutination.5. Typhi. B and C.2 ml of diluted serum from master dilution into the first and second tubes in all rows of the tubes.3 ml of saline in a test tube. and S. The antigens are stored at 4 °C before use. In this test the serum to be tested are diluted in series in test tubes and antigen suspensions of salmonella are added to it. Paratyphi B. Paratyphi A. S. 3 as AH and 4 as BH. Paratyphi A and B. and by Salmonella Paratyphi A. TH. each row containing 7 test tubes. 4 Add 0. S. III Specimen Serum sample to be tested Note: Serum samples may be stored at 4 °C for 10-15 days before testing. test tube racks. 40-1). 2 as TH. Typhi. Paratyphi A and S. What are the advantages and disadvantages of Widal test? Ans: Advantages: Simple and inexpensive test. AH and BH antigens. . Useful for cases in children. Sensitive test for the diagnosis of typhoid in endemic areas. The last tube showing agglutination is considered as the end point. If necessary. 6 O antibody titre rise indicates recent infection. 7 Add 0. FIGURE 40-1 Water bath. In case of H agglutinins large loose.2 ml of AH antigens to tubes from 1st to 7th in the row AH. If the dilution of the last tube showing agglutination is 1: 200.2 ml of serum from 2nd tube to the 3rd. 8 Add 0. The titer of the serum is the highest dilution of serum giving visible agglutination. 10 Add 0. OBSERVATIONS Observe the test tubes after 18 hours of incubation in waterbath at 56°C for agglutination and note the result. VIVA 1. No agglutination is seen as a small compact deposit (button) formation. 5 H agglutinins tend to persist longer than O agglutinins..2 ml from the 6th tube. 4 The serum of some uninfected subjects causes agglutinations at dilutions of about 1:50. so titers are considered significant when agglutination occurs in serum dilution above 100. 1:200. The serum of some uninfected subjects causes agglutinations (False positive reactions). A positive or a negative result in a single test is not significant.2 ml of TO antigens to tubes from 1st to 7th in the row TO. 1. test should be done later. AH and BH. 1:400. RESULTS AND INTERPRETATION 1 A progressive rise in the titer between first and third week after onset of fever is highly significant. then the titre is 200).2 ml of TH antigens to tubes from 1st to 7th in the row TH.Textbook of Practical Microbiology 117 5 In the row labeled as TO. The results are read by viewing the tubes under good light against a dark background with the aid of a magnifying lens. QUALITY CONTROL 7th tube in each row acts as a negative antigen control. 11 Incubate the racks in water bath at 37 °C for 18 hrs. Disadvantages: Less specific. who have a low prevalence of pre-existing antibody. Discard 0. The antibodies are detected only after 7 to 10 days of illness. Cases treated early with chloramphenicol may show a poor agglutinin response.50. 9 Add 0. 7 Persons immunized with TAB vaccine may show high titers of antibodies to all the antigens and so only a marked rise in titer is considered significant. 1. then from 3rd to 4th. TH. cotton and wooly clumps are formed and with O agglutinins only small granules are formed at the bottom of the tube. and so on through the 6th tube.2 ml of BH antigens to tubes from 1st to 7th in the row BH. The reciprocal of the dilution is considered as the titre (e. the tubes can be gently rotated. 8 Early treatment with antibiotics will alter antibody response. The same dilutions of a known positive serum is tested with TO. 6 Follow the same dilutions in the rows labeled as TH. 1:800. Note: The final dilution of the sera after addition of antigen are 1:25. to swirl up granules from the deposit.100. 3 Since the antibodies are detected only after 7 days to 10 days of illness. Persons immunized with TAB vaccine may show high titers of antibodies to all the antigens. mix and transfer 0.g. 2 A positive or a negative result in a single test is not significant. Allen SD. Medical Immunology.118 Widal Test KEY FACTS 1 In India enteric fever is caused commonly by S. FURTHER READINGS 1 Collins CH. Paratyphi B. Terr AI and Parslow TG. pp. 2 Koneman EW.. Color Atlas and Textbook of Diagnostic Microbiology. Paratyphi A. Typhi is only tested. Lippincott Williams and Wilkins. London. Janda WM. pp. Typhi. S. Typhi. Paratyphi A and S. 3 The last tube showing agglutination is considered as the end point. If the dilution of the last tube showing agglutination is 1: 200. Paratyphi B are tested separately. 902. then the titre is 200). . while H antigens of S. 1395. WC. 4 The reciprocal of the dilution is considered as the titre(e. Lyne PM and Grange JM. Butterworth. 2001. the O antigen of S. rarely by S. 10th Edition. 1997. 1995. Schreckenbergu PC and Winn Jr. 2 Since “ O” antigens are related to each other. Microbiological Methods. and S.g. 94-96. 5th Edition. 3 Stites DP. 2 Add 0. 4 Make doubling dilutions in the first row from the first tube(serum dilution 1:20) and discard 1 ml from the 6th tube (serum dilution 1:640). mooseri share alkali stable carbohydrate antigens with P.rickettsi. certain strains of Proteus are used instead of specific rickettsial pathogens as antigens. It is based on the principle that many patients infected with one of the Rickettsia produce antibodies that can agglutinate certain strains of bacteria of genus Proteus because of the presence of a common alkali stable carbohydrate antigen. REQUIREMENTS I Equipments Water bath. Preparation of master dilution of the serum 1 Add 2. 8 Add 0. and P. In this test. mirabilis OX K and R.3 ml of serum to the test tube which makes the master serum dilution as 1: 10. 5 In the same way. OX-2 of Proteus vulgaris and OX-K of P..85 % NaCl). OX-2 and P.5 ml of concentrated P. This test has been used for presumptive serological evidence of Rickettsial disease. 3 Add 1 ml of diluted serum from master dilution into the first tubes in all the three rows of the tubes. vulgaris OX-2 in to all 7 tubes in the second row of tubes.5 ml drop of concentrated P. Note: Leave the 7th tube as control without serum. tsutsugamushi with P. vulgaris OX-19 in to all 7 tubes in the first row of tubes. test tube racks. . Performance of the test 1 Arrange 3 rows of test tubes.Textbook of Practical Microbiology 119 LESSON 41 INTRODUCTION Weil-Felix Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Demonstrate the cross-reacting antibodies to Rickettsial antigens using Proteus strains OX-19.conori with P. vulgaris OX 19. and pipettes. Saline (0. 7 Add 0. mirabilis OX-K in to all 7 tubes in the third row of tubes.5 ml of concentrated P. PROCEDURE Weil-Felix is a serological test used for diagnosis of Rickettsial infection by demonstration of antibodies in the serum. II Reagents and glass wares Small test tubes. III Specimen Serum sample to be tested. The antigens are stored at 4°C before use. PRINCIPLE It is an agglutination test for cross-reacting antibodies. This test was developed from the observation that certain strains of Proteus isolated from the urine of patients with epidemic typhus were agglutinated by the sera of the typhus patients. Rickettsia prowazaki and R. vulgaris OX 2. 2 Add 1 ml of saline to all the tubes. followed by reincubating at 4°C for 18 hours. 6 Add 0. R. mirabilis by Weil-Felix test. each row containing 7 test tubes. mirabilis OX-K antigens. vulgaris OX-19. 9 Incubate the racks in water bath at 37°C for 2 hr. and R.7 ml of saline in a test tube. make doubling dilutions in the second and third row tubes. . 2001. in Borrelia infections and in severe liver disease. 3. 2 Proper precautions should be taken for standardization of antigens. pp. The last tube showing agglutination is considered as the end point. Color Atlas and Textbook of Diagnostic Microbiology. KEY FACTS 1 Since the Weil-Felix antigens also react with Proteus antibodies. 2 Koneman EW. 5th Edition. 1395. Microbiological Methods. FURTHER READINGS 1 Collins CH. 94-96. The same dilutions of a known positive serum are tested with Proteus OX 19. 1997. cold agglutination test for the diagnosis of primary atypical pneumonia. b.g. trench fever or Q fever as these persons do not develop Proteus agglutinins. If the dilution of the last tube showing agglutination is 1: 640. Schreckenbergu PC and Winn Jr. Allen SD. Lyne PM and Grange JM. OX 2 and OX K antigens.. The results are read by viewing the tubes under good light against a dark background with the aid of a magnifying lens. pp. 10th Edition. 902. Agglutination with Proteus OX K is suggestive of scrub typhus. Paul-Bunnel test for the diagnosis of Epstein-Bar virus infections and c. Butterworth. Janda WM. London. WC. Medical Immunology. 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. The reciprocal of the dilution is considered as the titre(e. in leptospirosis. OBSERVATIONS Observe the test tubes after overnight incubation for agglutination and note the result. The test is not helpful for the detection of antibodies in rickettsial pox. then the titre is 640). 1995. 3 Stites DP. Complete agglutination is demonstrated by complete clearing of the supernatant fluid and the formation of white flocculent masses in the bottom of tubes RESULTS AND INTERPRETATION A serum titer of 1: 80 and above is considered significant titer but four fold or greater increase of antibody between acute and convalescent sera is considered diagnostic. Agglutination with Proteus OX 2 is suggestive of murine typhus. Streptococcus MG agglutination test for the diagnosis of atypical pneumonia.120 Weil Felix Test QUALITY CONTROL 7th tube in each row acts as a negative antigen control. VIVA 1 What are heterophile antibodies? Ans: Antibodies produced in certain clinical conditions have the ability to cross react with more than one antigen called heterophile antibodies 2 What is the principle of Weil-Felix reaction? 3 List other tests detecting heterophilic antibodies Ans: a. Agglutination with Proteus OX 19 and OX 2 is suggestive of spotted fever. Lippincott Williams and Wilkins. Terr AI and Parslow TG. false positive reactions may occur in urinary tract infections with Proteus. microtitre plates. antigen-antibody reaction takes place and no hemolysis takes place. which contribute to its virulence.1:85. Many methods are available for detection of antistreptolysin in the serum. 3 Add 0. antigen control and a standard serum control always should be used with the test. 1:480. Streptolysin S is not antigenic. namely streptolysin O and streptolysin S. 2 Prepare serial two fold dilutions through sixth well. Serum dilutions for each serum sample are first row . If a patient’s serum contains antistreptolysin O antibodies. gently agitate.36. QUALITY CONTROL A cell control. Subsequent dilutions are carried out in microtitre plates. 2 Both serum and buffer should be placed at room temperature when preparing dilution.025ml of antigen to all tubes. 1:680. . 3 Standard serum of know titer is included in each days run to serve as positive control. test tubes. PRINCIPLE When streptolysin O in its reduced form is added to red blood cells hemolysis occurs.5% RBC’s in Alsever’s solution (human blood group O or rabbit erythrocytes). 1:60 and 1:85 are prepared in test tubes. Hence. 2. 1:120. 1:1360. phosphate buffered saline pH 6. III Specimen Serum. This antibody is known as antistreptolysin. Streptococci produce two haemolysins. PROCEDURE Serum dilution 1 The initial serom dilutions of 1:10. 1:960. 1:2760. 1:340.Textbook of Practical Microbiology 121 LESSON 42 INTRODUCTION Anti-Streptolysin O (ASLO) Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Demonstrate neutralizing antibodies to Streptococcus pyogenes haemolysin O antigen. 1:240. OBSERVATIONS Observe the microtitre plates for presence or absence of hemolysis. Str. II Reagents and lab wares Antigen (Streptolysin O). 1:170. REQUIREMENTS I Equipments Water bath. 1:1920. and incubate at 37°C for 15 minutes. second row . hence do not elicit production of any antibodies. Streptolysin O is oxygen labile and heat labile. Test procedure 1 Label microtiter plate so that each specimen is alligned in two rows (1:60 row and 1:85 row) with six wells per row. Streptolysin O is antigenic and elicits the production of specific antibodies against the antigen in an infected human host. It is inactive in the oxidized form but may be reactivated by treatment with mild reducing agents.1:60. It is lethal on intravenous injection into animals and has a specific cardiotoxic and leucotoxic activity. demonstration of antistreptolysin in the serum is an indirect indicator of infection.5. pipette. pyogenes produces several exotoxins and enzymes.025 ml of cold 2. it regularly appears in sera following streptococcal infection. 4 Add 0.5% red blood cells to all wells and incubate in water bath at 37° C for one hour. 2 Koneman EW. 902. FURTHER READINGS 1 Collins CH. Microbiological Methods. VIVA 1 Name the hemolysins produced by Streptococci. Lyne PM and Grange JM.122 Anti-Streptolysin RESULTS AND INTERPRETATION Reading is taken by observing for button formation and hemolysis in microtitre plate. Allen SD. hence do not elicit production of any antibodies. Butterworth. 2 Explain the principle of the LAT test. London. 1997. Janda WM. 2 Streptolysin S is not antigenic. 2001. pp. Lippincott Williams and Wilkins. KEY FACTS 1 Streptolysin O is antigenic and elicits the production of specific antibodies against the antigen in an infected human host. . 5th Edition. 94-96. 1395. Color Atlas and Textbook of Diagnostic Microbiology. Schreckenbergu PC and Winn Jr. Medical Immunology. pp. WC. The titer of ASLO is the highest serum dilution causing no hemolysis. 3 Stites DP. 10th Edition. Terr AI and Parslow TG. Significant titer is considered to be 200 or more. 1995. they will bind to each other to form an insoluble complex which results in visible precipitations. pH is adjusted to 6. Venereal Disease Research Laboratory (VDRL) test is an example of flocculation test to detect reaginic antibodies in serum of patients suffering from syphilis. and 10 gm of sodium chloride (NaCl). glass stoppered bottles. 2 Demonstrate the presence of reaginic antibodies in patient’s serum.The advantages and disadvantages of the VDRL test are listed in the table 43-1.5 ml of formaldehyde. 3.170 gm of potassium dihydrogen phosphate (KH2PO4). VDRL –ELISA is a modification of the VDRL test (Box 43-1). buffered saline solution. When cardiolipin antigen combines with reaginic antibodies in patient’s serum. II Reagents and glass wares VDRL antigen (It contains cardiolipin.3%. III Specimen Serum and CSF. all the sera are examined and those found to contain particulate debris are recentrifuged. 0. Preparation of un buffered saline solution: This is prepared by adding 1 gm of sodium chloride to 100 ml of distilled water.05 ml. PRINCIPLE PROCEDURE The VDRL slide flocculation test is a simple.Textbook of Practical Microbiology 123 LESSON 43 INTRODUCTION VDRL Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Perform the VDRL test. test. 0. Often the precipitate fails to settle down at the bottom of the container but remains suspended as floccules. saline. lecithin. 2H 2O). This is an example of standard test of syphilis in which cardiolipin antigen is used to detect non-specific reaginic antibodies in the serum. Temperature. in 1000 ml of distilled water. Preparation of buffered saline solution: This is prepared by mixing 0. tubes. I Equipments Water bath and VDRL shaker. The biological false positive reaction is a noted problem associated with the VDRL test (Box 43-2).037 gm of disodium hydrogen phosphate (Na 2HPO 4. The quantity of serum used in each test is 0. 0. The process of precipitation in the gel can be hastened by migration of antigens and antibodies under an electric field. REQUIREMENTS When soluble antigen and specific antibodies in the serum are combined in the right proportions. such reaction is known as flocculation.1. rapid convenient and economical test for serodiagnosis of syphilis.24% and cholesterol. it forms visible floccules. This is a nontreponemal test because no treponemal antigen is used in this Preparation of serum 1 Inactivate the serum in water bath at 56°C for 30 minutes. . This test is also used to detect antibodies in the CSF. pH and concentration of sodium chloride in the medium influence the reaction of precipitation.9%). pipettes and racks.0+ 0. 0. The precipitates settle at the bottom of the container. The precipitation reaction can be demonstrated in the liquid medium as well as in the solid media such as in gels. Cardiolipin antigen is an alcoholic extract of beef heart tissue to which cholesterol and lecithin are added. Each antigen pack usually consists of 10 ampoules of 5 ml buffered saline solution. VDRL glass slides (2”X3”) with depressions of 14 mm diameter each. Note: After removal from the water bath. 3 Blow the last drop of antigen and continue rotation of the bottle for 10 more seconds.5 ml of antigen. It helps in presumptive diagnosis of syphilis. These serum dilutions are tested as described above. 1:16. The antigen emulsion prepared ought to be used during the same day. Each dilution of the serum is treated as an individual serum. No clumps or very slight roughness: Non-reactive. Zone reactions are recognized by the irregular clumping. in qualitative serum test.0 pipette stopper the bottle and shake it vigorously for approximately 10 seconds. Medium and large clumps: Reactive. whereas these aggregate into clumps in reactive sera. Different dilutions of the serum are prepared in tubes as successive twofold dilutions (in the range of 1:2. Note: Slide must be thoroughly cleaned and then well dried before use. 4 Then add 4. It can be used as a prognostic test. 1:4. It is an inexpensive test.0 ml pipette graduated to tip. Small clumps: Weakly reactive. Note: Temperature of the buffered saline solutions and antigen should be preferably between 23°C to 29°C during the preparation of antigen emulsion. 2 Quantitative serum test Results are reported in terms of the highest dilution of the serum that produces a definite reaction. and the number and distribution of antigen particles per microscopic field in the negative sera should be optimum. which remain more or less evenly. directly on to the saline while rotating the bottle on a flat surface. Qualitative serum test 1 Pipette 0. Improper temperature of the laboratory.124 VDRL Test Preparation of antigen emulsion 1 Pipette 0. In such cases the results are reported on the basis of quantitative reaction done on the same serum. specimens or reagents contributes to the error in the test. Note: If rotated by hand on a flat surface this movement should roughly circumscribe a 2 inch diameter circle 120 times per minute.) with buffered saline solutions. The drop of antigen is transferred from an 18 gauze needle cut across and fitted to 1 ml syringe. 1:8. disperse in a nonreactive serum. Disadvantages It is a non-specific test. .1 ml of buffered saline from a 5.4 ml of buffered saline to the bottom of a 1 oz reagent bottle with flat or concave inner bottom surface. This test alone is not confirmative. drawn from an ampoule in to a 1. This test should present typically reactive and non-reactive results. etc. The antigen is added drop by drop but rapidly so that it takes approximately 6 seconds to complete the delivery. Maturation of the antigen: It increases the sensitiveness and this is almost complete in 15 minutes to 30 minutes. Table 43-1 Advantages and disadvantages of VDRL test Advantages It is a simple and rapid diagnostic test. OBSERVATIONS Observe for the formation of floccules immediately after rotation under a microscope with low power objective (10 x magnification). It can be used as qualitative and quantitative tests. 3 Rotate the slide for 4 minutes (180 rotations per minute). Preliminary testing of antigen emulsion: Each batch of prepared antigen emulsion should first be examined by testing known reactive and non-reactive sera. which are not compact. Quantitative serum test Quantitative test is performed on all reactive serum samples and on all samples showing weakly reactive or rough in the qualitative test. 2 One drop (1/60 ml) antigen emulsion is added on to the serum. 4 Read the result under 10 x objectives of the microscope. 2 Add 0. RESULTS AND INTERPRETATION 1 Qualitative serum test The results are reported on the basis of quantitative reaction done on the same serum. It shows biological false positive reactions.05 ml inactivated serum into the cavity of a VDRL slide. It is reproducible. Quality control Known reactive (with known titre) and non-reactive sera are included. The antigen particles are seen as small fusiform needles. 2001. As cardiolipin antigen is present both in T. Lyne PM and Grange JM. Maturation of the antigen increases sensitiveness of the test. 1995. In the indirect ELISA procedure. BFP reactions may be classified as acute or chronic. which are not compact. VIVA 1 2 3 4 What is the principle of the VDRL test? What is the antigen used in the VDRL test? What are the advantages and disadvantages of the VDRL test? List the biological false positive conditions shown by the VDRL test. The reactivity of this test disappears with treatment of the patient. Schreckenbergu PC and Winn Jr. pp. and not caused by technical faults. Microbiological Methods. which can measure IgG and IgM antibodies separately and is suitable for large scale testing of sera. London. 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. WC. 2 Koneman EW. Chronic BFP reactions persist for longer than six months and are typically seen in SLE and other collagen diseases. infectious mononucleosis. KEY FACTS 1 2 3 4 5 VDRL is an example of slide flocculation test and is an example of standard test of syphilis. This test has a sensitivity of 97% in untreated syphilis and specificity of 97%. Observe for the formation of floccules immediately after adding the antigen to the serum in a VDRL slide. in the absence of past or present treponemal infections. This accounts for BFP reactions. Medical Immunology. Butterworth. 1395. 94-96. Janda WM. . reaginic antibodies may be induced by treponemal or host tissue antigens. 7 VDRL test shows biological false positives in a variety of conditions. Allen SD. 5th Edition. Leprosy. They represent the nontreponemal cardiolipin antibody responses. Several hundred sera can be tested by VISUWELL Reagin test in a day. Cardiolipin antigen is used to detect non-specific reaginic antibodies in the serum. pallidum and in mammalian tissues. FURTHER READINGS 1 Collins CH. 1997. malaria. Quantitative test is performed on all reactive serum samples and on all samples showing weakly reactive or rough in the qualitative test. BOX 43-2 BIOLOGICAL FALSE POSITIVE REACTIONS OF VDRL TEST Biological false positive (BFP) reactions are defined as positive reactions obtained in tests using cardiolipin antigen. Color Atlas and Textbook of Diagnostic Microbiology. injuries or inflammatory conditions. VDRL antigen coats the wells of a microtiter plate. 3 Stites DP.Textbook of Practical Microbiology 125 BOX 43-1 VDRL –ELISA An automated VDRL-ELISA test has been developed using cardiolipin antigen. 10th Edition. Lippincott Williams and Wilkins. Acute BFP reactions last only for a few weeks or months and are usually associated with acute infections. The newest of the nontreponemal tests. 6 Zone reactions are recognized by the irregular clumping. relapsing fever. Clinically. Terr AI and Parslow TG. pp. is based on the Pedersen method. 902. hepatitis and tropical eosinophilia are examples of other conditions associated with BFP reactions. BFP reactions may occur in about one percent of normal sera. with negative results in specific treponemal tests. VISUWELL Reagin. graph may be plotted between diameter of the ring and the antigenic concentration and in that case a curved line with a convexity upwards is obtained. Bacto agar. As in the double diffusion method. holes are cut in the agar. purified agar. As the antigen diffuses out of the well. and antigen is placed in the well. which are visualized as a ring. syringes. water bath. acetic acid and physiological saline. a straight line or a line with concavity upwards indicates error in the technique. antiserum is incorporated into the agar gel during its preparation. III Specimen Serum. moist chamber. By constructing and using a standard curve. then circular precipitates develop. 2 Boil the mixture till the gel particles are dissolved fully and allow it to cool down to 45-50°C . Analytical balance. 4 Plot the graph between square of the diameters on the ordinate and the corresponding standard antigenic concentrations on abscissa using a linear graph paper. PROCEDURE Preparation of antibody containing gels 1 Make 2% agar solution in barbitone buffer. measuring template and test tubes. Note: A straight line is obtained by joining at least three reference points. In this test. amido black. Uses of gel diffusion tests are mentioned in the table 44-1. The size of the ring is proportional to the amount of antigen in the well. PRINCIPLE When the radial diffusion of the soluble antigens from the cylindrical wells occurs in to the antibody incorporated gel. microscope slides. If the antibody is evenly distributed in the uniformly thick gel and well size and volume of the antigen is kept constant then the diameter of the precipitate is directly proportional to the antigenic concentration. dark ground illuminating box. 3 Mix the appropriate quantity of the gel with the pre-warmed monospecific antibody (10% of the antibody is used). 10 and 15 md/dl) are selected and applied separately to the cylindrical wells using a suitable template. . Alternatively. 6.126 LESSON 44 INTRODUCTION Radial Immunodiffusion Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Quantitate the protein antigens in a test serum by single radial immuno diffusion. which indicates accuracy of the graph. II Reagents and glass wares Barbitone buffer. 2 Keep the gel in a moist chamber at 4°C . micropipettes. measuring cylinder. glass plates. REQUIREMENTS I Equipments Electrophoresis power supply. beakers. This technique helps in the quantification of human serum proteins feasible even in a moderately equipped laboratory. Calibration of reference graph 1 A set of five standards (5. it will form complexes with the incorporated antibodies. 7. Pasteur pipettes. 4 Then mix and pour the gel on microscopic slide and allow setting. 3 Measure the diameters of the circular precipitates under oblique illumination after 24 hours by using the special measuring template. Radial immunodiffusion is similar to the double diffusion method. the amount of antigen in each well can be quantitated. 3 Classify gel diffusion methods? Ans. Diagnosis of small pox. Schreckenbergu PC and Winn Jr. VIVA 1 What is the principle of gel diffusion test? 2 What are the advantages of gel diffusion? Ans. 2 The slide may be stained and preserved in a plastic bag for a permanent record. Janda WM. 5 Detection of a Fetoprotein antigen in serum. Lippincott Williams and Wilkins. 902. WC. Since there is a batch-to-batch variation the reference graph should be plotted for every new batch. 3 Transfer the gel to the moist chamber at 4°C. Double diffusion in one dimension (Oakley Fulthorpe procedure). 6 Detection of specific antigens of cryptococci and meningococci in cerebrospinal fluid.Textbook of Practical Microbiology 127 Testing unknown serum samples 1 Dilute the test sample in a ratio of 1 in 15 with physiological saline. b Precipitin band is stable and can be stained for preservation. KEY FACTS 1 2 3 4 Circular well with a clean edge is very important for a well-defined ring precipitate. London. Butterworth. 3 What are the uses of gel diffusion test? FURTHER READINGS 1 Collins CH. Rocket electrophoresis. Testing for normal and abnormal proteins in serum and in urine. Immunodiffusion Single diffusion in one dimension (Oudin procedure). The final value of the serum antigen level is derived by multiplying with the dilution factor (15). c The number of different antigens in the reacting mixture can be readily observed. 10th Edition. OBSERVATIONS Observe the gels for precipitin lines after 24 hours under oblique illumination for ring precipitates. Electroimmunodiffusion Countercurrentimmunoelectrophoresis. RESULTS AND INTERPRETATION 1 The antigen concentration is determined by taking the intercepts on the graph. Note: At least one check standard is employed on each occasion to test the reproducibility of the procedure while the remaining wells are charged with the test samples. Single diffusion in two dimensions (Radial immunodiffusion). Lyne PM and Grange JM. pp. Immunoelectrophoresis. 1997. Allen SD. Double diffusion in two dimensions (Ouchterlony procedure). . Table 44-1 Uses of gel diffusion tests 1 2 3 4 Screening sera for antibodies to influenza viruses. The advantages are as follows: a The reaction is visible as distinct band of precipitation. 94-96. 5 Measure diameter of the ring and record it. QUALITY CONTROL Known positive and negative samples. Terr AI and Parslow TG. 2 Koneman EW. Color Atlas and Textbook of Diagnostic Microbiology. Elek’s test for toxigenicity in diphtheria bacilli. The slope of the calibration graph is influenced by antibody concentration in the gel. 4 Observe the gel after 24 hours under oblique illumination for ring precipitates. 2 Punch out a total of 9 wells (2mm wide) in the gel using the suitable template. Medical Immunology. 1995. 1395. Microbiological Methods. 5th Edition. pp. The size of the ring is proportional to the amount of antigen in the well. 7 Purification and identification of proteins and nucleic acids. 2001. 3 Stites DP. 5 Place the slide on a graph paper. To coat the slide place two to three drops of the molten agar and spread over the surface of the slide.128 LESSON 45 INTRODUCTION Immunoelectrophoresis Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Demonstrate different antigens in antigen mixtures by immunoelectrophoresis. Note: The slide should be kept in such a way that the well containing antigen will be near the cathode. Punch two wells at about one third of its length.6). Note: the serum is mixed with the indicator dye (bromthymol blue)with a pipette. and then at 4°C in a refrigerator for 1 hour to set completely. glass plates. 9 Connect it to the power supply and run the electrophoresis for 60-90 min at 10-15 mA/slide till the bromthymol blue (indicator dye) spreads to the end of the slide. such as those in body fluids. and electrophoresis chamber. Diffusion is allowed to occur for 18-24 hours. Buffer (pH 8. glass slides (3” x 2”) and standard laboratory wares. 8 Fill the electrophoresis chamber with the buffer. The antiserum will diffuse laterally and react with the separated components. Immunoelectrophoresis is useful for demonstration of normal and abnormal serum proteins such as the myeloma proteins. Connect it to the buffer with Whatman 3 filter paper strips. purified agar or agarose. spread it uniformly and allow it to set at room temperature. 10 Remove the slides after turning off the power. Cut a trough between the two wells running parallel to the direction ofthe run. 6 Fill the two wells with the serum. amido black. 2 Put the coated slide on a horizontal work bench. are being analyzed in diffusion experiments like the double-diffusion system. Subsequently these antigenic components react specifically with their antibodies to form antigen-antibody precipitates that are formed in the gel. paper wicks and physiological saline. Suck out the agar plugs from wells and discard. Electrophoresis is carried out for one hour under electric current. Immunoelectrophoresis in principle is a method of combination of electrophoresis and diffusion. Human serum (antigen) and antihuman serum (antibodies) When mixtures. 3 Pipette 6 ml of agar to the slide. acetic acid. . II Reagents and glass wares Gel punch . REQUIREMENTS I Equipments Electrophoresis power supply. This will make the components move toward the anode. A rectangular trough is then cut parallel to the line of these separated proteins. PRINCIPLE Immunoelectrophoresis techniques is carried out on a glass slide layered with semisolid gel. Antiserum is placed in this trough. PROCEDURE 1 Take a clean glass slide. An aliquot of the antigen mixture is kept in the well cut out of the gel. 4 Remove the slide from the refrigerator. Note: These two wells should be 2 mm in diameter and I cm apart. By these methods antigens present in a mixture are separated from each other by agar gel electrophoresis. it is difficult to distinguish the individual antigenic components from each other. 7 Keep the slide in the electrophoresis chamber. III Specimen Patients serum. this procedure permits separation of proteins. Individual precipitation lines develop with each separated components of the antigen mixture. 10th Edition. RESULTS AND INTERPRETATION QUALITY CONTROL Known positive and negative samples are used in the test. 2 Koneman EW. Microbiological Methods. WC. Allen SD. OBSERVATIONS Observe the gels for precipitin lines after 24 hours at 4°C. Lippincott Williams and Wilkins. This will make the components move toward the anode. . Janda WM. 1 The antigen components in the mixture is noted by observing different lines of precipitations. 3. Color Atlas and Textbook of Diagnostic Microbiology. 14 Wash the slide and stain the slide. 1995. Lyne PM and Grange JM. 902. Immunoelectrophoresis is useful for demonstration of normal and abnormal serum proteins such as the myeloma proteins. 2 The slide may be stained and preserved in a plastic bag for a permanent record. VIVA 1 What is the principle of immunoelectrophoresis? 2 What are the advantages of immunoelectrophoresis? 3 List the applications of immunoelectrophoresis test. FURTHER READINGS 1 Collins CH. Butterworth. London. 12 Keep the slide in the moist chamber and allow the diffusion to take place at 4 °C for 24 hours. 13 Remove the slides and observe for the lines of precipitation. 94-96. Observe the gels for precipitin lines after 24 hours at 4°C. 1997. 1395. 2001. Keep the slide in the electrophoresis chamber in such a way that the well containing antigen will be near the cathode. 2. Medical Immunology.Textbook of Practical Microbiology 129 11 Remove the agar and fill the trough with the antiserum. Schreckenbergu PC and Winn Jr. KEY FACTS 1. 3 Stites DP. Terr AI and Parslow TG. pp. 5th Edition. pp. III Specimen Patients test serum.5 cm).7% acetic acid and Whatman filter paper No. To coat the slide place two to three drops of the molten agar and spread over the surface of the slide. purified agar or agarose. The slides are kept in an electrophoresis tank in such a way that the wells containing antigen will be on PROCEDURE 1 Take a clean glass slide.5 cm by 2. gel punch and standard laboratory wares. In this chapter the CIEP to detect hydatid antigen in the serum will be described.6). Preparation of agarose: This is prepared by adding 1gm agarose to 10 ml buffer and 90 ml distilled water. A line of precipitation visible to the naked eye is formed at a point between the antigen and antibodies. II Reagents and glass wares Glass plates. .3. One well is filled with antigen while the other well is filled with antibody.130 LESSON 46 INTRODUCTION Counter-current Immunoelectrophoresis Test the cathodic side and the well containing antibodies will be on the anodic side. Counter-current immunoelectrophoresis (CIEP) is a highly specific test used for demonstration of either antibodies or antigens in the serum. cryptococcal antigen in the CSF. glass slides (7. The CIEP test has the advantages of being simple. 2 Demonstrate antibodies or antigens in serum and other body fluids by counter-current immunoelectrophoresis (CIEP). CSF. amido black. template. The test is also highly specific test. hydatid antigens and antibodies in the serum. normal saline. hydatid antigen. PRINCIPLE 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. urine and other body fluids. 276 gm in 1000 ml of distilled water and dissolving it by heating. the result can be obtained within 30-45 minutes of performing the test. antiserum and its dilutions. and amoebic antigens and antibodies in the serum. Veronal buffer. staining of the slides and other variables if not properly standardised may affect final result of the test. buffer pH. voltage of the current. polyclonal hydatid antibodies raised in the rabbits Preparation of Veronal buffer 0. Disadvantages of the test is that it is a low sensitive test.45 gm sodium dimethyl barbiturate and 1 gm of sodium azide to the solution. This method was used earlier for diagnosis of many infectious diseases by demonstration of antigens or antibodies. This is followed by adding 15. LEARNING OBJECTIVES After completing this practical you will be able to: 1 Perform counter-current immunoelectrophoresis (CIEP). The pH is adjusted to 8.6. The test takes 30 minutes. The test is used for detection of hepatitis B antigen and antibodies in the serum. REQUIREMENTS I Equipment Electrophoresis power supply and electrophoresis chamber. and rapid test. so that during electrophoresis antibodies move towards the cathode and antigens move towards the anode. The agarose is melted by keeping it in a boiling water bath. The lines of precipitation in the slide can be stained by Amido black or 1% Coomassie brilliant blue stains.075 M (pH 8. 0.075 M (pH 8. control sera. The test is carried out on glass slides layered with the agarose with the wells cut out off the agarose. The supporting medium. The CIEP is also used to detect hydatid and filarial antigens in the urine specimens.6): The buffer is prepared by mixing dimethyl barbituric acid. 94-96. 8 Fill the electrophoresis chamber with the buffer. 3 Pipette 2. 3 Stites DP. Note: The slides can be kept at 4 °C for 24 hr and the result read and stained. London. Schreckenbergu PC and Winn Jr. Lippincott Williams and Wilkins. 15 After staining. 1997. Color Atlas and Textbook of Diagnostic Microbiology. 5th Edition. Medical Immunology. and then at 4°C in a refrigerator for 1 hour to set completely.5 ml of 1% agar to the slide. the result can be obtained within 30-45 minutes of performing the test. 1995. RESULTS AND INTERPRETATION 1 The line of precipitation is present in the control row : The test is performed correctly. QUALITY CONTROL Known hydatid antigen-positive and . With every slide known hydatid antigen is placed in the wells on the cathodic side and hydatid antibodies in the wells on the anodic side as control. 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. 5 Place the slide on a template. Butterworth.Textbook of Practical Microbiology 131 2 Put the coated slide on a horizontal work bench. . WC. Microbiological Methods. VIVA 1 2 3 4 5 What is the principle of counter current electrophoresis CIEP test? What are the advantages of CIEP test? What are the disadvantages of CIEP test? What are the dyes used for staining the slide after electrophoresis? List the applications of CIEP test. pp. 2 Line of precipitation is formed between the test serum and hydatid antibodies wells: It indicates the patient serum is positive for hydatid antigen by the CIEP test. Terr AI and Parslow TG. Connect it to the buffer with Whatman 3 filter paper strips. 14 Destain the slides with 7% acetic acid till the background stain is removed. 10 Remove the slides after turning of the power off. Then observe for the lines of precipitation in the test sera. Allen SD. Lyne PM and Grange JM. Remove the salts by washing the slides several times in distilled water for one hour. 6 Fill each well with 10µl of the patients serum to be tested and polyclonal hydatid antibodies. 1395. pp. 12 To stain the slides wash the slides first in saline for several hours. 9 Connect it to the power supply and run electrophoresis for 30 min at 10-15 mA/slide. 4 Remove the slide from the refrigerator. Note: The slide should be kept in such a way that the well containing test sera will be near the cathode and the wells containing the hydatid antibodies will be near the anodic side. and rapid test. OBSERVATIONS Observe the lines of precipitation after 30 min. 7 Keep the slide in the electrophoresis chamber. 2 Koneman EW. Punch out parallel rows of wells 4mm in diameter on the slides at distance of 3 mm from each other Note: Nine pairs of wells are punched out on each slide. 2001.negative serum samples. 902. First observe for the lines of precipitation in between the wells containing known hydatid antigen and known hydatid antibodies (Control row). spread it uniformly and allow it to set at room temperature. 3 No line of precipitation is formed between the test serum and hydatid antibodies wells: It indicates the patient serum is negative for hydatid antigen by the CIEP test. the lines of precipitation stains dark blue. 10th Edition. FURTHER READINGS 1 Collins CH. the lines of precipitation stains dark blue. 13 Stain the slides by immersing the slides in 1% Amido black in 7% acetic acid for 15-30 minutes. 11 Remove the slides and observe for the lines of precipitation. 3 After staining by Amido black. Janda WM. to the patients sera to demonstrate specific antibodies. The fluid is aspirated aseptically by syringe from an intact hydatid cyst. malaria amoebic liver abscesses. The protein content of the fluid is estimated by autoanalyser. Erythrocytes from different sources have been used by various workers in the IHA test. they settle quickly. The intact cyst should be collected as such without any rupture and sent to the laboratory immediately. Reverse passive haemagglutination test is a different test used to detect antigen in the serum and other body fluids (Table 47-1). phosphate buffer saline (pH7.132 LESSON 47 INTRODUCTION Indirect Haemagglutination Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Perform indirect haemagglutination test. The fluid obtained is sterilized by method of filtration using a Seitz filter or a membrane filter. Other sources are from human of ‘O’ blood group. Matt formation with test sera indicates that agglutination of RBCS has taken place. II Reagents Hydatid antigen. Formation of button with test sera indicate that agglutination of RBCS has not occurred. Preparation of hydatid antigen: Hydatid cysts removed surgically from humans or animals (cattle) are the source of hydatid fluid. The optimum sensitizing dose (OSD) of the antigen is determined for each dilution of stabilized chick cells by chequer board titration against the known positive control and negative PRINCIPLE This is based on the principle that the red blood cells act as carrier particles for the antigen (against which antibodies will be demonstrated). The fluid is then checked for sterility by inoculating in a blood agar and McConkey agar and incubating aerobically.4) and diluent (PBS pH 7.1% bovine serum albumin). so results of hemagglutinated chick cells could be observed within 30-45 min of agglutination. the chick RBCs are sensitized with hydatid antigen (fluid obtained from human hydatid cyst). Chick RBCs are stabilized by double aldehyde stabilization method. droppers (25µl) and diluters (25µl). The haemagglutination pattern of chick RBCs are observed after 30-45 min of the test. double aldehyde stabilized chick RBCs. The hydatid cyst should not be preserved in the formalin.2 with 0. echinococcosis and filariasis. Frequently and widely used erythrocytes are from sheep. This hydatid fluid is checked microscopically for the presence of any scolices or hooklets. they are heavier. leishmaniasis.2). The antigens is stored in -20ºC in 1ml aliquots. 2 Demonstrate antibodies in serum by the indirect haemagglutination (IHA) test.. In this chapter the IHA method for demonstration of hydatid antibodies in the serum for diagnosis of hydatid disease will be described. phosphate buffer saline (pH6. Indirect haemagglutination (IHA) test is a technically simple and inexpensive technique using antigen sensitized erythrocytes for demonstration of serum antibodies in a variety of parasitic diseases. REQUIREMENTS I Lab wares Microtitre plates. The test has been proved useful for diagnosis of various parasitic diseases viz. The RBCs sensitized with antigen are added . bovine and swine Avian cells are usually recommended because being nucleated. echinococcosis. The IHA using the chick cells have been used in the IHA for demonstration of specific serum antibodies in the diagnosis of amoebiasis. In IHA for hydatid disease. schistosomiasis. turkey. goose. The haemagglutination pattern of the antigen sensitized-RBCs with the sera are noted. 1ml of packed DAS chick cells in a centrifuge tube and wash with PBS 7. tuberculous meningitis and many other infectious diseases. 4 Dilute the sera upto the eleventh well leaving the last well as a serum free control.2). 7 Wash twice with PBS 7. With every test. The RBCs sensitized with antigen are added to the patients sera to demonstrate specific antibodies in serum. 1 Take 0. 3 Add 25µl volume of the serum to the first well of the appropriated row.1% BSA. KEY FACTS 1 2 3 4 5 The red blood cells from different sources have been used by various workers in the IHA test. The lowest amount of antigen which shows the maximum heamagglutination with the known positive control and negative reaction with the negative control is taken as OSD of the antigen for that batch.9ml of optimum sensitizing dose (OSD) of the hydatid antigen. 3 Add 0.2 and once with 0. 6 Next day mix the cells and keep the cells again in waterbath at 50°C for 10 minutes. 6 Agitate the plate gently for 2 min.2 with 1% bovine serum albumin) in each well of U bottomed microtitre tray. QUALITY CONTROL Known hydatid antibody-positive and .Textbook of Practical Microbiology 133 control. III Specimen Serum. The IHA test is carried out in a microtiter plate. 2 Dispense a 25µl volume of the diluent (PBS pH 7. 8 Make 1% suspension with 0. known hydatid antibody positive and negative serum samples are tested in the microtiter plate. The serum showing a titre of 1: 128 and above is considered positive for hydatid disease. Reverse passive haemagglutination test (RPHA) is used for demonstration of specific antigens for diagnosis of Hepatitis B infection. Matt formation in the cell control well rules out non-specific agglutination of RBCs. Matt formation with test sera indicates that agglutination of RBCs has taken place. RBCs act as carrier particles for polyclonal and monoclonal antibodies for demonstration of specific antigen in the serum. PROCEDURE Sensitisation of chick RBCs with OSD of the antigen.2 (twice) and centrifuge. 5 Keep the cells at 4°C overnight. The serum showing a titre of 1:128 and below is considered negative for hydatid disease. . 7 Incubate the plates at room temperature for 30 min and note the pattern of haemagglutination.negative serum samples. A well containing only sensitized chick cells (no sera is added to this well) is used as cell control. 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. 2 Wash with PBS 6.4 and centrifuge. Performance of the IHA test 1 Inactivate the test serum at 56ºC for 30 minutes before starting the test. BOX 47-1 REVERSE PASSIVE HAEMAGGLUTINATION TEST In this test. positive control serum and negative control serum. Matt formation in the cell control well rules out non-specific agglutination of RBCs. OBSERVATIONS Formation of button in the cells with test sera indicate that agglutination of RBCs has not occurred Matt formation with test sera indicates that agglutination of RBCs has taken place. The red blood cells act as carrier particles for the antigen (against which antibodies will be demonstrated). Japanese encephalitis.1ml of this packed chick cells to 0. 6 The serum showing a titre of 1: 128 and above is considered positive for hydatid disease. 5 Add 25µl volume of the 1% chick cells sensitized with the OSD antigen to each well. 4 Keep the chick cells in water bath for 5 minutes at 50°C with intermittent shaking at the end of each minute. 9 Store it at 4 °C until use. CSF and other body fluids.1% bovine albumin (prepared in PBS 7. RESULTS AND INTERPRETATION Formation of button in the cells with test sera indicate that agglutination of RBCs has not occurred. Terr AI and Parslow TG. What is reverse passive haemagglutination test? FURTHER READINGS 1 Collins CH. Butterworth. 3 Stites DP. 10th Edition. List the uses of IHA test. 902. 2001. Color Atlas and Textbook of Diagnostic Microbiology. .134 Indirect Haemagglutination Test VIVA 1 2 3 4 What is the principle of IHA test? List different RBCs which can be used in the IHA test. 1997. Lyne PM and Grange JM. 1395. 2 Koneman EW. WC. 5th Edition. 1995. London. pp. Lippincott Williams and Wilkins. Allen SD. Microbiological Methods. Medical Immunology. Schreckenbergu PC and Winn Jr. pp. 94-96. Janda WM. These reflected lights are visualized by a fluorescent microscope under ultra violet radiation. During this step any uncombined fluorescent antibody will be washed away. This test is most frequently used to detect rabies virus antigen in the tissue collected from the skin on the nape of the neck or face.2). Unknown mixed broth cultures of Group A S. Petri dishes. the same will bind the fluorescein labeled antibodies. Fluorescein isothiocyanate (FITC) is the most common dye used in the test. which will be observed by a fluorescent microscope using ultra violet radiations. and phycoerythrin are the other dyes used in the test. Advantages and disadvantages of the immunofluorescence test are summarized in the table 48-1. CSF. Neisseria gonorrhoeae. dansyl (yellow).Textbook of Practical Microbiology 135 LESSON 48 INTRODUCTION Immunofluorescence Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Demonstrate bacterial antigen by immunofluorescence test. U-shaped glass rods to fit into petri dishes. and Coplin jar and glass marking pencil. Evan’s blue counter stain (1:10000). .. Commercially available fluorescent antibody Streptococcus group A and fluorescent antibody Enterococcus group D. pyogenes. and bovine serum albumin. measles and mumps in various clinical specimens. the final reaction will be observed by a green fluorescence when observed under a fluorescent microscope. In a positive test.2): 10% glycerol in PBS. If antigen is present in the specimen. and is used to detect unknown antigen (e. The fluorescein labeled antibody is tagged to alcohol or acetone fixed bacterial smear. This is also used to detect Corynebacterium diphtheriae. PROCEDURE 1 Take three clean glass slides. Coli PRINCIPLE In this test specific antibodies raised against the antigen to be detected (e. anti-rabies antibodies to detect rabies antigen) is labeled with a fluorescent dye. REQUIREMENTS I Equipments Fluorescent microscope. buffered glycerol (pH 7. Direct fluorescent antibody test is employed to detect the specific antigens of bacteria.. phosphate buffered saline (pH 7. faeces. In this experiment you will use the method to demonstrate unknown bacterial antigens. and second slide as E. II Reagents and lab wares Acetone. viruses.g. urine. Rhodamine (red/orange). III Specimen Broth cultures of Group A Streptococcus pyogenes and Group D Enterococcus faecalis incubated at 35°C for 24 hours. tissues and other body fluids. ultra violet light) and reflect back the light of a different wavelength (visible light). Glass slides. parasites or other antigens in the serum.. and the antibody-bound fluorescein will emit a fluorescence. The fluorescent dyes have the property of absorbing the light of one wavelength (e. This is followed by washing the smear with physiologically buffered saline. faecalis.g. Divide the third slide in half and label as mixed unknowns. filter paper.faecalis / E.g. The dye emits a greenish/yellow light.. 2 With glass marking pencil label first slide as S. pyogenes/ Escherichia coli and Group D E. rabies antigen) in the specimen. Immunofluorescence assays involve the use of either antigen or antibody labeled with a fluorescent substance. First slide with S. label one side FA-A and the other slide FA-D and add one drop of fluorescent antibody Streptococcus group A to the side FA-A and one drop of fluorescent antibody Enterococcus group D to the side FA-D. What are the advantages and disadvantages of the direct fluorescent antibody test? . KEY FACTS 1 2 3 4 5 Immunofluorescence assays involve the use of either antigen or antibody labeled with a fluorescent substance. pyogenes on the first slide. Fluorescent dye fades faster. pyogenes. Requires UV radiation for visualization of the test result. 4 Add one drop of fluorescent antibody Streptococcus group A on to the slide one and fluorescent antibody Enterococcus group D to the second slide and spread gently over the surface of the smear. faecalis on the second slide and of mixed unknowns on the third slide. of E. The smears should not be allowed to dry at any stage. the side labeled FA-A : Third slide. the side labeled FA-D : Positive for fluorescence. Improper washing steps cause a very high background. Slides should be cleaned before use. 5 In the third slide. Rapid method. 11 Add one drop of buffered glycerol to each slide and cover with a cover slip. Positive for fluorescence. To minimize non-specific binding of proteins to cells or tubes. It can avoid the danger of radiation-based hazards. 9 Put the slides in a Coplin jar containing 1% buffered saline for 10 minutes at 25°C. Requires expertise personnel. Direct fluorescent antibody test is employed to detect the specific antigens. pyogenes and E. Requires expensive equipment. RESULTS AND INTERPRETATION The unknown specimen contains the bacteria E. Positive for fluorescence.136 Radial Immunodiffusion Test 3 Prepare alcohol or acetone fixed smears of S. 8 Remove the slides from the petri dish and wash with 1% buffered saline to remove away excess antibody. Disadvantages Difficult to quantitate the test. More specific when fluorescent dye is labeled to monoclonal antibodies. Table 48-1 Advantages and disadvantages of the immunofluorescence test Advantages More sensitive. VIVA 1 2 3 4 What is the principle of the direct fluorescent antibody test? What are the uses of direct fluorescent antibody test? Give examples of fluorescent dyes. Useful for the identification of viruses.1 – 1% BSA. 12 Examine under a fluorescent microscope. all dilutions and washings should be done in PBS containing 0. Negative for fluorescence. 10 Blot dries the slides with paper. faecalis treated with fluorescent antibody Streptococcus group A and fluorescent antibody Enterococcus group D are used as known positive controls. pyogenes : Second slide with E. 7 Place the prepared slides on the U-shaped glass rod kept in a petri dish and incubate at 25°C for 30 minutes. QUALITY CONTROL The slide with S. faecalis but does not contain S. OBSERVATIONS The slides are observed for the absence or presence of fluorescence in all the slides. faecalis : Third slide. 6 Allow to spread gently over the surface of the smear. False positive reactions may occur. 6 1-10 µg/ml of phenylene diamine could be added to the mounting medium to prevent the fading of fluorescence of fluorescein. Note: 1-10 µg/ml of phenylene diamine could be added to the mounting medium to prevent the fading of fluorescence of fluorescein. Textbook of Practical Microbiology FURTHER READINGS 137 1 Collins CH. Lyne PM and Grange JM. pp. Butterworth. Janda WM. 10th Edition. London. pp. 2 Koneman EW. Allen SD. 5th Edition. 2001. Color Atlas and Textbook of Diagnostic Microbiology. 1997. 902. Lippincott Williams and Wilkins. Microbiological Methods. Terr AI and Parslow TG. 94-96. Medical Immunology. 1995. Schreckenbergu PC and Winn Jr. 1395. . WC. 3 Stites DP. if the solution changes color). the indirect ELISA for antibodies. bacterial (syphilis. To detect the reaction a goat antihuman immunoglobulin antibody conjugated with enzyme is added. and c. beads. microplate. lymphatic filariasis.). A substrate is added to detect the binding and in a positive reaction the enzyme acts on the substrate to produce a change of colour. Competition occurs between these two antibodies for the same antigen. etc. The specimen such as serum to be tested are added to the coated wells. salmonellosis. In a positive test. The walls of the microtiter plates are coated with specific antibody against the antigen to be detected. The enzyme conjugated antihuman immunoglobulin binds to the antibody. they will have bound to the antigen. 2 Perform the ELISA test and interpret the results. aspergillosis. PRINCIPLE 1. The conjugated antibody combines with antibody that has combined with the antigen. ELISA is a heterogenous enzyme assay that uses solid phases e. in fact. which can be read by spectrophotometer or ELISA reader. An aliquot of the serum to be tested is added to each tube and is incubated. If antibodies are present in the serum. followed by washing. etc. etc. Then the enzyme-linked antiglobulin is added. Enzyme-linked immunosorbent assay (ELISA) is a widely used method for demonstration of specific antigens or antibodies in the serum and other body fluids. This is used for demonstration of HIV antibodies in the serum. . brucellosis. If antigen is present in the serum. A substrate is added to detect the binding of conjugated antibody to antigen-antibody complex. horseradish peroxidase and ßgalactosidase. respiratory syncytial infection.g. parasitic (amoebiasis. contain the antibodies of interest. The color change can be quantified using a spectrophotometer. which means that the serum did. and membranes such as cellulose acetate or cellulose nitrate. The indirect ELISA for antibodies: This is a frequently used method to detect antibodies. 3. Add the substrate for the enzyme. Depending on the nature of the antigen used. 2. Polystyrene tubes or wells in polystyrene plates are coated with the antigen solution. They are used extensively for diagnosis of a variety of human diseases including many viral (AIDS. the sandwich ELISA for antigen.). hence is called the competitive ELISA. Enzyme activity is generally determined by the change of colour. cysticercosis. the positive reaction is tested by the change of colour. this means that the enzyme-linked antiglobulin was bound. leishmaniasis. then it will combine with the coated antibody. The sandwich ELISA for antigen: This method to detect antigen is very sensitive. If the reaction occurs (for example. plastic tubes. hepatitis. One antibody is conjugated with enzyme.) and fungal (histoplasmosis. which has bound to the plate. the only difference being that an enzyme is used instead of fluorescent dye. Competitive ELISA for antibodies: This involves the use of two specific antibodies. The principle of ELISA in essence is similar to that of the fluorescence. the ELISA can also be classified as 1st. which can be read by spectrophotometer or ELISA reader. rubella.138 LESSON 49 INTRODUCTION Enzyme-linked Immunosorbent Assay LEARNING OBJECTIVES After completing this practical you will be able to: 1 Know the importance of various steps involved in performing enzyme-linked immunosorbent assay (ELISA). Antibody conjugated with enzyme is added to detect the antigen-antibody reactions. competitive ELISA for antibodies.). 2nd and 3rd generation ELISA as used in HIV infections (Box 49-1). and now the antiglobulin will bind to the primary antibodies. The most commonly used enzymes are alkaline phosphatase. etc. The enzyme immunoassay can be broadly of three types: a. Enzymes and substrates used in the ELISA are listed in the table 49-1. where as the other antibody is present in the serum to be tested. b. polyvinyl chloride plates. Textbook of Practical Microbiology 139 The Dot ELISA is a rapid visually read microassay form of the ELISA (Box 49-2). The advantages and disadvantages of ELISA tests are mentioned in the table 49-2. In this experiment indirect ELISA to detect antibodies and sandwich ELISA to detect antigen will be described. 7 Add 100 µl of conjugate to each well and incubate at 37°C for 1 hour. 8 Wash the wells thrice and add 100 µl of substrate solution and incubate for 30 minutes at 37°C in dark. 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 I Equipments Micro titer plate, ELISA reader and ELISA washer. Appropriate controls, such as controls with positive and negative sera, buffer control, conjugate control and substrate control should be tested along with every plate. II Reagents and glass wares Carbonate buffer (pH 9.6), washing buffer (PBS-Tween 20), conjugate, citric acid phosphate buffer (0.1M) pH 5.0, substrate, 4% bovine serum albumin (BSA) in PBS-tween 20 and 3N H2SO4, micro pipettes, tips, glass beakers, and filter paper. 1 Carbonate buffer (pH 9.6) Solution A: 5.3 g sodium carbonate (Na2CO3) is dissolved in one liter of double distilled water. Solution B: 1.2 g sodium bicarbonate (NaHCO3) is dissolved in one litre of double distilled water. Mix 16 ml of solution A with 34 ml of solution B and make volume to 100 ml with distilled water and adjust pH to 9.6. 2 Washing buffer (PBS-Tween 20): Phosphate buffered saline (0.01M) pH 7.2 with 0.05% tween 20. 3 Conjugate: Goat antimouse immunoglobulin conjugated with horse radish peroxidase. 4 Citric acid phosphate buffer (0.1M) pH 5.0: Weigh and dissolve 7.3 g of citric acid and 11.86 g of disodium hydrogen phosphate (Na2HPO4) in one liter double distilled water and adjust pH to 5.0. 5 Substrate: Dissolve 8 mg of Orthophenylene diamine in 15 ml of citrate buffer. Then add and mix 15 ml of Hydrogen peroxide just before to use. OBSERVATIONS Observe the microtiter plate for the development of colour. Observe control (negative, positive and blank) wells. RESULTS AND INTERPRETATION Development of yellow colour indicates the test serum contains antibodies to tested antigen. No colour is negative reaction (Fig. 49-1). SANDWICH ELISA TO DETECT ANTIGEN REQUIREMENTS I Equipments Micro titer plate, ELISA reader and ELISA washer. II Reagents and glass wares Carbonate buffer, blocking solution, washing buffer, monoclonal antibodies (1-10 µg/ml in carbonate buffer) and substrate solution, micropipettes, tips, glass beakers, and filter paper. III Specimen Serum, CSF, tear, etc. PROCEDURE 1 Make up antigen to optimal dilution in carbonate buffer and add 100 µl per well in 96 well microtiter plate and incubate at 37°C for 3 hours or at 4°C overnight. 2 Decant the antigen solution and wash the plates thrice with washing buffer. Remove all residual fluid. 3 Add 100 µl of 4% BSA in washing buffer to each well and incubate at 37°C for 1 hour. 4 Wash thrice with wash buffer and remove all residual fluid. 5 Add 100 ul of diluted serum samples and incubate for 1 hour at 37°C . 6 Wash the wells with washing buffer and remove all residual fluid. III Specimen Serum, CSF, urine etc. PROCEDURE 1 Coat the ELISA plates with monoclonal antibody (mAb) in carbonate buffer by adding 100µl to each well, and incubate overnight at 4°C . 2 Remove monoclonal antibody from ELISA plate and add 100µl of blocking solution. Incubate for 30 minutes at 37°C. Wash the plates thrice with washing buffer. Remove all residual fluid. 3 Add 100µl of 4% BSA in washing buffer to each well and incubate at 37°C for 1 hour. 140 Enzyme-linked Immunosorbent Assay 4 Wash thrice with wash buffer and remove all residual fluid. 5 Add 100µl of diluted serum samples and incubate for 1 hour at 37°C. 6 Wash the wells with washing buffer and remove all residual fluid. 7 Add 100µl of conjugate to each well and incubate at 37°C 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. RESULTS AND INTERPRETATIONS Development of yellow colour indicates the test serum contains antigens to tested antibody. Development of no colour inducates negative reaction. QUALITY CONTROL Appropriate controls, such as controls with positive and negative sera, buffer control, conjugate control and substrate control should be tested along with every plate. OBSERVATIONS Observe the microtiter plate for the development of colour. Observe control (negative, positive and blank) wells. FIGURE 49-1 ELISA test. BOX 49-1 1ST, 2ND AND 3RD GENERATION ELISA 1st generation ELISA: It is an ELISA in which crude antigen is used for the detection of antibodies. 2nd generation ELISA: It is an ELISA in which semi-purified antigen is used for the detection of antibodies. 3rd generation ELISA: It is an ELISA in which recombinant antigens or synthetic peptides are used for the detection of antibodies. BOX 49-2 DOT ELISA Dot ELISA is a rapid visually read microassay. The principle is similar to that of ELISA where enzyme is used as a marker or label to detect the binding of antigen and antibody. The test is performed on a nitrocellulose membrane. Enzyme converts colourless substrate to a coloured product, which indicates the presence of antigen-antibody binding. When the test serum is layered on to the membrane, specific antibodies if present, will bind to corresponding dot of the antigen. Addition of a labeled serum antibody (conjugate) and the subsequent development of the colour allow the detection of the presence of antibodies based on the pattern of the antigen sites. Dot-ELISA can be used for the detection of antibodies as well as antigen. Table49-1 Enzymes and substrates used in the ELISA Enzymes Horseradish peroxidase. Alkaline phosphatase. b-D galactosidase, Glucose 6-phosphate dehydrogenase (G6PD). Acetylcholinesterase. Table 49-2 Advantages and disadvantages of ELISA test Advantages Highly sensitive Specific Rapid Disadvantages Requires specialized expert personnel. Requires expressive laboratory equipment. Enzymes require cold environment for storage. Substrates Hydrogen peroxide (H2O2). Nicotinamide adenine dinucleotide phosphate (NADP+). 2-nitrophenyl-b-D-galactoside. Glucose-6 phosphate. Acetylcholine. Textbook of Practical Microbiology 141 KEY FACTS 1 2 3 4 Preliminary chequer board titrations are carried out for standardizing antigen and antibody concentration. Read all plates at a standard time after addition of the substrate because colour is not stable. Substrate should be made just before use as it decomposes spontaneously and is unstable. 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 UNIT 143 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 methods such as isolation of plasmids, polyacrylamide gel electrophoresis (PAGE), isolation of antibiotic resistant mutants and conjugation in bacterial systems. Genetic information is stored in DNA of the bacteria as a sequence of nucleotide bases (adenine, cytosine, guanine, thymine, abbreviated as A, C, G, T respectively) read sequentially in a 5' to 3' direction (or in RNA, with uracil, abbreviated U, replacing thymine). The most common form of DNA (present in all cellular genomes, as well as many viral genomes) is double stranded. The 5' to 3' polarity of the two strands is opposite, and they are held together by hydrogen bonding between nucleotide base pairs, A to T and G to C. The sense strand carries the coded genetic information. The antisense strand consists of a complementary sequence of bases oriented in the opposite 5' to 3' direction. During DNA replication, the two strands separate and each is used as a template for synthesis of a new complementary strand. This allows genetic information to be replicated with a high level of precision. Because replication is bidirectional, new DNA can only be synthesized in a 5' to 3' direction, the overall pattern of replication is rather complex. Genetic information is transcribed from DNA to RNA, with the antisense strand of the DNA serving as a template for synthesis of RNA with the same base sequence (5' to 3') as the sense strand of the double helical DNA, except that uracil (U) replaces thymine (T). Genetic information contained in messenger RNA (mRNA) is translated into a sequence of amino acids in a polypeptide chain during protein synthesis (translation). A redundant nucleotide triplet code, read 5' to 3' on the mRNA (and on the sense strand of the DNA), specifies the amino acid sequence of the protein, read from N-terminal to C-terminal. The study of genetic materials helps to a). demonstrate mutations, b). study the gene transmission among progeny or generations, c) treat genetic disorders, and d) in diagnosis and research. FREQUENTLY USED TERMINOLOGY IN GENETICS Phenotype: Phenotype refers to “the observable outward appearance of an organism, which is controlled by the genotype and its interaction with the environment”. Genotype: Genotype refers to “the genetic makeup of an individual organism”. Transcription: The “central dogma” of molecular biology describes the transcription of genetic information from a DNA nucleotide triplet code to an RNA triplet code, followed by translation to specific amino acid sequences in protein. Prototrophs: A wild type strain that has minimal requirements for exogenously supplied nutrients is referred to as a prototroph. Auxotrophs: A mutant strain that has lost the ability to synthesize its own supply of a particular nutrient, such as histidine or adenine or thiamine is called an auxotroph. Recombinant DNA technology: Recombinant DNA in this context refers to the creation of a new combination of DNA segments that are not found together naturally. Such technology is now widely used in many practical applications ranging from basic research on control of gene expression to forensic medicine to biotechnology. Restriction endonucleases: An endonuclease is an enzyme that can cleave the phosphodiester bonds of a nucleic acid at an internal site (as opposed to cleavage by an exonuclease, which can only remove nucleotides from one of the ends of a nucleic acid). Example: Eco RI G|AATTC. Hybridization probes: Complementary strands of DNA, RNA, or DNA plus RNA hybridize readily to form double stranded helical structures when placed under suitable annealing conditions. This property is used extensively in molecular genetics to identify specific nucleic acid sequences. A probe consisting of radioactively labeled DNA (or RNA) is hybridized to denatured DNA (or naturally single-stranded RNA) immobilized on a support, such as a nitrocellulose membrane. Hybridization is normally done at a temperature about 25°C below the melting (denaturation) temperature for the DNA. 28.0. often for antibiotic resistance. the plasmid DNA is released from the folded chromosomal complex by a shearing step or by RNase treatment. Preparation of Luria-Bertani medium (LB medium): The medium is prepared by adding the following ingredients: Bactotryptone. 10 g. Plasmids that carry appropriate genes are capable of making bacteria resistant to antibiotics(Box 50-1). 1000 ml. micropipettes. Solution B 0. Eppendorf tubes. chromosomal DNA concentrations are reduced or eliminated. 25 mM Tris chloride (ph 8). The medium contains the following solutions: Solution A 50 mM glucose. II Reagents and lab wares Ethidium bromide solution. Bacto yeast extract. 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.Textbook of Practical Microbiology 145 LESSON 50 INTRODUCTION Isolation of Plasmids LEARNING OBJECTIVES After completing this practical you will be able to: 1 Isolate the plasmid from bacteria. 60 ml. Luria-Bertani medium. They usually contain only a few thousand base pairs of DNA and carry only a few genes.5 ml. PROCEDURE Extraction of plasmid 1 Inoculate a single bacterial colony into 2 ml of LB medium containing antibiotic ampicillin in a 15 ml test tube and incubate over night with vigorous shaking. The plasmid DNA can be resolved as covalently closed circular molecules by centrifugation in ethanol containing ethidium bromide. sodium chloride (NaCl). circular. Just before extraction. 11. such that they can multiply independently within the bacterial cells. 10mM EDTA (pH 8).2 N sodium hydroxide (NaOH). . 10 g. The DNA is freed of RNA and proteins by RNase and protease treatments. and distilled water. Under these conditions. Distilled water. Glacial acetic acid. 1% SDS. PRINCIPLE This method utilizes the molecular characteristics of covalently closed circular deoxyribonucleic acid (DNA) that is released from cells under conditions that denature DNA by using alkaline sodium dodecyl sulfate. 5 g. extra chromosomal DNAs that are capable of autonomous replication within bacterial cells. Preparation of ethidium bromide working solution: The solution is prepared bu adding 10µl ethidium bromide stock solution in 100 ml of agarose solution. III Specimen Bacterial strain: Plasmid containing strain PHSV 106 or equivalent strain is grown in a medium containing 20µg/ml ampicillin. Solution C 5M potassium acetate. Plasmids have their own origins of replication. REQUIREMENTS I Equipments Centrifuge and inoculating chamber. The clarified extract is used directly for electrophoretic analysis.5 ml. The pH is adjusted to 7. and Bunsen burner. Bacterial plasmids are small. which makes it possible to select for bacteria that have taken up the plasmids. 3 Care must be taken while decanting the supernatant after centrifugation. some exotoxins. 5 Run electrophoresis at 50-70 V/gel till the tracking dye reaches the end. such as the tetanus exotoxin and Escherichia coli enterotoxin are also coded for by plasmids. VIVA 1 What are plasmids? 2 What are the properties and functions of plasmids? Ans. 2 Care must be taken at every step of the procedure for extraction of the plasmid DNA and electrophoresis on agarose gel. covalently closed circular DNAs. 8 Precipitate the DNA with two volumes of 100% ethanol at room temperature. found in some Gram-negative bacteria. circular. remove the comb and pour the TE buffer until it fully covers the gel surface. RESULTS AND INTERPRETATION Plasmid appears as a sharp orange band. Observe the bands of molecular weight marker. 4 Load the wells with 5 µl plasmid preparation and 2 µl bromophenol blue tracking dye and a molecular weight marker in other well. Mix well and allow it to stand for 2 minutes. Through a process called conjugation. making them also multiple antibiotics resistant and able to produce a conjugation pilus. Undigested RNA appears as hazy opaque area along the lanes. Plasmids that carry appropriate genes are capable of making bacteria resistant to antibiotics. 4 Add 200 µl of solution B and mix the contents by inverting the tube rapidly several times. extra chromosomal DNA that is capable of autonomous replication within bacterial cells. often for antibiotic resistance. 000 g for 2 min at 4°C and discard the supernatant. the conjugation pilus enables the bacterium to transfer a copy of the R-plasmids to other bacteria. which makes it possible to select for bacteria that have taken up the plasmids. 10 Resuspend the pellet in 50 µl of TE (pH 8) containing RNAse (20 µg/ml). 7 Centrifuge at 12. 6 Remove the gel carefully and place on UV transilluminator and examine for the bands. 000 g for 5 minutes at 4°C and transfer the supernatant to a fresh tube. 3 Resuspend the pellet in 100 µl of ice-cold solution A and vortex vigorously. 3 Place the gel in electrophoresis tank. R-plasmids. 11 Run in agarose gel containing ethidium bromide and view under UV transilluminator. Plasmids code for synthesis of a few proteins not coded for by the nucleoid. 6 Store the tube in ice for 5 minutes.146 Isolation of Plasmids 2 Take 1. 2 Pour 20 ml -25 ml of molten agarose in the gel chamber after placing the comb and allow it to set. 5 Add 150 µl of ice-cold solution C and mix the contents by inverting the tube. 9 Centrifuge at 12000 g for 5 minutes at 4°C and remove supernatant. BOX 50-1 ROLE OF PLASMIDS IN DRUG RESISTANCE Bacterial plasmids. often have genes coding for both production of a conjugation pilus and multiple antibiotic resistance. KEY FACTS 1 Plasmid is small. 3 How do you extract plasmids from bacteria? . In addition. Molecular weight marker must be used in the analysis. QUALITY CONTROL A parallel procedure should be carried out using standard strain of bacteria. For example. They usually contain only a few thousand base pairs of DNA and carry only a few genes. OBSERVATIONS Observe the stained gel under UV illumination for DNA band.5 ml of culture into a Microfuge tube and centrifuge at 12. have their own origins of replication and are capable of autonomous replication. Electrophoresis on agarose gel 1 Prepare 1% agarose in 1 X TE buffer. melt in a boiling water bath and add 1 µl of ethidium bromide. pp.. Balows A.Textbook of Practical Microbiology 147 FURTHER READINGS 1 Collier L. London. Microbiological Methods. Lyne PM and Grange JM. Systemic Bacteriology. . 1996. 2002. Topley and Wilson’s Microbiology and Microbial Infections. 9th Edition. 3 Mackie and McCartney. 921. Volume 2. 1995. Butterworths. Sussman M. pp 1501. pp 94-96. 2 Collins CH. 14th Edition. Practical Medical Microbiology. Churchill Livingstone. Arnold publishers. pp. 8) 4ml 10% SDS 21ml distilled water 4 Stacking gel buffer (4x) 50ml 1M Tris-HCl (pH 6.8) 4ml 10% SDS 46ml distilled water : 100ml. II Reagents and lab wares Micropipette or Hamilton syringes (50µl and 100µl). 5 10% Ammonium persulfate (APS) : 5ml.5M. : 0. 2 Assemble the electrophoresis apparatus and power pack.5M. N’-tetramethylene-ethylenediamine (TEMED). boiling water bath. : 1. : 0. Other types of electrophoresis in gels are listed in the table 51-1.8) : 100ml. Add distilled water to make 100ml and stir until completely dissolved. REQUIREMENTS I Equipments Minigel/maxigel apparatus. Bisacrylamide : 0. 3 10 %(w/v) SDS (store at room temperature). N’. 500mA). (solution filtered after preparation to remove aggregated dye).5g APS dissolved in 5ml distilled water. 3 Separating gel buffer (4x) 75ml 2M Tris-Hcl (pH 8. power supply (capacity 200V. . Their rate of migration depends on the strength of the field. and so on. 1 Stock solutions 1 2M Tris-Hcl (pH 8. microcentrifuge and rocking/ rotatory shaker. size and shape of the molecules and also on the ionic strength. on the net charge. Few examples include analysis of antigen (protein) fractions from different sources for immunological and molecular biological studies. Advantages and disadvantages of PAGE are mentioned in the box 51-1.and SDS binds to proteins fairly specifically.2g. hence SDS confers a negative charge to the polypeptide in proportion to its length.4%. dithiothreitol. Acrylamide : 29.148 LESSON 51 INTRODUCTION Polyacrylamide Gel Electrophoresis LEARNING OBJECTIVES After completing this practical you will be able to: 1 Prepare agarose gel.4%. 6 N. 8 Glycine. 2 Working solutions Acrylamide stock (30%) :100ml.8): 100ml. 2 1M Tris-Hcl (pH 6. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is used in a wide variety of applications for characterizing proteins. taxonomical applications by analysis of whole cell /cell envelope protein of the organism. : 100ml(Table 51-2). by separating and characterizing based on the molecular weight of the protein. PRINCIPLE Electrophoresis is the migration of charged molecules in solution in response to an electric field. N. 0. 7 2-mercaptoethanol or. Sodium dodecyl sulphate (SDS) is an anionic detergent which denatures proteins by “wrapping around” the polypeptide backbone . 4 50% (v/v) glycerol: 100ml. 5 1 %(w/v) bromophenol blue-10ml. : 0. The disulphide bridges in proteins are reduced with 2-mercaptoethanol.8g. 3 Analyse the protein profile from the desired source. Polyacrylamide acts as a support matrix for running the sample. viscosity and temperature of the medium in which the molecules are moving. 1%. 450ml methanol.4mM. 13 Run the electrophoresis at a constant current of 20mA till the dye front reaches the separating gel and then increases it to 25mA. Then remove the comb carefully and rinse the wells with distilled water. 4 The bands can also be transferred by blotting and confirmed by coupling with appropriate antisera containing complimentary antibodies for the antigen (electro-immuno transfer blot). 10 Now remove the casted gel plate from the gel casting stand/ clamps and detach the bottom spacer. melted wax on all three sides leaving the topside so that the assembly is leak proof. 8 Prepare the stacking gel and cast over the separating gel as mentioned above and insert the comb. 3 Seal the glass plate assembly with 2% agar or. 1g SDS : 0. 12 Switch off power supply and load the wells with your protein samples (10-50µl) diluted in sample buffer along with a molecular weight marker (5-10µl) using a micropipette/ syringe.9ml distilled water 8 Staining solution 1. : 1000ml. 14. : 25%.0g Coomassie blue R-250. 7 Overlay the acrylamide with n-butanol.10 minutes depending on the nature of the sample. 2 Assemble the clean glass plates by placing the spacerstwo on either sides.5ml 2-mercaptoethanol 1ml 1% Bromophenol blue 0. which helps to keep the gel surface flat.Textbook of Practical Microbiology 149 6 Electrophoresis / Running Buffer (1x) : 1000ml. RESULTS AND INTERPRETATION 1 The protein bands separated according to the molecular weight and charge are visualized directly.8) 5ml 50% glycerol 2ml 10% SDS 0. Place the gel assembly into electrophoresis chamber with the notched plates facing inside and fill the upper and lower tanks with running buffer. Northern blotting. 15 The gel may be stained using the Coomassie staining solution for 1-2hrs and then destained using the destaining solution for overnight in a rocking shaker. After polymerization remove the butanol and rinse with water. 6 Add TEMED finally to the mixture and pour immediately between glass plates upto 2cm below the notch and allow the gel to polymerize for 30-60 minutes at room temperature.6ml 1M Tris-HCl (pH 6. Observe the control protein bands.1%. Observe and analyze the test protein bands. 14 Now remove the side spacers from the glass plates and carefully scoop the gel using a spatula. 4 Prepare and cast the separating gel using a small beaker. An alternate silver staining procedure too can be followed who is sensitive but a little expensive. Southern blotting. 800ml distilled water. using a syringe. 3g Tris : 25mM. : 1000ml. : 10ml. The gel may be stained and destained for visualization or. OBSERVATIONS PROCEDURE 1 Mix protein sample (20µl) with sample buffer (5µl) in an Eppendorf tube and heat it at 75-100°C for 2. Table51-1 List of other types of electrophoresis in gels Electroimmunodiffusion. . 11 Connect the electrodes to the power pack and make prerun at a constant current of 20mA for 10-15minutes. Counterimmunoelectrophoresis. : 60mM. or one along the bottom edge and fix the whole assembly tightly with clamps or gel casting stand. : 2%. 5 Mix the components of the separating gel mixture without TEMED and de aerate. 100ml glacial acetic acid. 2 Check out for the desired bands by comparing with a standard marker. 9 Destaining solution 100ml methanol. used as such in blotting experiments without staining. 450ml distilled w.Stop the run when the dye front reaches the bottom of the gel. III Specimen Protein suspension to be separated. Note: Remove air bubbles in the wells if any. : 0. 9 Allow to polymerize for 15-30minutes. QUALITY CONTROL Known protein ladder is loaded in each run. Distilled water to make 1000ml 7 Sample buffer 0. 3 Interpret the desired band based on experimental and literature analysis by either visually or using a gel documentation system with appropriate software. : 14.4g glycine : 192mM.ater 100ml glacial acetic acid. Rocket electrophoresis. d) in purification of antigen (protein) fractions.2ml.: whether 7. 3 Write a brief note on electro immuno transfer blot.SDS-PAGE can be used a) in a wide variety of applications for characterizing proteins. from different sources for immunological and molecular biological studies. 3 Transfer of the separated polypeptides to membrane support. Acrylamide stock x /3 ml Stacking / separating gel buffer 2. EITB combines the resolution of gel electrophoresis with the specificity of immunochemical detection. i. 10 ml. also known as Western blot is an immunological technique. Protein bands are stable and can be stained for preservation. 50µl. 5% Stacking gel Acrylamide stock Distilled water 10% APS TEMED Total volume 1.5ml Distilled water (7. Highly sensitive.150 Polyacrylamide Gel Electrophoresis Table 51-2 Calculation for X % separating or. 5µl.5ml BOX 51-1 ADVANTAGES AND DISADVANTAGES OF PAGE Advantages Efficient tool for characterizing proteins. 12.9ml. depends on ones experimental need and the desired quantity of stock solutions to cast the gel are calculated using the following formula.e. 5µl. c) in analysis of antigen (protein) fractions. The EITB technique involves six steps: 1 Preparation of antigen sample. Very expensive. which is used to detect a protein immobilized on a matrix.3 ml. 5 Addition of antibody.6 ml. Electro-immuno transfer blot (EITB). stacking gel The gel concentration percentage. 50µl. 4 What is the action of sodium dodecyl sulphate on proteins? . and e) in taxonomical applications by analysis of whole cell /cell envelope protein of the organism and categorizing accordingly. 5. Prior to immunoblot. Trained professional and well equipped laboratory is required. Rapid method. Ans.5-x /3) ml 10% APS 50µl TEMED 5µl (10µl if x < 8%) Total volume 10 ml 10% Separation gel Acrylamide stock Distilled water 10% APS TEMED Total volume 3. 4.5ml Stacking gel buffer 2. the protein of interest from a complex mixture is separated based on their molecular weight by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE). 10 ml. The number of different antigens can be readily observed.5 or 15 %. b) in the diagnosis of infectious diseases.5. Disadvantages Chemicals are neurotoxic. The limit of detection by EITB is of the order 10 picogram with horse radish peroxidase labeling. Since the loading capacities of the gels are limited. an antigen cannot be detected when its concentration falls below 1 ng/sample. 4 Blocking of nonspecific binding sites on the membrane. 10. 6 Detection. Separating gel buffer 2. VIVA 1 What is the principle of electrophoresis? 2 What are the applications of PAGE in microbiology? Ans. 2 Resolution of the sample by gel electrophoresis. Microbiological Methods. London. pp 1501. 14th Edition. 921. 9th Edition. 2 Collins CH. Balows A. pp. Volume 2. pp. 1995. Butterworths. Churchill Livingstone. 1996. Always freshly prepared buffers and reagents should be used. Arnold publishers. Systemic Bacteriology.. Observe for complete circuit of electricity (use sufficient levels of buffer) throughout the procedure. . pp 94-96. FURTHER READINGS 1 Collier L. 3 Mackie and McCartney. Topley and Wilson’s Microbiology and Microbial Infections. Lyne PM and Grange JM. Sussman M. Avoid introducing air bubbles in to the gel. 2002. Practical Medical Microbiology.Textbook of Practical Microbiology 151 KEY FACTS 1 2 3 4 Care must be taken for spillage or direct exposure to reagent while casting a gel. However. Hence. micropipettes. including changes that affect expression of genes without altering their coding sequences and changes that do not cause any detectable phenotypic difference (silent mutations). Survival and stability of each species is dependent on faithful replication of genetic information for use by each new generation. mutational changes provide the ability for species to adapt to changing conditions and challenges. II Reagents and lab wares Eppendorf tubes. 5 Add 0. two 10 ml trypticase soy agar in 20 ml test tubes and streptomycin solution (10 mg per 100 ml distilled water). as well as the development of biological diversity (Box 52-3). PRINCIPLE After inoculation of the prototrophic test culture in to trypticase soy agar medium containing streptomycin by spread plate . 3 Add 0. MullerHinton agar). mutation can occur at many different structural levels and can be classified in many different ways. a low level of mutational change is highly desirable. REQUIREMENTS I Equipments Micro centrifuge. 2 Place a glass rod under one end of a sterile Petri dish.2 ml of E. to be tested for antibiotic resistance is used as specimen. coli test culture with a sterile pipette and spread the culture with a sterile bent rod on entire agar surface. PROCEDURE 1 Melt two trypticase soy agar tubes in a water bath and cool to 45°C. The importance of mutation Genes are stable repositories of the information needed for synthesis of all of the RNA and proteins in a living organism. pour the molten agar until it cover the entire bottom surface and allow to solidify in the same position. Different types of mutations and their role in microbial infections are summarized in the table 52-1. Over an extended period of time. colonies appearing in a region of high streptomycin concentration are indicative of streptomycin-resistant mutants. Mutation is any change in genetic information relative to a reference “wild-type” genome. III Specimen Pure growth of Escherichia coli from solid media preferably from non-blood agar plates (Examples: nutrient agar.152 LESSON 52 INTRODUCTION Isolation of Antibiotic Resistant Mutant LEARNING OBJECTIVES After completing this practical you will be able to: 1 Isolate the antibiotic resistant mutants from a prototrophic bacterial population.Mechanisms of mutations are many (Box 52-2). mutational changes serve as the raw material for selective survival and the evolution of more advanced and efficient species. 4 Place the dish in horizontal position and pour the molten agar medium containing streptomycin medium until it fully covers the gradient agar layer and allow to solidify. Mutations are mainly classified into two types: Point mutations and large-scale mutations (Box 52-1). In a complex organism. method.1 ml of the streptomycin solution to a second tube of molten trypticase soy agar tube using a sterile pipette and mix gently. including deletions. Missense mutations Most base pair substitutions change the amino acid specified by the codon in which they occur. base pair substitutions generate a different codon for the same amino acid. missense mutations may have a variety of effects. UGA) generated by reading the coding sequence out-of-frame. . which are a special subclass of point mutations. However. which is often prematurely truncated by stop codons (UAG. even at the level of protein amino acid sequence. Such mutations. 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. 1 Presence of colonies after first incubation indicates that the strain is resistant to streptomycin. RESULTS AND INTERPRETATION OBSERVATIONS Observe the colonies after first and second incubation steps. are referred to as frameshift mutations. Point mutation can result in missense (amino acid substitution). 8 Incubate the plate for 24-48 hours at 37°C. with no biological effect whatsoever. or frameshift (either positive or negative).Textbook of Practical Microbiology 153 6 Incubate the plate in an inverted position for 48 hours at 37°C. Addition of one base pair (or loss of two base pairs) shifts the reading frame behind by one base. 2 Sensitive strains do not produce colonies after first incubation. Depending on the nature of the amino acid substitution and its location within the protein. nonsense (insertion of a stop codon). translocations from one chromosome to another. because they have no phenotypic effect. 3 Presence of colonies after second incubation denotes the resistant strains. Large scale mutations Mutations that involve larger changes in chromosomes. and extra or missing chromosomes are large-scale mutations. and thus result in a completely different amino acid sequence. duplications. inversions. Silent mutations In some cases. Such mutations are described as missense mutations because they cause an amino acid substitution in the coded protein. and is often referred to as a positive frameshift. BOX 52-1 POINT MUTATIONS AND LARGER SCALE MUTATIONS In classical genetics. 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. they are called silent mutations. 7 Select one or two isolated colonies from the middle of the streptomycin concentration gradient with a sterile loop and streak toward the high concentration end of the plate. 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. Table 52-1 Different types of mutations and their role in microbial infections Type of mutation Point mutations Large scale mutations Role in microbial infections Avian sarcoma/leukemia virus HIV virus Leishmania donovani Neisseria gonorrhoea MRSA Gyrase risistance gene in Salmonella Typhi QUALITY CONTROL The parent strains (streptomycin sensitive strains) are included as controls. Deletion of a single base pair results in moving ahead one base in all of the downstream codons. 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 is often referred to as a negative frameshift. UAA. ranging from complete loss of biological activity to reduced activity or temperature-sensitive activity or no functional effect at all. resulting in a transition. Transitions A tautomeric shift in any of the four DNA bases can lead to mispairing of A to C or G to T. Volume 2. tautomerization of one of the bases and rotation of the other to yield a purine: purine pairing. During the next round of DNA synthesis. proton shifts can convert the amino groups in adenine and cytosine to imino groups. as well as the development of biological diversity. VIVA 1 Define and classify mutations. Over an extended period of time. 2002. However. and transversions. London. 2 Sufficiently cool the molten agar before adding antibiotic solution. 14th Edition. and the keto groups in guanine and thymine to enol groups. two events are thought to be involved. Churchill Livingstone. transitions. Balows A. mutational changes provide the ability for species to adapt to changing conditions and challenges. a low level of mutational change is highly desirable. 5 What are different types of mutations and their role in microbial infections? FURTHER READING 1 Collier L. Arnold publishers. 3 Presence of colonies after second incubation denotes the resistant strains. Survival and stability of each species is dependent on faithful replication of genetic information for use by each new generation. The tautomeric state can occur either in the template base or the incoming base. in which an AT base pair replaces a GC or a GC replaces an AT. These changes. which is lower than that of transitions. Butterworths. pp 1501. involve momentary expression of rare alternative molecular configurations that exist in equilibrium with the more common forms. Practical Medical Microbiology. and thus serve as the raw material for selective survival and the evolution of more advanced and efficient species.154 Isolation of Antibiotic Resistant Mutant BOX 52-2 MECHANISMS OF MUTATION Tautomerization Spontaneous mutations that involve base pair substitutions are caused primarily by configurational changes within the individual bases that result in mispairing. 1996. A second possible mechanism for transversions is the formation of an apurinic site. 2 Collins CH. The frequency of spontaneous transversions. 9th Edition. Systemic Bacteriology. Lyne PM and Grange JM. appears to be consistent with this interpretation. the mispaired base will pair with its normal partner. 2 Write a short note on point mutations. 921. which can result in replacement of the original purine with any of the four bases. pp. 3 What are the mechanisms of mutations? Ans. Transversions To achieve a transversion. BOX 52-3 THE IMPORTANCE OF MUTATION Genes are stable repositories of the information needed for synthesis of all of the RNA and proteins in a living organism. pp 94-96. Tautomerization. 1995. pp. Transitions are the most common type of mutation resulting from spontaneous mispairing due to tautomerization. Microbiological Methods. in which the positions of purine and pyrimidine are reversed in the DNA double helix. Specifically. . Sussman M. which are called tautomeric shifts. KEY FACTS 1 Properly mix streptomycin solution to molten trypticase soy agar. Topley and Wilson’s Microbiology and Microbial Infections. with no change in the purine: pyrimidine polarity of the base pair. 4 Explain the mechanism of isolation of antibiotic resistant mutant strains. 3 Mackie and McCartney. it is due to random chromosomal integration of the F factor. Thiamine acts as a nutrient supplement to thiamine negative recombinant cells. PRINCIPLE After inoculation and incubation of F– and Hfr strains together in a minimal growth medium containing thiamine and streptomycin. During conjugation of an F + cell with an F –. The bacterial genes carried on the F’ plasmid are easily transmitted into F– cells by conjugation. Hfr strains In certain F+ strains. . 3 Incubate for 30 min at 37°C. and streptomycin sensitive Hfr E. coli cultures into a sterile test tube. if it does happen. and test tubes. Donor strains contain an additional genetic element. 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. F’ factors and sexduction Sometimes an integrated F+ factor from an Hfr strain will escape from the bacterial chromosome carrying a few chromosomal genes with it in its circular plasmid DNA. recombinant strains of the bacteria only grow.3 ml of Hfr E. the F factor has become integrated into the bacterial chromosome. Bacterial conjugation can be viewed as a primitive form of sex. However. III Specimen F– E. Parental F– strains cannot grow due to the lack of threonine and leucine. sterile pipettes.Textbook of Practical Microbiology 155 LESSON 53 INTRODUCTION Bacterial Conjugation LEARNING OBJECTIVES After completing this practical you will be able to: 1 Demonstrate genetic recombination in bacteria by conjugation. PROCEDURE 1 Take nutrient agar medium. coli culture. in which a cell from a donor strain injects DNA into a recipient cell. 2 Add 1 ml of F– E. Such a plasmid is called an F-prime (F’) factor. 4 Vigorously agitate the mixed culture to terminate the genetic transfer. When this happens. Mechanisms of DNA transfer are highlighted in the Box 53-1. coli strain. 24 hour nutrient broth cultures of streptomycin resistant F– Escherichia coli strain which require threonine. Streptomycin inhibits the growth of parental streptomycin sensitive Hfr strains. the frequency of transformation for any genes other than the F factor is very low. called the fertility factor (F). REQUIREMENTS I Equipments Incubator. Differences between F+ strain and Hfr strain are listed in the Table 53-1. leucine and thiamine. Cells that are receptive to conjugation lack the F factor and are sometimes designated F –. coli culture and 0. usually in the form of a plasmid. where it can undergo recombination and become part of the recipient’s genome. Bacterial strains with integrated F factors are referred to as Hfr strains (“high frequency of recombination”). nutrient agar plates and 95% ethanol. II Reagents and glass wares Bend glass rod. threonine and leucine. Table53-1 Differences between F+ strain and Hfr strain F+ strain 1. BOX 53-1 MECHANISMS OF DNA TRANSFER The DNA transfer that occurs in conjugation begins as a single strand break in the donor chromosome (or plasmid). QUALITY CONTROL Prepare control plates of parental Hfr and F– and aseptically add 0. F factor has become integrated into the bacterial chromosome. There are two classes of transduction. 2. KEY FACTS 1 Young cultures of bacterial strains must be used.1 ml of each strain to its agar plate and spread with a bent gloss rod. 3 Avoid contamination. RESULTS AND INTERPRETATION Presence of colonies in the test medium indicates the transfer of genetic material. In specialized transduction. 2 Mix the culture properly to terminate the genetic transfer. Transduction Transduction refers to a genetic exchange in which bacteriophages carry bacterial genes from one bacteria cell to another. with only one strand transferred through the F– pilus to the recipient cell. Low frequency of recombination. 3.1 ml of the mixed culture to a minimal growth medium containing streptomycin and thiamin and spread over the entire surface agar surface. 3. As a result of receiving new DNA from the donor. In generalized transduction. The single strand left behind in the donor and the one that is transferred to the recipient are both converted into double strands. OBSERVATIONS Observe the culture plates for colonies. a lysogenic phage undergoes recombination with the host genome and later when it is excised to become an independent phage genome.156 Bacterial Conjugation 5 Add 0. 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. . thereby giving the bacterial cell new genetic properties. Hfr strain 1. the recipient is temporarily partially diploid. restoring the donor chromosome and generating double stranded donor DNA in the recipient. fragments of host DNA are mistakenly packaged into a bacteriophage in place of its own DNA. Any DNA that is not integrated is soon destroyed. which can be transduced into a new host cell and recombined into the genome of that cell. Transformation Transformation refers to the ability of extracellular DNA to enter a bacterial cell and recombine with the bacterial genome. Only F factor transfer occurs in conjugation. 2. 6 Incubate the plates for 48 hours at 37°C. it carries one or more host genes with it. High frequency of recombination. Recombination can then integrate parts of the transferred DNA into the recipient genome. specialized and generalized. F factor usually present in the form of a plasmid. Differentiate between F+ and Hfr strains. Arnold publishers.Textbook of Practical Microbiology 157 VIVA Define bacterial recombination. Systemic Bacteriology. Volume 2. London. This strand passes in to the recipient cell. The F1 factor is formed by the excision of the F factor from the chromosome along with a segment of the chromosome. Microbiological Methods. pp 94-96. often for antibiotic resistance. pp. . Balows A. Sussman M. pp. and e Production of recombinant strains of bacteria with required characteristics. During conjugation there is a break in one of the two strands of the plasmid. Discuss the role of conjugation in acquiring drug resistance. 5. The release of F factor occurs by breakage and reunion at a point different from the point of plasmid insertion. 9th Edition. pp 1501. Practical Medical Microbiology. 1996. b Production of recombinant vaccine proteins. Donor cell retains one strand. 3 Mackie and McCartney. What are the methods of transfer of genetic material in bacteria? How do you demonstrate bacterial conjugation? What are the applications of bacterial recombination? Ans: Applications of bacterial recombination include: a Production of recombinant antigens for diagnosis. c Gene therapy. 14th Edition. Ans: Plasmids carry only a few genes. 6. 2 Collins CH. The plasmid is infectious and can transfer itself by conjugation. 1995. 1 2 3 4 FURTHER READINGS 1 Collier L. The plasmid (F factor) is present as an independent double stranded ring of DNA distinct from chromosome. This results in an interchange of plasmid and chromosomal DNA segments. Butterworths. Plasmids that carry appropriate genes are capable of making bacteria resistant to antibiotics. d Transfer of antibiotic resistance. 921. 2002. Topley and Wilson’s Microbiology and Microbial Infections. Churchill Livingstone. Donor and recipient cells synthesize complimentary strands to form intact duplexes and recipient cell becomes a potential donor cell. Lyne PM and Grange JM. 158 . Textbook of Practical Microbiology UNIT 159 VIII Bacteriology Lesson 54 Normal Microbial Flora of the Mouth Lesson 55 Normal Microbial Flora of the Throat Lesson 56 Normal Microbial Flora of the Skin Lesson 57 Identification of Staphylococcus aureus Lesson 58 Identification of Streptococcus pneumoniae Lesson 59 Identification of b-haemolytic Streptococci Lesson 60 Identification of Corynebacterium diphtheriae Lesson 61 Identification of Lactose Fermenting Enterobacteriaceae Lesson 62 Identification of Vibrio cholerae Lesson 63 Identification of Pseudomonas aeruginosa . two blood agar and chocolate agar plates. coliform bacilli. Staphylococcus. Observe and record morphology of the bacteria. the mouth contains usually the organisms that are present in mother’s vagina (e. The colonies grown on these plates are studied further for their identification. III Specimen Saliva. Döderlein’s bacilli. Gram stain reagents (methyl violet. normal oral and nasopharyngeal flora appear. PROCEDURE 1 Collect the saliva into a sterile sample collection bottle. Some of these organisms can cause opportunistic infections. 3 Record your observations. 2 Observe all the colonies on both plates. which inhabit the skin and mucous membrane of the normal human body. These are Streptococcus viridans. Fusiform species.). 95% alcohol and dilute carbol fuchsin). 2 Incubate uninoculated blood agar and chocolate agar plates along with the inoculated ones. Normal flora usually denotes to microorganisms. nonpathogenic Neisseria. anaerobic spirochaetes.160 LESSON 54 INTRODUCTION Normal Microbial Flora of the Mouth LEARNING OBJECTIVES After completing this practical you will be able to: 1 Demonstrate various microorganisms which are present as part of the normal flora in the mouth. Within a few hours of birth. There are many microorganisms which are present as part of the normal flora of the mouth and teeth. inoculation loop. filter paper containing 1% tetramethyl paraphenylene diamine dihydrochloride. micrococci. The normal microbial flora offers many benefits to the human (Box 54-1). pencil. Many of these bacteria can be demonstrated in the saliva. PRINCIPLE Saliva is collected from the mouth and inoculated onto the surface of the agar plates. etc. as they acquire these organisms during their birth through the vaginal canal. 2 Inoculate the saliva onto blood agar and chocolate agar plates. QUALITY CONTROL 1 Test all agar plates for sterility before inoculation. . streptococci. 3 Incubate the chocolate agar plate in CO2 incubator for 48 hours at 37°C. and burner. iodine. glass slides. Streptococcus mutans. II Reagents and lab wares Petri dishes. OBSERVATIONS 1 Look for the presence of haemolysis on blood agar plate by viewing the plate under transmitted light. REQUIREMENTS I Equipments Microscope and CO2 incubator. etc. In infant at the time of birth. 4 Perform Gram stain. 4 Incubate one blood agar plate aerobically for 48 hours at 37°C. sample collection bottle. These organisms however diminish in number during first week after birth and are replaced by the normal bacterial flora present in the mouth of the mother.g.. 23rd Edition. (The CV Mosby Company.Textbook of Practical Microbiology 161 RESULTS AND INTERPRETATION 1 On blood agar streptococci produce pinpoint a-hemolytic colonies where as staphylococci produce pinhead colonies with b . Neisseria are oxidase positive. Louis) 2002. 14th Edition. Murray PR. VIVA 1 What are the microorganisms present in the mouth as normal flora? FURTHER READINGS 1 2 3 4 5 Collins CH. Melnick and Adelberg. 94-96. 2003. Rosenthal KS. Butterworths. they are Gram positive cocci arranged in chains. Microbiological Methods. 2002. Kobayashi GS and Pfaller ME. Staphylococci are arranged in clusters. Churchill Livingstone. St. 3 On chocolate agar. colonies grown are tested for the presence of the enzyme oxidase (refer chapter 21). Medical Microbiology. 2 Colicins produced by some bacteria lyse some pathogenic bacteria. Mackie and McCartney. 1996. Sahm DF and Weissfeld AS. London. KEY FACTS 1 There are many microorganisms which are present as part of the normal flora in the mouth and teeth. They turn the oxidase paper into deep purple in color when they are streaked on the surface of the filter paper. Lyne PM and Grange JM. Forbes BA. streptococci are identified based on the arrangement. Jawetz.haemolysis. Mosby Inc. BOX 54-1 BENEFICIAL EFFECTS OF NORMAL FLORA 1 They suppress the colonization of the body by pathogens. 3 Antibodies produced against some commensals show cross reaction with some pathogens. 3 Saliva sample should be processed immediately after collection. 2 Saliva sample should be collected in a sterile bottle. 2 On Gram stain. Medical Microbiology. thereby enhancing immune status of the host. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. McGaw Hill. Practical Medical Microbiology. 5th Edition. 4 Endotoxins produced by some bacteria facilitate complement mediated defence system of the humans. 1995. . 95% alcohol and dilute control fuchsin). 3 Look for the presence of black colored colonies on potassium tellurite agar. 1 Look for the presence of hemolysis on blood agar plate by viewing the plate under transmitted light. III Specimen Throat swab. 2 On Gram stain. 4 Incubate the chocolate agar plate in CO2 incubator for 48 hours at 37°C. they are Gram positive cocci arranged in chains. PROCEDURE 1 Place a tongue depressor on the extended tongue and collect the specimen by using a sterile cotton swab. These are a -hemolytic streptococci. chocolate agar and potassium tellurite agar plates.162 LESSON 55 INTRODUCTION Normal Microbial Flora of the Throat LEARNING OBJECTIVES After completing this practical you will be able to: 1 Demonstrate various microorganisms which are present as part of the normal flora in the throat and respiratory tract. Many microorganisms are present in the throat and upper respiratory tract. 2 Emulsify the throat swab in sterile saline and mix to form a uniform suspension. Neisseria. two blood agar plates. REQUIREMENTS I Equipments Microscope and CO2 incubator. . a. Staphylococci are arranged in clusters. diphtheroids and spirochaetes. Gram staining reagents (methyl violet. tongue depressor. They continue to remain as part of important microbial flora through out life. 5 Perform Gram stain. Iodine. sterile cotton swabs. Branhamella. Streptococcus pneumoniae. by rotating the cotton swab gently over pharyngeal tonsils. streptococci produce pinpoint a-hemolytic colonies where as staphylococci produce pinhead colonies with b-hemolysis. The colonies grown on these plates are studied further for their identification. 3 Inoculate the specimen onto blood agar. one potassium tellurite agar plate. OBSERVATIONS PRINCIPLE Specimen taken from the throat is inoculated onto the surface of the media plates. streptococci are identified based on the arrangement. RESULTS AND INTERPRETATIONS 1 On blood agar. glass slides and pencil. II Reagents and lab wares Bunsen burner. 5 Incubate one blood agar plate and potassium tellurite agar plates aerobically for 48 hours at 37°C. and one 5ml sterile saline tube. Observe and record morphology of the bacteria. The microbial flora present in the pharynx and trachea are similar to that present in the mouth. 4 Record your observations. Staphylococcus.haemolytic streptococci are present in the upper respiratory tract within 12 hours of the birth. 2 Perform oxidase test for the colonies grown on chocolate agar. Haemophilus . filter paper containing 1% tetramethyl paraphenyline diamine dihydrochloride. Louis) 2002. 1995. St. Mackie and McCartney. 4 Diphtheroids when grown on potassium tellurite agar produce pinpoint black colored colonies. colonies grown are tested for the presence of the enzyme oxidase (refer chapter 21). 1996. Bailey and Scott’s Diagnostic Microbiology. 3 Throat swab should be processed immediately after collection. Medical Microbiology. Some organisms also form symbiotic association with the host. Murray PR. McGaw Hill. Practical Medical Microbiology. the normal microbial flora of human is helpful? Ans. (The CV Mosby Company. Medical Microbiology. Oxidase negative bacteria do not produce color when the colonies are streaked on oxidase paper. Mosby Inc. Rosenthal KS. 11 th ed. 23rd Edition. London. Churchill Livingstone. Sahm DF and Weissfeld AS. 2 Throat swab should be collected in a sterile bottle. KEY FACTS 1 There are many microorganisms which are present as part of the normal flora in the throat and upper respiratory tract. Melnick and Adelberg. 5th Edition. 14th Edition. 94-96. Kobayashi GS and Pfaller ME. Lyne PM and Grange JM. Neisseria are oxidase positive. 2003.Textbook of Practical Microbiology 163 3 On chocolate agar. Forbes BA. Microbiological Methods. They turn the oxidase paper into deep purple in color when they are streaked on the surface of the filter paper. VIVA 1 In which respect. Butterworths. . 2002. The normal microbial flora of human can reduce the infections by competing with pathogens for nutrients and binding receptors. FURTHER READINGS 1 2 3 4 5 Collins CH. Jawetz. diphtheroids. 1 Look for the presence of hemolysis on blood agar by viewing the plates under transmitted light. 3 Record your observations. The microbial flora present in the pharynx and trachea are similar to that present in the mouth. Sabouraud’s RESULTS AND INTERPRETATION 1 Perform Gram stain of the colonies. II Reagents Bunsen burner. The colonies are studied further for their identification. normally harbours many bacteria. dextrose agar plate. 2 Perform LPCB stained smears of colonies grown on Sabouraud’s dextrose agar. mannitol salt agar plate. 2 Place the swab in a tube containing sterile saline and mix it. sterile cotton swabs. VIVA 1 What is the role of resident flora of skin? . Staphylococcus epidermidis and diphtheroids are predominant bacteria. Candida species. Streptococcus viridans. etc. aureus. REQUIREMENTS I Equipments Microscope and incubator. micrococci. Skin as part of its normal flora. OBSERVATIONS PRINCIPLE Swab is collected from surface of the skin and is inoculated on agar plates and incubated. It contains 102 to 104 organisms per cm2. peptostreptococci. glass slides and pencil. 3 Inoculate a loopful suspension on one plate each of blood agar. Malasezia furfur . are the other microorganisms. 3 Look for the presence of yellow color of the medium surrounding colonies grown on mannitol salt agar medium. mannitol salt agar and Sabouraud’s dextrose agar 4 Incubate the Sabouraud’s agar plate for 48 hours at 25°C and the remaining plates for 48 hours at 37°C. lactophenol cotton blue and reagents for Gram stain. enterococci. Yellow color colony is suggestive of S. blood agar plate. 2 Look for the presence of mold like or moist growth on Sabouraud’s agar plate. b -haemolytic streptococci. PROCEDURE 1 Collect the swab from surface of the skin by rubbing the cotton swab after moistening the swab in sterile saline. propionibacterium.164 LESSON 56 INTRODUCTION Normal Microbial Flora of the Skin LEARNING OBJECTIVES After completing this practical you will be able to: 1 Demonstrate various microorganisms which are present as part of the normal flora on the skin. III Specimen Skin swab. 2003. Jawetz. 23rd Edition. Louis) 2002. 11 th ed. 94-96. St. Kobayashi GS and Pfaller ME.Textbook of Practical Microbiology 165 KEY FACTS 1 Skin as part of its normal flora. 1996. 1995. Medical Microbiology. Sahm DF and Weissfeld AS. Mosby Inc. Lyne PM and Grange JM. Churchill Livingstone. (The CV Mosby Company. 5th Edition. Mackie and McCartney. Bailey and Scott’s Diagnostic Microbiology. . 14th Edition. Rosenthal KS. 2 The normal resident flora of skin can reduce the infections by competing with pathogens for binding receptors. London. 2002. Medical Microbiology. McGaw Hill. normally harbours many bacteria. Melnick and Adelberg. Butterworths. FURTHER READINGS 1 2 3 4 5 Collins CH. Forbes BA. Murray PR. Practical Medical Microbiology. Microbiological Methods. non-spore forming bacteria Staphylococci are part of the indigenous flora of skin surfaces. aureus (refer chapter 22). On blood agar. They usually cause suppurative lesions such as pustule. In this chapter the following tests will be described which are employed routinely for identification of S. Golden yellow pigment is demonstrated on nutrient agar (Fig. They are catalase positive. deoxyribonuclease is used to reconfirm the identification of S. blood agar and MacConkey agar plates and kept for aerobic incubation at 37°C.2% DNA). After incubation. toxic shock syndrome and staphylococcal scalded skin syndrome. Phenol red is used as an indicator. and 7. furuncle or carbuncle and can also involve muscles and bones. and wound swab. aureus also produce the enzyme deoxyribonuclease enzyme. The medium is used to test mannitol fermenting ability of .8 µm to 1 µm in size arranged in clusters. aureus. aureus. 57-3). On MacConkey agar they produce minute pink colored colonies. 2 Know the procedures used to identify Staphylococcus aureus and differentiate it from other staphylococcal species. They are less commonly associated with systemic infection. Absence of halo around the growth indicates DNase-negative S. The test organism is streaked onto the DNA agar medium (0. 3. aureus hydrolyze the DNA resulting in production of halo around the growth. It contains nutrient agar with 1% mannitol. Staphylococcus species are Gram positive spherical shaped bacteria measuring 0. demonstrate the presence of Gram positive cocci arranged in clusters along with pus cells (Fig.6% of hydrochloric acid (1N HCl) is added to the medium to determine the DNase activity.5% sodium chloride. TESTS FOR THE IDENTIFICATION OF STAPHYLOCOCCUS AUREUS 1 Direct examination Gram stain Gram stain of smears of clinical specimens is carried out to 5 Mannitol salt agar This is a selective medium used to isolate staphylococci from clinical specimens. DNase positive S. S.166 LESSON 57 INTRODUCTION Identification of Staphylococcus aureus LEARNING OBJECTIVES After completing this practical you will be able to: 1 Know about the clinical importance of staphylococci. 4 Deoxyribonuclease test Coagulase positive S. 57-2). 3 Coagulase test Coagulase test is most important test used for identification of S. aureus which are also coagulase negative. SPECIMENS Clinical specimens like pus swab. However they produce a variety of exotoxins causing various toxin-mediated diseases such as food poisoning. Detection of the presence of the enzyme. circular colonies with butyrous consistency. Then the DNA plate is incubated overnight at 37°C. they produce bhaemolytic golden yellow or white colonies (Fig. aureus produces opaque. Laboratory tests for differentiation of staphylococcal species are listed in the table 57-1. 2 Culture Clinical specimens are inoculated onto nutrient agar. 57-1). mucous membranes and upper respiratory tract. followed by application of a 5µg novobiocin disc on the surface of the agar. x 1000. aureus.Textbook of Practical Microbiology 167 S. saprophyticus. In acidic pH the indicator. epidermidis from S. Both S. 6 Novobiocin sensitivity Novobiocin sensitivity testing is used to detect sensitivity or resistance of S. epidermidis are novobiocinsensitive where as S. novobiocin. This test is used to differentiate S. FIGURE 57-3 Staphylococcus aureus colonies on blood agar. aureus and S. aureus S. . aureus and S. In this test. S. The yellow colour is seen due to the fermentation of mannitol with production of acid by S. FIGURE 57-1 Pus smear showing Gram positive cocci in clusters. The presence of zone of inhibition (17 mm) around the disc indicates novobiocinsensitive S. aureus and S. aureus to tolerate the high salt concentration in the medium and grow readily. produces yellow colour. It also tests ability of S. staphylococci strain to be tested is inoculated onto the surface of a Mueller Hinton agar plate. saprophyticus is novobiocin-resistant. aureus produces yellow colonies on the medium while other staphylococcal species do not. epidermidis S. Table 57-1 Laboratory tests for differentiation of staphylococcal species Test Growth on Mannitol salt agar Colonial pigmentation Coagulase test DNAase test Hemolysis on blood agar Novobiocin sensitivity S. aureus to the antibiotic. aureus. saprophyticus + Golden yellow + + Beta Sensitive White Sensitive White Resistant FIGURE 57-2 Staphylococcus aureus colonies on nutrient agar. The plate is then incubated overnight at 37°C. saprophyticus. epidermidis and no zone of inhibition around the disc indicates novobiocin-resistant S. phenol red. thereby reducing pH of the medium to the acidic. Mackie and McCartney.schleifer VIVA 1 What are the tests to identify S. 23rd Edition. 2003. Mosby Inc. 11 th ed. aureus? 2 List novobiocin-sensitive staphylococci. 5th Edition. McGaw Hill. Murray PR. aureus. Churchill Livingstone.hydenensis and S. Microbiological Methods. 1995. Medical Microbiology. 1996. 94-96. Jawetz. bacitracin and furazolidone. Lyne PM and Grange JM. Sahm DF and Weissfeld AS. 14th Edition. Bailey and Scott’s Diagnostic Microbiology. FURTHER READINGS 1 2 3 4 5 Collins CH. Melnick and Adelberg. Practical Medical Microbiology. 2002. 3 Some species of staphylococci produce only bound coagulase (clumping factor) such as S. Rosenthal KS. St.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. 2 Some coagulase negative staphylococci can produce beta hemolysis on blood agar similar to coagulase positive staphylococci. Kobayashi GS and Pfaller ME. . Medical Microbiology. (The CV Mosby Company. Butterworths. hence should not be confused with that of S. Louis) 2002. Forbes BA. London. Clearing of turbidity indicates positive test whereas persistence of turbidity indicates negative test. S. conjunctivitis. . pneumoniae undergo autolysis in the presence of surface active agents such as sodium deoxycholate whereas other alpha hemolytic streptococci are bile insoluble hence is not lysed by these bile salts. SPECIMENS Clinical specimens such as sputum. pneumoniae are Gram positive cocci arranged in pairs and individual coccus is lanceolate shaped.5 ml of sodium deoxycholate solution into each tube. This is due to the autolysis of the bacteria in old colony. TESTS FOR THE IDENTIFICATION OF STREPTOCOCCUS PNEUMONIAE 1 Direct examination Gram stain Gram stain of smears from clinical specimens is carried out to demonstrate the presence of Gram positive cocci. 58-1). S. CSF. These are 4 Optochin test S. etc. pneumoniae can be differentiated from other alpha hemolytic streptococci by many tests (Table 58-1). small. Label two brain heart infusion broth tubes. On prolonged incubation.Textbook of Practical Microbiology 169 LESSON 58 INTRODUCTION Identification of Streptococcus pneumoniae LEARNING OBJECTIVES After completing this practical you will be able to: 1 Know about the clinical importance of pneumococci. S. Add loopful of test organisms in the broth to give a heavy suspension. Gram stain of the CSF specimen from a case of meningitis shows both intracellular as well as extra cellular cocci. pneumoniae produce a haemolysis on blood agar. Then add 0.pneumoniae. In this chapter the following tests will be described which are employed routinely for identification of S. these colonies may show a depressed center with an elevated rim (Draughtsman’s colonies).pneumoniae produces small. slightly elongated cocci arranged in pairs (diplococci) with the broad ends in apposition. shiny dome-shaped and translucent colonies which are surrounded by alpha hemolysis (Fig. Each coccus has one end pointed and other end broad or rounded (lanceolate shaped). pleural exudate. pneumoniae is bile solubility positive. pneumoniae are sensitive to Optochin (ethyl hydrocupreine hydrochloride) and produce a zone of inhibition measuring 15 mm and more. Incubate the tubes in a water bath at 37°C for one hour. They are encapsulated and are an important cause of lobar pneumonia. 2 Know the procedures used to identify Streptococcus pneumoniae and differentiate it from other alpha hemolytic streptococci. 3 Bile solubility test S. pneumoniae (positive control) and the other tube as S. peritonitis. S. mitis (negative control). They also cause meningitis and other infections such as otitis media. Other alpha hemolytic streptococci are resistant to Optochin which produce a zone of inhibition of less than 15 mm (Fig. one as S. blood. S. 58-2). 2 Culture The specimens are inoculated onto blood agar and kept for aerobic incubation at 37°C for 24 hours in the presence of C02. sinusitis and suppurative arthritis. mitis. 5 Str. 3 Str. 5 Inulin fermentation S. 2 Gram stain of the CSF from a case of meningitis shows both intracellular as well as extra cellular cocci. Mouse virulence Streptococcus pneumoniae + + + + + Streptococcus mitis - KEY FACTS 1 Streptococcus pneumoniae are Gram positive cocci arranged in pairs or as short chains and individual coccus is lanceolate shaped. Using a sterile cotton swab. pneumoniae are sensitive to Optochin. FIGURE 58-2 Optochin sensitivity test. pneumoniae is capable of fermenting the sugar. followed by application of 0. 4 Str. Positive test is indicated by change of color of the media from red to yellow. inulin whereas other alpha hemolytic streptococci do not ferment inulin.170 Identification of Streptococcus pneumoniae A blood agar plate is divided into two halves by glass marking pencil. FIGURE 58-1 Streptococcus pneumoniae on blood agar. test strains are inoculated onto the surface of blood agar. . Zone of inhibition less than 15 mm shows the test to be negative. Negative test is indicated by no color change. pneumoniae is bile solubility test positive. Optochin sensitivity 3. pneumoniae while the other half is labeled as S. The plate is incubated aerobically at 37°C under 10% CO2 environment for 24 hours. inulin whereas other alpha hemolytic streptococci do not ferment inulin. Inulin fermentation 4. Development of zone of inhibition of 15 mm and more shows the test to be positive. One half is labeled as S. Table58-1 Differences between Streptococcus pneumoniae and Streptococcus mitis Tests 1. pneumoniae is capable of fermenting the sugar. and medium continues to appear as red.05 units Optochin disc over the inoculated surface by using forceps. Bile solubility 2. Quellung reaction 5. The test strain is inoculated into the inulin sugar medium (serum sugar) and is incubated over night at 37°C. 3 What is the strength of the routinely used Optochin disc? 4 What are the rapid diagnostic methods for pneumococcal meningitis? Ans. Capsular antigen detection in the CSF by latex agglutination and CIEP. India ink preparation. 1995. 2 What is the mechanism of autolysis? Ans. Forbes BA. 1996. 14th Edition. 2002. Bailey and Scott’s Diagnostic Microbiology. London. Louis) 2002. Medical Microbiology. 5th Edition. pneumoniae? Ans. 23rd Edition. St. Microbiological Methods. Lyne PM and Grange JM. Butterworths. Practical Medical Microbiology. Intracellular autolytic enzyme mediated. 94-96. Kobayashi GS and Pfaller ME. 11 th ed. . Murray PR. Sahm DF and Weissfeld AS. 2003. McGaw Hill. Mackie and McCartney.Textbook of Practical Microbiology 171 VIVA 1 How do you demonstrate capsules of S. Rosenthal KS. Jawetz. Mosby Inc. Melnick and Adelberg. (The CV Mosby Company. Churchill Livingstone. FURTHER READINGS 1 2 3 4 5 Collins CH. Medical Microbiology. This classification is known as Lancefield classification. S. SPECIMENS Throat swab. genital and other deep infections. b -haemolytic streptococci produce a clear zone of haemolysis on the blood agar surrounding the colonies. and c) g haemolysis: It is described as absence of any hemolysis around the colony. excepting I and J). The lysis is caused by two haemolysins. within which erythrocytes are completely lysed. They are cocci arranged in chains. streptolysin O and streptolysin S. These are: a) a hemolysis: It is an incomplete form of hemolysis where a green zone is produced around the colonies. This appears as a clear zone around the colonies. On blood agar three types of haemolysis are observed.172 LESSON 59 INTRODUCTION Identification of b-haemolytic Streptococci LEARNING OBJECTIVES After completing this practical you will be able to: 1 Know about clinical significance of b -haemolytic streptococci. g-haemolytic streptococci are avirulent. also known as Streptococcus pyogenes is the important species causing most human infections.04 units is placed on the inoculated plate. b-haemolytic streptococci on basis of their carbohydrate C antigen are classified in to 20 groups (from A to V. group B streptococci and group D streptococci. b-haemolytic streptococci are frequently associated with pathogenicity. b) b hemolysis: It is a complete form of hemolysis. 2 Perform important tests to identify group A streptococci. It also causes non-suppurative infections such as rheumatic fever and acute glomerulonephritis. Str. 59-1). All members of b-haemolytic streptococci are Gram positive and fastidious. streptococci cause neonatal infections such as septicemia and meningitis. Streptococci. The plate is incubated at 37°C for . Group D streptococci may cause urinary tract infections and wound infections. The test organism is streaked on to the surface of the blood agar. They appear as circular translucent pinpoint colonies on blood agar. 2 Culture The specimens are inoculated onto 5% sheep blood agar and kept for aerobic incubation at 37°C for 24 hours in the presence of C02. In this chapter the following tests will be described which are employed routinely for identification of S. skin. Bacitracin disc when placed on the surface of blood agar plate streaked with test organism inhibits its growth and forms a zone of inhibition. 3 Bacitracin sensitivity test Bacitracin test is a frequently used test to identify and differentiate group A streptococci from non.Group A streptococci. Group A streptococci. Then a bacitracin having strength of 0. pyogenes. both aerobic and anaerobic are classified on the basis of their haemolysis on blood agar. 59-2). Group B TESTS FOR IDENTIFICATION OF STREPTOCOCCUS PYOGENES 1 Direct examination Gram stain Gram stain of smears from clinical specimens like throat swab is carried out to demonstrate the presence of Gram positive cocci arranged in short chains (Fig. pyogenes causes a wide variety of suppurative infections of the respiratory tract.pyogenes produces pinpoint colonies which are surrounded by a large zone of b-hemolysis (Fig. aureus growth. FIGURE 59-1 Streptococci in short chains. Positive test is indicated by change of the brown colour of the medium in to black. aureus streak line leaving 1 cm space. Most strains of S. Test strain is inoculated onto the surface of the bile aesculin agar and kept for aerobic incubation at 37°C for 24–48 hr. 7 dihydroxy-coumarin which FIGURE 59-2 Beta hemolytic streptococci colonies on blood agar. CAMP substance is a peptide produced by group B streptococci which acts synergistically with the b-hemolysis produced by some strains of Staphylococcus aureus enhancing the effect of haemolysis. The negative test is indicated by no zone of inhibition around the bacitracin disc. x 1000. 59-3). 5 Bile aesculin test This test is carried out to identify Group D streptococci. Positive test is indicated by an arrow head zone of hemolysis near the S. negative control (group A streptococci) and test strains are also inoculated as a straight lines parallel to S. Atkins and Munch-Peterson) test is a test used to identify Group B streptococci. The plate is incubated aerobically at 37°C overnight in 5–10% CO2 environment. Negative test is indicated by no brown or black discoloration of medium. Atkins and Munch-Peterson) test? What is the principle of the bile aesculin test? . Negative test is indicated by the absence of arrow head zone of hemolysis (Fig. pyogenes are bacitracin sensitive. aureus strain as a straight line on the surface of the sheep blood agar. Positive test is indicated by a zone of inhibition around the bacitracin disc. VIVA 1 2 3 4 What are different types of haemolysis produced by streptococci? What is the principle of the bacitracin sensitivity test? What is the principle of the CAMP (Christie. 4 CAMP (Christie. FIGURE 59-3 CAMP Test. Known positive control (Group B streptococci). The cocci hydrolyse aesculin into 6. Atkins and Munch-Peterson) test CAMP (Christie. The test is performed by inoculating S. when reacts with iron salts in the medium causes brown to black discolouration of medium following incubation.Textbook of Practical Microbiology 173 overnight. St. Mackie and McCartney.174 Identification of b-haemolytic streptococci KEY FACTS 1 b-haemolytic streptococci produce a clear zone of haemolysis on the blood agar surrounding the colonies. Jawetz. 3 Blood agar plate for CAMP test should be incubated in the environment of 5–10% CO2.Group A streptococci. 11 th ed. Murray PR. Microbiological Methods. 1996. 14th Edition. heat resistant test and salt tolerance test. McGaw Hill. FURTHER READINGS 1 2 3 4 5 Collins CH. Mosby Inc. Forbes BA. 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. (The CV Mosby Company. Rosenthal KS. 23rd Edition. Practical Medical Microbiology. Medical Microbiology. 2002 . 1995. Butterworths. 5th Edition. Melnick and Adelberg. Bailey and Scott’s Diagnostic Microbiology. 2003. Lyne PM and Grange JM. London. Churchill Livingstone. Medical Microbiology. 94-96. Sahm DF and Weissfeld AS. 2 Bacitracin test is a frequently used test to identify and differentiate group A streptococci from non. within which erythrocytes are completely lysed. Kobayashi GS and Pfaller ME. Louis) 2002. Though diphtheria is a disease of respiratory tract this can also affect skin. and trehalose) for the detection of acid. conjunctiva. the swab specimen is inoculated on the surface of Macleod’s potassium tellurite agar media (0. and incubated at 37°C for 24 hours. These bacilli have metachromatic granules towards the end of the logarithmic growth period. The smear is examined under oil immersion (100 x) objective (Fig. because no acid is produced. They are arranged in angled pairs or in angular fashion which resemble Chinese letters. maltose and sucrose with TESTS FOR THE IDENTIFICATION OF CORYNEBACTERIUM DIPHTHERIAE 1 Direct examination . Negative test is indicated by the persistence of red colour. ear. The specimen is collected from the surface of the pseudomembrane by a sterile cotton swab. 60-2). larynx and trachea. diphtheriae. The colonies grown on blood agar are inoculated on to serum sugars and gelatin. The swab containing the specimen is rolled on the surface of the clean glass slide. diphtheriae. diphtheriae reduces potassium tellurite into metallic tellurium which results in production of black colored colonies on the medium (Fig. nasal passages. diphtheriae. Colonies grown on this medium are stained by Albert’s staining for demonstrating metachromatic granules. These granules are the storage depots of polymetaphosphate.3 µm in size. Loeffler’s serum slope is inoculated with the throat swab and incubated at 37°C for 6 hours. C. Positive test is indicated by the change of media to yellow colour due to production of acid. 60-3). C.04%) and is incubated at 37°C for up to 48 hours. 3 Biochemical tests SPECIMENS Throat swab. diphtheriae ferments glucose.Textbook of Practical Microbiology 175 LESSON 60 INTRODUCTION Identification of Corynebacterium diphtheriae LEARNING OBJECTIVES After completing this practical you will be able to 1 Know about the clinical importance of Corynebacterium diphtheriae. lactose. diphtheriae is made based on the presence of metachromatic granules by Albert’s stain C. 60-1). In this chapter the following tests will be described which are employed routinely for identification of C. posterior pharyngeal wall. diphtheriae produces minute smooth colonies after 6 hours of incubation (Fig. Commonest sites affected are tonsils. diphtheriae are thin. C. non-sporing. maltose. These bacilli have typical club-shaped swellings at one or both ends of bacilli. Similarly. The following biochemical tests are carried out: a. Fermentation of serum sugars (glucose. They produce diphtheriae toxin that causes local tissue necrosis which results in the formation of grayish white membrane at the affected site. 2 Know the procedures used to identify C.Presumptive identification of C. diphtheriae causes natural infection only in man. etc. C. Albert’s stain Albert’s stain is done to detect the presence of metachromatic granules in C. non-capsulated and nonmotile Gram positive bacilli which measure approximately 3 µm × 0. The colour change in the serum sugar media is looked for. sucrose. diphtheriae and differentiate it from other non pathogenic Corynebacterium species. 2 Culture Loeffler’s serum slope and potassium tellurite agar are the two media frequently used for culture of C. mannitol. The smear is heat fixed and Albert’s stain is carried out (refer chapter 9). x 1000. ulcerans hydrolyses urea. . which have metachromatic granules. Ans. Positive gelatin hydrolysis test is indicated by free flowing of medium on inverting the gelatin tube. include urea hydrolysis and gelatin hydrolysis. C. 2 Why serum sugars are used for testing acid production by C.176 Identification of Corynebacterium diphtheriae production of acid only. diphtheriae. These organisms are fastidious. diphtheriae do not hydrolyse urea and gelatin whereas non-pathogenic C. If any delay is anticipated Amie’s transport medium can be used to transport the specimen. maltose and sucrose but do not ferment lactose. b. 3 Throat swab should be processed immediately without delay. mannitol. Bordetella and Brucella. Other biochemical tests which are used primarily to differentiate C. Negative urea hydrolysis test is indicated by no colour change. ulcerans hydrolyses urea. They do not ferment lactose. 6 C. diphtheriae in causation of disease than just identification of C. FIGURE 60-3 Black coloured colonies on potassium tellurite agar. 3 Name other organisms. diphtheriae. KEY FACTS 1 C. diphtheriae do not hydrolyse urea and gelatin whereas non-pathogenic Corynebacterium species such as C. 5 Loeffler’s serum slope and potassium tellurite agar are the two media frequently used for culture of C. diphtheriae is made based on the presence of metachromatic granules by Albert’s stain. Neisser’s and Ponder’s stains. mannitol and trehalose. Ans. They cannot grow in ordinary sugar media. diphtheriae causes natural infection only in man. No free flowing on inversion of tube indicates negative test. and trehalose. 7 C. diphtheriae ferments glucose. 2 Toxin detection is important to prove the role of C. VIVA 1 Name other staining methods used to demonstrate volutin granules. FIGURE 60-1 Albert’s stain showing volutin granules. diphtheriae? Ans. diphtheriae from other Corynebacterium spp. Positive urea hydrolysis test is indicated by change in colour of the medium into red. FIGURE 60-2 Loeffler’s serum slope. 4 Presumptive identification of C. Textbook of Practical Microbiology 177 FURTHER READINGS 1 2 3 4 5 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 94-96, 1995. Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002. Jawetz, Melnick and Adelberg. Medical Microbiology. 23rd Edition. McGaw Hill. 2003. Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. 1996. Murray PR, Rosenthal KS, Kobayashi GS and Pfaller ME. Medical Microbiology. 5th Edition. Mosby Inc. 2002 178 LESSON 61 INTRODUCTION Identification of Lactose Fermenting Enterobacteriaceae Gram’s stain Gram stain is indicated in wound infection where pus ells are detected along with Gram negative bacilli. Both centrifuged and un centrifuged urine are subjected for microscopic examination for the detection of pus cells. LEARNING OBJECTIVES After completing this practical you will be able to 1 Know the procedures used to identify lactose fermenting Enterobacteriaceae and differentiate it from other members of the family Enterobacteriaceae. 2 Perform the tests to identify Escherichia coli and Klebsiella species. 2 Culture Stool specimen is inoculated onto MacConkey agar and CLED media and wound swabs are processed using blood agar and MacConkey agar. Inoculated plates are kept for aerobic incubation at 37 °C for 24 hours. Pink colored lactose fermenting colonies of E.coli and K.pneumoniae appear on MacConkey agar plates (Fig. 61-1 and 61-2). On CLED medium E.coli produces lactose fermenting yellow colonies (Fig. 61-3). These are subjected to testing by various biochemical tests to identify the bacteria. Enterobacteriaceae are the part of the normal intestinal flora of animals and humans. They are aerobes and facultative anaerobes. They are non-fastidious and reduce nitrate to nitrites. They are catalase positive and oxidase negative. Based on their action on lactose they are classified into lactose fermenters and non-lactose fermenters. Many of commensal floras of the intestine are lactose fermenters. Late lactose fermenting bacteria are called as para colon bacilli and with the exception of Shigella sonnei all others are commensals. These organisms cause gastrointestinal infection, urinary tract infection, pneumonia and septicaemia. Lactose fermenters such as E. coli, Enterobacter, Klebsiella, Citrobacter and enterotoxigenic E. coli cause watery diarrhoea. The tests used for identification of E. coli and Klebsiella species are listed in the boxes 61-1 and 61-2 respectively. 3 Biochemical tests Biochemical tests and other tests, as summarized in the tables are performed to identify E. coli (Fig. 61-4) and Klebsiella species (Fig. 61-5). 4 Antibiotic susceptibility testing Antibiotic susceptibility testing is carried out for the identified bacteria which provide the important information of the susceptibility pattern of the organism. SPECIMENS Stool, urine, wound swab, sputum, etc. In this chapter, following tests will be described which are routinely used for identification of E.coli and Klebsiella spp. TESTS FOR IDENTIFICATION OF E. COLI AND KLEBSIELLA SPP. 1 Direct examination FIGURE 61-1 Mc Conkey agar with lactose fermenting pink colonies of Escherichia coli. Textbook of Practical Microbiology 179 + FIGURE 61-2 Mc Conkey agar with lactose fermenting pink mucoid colonies of Klebsiella pneumoniae. + – – FIGURE 61-4 IMViC test for Escherichia coli. – FIGURE 61-3 CLED Medium with lactose fermenting yellow colonies of Escherichia coli. – + + FIGURE 61-5 IMViC test for Klebsiella pneumoniae. BOX 61-1 IDENTIFICATION OF ESCHERICHIA COLI Gram stain Gram negative bacilli. Motility by hanging drop preparation method Motile. Biochemical tests Catalase test: Positive. Oxidase test: Negative. Nitrate reduction test: Reduced to Nitrite. Kligler’s iron agar medium: Acid/Acid. No H2S gas is produced. Fermentation of sugars (glucose, lactose and mannitol): Acid and gas. Methyl red test: Positive. Voges-Proskauer test: Negative. Indole test: Positive. Citrate utilization test: Negative. Urease test: Negative. Lysine decarboxylation test: Positive. BOX 61-2 IDENTIFICATION OF KLEBSIELLA SPECIES Gram stain Short thick gram negative bacilli. Motility by hanging drop preparation method Non motile. Biochemical tests Catalase test: Positive. Oxidase test: Negative. Nitrate reduction test: Reduced to Nitrite. Kligler’s iron agar medium: Acid/Acid. gas is present. Fermentation of sugars (glucose, lactose and mannitol): Acid and gas. Methyl red test: Negative. Voges-Proskauer test: Positive. Indole test: Negative. Citrate utilization test: Positive. Urease test: Positive (only K. pneumoniae). Lysine decarboxylation test: Positive. 180 Identification of Lactose Fermenting Enterobacteriacae KEY FACTS 1 Enterobacteriaceae is the part of the normal intestinal flora of animals and humans. 2 Lactose fermenters such as Enterobacter, Klebsiella and Citrobacter and enterotoxigenic E. coli causes watery diarrhoea. 3 Antibiotic susceptibility testing should be done for the identified organism which will give the important information of the susceptibility pattern of the organism. VIVA 1 Name the indicator used in MacConkey agar medium? Ans. Neutral red. 2 Why some bacteria are late lactose fermenters? Ans. They lack the enzyme lactose permease. 3 What is the color of the colonies of lactose fermenting organism on CLED medium? Ans. Yellow colour. 4 Lactose fermenting colonies grown on MacConkey agar plate are not picked up for oxidase test why? Ans. Acidic pH of the colonies will give false positive reactions. 5 Differentiate between E. coli and Klebsiella species. FURTHER READINGS 1 2 3 4 5 Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths, London, 94-96, 1995. Forbes BA, Sahm DF and Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11 th ed. (The CV Mosby Company, St. Louis) 2002. Jawetz, Melnick and Adelberg. Medical Microbiology. 23rd Edition. McGaw Hill. 2003. Mackie and McCartney. Practical Medical Microbiology. 14th Edition. Churchill Livingstone. 1996. Murray PR, Rosenthal KS, Kobayashi GS and Pfaller ME. Medical Microbiology. 5th Edition. Mosby Inc. 2002 Textbook of Practical Microbiology 181 LESSON 62 INTRODUCTION Identification of Vibrio cholerae LEARNING OBJECTIVES After completing this practical you will be able to 1 Know about the clinical importance of Vibrio cholerae. 2 Perform the tests to identify V. cholerae and differentiate it from other Vibrio species. the corners of cover slip. A loopful of stool specimen is collected by a heat sterilised wire loop and transferred it on to the middle of the cover slip. This is placed over a clean cavity slide in such a way that the drop hangs from the cover slip to cavity of the slide. This preparation is observed first under 10x followed by 40x, for the presence of characteristic darting motility of V.chloerae. Observation of darting motility in stool sample helps in presumptive diagnosis of cholera, which should be confirmed by biochemical and serologic methods. V. cholerae are short curved or straight Gram negative bacilli measuring about 1.5 µ × 0.2 to 0.4 µ. These organisms are actively motile by means of a single sheathed polar flagellum. They are strongly aerobic and they grow in alkaline pH (7.4 to 9.6) media. They cause cholera, a toxin mediated disease. Cholera occurs in many forms sporadic, endemic, epidemic or pandemic. Cholera is an exclusively human disease. Infections are spread by feco-oral transmission. List of tests for identification of V.cholerae is summarized in the box 62-1. 2 Culture Alkaline peptone water is used as enrichment medium. DCA and TCBS are employed as the selective media for V. cholerae. A loopful of stool is inoculated into alkaline peptone water and incubated at 37°C for 6 hours. Then this is sub cultured on to TCBS medium, and incubated at TCBS at 37°C overnight. After overnight incubation the presence of yellow colonies on TCBS medium and non lactose fermenting colorless colonies on DCA is looked for. The biochemical tests of these colonies are performed for identification of V. cholerae. SPECIMENS 3 Biochemical tests Stool (rice watery stool) and rectal swab. In this chapter the following tests will be described which are employed routinely for identification of V. cholerae. Biochemical tests such as lysine and ornithine decarboxylase and arginine dihydrolase (Fig. 62-1) and other tests performed to identify V. cholerae are summarized in the box 62-1. Biochemical tests like Voges Proskauer, polymyxin-B (50 U) sensitivity (Fig. 62-2), sheep RBC hemolysis and chick cell agglutination are done further for the differentiation of two biotypes Classical and Eltor (Table 62-1). TESTS FOR IDENTIFICATION OF VIBRIO CHOLERAE 1 Direct examination Hanging drop preparation This test is carried out to demonstrate motility of V.chloerae, which are actively motile. A clean cover slip is taken and vaseline is applied over all 4 Antibiotic susceptibility testing Antibiotic susceptibility testing is carried out for the identified bacteria which provide the important information of the susceptibility pattern of the organism. 182 Identification of Vibrio cholerae BOX 62-1 IDENTIFICATION OF VIBRIO CHOLERAE Gram stain Gram negative curved or straight rods. Motility by hanging drop preparation method Actively motile. Biochemical tests Catalase test: Positive. Oxidase test: Positive. 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 62-1 Lysine and ornithine decarboxylase and arginine dihydrolase tests for Vibrio cholerae. FIGURE 62-2 Polymyxin B sensitivity of Vibrio cholerae. Table62-1 Differences between V. cholerae biotype classical and V. cholerae biotype El Tor Test Voges-Proskauer (VP) test Sheep RBCs haemolysis Chick RBCs agglutination Polymyxin B sensitivity Susceptibility to Mukherjee’s phage IV Sensitivity to Vibriostatic (O/129) agent Susceptibility to group V phage V. cholerae biotype classical + + + 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. Jawetz. Practical Medical Microbiology. Louis) 2002. Medical Microbiology. Rosenthal KS. Butterworths. cholerae? Ans. St. Sodium deoxycholate (0.Textbook of Practical Microbiology 183 VIVA 1 What are non agglutinable vibrios? 2 Mention the application of vibriostatic O/129 reagent. . 4 What is the chemical agent used for string test for V. which converts bromo thymol blue to yellow colour. Microbiological Methods. 5 How the haemodigestion of V. 2003. Sahm DF and Weissfeld AS.5 %). Forbes BA. cholerae? Ans. 94-96. 14th Edition. London. Churchill Livingstone. 5th Edition. Haemodigestion is not toxin mediated. McGaw Hill. 23rd Edition. Lyne PM and Grange JM. 1996. Medical Microbiology. Murray PR. 3 Why yellow colonies are produced on TCBS while culturing V. Sucrose fermentation degreases pH. 2002. It is mediated by peroxides. Bailey and Scott’s Diagnostic Microbiology. Mackie and McCartney. FURTHER READINGS 1 2 3 4 5 Collins CH. Kobayashi GS and Pfaller ME. Mosby Inc. (The CV Mosby Company. Melnick and Adelberg. 11th ed. cholerae differs from hemolysis of other bacteria? Ans. 1995. aeruginosa. 3 Oxidase test P. By a platinum wire.5 µm to 3µm by 0. They utilise carbohydrates oxidatively. . aeruginosa produces pigments such as pyocyanin. pyoverdin and pyorubrin. 2 Culture Pus swab is inoculated on to blood agar. Moist hemolytic colonies are produced on blood agar (Fig. In this chapter the following tests will be described which are employed routinely for identification of P. The tests for identification of P. They grow at a temperature range of 6°C to 42°C. aeruginosa colonies produce earthy or grape juice like odour. aeruginosa are mentioned in the box 63-1. P. P. It produces the enzyme cytochrome oxidase. the colonies on nutrient agar are picked up and streaked on the surface of the filter paper. 5 Antibiotic susceptibility testing 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). MacConkey agar and nutrient agar and the plates are incubated at 37°C overnight. 63-1). 2 Perform the tests to identify P. aeruginosa are non-fermentative slender Gram negative bacilli that measure about 1. 63-2). They produce opaque irregular colonies with earthy smell and form non-lactose fermenting colonies on MacConkey agar. 63-3). P. The enzyme in the presence of oxygen oxidizes 1% tetramethyl paraphenylene diamine dihydrochloride and produces a deep purple colour. Positive test is indicated by appearance of deep purple color.5–3 µm by 0. aeruginosa. Pseudomonas causes infection of burns and wounds and is a common cause of nosocomial infections. aeruginosa is an oxidase positive bacterium. They break down glucose oxidatively with production of acid only (Fig. Pseudomonas and other nonfermentative Gram negative bacilli are oxidase positive whereas all Enterobacteriaceae bacteria are oxidase negative. A filter paper strip impregnated with 1% tetramethyl paraphenylene diamine dihydrochloride is taken. Greenish blue pigmented colonies are produced on nutrient agar (Fig. 4 Biochemical tests They are non-fermenters.5 µm in size. SPECIMENS Pus swab.5 µm in size. They are obligate aerobes.184 LESSON 63 INTRODUCTION Identification of Pseudomonas aeruginosa LEARNING OBJECTIVES After completing this practical you will be able to 1 Know the clinical importance of Pseudomonas aeruginosa. TESTS FOR IDENTIFICATION OF PSEUDOMONAS AERUGINOSA 1 Direct examination Gram stain Gram stain of the pus swab reveal thick Gram negative bacilli measuring 1. In liquid media they form a surface pellicle. and negative test by no color change (refer chapter 21). FIGURE 63-2 Pseudomonas colonies on nutrient agar. Oxidase test: Positive. London. aeruginosa. 2003. 1995. Cetrimide agar.Textbook of Practical Microbiology 185 BOX 63-1 IDENTIFICATION OF PSEUDOMONAS AERUGINOSA Gram stain Gram negative bacilli. Microbiological Methods. Indole test: Negative. aeruginosa produces greenish blue pigmented colonies on nutrient agar. Sahm DF and Weissfeld AS. P. aeruginosa are non-fermentative slender Gram negative bacilli. McGaw Hill. VIVA 1 Name the selective media used for the isolation of P. Lysine decarboxylation test: Negative. Churchill Livingstone. Urease test: Variable. KEY FACTS 1 2 3 4 P. Butterworths. 2 Name different pigments produced by P. 14th Edition. (The CV Mosby Company. 2002. Bailey and Scott’s Diagnostic Microbiology. – FIGURE 63-1 Non hemolytic Pseudomonas colonies on blood agar. 94-96. Mackie and McCartney. Louis) 2002. 5th Edition. Arginine dihydrolase test: Positive. Biochemical tests Growth at 42°C: Positive. Motility Motile. Ans. Nitrate reduction test: Reduced to nitrite. P. mannitol. Jawetz. 11 th ed. Catalase test: Positive. Citrate utilization test: Positive. Forbes BA. Kligler’s iron agar median: K/K Oxidative breakdown of sugars glucose: acid only. They utilise carbohydrates oxidatively. sucrose. Kobayashi GS and Pfaller ME. aeruginosa is an oxidase positive bacteria. Practical Medical Microbiology. St. Murray PR. Medical Microbiology. + FIGURE 63-3 OF test showing oxidative utilization of glucose. Mosby Inc. 1996. Rosenthal KS. Medical Microbiology. maltose: No acid. Lyne PM and Grange JM. . aeruginosa? FURTHER READINGS 1 2 3 4 5 Collins CH. 23rd Edition. lactose. Melnick and Adelberg. 186 . Textbook of Practical Microbiology UNIT 187 IX Parasitology Introduction Lesson 64 Saline Wet Mount of Stool Lesson 65 Lesson 66 Iodine Wet Mount of Stool LPCB Wet Mount of Stool Lesson 67 Acid-fast Staining of Stool Smears Lesson 68 Leishman’s Staining of Peripheral Blood Smears Lesson 69 Concentration of Stool for Parasites Lesson 70 Culture of Stool for Entamoeba histolytica . . It becomes even more important to provide well-written lab protocols and to standardize test methods for consistency. schistosomiasis. arthropods) are antigenically and biochemically complex. 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. During past few years. clinicians are becoming increasingly able to diagnose and treat diseases at the molecular level. there has been an increased awareness of the importance of trained and qualified personnel to perform diagnostic procedures. The diseases caused by parasites constitute major human health problems throughout the world. as are their life histories and the pathogenesis of the diseases they cause.g.g. The basis for effective treatment and cure of a patient is the rapid diagnosis of the disease and its causative agent. cryptosporidiosis. Therapy based on patient history and symptoms is not generally recommended in cases of parasitic infections. parasitic helminths (worms). malaria) have increased rather than decreased in recent years. . Other parasitic illnesses have increased in importance as a result of the AIDS epidemic (e. and those arthropods that directly cause disease or act as vectors of various pathogens. and strongyloidiasis). As we are in the 21st century. The unicellular parasites (protozoa) and multicellular parasites (helminths. Pneumocystis carinii pneumonia. The incidence of many parasitic diseases (e. Infections of humans caused by parasites number in the billions and range from relatively innocuous to fatal. which is based on the analysis of the clinical symptoms in combination with laboratory tests.188 Introduction Parasitology traditionally includes the study of three major groups of animal parasites: parasitic protozoa. though not distinct. demonstrate the presence of trophozoites by demonstrating the motility and b differentiate between bile stained and non bile-stained eggs. larva and helminthic eggs are prepared.5 gm of sodium chloride and dissolving it in 1000 ml of distilled water. 5 Examine the preparation first under the low power (10x) and then under high power (40x) objective of the microscope. and applicator stick Preparation of physiological saline: Normal saline is also called physiological saline. The saline wet mount is a colourless preparation that highlights the staining property of the egg. helminthic eggs and larva based on the recognition of specific morphological characters. PRINCIPLE The saline wet mount preparation is prepared by mixing a small quantity of faeces with physiological saline. These preparations are observed and compared with the saline and iodine wet mount preparations of the test stool specimen for various morphological forms of the parasite and identified. colourless structure seen with four nuclei. 2 With help of a glass dropper. detects motility of trophozoites. It is prepared by weighing 8. II Reagents and glass wares Microscopic slides.Textbook of Practical Microbiology 189 LESSON 64 INTRODUCTION Saline Wet Mount of Stool LEARNING OBJECTIVES After completing this practical you will be able to: 1 Perform saline wet mount preparation of faeces for demonstration of intestinal parasites. REQUIREMENT I Equipments Compound light microscope. trophozoites. take a small portion of the stool specimen (match stick head size) and emulsify it in the drop of saline. cover slips. Stool microscopy is an easy and rapid method employed for detection of intestinal ova and cysts with the aid of wet mount preparations. 2 Identify intestinal protozoal cysts. lacto-phenol cotton blue (LPCB) wet mount preparation is also used.85% sodium chloride in distilled water. Of late. The wet mount preparations most commonly used for detection are the i) saline wet mount preparation and ii) iodine wet mount preparation. 3 With the help of an applicator stick. 4 Put a cover slip over the saline suspension of the stool. III Specimen Fresh stool specimen is required for stool microscopy. and facilitates demonstration of chromatoidal bodies in the cyst. The saline preparation is mainly used to a. QUALITY CONTROL A saline and iodine wet mount preparation of known positive stool specimen for protozoal cysts. physiological saline. It is 0. 6 Record the findings with description of the morphological characteristics. whether bile or non-bile stained. PROCEDURE 1 Take a clean glass slide. The wet mount preparations of stool specimen are screened first under the low power objective (10x) and then under high power (40x). . OBSERVATIONS 1 A 10–15 µm sized round refractile. in the stool specimen. put a drop of saline on the glass slide. FIGURE 64 -1 Cyst of Entamoeba histolytica/dispar. Egg of Trichuris trichiura. round refractile. 6 A 25µm × 50µm sized. RESULTS AND INTERPRETATION 1 Cyst of Entamoeba histolytica/dispar (Fig. 3 4 5 6 Cyst of Giardia intestinalis (Fig. Fertilized egg of Ascaris lumbricoides (Fig. colourless structure seen with transparent hyaline shell membrane and segmented ovum with blastomeres. x 400. x 400. FIGURE 64 -2 Cyst of Giardia intestinalis. Hook worm egg (Fig. x 400. x 100. though not distinct. colourless structure seen with at least eight nuclei. It contains a very large conspicuous unsegmented ovum with a clear space at each pole. round oval shaped. A clear space is visible between the thin hyaline shell membrane and blastomeres. . 64-4). 64-5). non-bile stained. bile stained structure seen with thick translucent shell with an albuminous coat. 5 A 60–75µm × 40–50µm sized. 2 Cyst of Entamoeba coli. 64-3). 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 -5 Eggs of Trichuris trichiura. bile coloured. (Fig. 3 A 6–10µm sized oval shaped colourless structure seen with distinct wall and faint axostyle.190 Saline Wet Mount of Stool 2 A 15–30µm sized. FIGURE 64 -4 Fertilized egg of Ascaris lumbricoides. FIGURE 64 -3 Hook worm egg. 64-2). oval shaped. x 400. 64-1). 4 A 60µm × 40µm sized. 1998. . rice starch. iodine wet mount. 2 Parija SC. Stool Microscopy. 2006. muscle fibres. The preparation should be such that. plant epidermal hairs. The normal constituents of stool are yeast cells.C. Ans. VIVA 1 List the normal constituents of stool. 2 What are the different wet mount preparations of stool? Ans. Diagnostic Medical Parasitology. LPCB wet mount. Washington D. pollen. Dharan. stone cell. 3 What are the uses of saline wet mount? FURTHER READINGS 1 Garcia LS. oil drops. 3 Trophozoites and larvae are visualised best in the saline wet mount. starch cell. Textbook of Medical Parasitology. corn starch. 3 Parija SC. Nepal. Different wet mount preparations of stool are saline wet mount. 2003. 4th Edition. All India Publishers and Distributors. and buffered methylene blue. ASM press. BPKIHS. moulds and bacteria. 2 A wet mount preparation should neither be too thick nor thin. cotton fiber. leucocytes. fungal spores. potato parenchymal cell. potato starch. The wet mount preparation must not overflow. a printed letter should be read through it.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. 3rd Edition. 4 Bile staining property of the parasitic egg can be appreciated. plant cells. vascular structure of plants. It is mixed well and Dobell’s iodine solution is prepared. QUALITY CONTROL A saline and iodine wet mount preparation of known positive stool specimen for protozoal cysts. It contains iodine and potassium iodide in distilled water. Bile staining property of the helminthic eggs cannot be appreciated in the iodine preparation. Preparation of Dobell’s iodine: Dobell’s iodine is used for making iodine wet mount of stool. put a drop of iodine on the glass slide. Lugol’s’ iodine and D’ Antonie’s iodine. . cover slips. II Reagents and glass wares Microscopic slides. Different types of iodine solution are used. applicator stick and Dobell and O’Connor’s iodine. Iodine wet mount is mainly used for protozoal cysts. These are Dobell and O’Connor’s iodine. It is prepared by weighing 2 grams of iodine crystals and dissolving it in 100 ml of distilled water. The motility of trophozoites is also inhibited in the iodine wet mount. 3 With the help of an applicator stick. These preparations are observed and compared with the saline and iodine wet mount preparation of the test stool specimen for various morphological forms of the parasite and identified. 5 Examine the preparation first under the low power (10x) and then under high power (40x) objective of the microscope. as it is already coloured. Iodine wet mount is prepared by using a drop of saline for preparation of wet mount of stool. 2 Identify intestinal protozoan cysts. List of bile-stained and non-bile stained eggs are provided in the table 65-1. yellowish cytoplasm and brown glycogen material within the cysts.192 LESSON 65 INTRODUCTION Iodine Wet Mount of Stool LEARNING OBJECTIVES After completing this practical you will be able to: 1 Perform saline wet mount preparation of faeces for demonstration of intestinal parasites. The preparation clearly demonstrate the presence of nuclei in protozoan cyst as brown dots. 2 With the help of a glass dropper. 4 Put a cover slip over the iodine suspension of the stool. PROCEDURE 1 Take a clean glass slide. Then 4 grams of potassium iodide is weighed and added to the solution prepared. 6 Record the findings with description of the morphological characteristics. larva and helminthic eggs are prepared. and also demonstrates the presence of glycogen mass in protozoan cyst. trophozoites. in the stool specimen by iodine wet mount. PRINCIPLE The iodine wet mount preparation is a brown coloured preparation that highlights the presence of pale refractile nuclei. take a small portion of the stool specimen (match stick head size) and emulsify it in the drop of iodine. III Specimen Fresh stool specimen is required for stool microscopy. helminthic eggs and larva based on the recognition of specific morphological characters. REQUIREMENTS I Equipments Compound light microscope. 5 A 60–75µm×40–50µm sized. 3 A 6–10µm sized oval shaped brown coloured structure seen with visible axostyle and distinct cyst wall surrounded by a halo. Fasciola hepatica. round. and Clonorchis sinensis. Schistosoma japonicum. 65-1). 65-4). Schistosoma hematobium. x 400. structure seen with brown coloured 1–4 nuclei and glycogen mass. Enterobius vermicularis and Hymenolepis nana. Fasciolopsis buski. Non-bile stained eggs Hook worm. barrel-shaped structure seen with lightly stained mucus plugs at each pole. yellow coloured structure seen with yellow stained outer corticated thick cell wall. 65-2). yellow coloured. oval shaped. Eggs of Trichuris trichiura (Fig. A clear space is visible between the stained shell membrane and blastomeres. 2 A 15–30µm sized. 65-3). round. The unsegmented ovum and also the space between the shell and ovum at each pole are stained yellow. RESULTS AND INTERPRETATION 1 2 3 4 5 6 Cyst of Entamoeba histolytica/dispar (Fig. x 400. Fertilized egg of Ascaris lumbricoides (Fig. Diphyllobothrium latum. yellow coloured structure seen with brown coloured 1–8 nuclei and diffuse glycogen mass. Echinococcus species. yellow coloured structure seen with lightly stained shell membrane and segmented ovum with light yellow stained blastomeres. Schistosoma mansoni. FIGURE 65. 4 A 60µm × 40µm sized. Hook worm egg (Fig. FIGURE 65-2 Cyst of Giardia intestinalis. Trichuris trichiura. The egg shell is stained brown and encloses the light yellow stained unsegmented ovum. Table 65-1 List of bile-stained and non-bile stained eggs Bile stained eggs Ascaris lumbricoides.Textbook of Practical Microbiology 193 OBSERVATIONS 1 A 10–15µm sized. Paragonimus westermani. 65-5). round oval shaped. 6 A 25µm × 50µm sized yellow coloured. x 400. Taenia species. . Cyst of Entamoeba coli.1 Cyst of Entamoeba histolytica/dispar. Cyst of Giardia intestinalis (Fig. FIGURE 65-3 Hook worm egg. Ans. 3 Parija SC. ASM press. 2006. 4th Edition. 4 List bile stained eggs. Iodine solution should always be handled with care. Dharan. VIVA 1 What are the uses of iodine wet mount? 2 What are the different types of iodine solution that can be used for stool wet mount preparation? 3 List the advantages and disadvantages of iodine wet mount. KEY FACTS 1 2 3 4 5 A stool wet mount should always be screened immediately. . b It also demonstrates the yellowish cytoplasm and brown glycogen mass present in the cysts.C. as it proves injurious if inhaled or comes in contact with eyes. Nepal. Iodine wet mount kills both living trophozoites of protozoa and larvae of worms. Bile staining property of the parasitic egg cannot be appreciated in the iodine preparation as it is already coloured. 2 Parija SC. FURTHER READINGS 1 Garcia LS. Diagnostic Medical Parasitology.4 Fertilized egg of Ascaris lumbricoides. All India Publishers and Distributors. BPKIHS. c Chromatoidal bars in protozoan cysts are not clearly demonstrable. Washington D. Stool Microscopy. 2003.194 Iodine Wet Mount of Stool FIGURE 65. 3rd Edition. Textbook of Medical Parasitology. Disadvantages a Trophozoites are killed by iodine. b The bile staining property of the helminthic eggs cannot be made out. hence cannot be demonstrated in iodine wet maint. 5 List non-bile stained eggs. A wet mount preparation should neither be too thick or thin. 1998. Advantages a The number of nuclei present in the protozoan cyst can be clearly distinguished. x 400. x 400. FIGURE 65 –5 Eggs of Trichuris trichiura. Vegetable cells. It contains cotton blue which stains both the helminthic ova and protozoal cysts deep blue. helminthic eggs and larva based on the recognition of specific morphological characters. Measure 20 ml of distilled water in a measuring cylinder and add to the phenol. the intestinal coccidian parasites. in the stool. in the wet mount preparation of LPCB. Mix it well. 5 Examine the preparation first under the low power (10x) and then under high power (40x) objective of the microscope. Then allow it to be cooled. staining and clearing agent. Then transfer to the bottle. and mix it well. muscle fibres and other artifacts are clearly stained with LPCB stain. Additional advantage of the LPCB is that it can also detect blue coloured Cyclospora and Isospora. Of late.Textbook of Practical Microbiology 195 LESSON 66 LPCB Wet Mount of Stool LEARNING OBJECTIVES After completing this practical you will be able to: 1 Perform lacto-phenol cotton blue (LPCB) wet mount preparation of faeces for demonstration of intestinal parasites. ova and cysts of the intestinal parasites. . blue-colour stained cysts of intestinal protozoa and ova of eggs could easily be detected and identified. II Reagents and glass wares Microscopic slides. Measure 40 ml of glycerol and then transfer to the bottle. Glycerol in the LPCB provides a semi-permanent preparation. take a small portion of the stool specimen (match stick head size) and emulsify it in the drop of LPCB. 2 Identify intestinal protozoal cysts. INTRODUCTION Lacto-phenol cotton blue (LPCB) is a staining reagent which is extensively used in the examination of clinical specimens for demonstration of fungi and fungal elements. trophozoites. put a drop of LPCB on the glass slide. PRINCIPLE The LPCB is a combined fixative. the LPCB has also been used in the direct wet mount preparation of stool specimens for demonstration of larvae. cover slips. Label the bottle and mark it.05 gm of cotton blue. Measure 20 ml of lactic acid. Therefore. in the stool specimen by the LPCB wet mount REQUIREMENTS I Equipments Compound light microscope. applicator stick and lactophenol cotton blue (LPCB) reagent. III Specimen Fresh stool specimen is required for stool microscopy. Helminthic ova are stained such a deep blue that it is difficult to miss them even during screening with a low power objective. For daily use. mucus. leak proof. Add this to the solution and mix it well. Transfer the LPCB solution to a clean. students can transfer about 10 ml of LPCB solution to a small brown dropper bottle or insert a dropping pipette through the cap of a small brown bottle. and transfer it to a beaker (100 ml capacity). PROCEDURE 1 Take a clean glass slide. brown bottle. Weigh 0. 6 Record the findings with description of the morphological characteristics. Preparation of LPCB solution: Weigh 20 gm phenol crystals. 4 Put a cover slip over the LPCB suspension of the stool. 3 With the help of an applicator stick. Note: Examine at least 30 minutes after preparation of the wet mount. 2 With help of a glass dropper. It contains phenol and lactic acid which clears faecal debris. They are still recognisable as artifacts when the LPCB stain is used. Dissolve the solution by heating gently over a spirit flame. round.196 LPCB Wet Mount of Stool QUALITY CONTROL A LPCB wet mount preparation of known positive stool specimen for protozoal cysts.3 Hook worm egg.4 Fertilzed egg of Ascaris lumbricoides. 4 A 60µm × 40µm sized. The egg shell is stained blue and encloses the blue stained unsegmented ovum. structure seen with blue coloured 1–4 nuclei and glycogen mass. round oval shaped. FIGURE 66 . The unsegmented ovum and also the space between the shell and ovum at each pole are stained blue. . deep blue coloured structure seen with lightly stained shell membrane and segmented ovum with deep blue stained blastomeres. larva and helminthic eggs are prepared. 66-1). FIGURE 66 . 3 A 6–10 µm sized oval shaped deep blue coloured structure seen with visible axostyle and distinct cyst wall surrounded by a halo. Cyst of Entamoeba coli. FIGURE 66 .2 Cyst of Giardia intestinalis. 5 Fertilized egg of Ascaris lumbricoides (Fig. deep blue coloured. 6 Egg of Trichuris trichiura. histolytica / dispar. FIGURE 66 . 6 A 25µm × 50µm sized blue coloured. barrel -shaped structure seen with lightly stained mucus plugs at each pole. deep blue coloured structure seen with blue coloured 1–8 nuclei and diffuse glycogen mass. RESULTS AND INTERPRETATION 1 2 3 4 Cyst of Entamoeba histolytica/dispar (Fig. deep blue coloured structure seen with blue stained outer corticated thick cell wall. Hook worm egg (Fig. These preparations are observed and compared with the LPCB wet mount preparation of the test stool specimen for various morphological forms of the parasite and identified. 66-2). round. 66-3). 5 A 60–75 µm × 40–50µm sized. x 400. x 100. 66-4). 2 A 15–30 µm sized.1 Cyst of E. OBSERVATION 1 A 10–15 µm sized. A clear space is visible between the stained shell membrane and blastomeres. oval shaped. x 400. Cyst of Giardia intestinalis (Fig. x 400. A wet mount preparation should neither be too thick nor thin. Diagnostic Medical Parasitology.Textbook of Practical Microbiology 197 KEY FACTS 1 2 3 4 LPCB wet mount of stool is always examined at least 30 minutes after preparation of the wet mount. Dharan. Stool Microscopy. . 4th Edition. 2 Parija SC. Nepal. ASM press. BPKIHS. 2006.C. both bile-stained and non bile-stained helminthic eggs are stained blue. Giardia and Trichomonas. 2003. 3rd Edition. Washington D. LPCB kills the trophozoites of Entamoeba. hence can not be demonstrated by LPCB. 1998. VIVA 1 What are the uses of LPCB mount? 2 List the advantages and disadvantages of the iodine wet mount. 3 Parija SC. In LPCB preparation. Textbook of Medical Parasitology. All India Publishers and Distributors. FURTHER READINGS 1 Garcia LS. They do not get decolourised with the acid alcohol and hence do not take up the counter stain methylene blue. 7 Wash the slides with water to remove all traces of acid. 11 Record the observations in the note book.198 LESSON 67 INTRODUCTION Acid-fast Staining of Stool Smears LEARNING OBJECTIVES After completing this practical you will be able to: 1 Perform modified acid fast staining of faeces for demonstration of intestinal coccidian parasites. as counter stain. Both hot and cool modified acid stains have been used with equal sensitivity. and then under oil immersion (100x) objective. for 30 seconds. 6 Cover the smear with 5% aqueous sulphuric acid. The stool material in the background gets decolourised easily and take up the counter stain appearing blue. 10 Observe the smear first under low power (10x) objective. the diagnostic morphological form of the parasite. III Specimen Stool specimen. before declaring the smear negatives. Findings are recorded. 5 Wash the smears with tap or distilled water. The slide is allowed to stain for 9 minutes. 1 Make a smear of stool on a clean glass slide. The stained smear is compared with the control smear for appropriate oocyst morphology and staining appearance. REQUIREMENTS I Equipments Compound light microscope and Bunsen flame. are usually excreted in the human faeces.3% methylene blue (counter stain). 2 Heat fix the smears by heating at 70°C for 10 minutes. These oocysts are demonstrated by the modified acid fast staining technique as pink-coloured acid fast structures against a blue background. 8 Cover the smear with methylene blue. 3 Put the smears on a slide rack and flood the smear with carbol fuchsin. II Reagents and lab wares Inoculation loop. and 0. Cyclospora cayetanensis and Isospora belli cause infection of the gastrointestinal tract of humans. 5% aqueous sulphuric acid (decolouriser). which may be serious in the immunocompromised patients. 9 Rinse the smears again under tap water and air dry it. Oocysts. Note: The smear should be examined following a zig-zag pattern for at least 10–30 minutes. Do not allow the stain to dry on the slide. together with grading of the positive smear. as decolouriser. for 1 minute. and if necessary add more carbol fuchsin to cover the smear. parvum. PRINCIPLE The principle of modified acid fast staining is based on the fact that the oocyst of these coccidian parasites are acid fast and retain the basic dye (dilute carbol fuchsin)appearing pink. carbol fuchsin. namely Cryptosporidium parvum. PROCEDURE Intestinal coccidian parasites. 2 Detect and recognize the presence of acid fast oocysts in the stool smear stained by acid-fast method. In this chapter hot modified acid fast technique will be described. The infection causes diarrhoea. Acid fast parasites and parasitic components are listed in the box 67-1. 4 Heat the slides from below intermittently by Bunsen flame until the steam rises. . QUALITY CONTROL A known positive control stool smear stained by the modified acid fast staining method containing the pink coloured acid fast oocysts of C. Other parasitic components Scolices of Echinococcus granulosus. The non. 2 The acid fast structure is the oocyst of I. FIGURE 67. round to ovoid shaped acid fast structure measuring 8µm–10µm diameter seen against a blue background of stool smear. x 1000.Textbook of Practical Microbiology 199 BOX 67-1 ACID FAST PARASITES AND PARASITIC COMPONENTS The intestinal coccidian parasites Cryptosporidium parvum. x 1000. Some structures are acid fast while others are not. parvum. belli.2 Oocyst of I. 67-1). x 1000.intestinal coccidian parasites Toxoplasma gondii. FIGURE 67. KEY FACTS 1 Modified acid fast staining is a widely used method for the demonstration of the oocysts of intestinal coccidian parasites in stool. as infection is caused by ingestion of oocysts from hands. 67-2).3 Oocyst of C. Sarcocystis hominis. 3 Stool specimens should be handled carefully. . Eggs of Taenia saginata. parvum (Fig. 67-3). Note: Cyclospora oocysts are variably acid fast. 3 The acid fast structure is the oocyst of C. 6 Cryptosporidium oocysts are to be distinguished from yeast cells which are non acid fast and show budding and variable sizes. 5 Cyclospora oocysts are twice the size of the Cryptosporidium oocysts. belli (Fig. Cyclospora cayetanensis.1 Oocyst of C. FIGURE 67. 2 Red coloured elliptical shaped acid fast structure measuring 20µ m x 10µm seen against a blue background of stool smear. cayetanensis (Fig. 4 Oocysts of Cryptosporidium and Isospora are consistently acid fast in nature while Cyclospora is variably acid fast. Spores of Microsporidia. OBSERVATIONS 1 Red coloured spherical acid fast structure measuring 4-6µm diameter seen against a blue background of stool smear. RESULTS AND INTERPRETATION 1 The acid fast structure is oocyst of C. 2 It is an example of permanent staining of stool. cayatenensis. 3 Red coloured. Isospora belli. 3 Name the coccidian parasite associated with traveler’s diarrhoea. Textbook of Medical Parasitology. 4th Edition. Stool Microscopy. Sheather’s sucrose floatation method. 1998. Ans. Diagnostic Medical Parasitology. FURTHER READINGS 1 Garcia LS. 4 Name the stool concentration method for intestinal coccidian parasites? Ans.C. Nepal. 5 List all acid fast parasitic components. . Cyclospora cayetanensis and Cryptosporidium parvum. 2006. Washington D. 3rd Edition. 2 How are oocysts of Cryptosporidium differentiated from yeast cells in this staining technique? Ans. All India Publishers and Distributors. BPKIHS. 2003. Dharan. 3 Parija SC.200 Acid-fast Staining of Stool Smears VIVA 1 List the intestinal and non intestinal coccidian parasites. 2 Parija SC. ASM press. The features that differentiate the oocyst of Cryptosporidium from that of yeast cells in modified acid-fast staining are that oocyst of Cryptosporidium are acid-fast while that of the yeast cells are non-acid fast and are of variable sizes with budding. 4 ml is aliquoted into tubes and the water is evaporated. babesiosis. EDTA anticoagulated blood. 2 Detect and identify important parasites of blood such as malarial parasites (various morphological forms) and microfilaria of filarial worm. These include i). One type of stains (e.Textbook of Practical Microbiology 201 LESSON 68 INTRODUCTION Leishman’s Staining of Peripheral Blood Smears LEARNING OBJECTIVES After completing this practical you will be able to: 1 Perform Leishman’s staining of peripheral blood smear for malaria parasites and microfilariae. the number of parasites per field too are much less than that of thick blood smear. Then 0. Field. Leishman’s. These blood smears. Leishman’s stain. the thin films need prior fixation before staining with any of these stains. in addition to species identification. Thick and thin blood smears stained with Romanowsky’s stains are the permanent staining methods widely used for demonstration of blood parasites. African trypanosomiasis and leishmaniasis. The bottle is shaken until the powder is dissolved and allowed to stand for 48 hours with frequent shaking in between. etc. Haemoparasites are found in the peripheral blood smears of many parasitic diseases. their correct and accurate identification is possible only after permanent staining of blood smear. Thin blood films are of monolayer thickness. Examination of permanent stained blood smears is essential for detection and specific identification of blood parasites. PRINCIPLE As a rule thin blood smears are fixed by alcohol or other fixatives before staining.15 gram of Leishman’s dry powder in 100 ml of absolute methyl alcohol in a bottle. buffered distilled water and methyl alcohol. Chagas’ disease. Thick blood smear is primarily used as a screening procedure.g. detection and identification of these haemoparasites. on basis of their shape and motility. Therefore. The other type of reagents (e. The absence of fixation of the smears by methanol allows lysis of red blood cells and dehaemoglobinisation by aqueous stain solution. Preparation of EDTA anticoagulated blood: It is prepared by dissolving 5 grams of EDTA in 100 ml of distilled water. Two types of stains are used. II Reagents and lab wares Bunsen flame and clean glass slides. REQUIREMENTS I Equipments Compound light microscope. Microfilariae although can be identified in wet mount preparation of fresh blood. Preparation of Leishman’s stain: Leishman’s stain is prepared by dissolving 0. 70% ethyl alcohol. Thin blood films are used primarily for identification of species of malarial parasites. JSB stains) has only staining reagents. Blood can then be added to the anticoagulant or 20 mg of dry EDTA too can be added per tube. The parasites found in the peripheral blood smear are listed in the box 68-1. protozoal infections such as malaria. For malarial parasites. Wright’s. blood films help quantitate parasitaemia. as it allows examination of larger amount of blood. allows better identification of species. The procedure followed for staining thick smears are essentially the same as for thin smears except that the initial steps of fixation smears by methanol is not done. . Then blood can be added to it.) has both fixatives and staining reagents. Thick blood smears are several layers thick. It is a sensitive procedure for detection of parasites compared to thin smear. and loiasis. Disadvantage of thick blood smear is that species identification of parasites cannot be made. Giemsa. although simple and easy to prepare require experienced eyes for the screening. and ii) helminthic infections such as lymphatic filariasis. allows only detection of parasites.g. The disadvantage of thin blood smear is that as less amount of the blood is examined. 4 The Leishman stained thick blood smear shows the presence of coiled structure with a sheath and the absence of nuclei in the tail end. some of the enlarged RBC’s show within it. OBSERVATIONS 1 The blood film shows the presence of RBC’s identified as pale round cells with dense periphery and lighter core. c Monocytes have kidney shaped nucleus. b Lymphocytes are identified as having big nucleus pushing the cytoplasm to the periphery. neutrophils. . 2 Place a small drop of fresh blood on the clean microscopic slide. it may be helpful to prepare slides with both the thick and thin film on the same slides. These structures have rounded or pointed ends and are about one and half-time larger than the RBC.5 cm from the end of the slide. dilute the stain by adding twice as many drops of buffered distilled water. lyse the thick blood film with distilled water for dehaemoglobinisation. they should be first cleaned with detergent and then with 70% ethyl alcohol.202 Leishman’s staining of Peripheral Blood Smears III Specimen Fresh blood obtained by finger prick (or) EDTA anticoagulated blood. 12 to 24 darkly stained merozoites are found within the RBC’s. over an area of about 2 cm in diameter. 7 Examine the stained smear under oil-immersion objective (100 x) for detection of malarial parasites and microfilaria. 3 The blood film shows the presence of RBC’s. Note: If blood containing anticoagulant is used. In addition. typical crescent (banana) shaped structures are seen with dark blue cytoplasm. lymphocytes and monocytes. pigment and central chromatin. 2 Spread and mix the drops of blood with the corner of another slide. 4 After drying. In addition. 3 Allow the thick blood film to air-dry at room temperature in a dust free area. along the surface of the horizontal slide to the far end forming a ‘tongue shaped’ thin film. lymphocytes and monocytes. large amoeboid coarse haemazoin pigments occupying the entire RBC. and rinse it dry. PROCEDURE Leishman’s staining Preparation of thin blood smear 1 Take an alcohol cleaned and grease free slide. Note: With this type of preparation: a Sufficient time should be allowed for the thick portion of the smear to dry before staining. neutrophils. air-dry the film in a vertical position. 3 Allow the solution to stand for 15–20 minutes. 4 Push the second slide or the spreader across at about a 30° angle. 2 After 2 minutes. Preparation of combined thick and thin films In field surveys. Leishman stained thick and thin blood films for malarial parasites and microfilaria. and occupy the central area of the slide. 1 Flood the blood smear with 5–10 drops of the Leishman’s stain and allow to stand for 2 minutes. feathered end should be at least 2 cm long. Often 2 or more ring forms of the parasite are found inside a single RBC. 5 Allow the thin blood film to dry. lymphocytes and monocytes are also seen. b The thin film only must be fixed in absolute methanol before staining. The mixing is continued for about 30 seconds to prevent formation of any fibrin strands which may conceal the parasites in a stained smear. Note: The thin. stain the film with Leishman’s staining. with free margins on both sides. Note: If old slides are to be used. a Neutrophils in the blood films are identified by their multilobed nucleus. compact nucleus. neutrophils. 3 Touch the drop of blood with the edge of another slide and allow the blood to spread along the edge. Some of the normal sized RBC’s show blue ring-shaped parasite cytoplasm surrounding a central vacuole with a red coloured nucleus (1 chromatin dot) present at its centre. 2 or 3 drops maybe spread over an area of about 1 cm in diameter. is compared with the stained test blood films for similar morphology of the parasites. 2 The blood film shows the presence of normal RBC’s. Preparation of thick blood smear 1 Place two or three small drops of fresh blood (without any anticoagulant) on a grease-free alcohol cleaned slide. QUALITY CONTROL A known positive control. RBC’s. 6 After staining. Allow the solution to mix well. c Both the thick and thin blood films should be stained simultaneously. 5 After the step of dehaemoglobinisation. and then stain the smear with Leishman’s stain. about 1. 4 Wash the slide with buffered distilled water. 7 First screen the film with the low-power objective of the microscope for detection of microfilaria and examine at least two hundred microscopic fields using 100 x objectives for the presence of malarial parasites. In addition. 5 Examine the stained slide under the oil immersion objective (100 x) of the microscope. It does not serve the purpose of species identification. RESULTS AND INTERPRETATION 1 The blood film shows the presence of ring forms of Plasmodium falciparum (Fig. Plasmodium malariae. 3 Thick blood films should not be fixed because fixation with methanol prevents lysis of RBC’s and dehaemoglobinisation. 68-1). 4 Leishman stained thick blood smear with microfilaria of Wuchereria bancrofti (Fig.g. x1000. 4 Thick blood films helps in detection of microfilaria and malarial parasites. FIGURE 68-3 Erythrocytic schizont stage of Plasmodium vivax in the blood films. Microfilaria of Wuchereria bancrofti. Amastigote forms of Leishmania species. FIGURE 68-4 Leishman stained thick blood smear with microfilaria of Wuchereria bancrofti. 68-3).Textbook of Practical Microbiology 203 BOX 68-1 THE PARASITES FOUND IN THE PERIPHERAL BLOOD SMEAR In RBC’s Plasmodium vivax. Babesia species. 68-4).g. Giemsa. x1000.. 5 Thin blood films allows identification of malarial parasite to the species level. Jaswant Singh Bhattacharjee stain. x1000. Amastigote forms of Trypanosoma species. Field’s. Plasmodium ovale. Leishman stains. Plasmodium falciparum. In leucocytes In plasma FIGURE 68-2 The blood film shows the presence gametocyte of Plasmodium falciparum. 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. Wright’s. FIGURE 68-1 The blood film shows the presence of ring forms of Plasmodium falciparum. 2 The blood film shows the presence of gametocyte of Plasmodium falciparum (Fig. . and ii) Other type of stain which has only staining reagents e. Microfilaria of Mansonella perstans. 3 Erythrocytic schizonts of Plasmodium vivax in the blood films (Fig. Hence with the use of these stains. 68-2). x1000. Microfilaria of Brugia timori. Microfilaria of Loa loa. KEY FACTS 1 Always thin blood films should be fixed by alcohol or other fixatives before staining. Microfilaria of Brugia malayi. the thin films should be fixed before staining. FURTHER READINGS 1 Garcia LS. Stool Microscopy. . Chagas disease. Leishmaniasis. Wright’s stain. 4th Edition. Washington D. Clinical conditions in which examination of peripheral blood may be useful include: Protozoal infections Malaria.204 Leishman’s staining of Peripheral Blood Smears VIVA 1 Name different Romanowsky’s stains. Dharan. Thin smear: Allows examination of small amount of blood. BPKIHS. All India Publishers and Distributors. Smear prepared with 2-3 small drops of fresh blood spread over 2 cm diameters. Helminthic infections Lymphatic filariasis. 2006. Babesiosis. Made up of monolayer of RBCs. Diagnostic Medical Parasitology. It is several layers thick. Ans. 5 What are differences between thick and thin smears? Ans. Nepal. Different Romanowsky’s stains are Leishman’s stain. Ans. Jenner’s stain and Giemsa stain. 2 Name the parasites that can be found in the peripheral blood smear. Smear prepared with a small drop of blood spread along the edge. 3 Name two intra-erythrocytic parasites. 3 Parija SC. Plasmodium and Babesia. 2003. 1998. 3rd Edition.C. Textbook of Medical Parasitology. 4 What are the clinical conditions in which examination of a peripheral blood smear may be helpful? Ans. 2 Parija SC. ASM press. Thick smear: It allows examination of larger amount of blood. Loiasis. African trypanosomiasis. wooden spatula for both salt solution flotation and formalin-ether sedimentation procedure. After all the salt has dissolved completely without a trace. REQUIREMENTS I Equipments Microscope. III Specimen Stool. saline (for formalin-ether method) Preparation of saturated sodium chloride solution: Saturated sodium chloride solution is prepared by dissolving a spoonful of sodium chloride in 100 ml of distilled water. ether. a liquid with high specific gravity is used. glass funnel. applicator sticks. However. sodium chloride powder (for salt solution method). The concentrated parasites can be demonstrated in the wet mount preparation of the sediment. centrifuge and discarding jar. 30 ml glass vials. Broadly. PRINCIPLE In the standard salt solution flotation method. trophozoites cannot be concentrated by any of the concentration methods as they get killed. . In this method. more salt is added till it remains undissolved. 2 Formalin-ether sedimentation method for concentration of stool for parasitic ova and cysts. for formalin-ether method alone. measuring cylinder. and protozoal cysts can be concentrated. centrifuge tubes. Concentration of stool is recommended when direct examination of stool wet mounts fail to demonstrate any parasites particularly when the number of parasites in stool specimens are low. Pasteur pipette. Other floatation methods for stool concentration are listed in the table 69-1. Various concentration methods are available. This makes saturated sodium chloride solution. By concentration methods the helminthic ova and larvae. i) Lane’s saturated salt solution floatation method and ii) formalin-ether sedimentation method will be described. broad cover slips. The salt is dissolved in water using a spatula or a shaker. the two commonly used concentration methods. These two approaches are based on the principle of separating parasite from faecal debris and other materials by their differences in the specific gravity. This helps in separation of protozoal cysts and helminthic eggs from faecal debris.Textbook of Practical Microbiology 205 LESSON 69 INTRODUCTION Concentration of Stool for Parasites LEARNING OBJECTIVES After completing this practical you will be able to perform: 1 Saturated salt solution flotation method for concentration of stool for parasitic ova and cysts. In addition to the above. The principle of formalin ether sedimentation method is based on recovering helminthic eggs and protozoal cysts in the sediment of the faeces using centrifugation. the stool sample to be tested is mixed with formalin ether in a test tube and centrifuged. The advantages and disadvantages of both the salt solution flotation method and formalin ether sedimentation methods are summarized in the box 69-1. The eggs and cysts. In this method the eggs and cysts of many parasites float because specific gravity of these eggs and cysts are less than specific gravity of the saturated salt solution. II Reagents and lab wares Microscope slides. because of the centrifugal force settle down at the bottom of the centrifuge tube and form sediment. surgical gauze and stopper are required. In this chapter. they can be classified into two groups: flotation techniques and sedimentation techniques. saturated salt solution. Distilled water. 5%-10% formalin. The faecal debris are found at the bottom of the container. RESULTS AND INTERPRETATION 1. until the container is nearly full with continuous stirring. OBSERVATIONS 1 A 10-15µm sized spherical structure seen possessing 1-4 nuclei. i) first sediment layer at the bottom of the tube containing parasitic cysts or eggs. 6 Then place a broad cover slip over the glass vial. 3 A 6-10µm size oval ellipsoidal shaped structure seen with 4 nuclei remains of axoneme and flagella. 1 gram) in a 30 ml glass vial. Then with a Pasteur pipette. four layers are formed namely. ii) second layer of formol saline.206 Concentration of Stool for Parasites PROCEDURE Saturated salt solution flotation method 1 Take a walnut sized stool (approx. iii) third layer of faecal debris on the top of the formol saline layer and iv) lastly a top layer of ether. 7 Close the tube with a stopper and shake it well for 30 seconds. Add saline to the tube to bring the fluid level within several millimeters of the rim of the tube. and clear space between the shell membrane and blastomeres. aspirate a little of the sediment fluid and examine by making the saline and iodine wet mount preparations. 5 Centrifuge the tube again for 10 min at 500 g. 5 A 60-75µm × 40-50µm size rounded. 12 Mix the sediment with the fluid using an applicator stick. 2 Filter the faecal suspension through two layers of gauze in a funnel into a 15 ml centrifuge tube. and 3 ml of ether. 10 Remove the plug of faecal debris by piercing all around with an applicator stick. non bile stained structure seen with a thin transparent hyaline shell membrane enclosing 7-8 blastomeres. Suspend the sediment in saline. 11 Discard all the fluid into the discarding jar by one firm swing. Table 69-1 Other floatation methods for concentration of stool Zinc sulphate floatation method. 8 Remove the stopper carefully. Allow the tube to stand 5 min. Magnesium sulphate floatation method. 2. Fertilized egg of Ascaris lumbricoides. . 5. Formalin –ether sedimentation method 1 Take a half teaspoon of stool in a 15ml centrifuge tube containing 10 ml of 10% formalin. QUALITY CONTROL Wet mounts are prepared from a stool specimen known to be positive for ova and cysts and compare with the wet mounts obtained after the formol ether sedimentation technique for similar morphology of eggs and cysts. Cyst of Giardia intestinalis. Then focus under the high power objective (40x) to study the morphology and identification of the egg. Note: After centrifugation. 2 Add a few ml of saturated sodium chloride solution to the stool and mix it with a broomstick. 13 Examine the wet mount preparation first under low power objective (10 x) of the microscope for parasite eggs and cysts. Cyst of Entamoeba histolytica /dispar. 10 Examine the wet mount preparation first under low power objective (10x) of the microscope for parasite eggs. 9 Make a wet mount preparation with one or two drops adherent to the cover slip. nearly filling up to the brim of the tube. add a few more ml of salt solution and mix. 4 A 60µm × 40µm size oval shaped. Then focus under the high power objective (40x) to study the morphology and identification of the egg and cysts. 4. Note: Hold the tube in such a way that the stopper is held away from the face. Cyst of Entamoeba coli. 4 Discard the supernatant. 7 Allow it to stand for 20 minutes to 30 minutes. 3 Centrifuge the tube at 500 g for 10 min. so that it is in contact with the top of the meniscus. 3 When the stool suspension is smooth. 4 Remove any coarse material found floating by a broomstick 5 Add more salt solution little by little using a Pasteur pipette. until a convex meniscus is formed at the top of the container. taking care not to disrupt the debris. 8 Lift the cover slip from the meniscus with a steady but rapid movement and place on a clean microscope slide. 9 Centrifuge the tube at 500 g for 10 minutes. 3. leaving behind one or two drops of fluid with the sediment. Egg of hook worm. Note: The steps 2 and 3 are repeated. and allow it to stand for 30 minutes. Sheather’s sucrose floatation method. 6 Resuspend the sediment in 7 ml of 10% formalin. bile stained structure seen with a thick translucent shell with an albuminous coat containing a large conspicuous unsegmented ovum with a clear space at each pole. 2 A 15-30µm sized spherical structure seen possessing 1-8 nuclei. Textbook of Practical Microbiology 207 BOX 69-1 ADVANTAGES AND DISADVANTAGES OF THE CONCENTRATION METHODS Saturated salt floatation method Advantages Simple procedure. 3rd Edition.C. The stool concentration method employed for the recovery of coccidian parasites is the Sheather’s sucrose floatation method. 1998. formol saline is used to fix the cysts of protozoa and eggs. It increases sensitivity of stool microscopy. Textbook of Medical Parasitology. Disadvantages Not suitable for demonstration of unfertilized eggs of Ascaris. Stool Microscopy. BPKIHS. 5 In formol ether procedure. 2 Parija SC. . Diagnostic Medical Parasitology. Good method for concentration of parasitic eggs. 3 Parija SC. Good method for both cysts and ova. 4th Edition. ASM press. FURTHER READINGS 1 Garcia LS. These also kill the parasitic eggs and cysts. Dharan. Which is the stool concentration method employed for recovery of coccidian parasites? Ans. High specific gravity of the fluid may cause distortion of the morphology of the parasitic eggs and cysts. KEY FACTS 1 Concentration of stool is the method of choice. 3 Stool concentration by salt floatation and formol ether sedimentation are the two reliable methods of concentration of parasites in stool. operculated eggs of trematodes. Formalin-ether sedimentation method Advantages More sensitive method. Can be done at any level of the laboratory. Washington D. 2003. Disadvantages Technically cumbersome. thus unaltering morphology of these structures. Inexpensive method and easy to perform. 4 Salt floatation is a better method for concentration of helminthic eggs. 2 Trophozoites of protozoa cannot be concentrated by any of the concentration methods. Morphology of eggs and cysts preserved. larvae of Strongyloides and eggs of Taenia species. VIVA 1 2 3 4 5 6 What are the advantages of floatation method? What are the disadvantages of floatation method? What are the advantages of sedimentation method? What are the disadvantages of sedimentation method? Name the other floatation methods that can be employed as stool concentration method. Ether is used to dissolve and extract the fat and other debris in the stool. Least subjective to technical error. when the number of parasites in the stool specimens are less and cannot be demonstrated in stool wet mount preparation. Ether is highly inflammable and not easily available. Nepal. All India Publishers and Distributors. 2006. and antibiotics solution. 5 Autoclave the tubes at 15 lb pressure for 20 minutes. . 2 Then add 50 ml of Locke’s solution and shake the mixture until homogenous. Trophozoites of E. 10 Add antibiotics solution (penicillin. 6 Prepare a mixture of 8 parts of sterile Locke’s solution with 1 part sterile inactivated bovine serum. Balamuth’s. Axenic culture is not used for diagnosis by isolation of amoebae from the stool. Boeck and Drbohlav’s Lockeegg serum (LES) medium is a polyxenic medium commonly used for culture and isolation of the amoebae from the stool specimens for diagnostic purposes.02%) to the medium to inhibit the overgrowth of commensal bacteria present in the stool. histolytica usually appear in large numbers within 48 hours of inoculation. REQUIREMENTS I Equipments Bacteriological incubator and inspissator. 3 Dispense the medium in tubes. Axenic culture is a bacteria-free culture medium. In this chapter polyxenic culture of E. Preparation of polyxenic Boeck and Drbohlav’s medium: 1 Break four eggs into a sterile flask containing glass beads. which provide nourishments for the growing parasites.5 inches are produced at the bottom of the tube. such that a slant of 1 to 1. The polyxenic media are Boeck and Drbohlav’s Locke-egg-serum (LES) medium. after that they are washed and shells are wiped dry with 70% alcohol. For the cultivation of Entamoeba histolytica various media have been used. 4 Plug the tubes and place the tubes in a slant position in an inspissator at 70°C until the slant solidifies. This is used routinely for diagnosis of intestinal amoebiasis by isolation of the amoeba from the stool. The media have been classified broadly into two groups: polyxenic (polybacterial culture) and axenic (bacteria –free culture) medium. ii) testing antiamoebic drugs in vitro.hsistolytica in Boeck and Drbohlav’s medium will be described. PRINCIPLE Polyxenic culture medium is usually composed of egg and Locks’ solution mixture supplemented with serum. heat inactivated bovine serum. 8 Cover the slants of the medium to a depth of 1 cm with 6 ml to 8 ml of the sterile lock’s solutions. Nelsons’ or Robinson’s medium. screw cap tubes. II Reagents and lab wares Boeck and Drbohlav’s Locke-egg-serum (LES) medium. 1000 units/ml. starch and bacterial flora. III Specimen Stool in cases of suspected amoebic dysentery. 7 Sterilise the mixture by filtration and incubate at 37°C for 24 to 48 hours as a sterility check before use. This is used to study i) the pathogenicity of amoebae. and pus in cases of amoebic liver abscess.1 ml of 0. 9 Add a loopful of sterile rice powder/starch to each tube along with loopful of Escherichia coli colony. 0. streptomycin 2 mg/ml and acriflavine. iii) immunological properties of amoebic antigen and iv) prepare axenic amoebic antigen for use in the immunodiagnosis of amoebiasis.208 LESSON 70 INTRODUCTION Culture of Stool for Entamoeba histolytica LEARNING OBJECTIVES After completing this practical you will be able to: 1 Culture stool for Entamoeba histolytica. Textbook of Medical Parasitology. Axenic culture medium is employed to study pathogenicity of amoeba. OBSERVATIONS Presence of 8–30 µm structure. Dharan. 4th Edition. 1998. histolytica? List the polyxenic culture media and the uses of polyxenic culture of E. histolytica. 2003. KEY FACTS 1 2 3 4 5 Cultivation of E. What are the uses of axenic culture of E. ASM press. Washington D. QUALITY CONTROL A known positive culture of E. RESULTS AND INTERPRETATION Trophozoites of E. Axenic culture medium is not used routinely for culture and isolation of the amoebae from stools specimens. 2006.Textbook of Practical Microbiology 209 PROCEDURE 1 Inoculate a loopful of the stool sample or liver pus on to the slant of the two medium tubes. Stool Microscopy. leucocytes and tissue debris. histolytica is grown in the culture. 4 Examine the growth of amoebae by a wet mount preparation of culture fluid. 3rd Edition. FURTHER READINGS 1 Garcia LS. BPKIHS. and cytoplasmic inclusions such as red blood cells.1 ml of sediment under light microscope for characteristic motility of the trophozoites. Nepal. histolytica. All India Publishers and Distributors.C. finger-shaped pseudopodia. In polyxenic culture medium (polybacterial culture) bacterial flora serve as rich source of nutrients for the feeding amoebae. Note: Although the initial culture may appear negative. 2 Parija SC. Define axenic and polyxenic culture medium. addition of special vitamin mix provides a rich source of nutrition. 3 and 4 days by examining 0. 2 Incubate the tubes in an incubator at 37°C for 24 hours to 48 hours. clearly differentiated cytoplasm into ectoplasm and endoplasm. VIVA 1 2 3 4 Name the axenic culture medium used for culture of E. actively motile with hyaline. 3 Parija SC. In axenic culture medium (bacteria-free culture). histolytica already maintained by serial sub cultivation in the culture medium. 3 Observe the cultures regularly at 2. . subcultures may reveal amoebae. in vitro testing of efficacy of anti-amoebic drugs and preparation of axenic amoebic antigen for immunodiagnosis of amoebiasis. Diagnostic Medical Parasitology. histolytica can be done using both polyxenic and axenic culture medium. 210 . Textbook of Practical Microbiology UNIT 211 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 . The aged patients. 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 symbiotic relation of the fungi. Wet mount of clinical specimens is a very useful method for demonstration of fungi in clinical specimens. The fungal infections are not usually transmitted sexually like those of the viral. In addition many factors directly or indirectly decrease the accuracy of data on fatal mycoses cases. . like other organisms. commensalisms and parasitism. bacterial or parasitic diseases. Till date there is no ideal vaccine available to control any fungal infections. 3 Subcutaneous mycoses: The infection is due to the pathogenic organism of low virulence and usually following traumatic injury. Nosocomial blood stream infections are particularly serious and there is evidence to suggest that these infections are becoming more common. muscles and fasciae. Direct methods include the demonstration of fungi or their components in body tissues or fluids. Various wet mount preparation used in a mycology laboratory include lactophenol cotton blue (LPCB) wet mount. Fungal infections can be diagnosed by their demonstration.212 Introduction The fungi are now recognized as significant causes of morbidity and mortality. The immune response is rarely induced. However. the incidence of the fungal infections has increased enormously. the reported deaths from the fungal infections have maintained an approximately constant numerical ratio to the general increase in the population. subcutaneous tissues. Diagnosis of fungal infections is made by direct and indirect methods. isolation and final identification from clinical specimens. The prognosis among these patients is very poor and therefore an early diagnosis and treatment is essential. there has been comparatively little progress in understanding the pathogenesis of nosocomial fungal infections or in their prevention. mostly involving host debilitation. It involves the dermis. despite this increase. They have emerged as important etiological agents of opportunistic infections and full-fledged diseases. The nosocomial fungal infections have been recognized as a significant ground for adverse patient outcome and a major public health problem. diagnosis and treatment. can be divided into three modes of their existence namely mutualism. 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. Nosocomial mycoses have developed especially in association with or as a consequence of the extraordinary progress in the management of seriously ill patients. 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. potassium hydroxide (KOH) wet mount and India ink preparation. 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. Today. Simultaneously. due to underlying predisposing factors such as immunocompromised situations. autoclave/hot air oven for sterilization. urine. 71-1). PRINCIPLE Clinical specimens should be processed promptly and plated on to isolation media as a means to recover fungi that may be causing disease. hair. RESULTS AND INTERPRETATION Growth of fungal colonies in the tube inoculated with test specimen indicates the patient is infected with fungus (Fig. OBSERVATIONS Observe both the test tubes and control tubes everyday for upto 30 days. glass marking pencil. biopsied materials and CSF. The control strains should be used to grow and see if growth is perfect in prepared medium. bronchial washings. . 2 Take processed specimen and inoculate a loop full of specimen in each tube. Sabouraud’s dextrose agar (SDA) (Fig. III Specimen Skin and nail scrapings. inoculating loop. QUALITY CONTROL Prepared media should be incubated and then used to ensure sterility. 2 Isolate fungi from clinical specimens in the laboratory. Quality of sterilization should be checked periodically and appropriate pH should be adjusted for best results. Bunsen burner. REQUIREMENTS I Equipments Water bath. Appropriate media and incubation temperatures are selected to allow for the growth of pathogenic and opportunistic yeasts and fungi. appropriate sterile media (Table 71-1). sputum.Textbook of Practical Microbiology 213 LESSON 71 INTRODUCTION Cultivation of Fungi LEARNING OBJECTIVES After completing this practical you will be able to: 1 Select appropriate medium to grow fungi from clinical specimens. pH meter or pH paper. 1 Observe for fungal colonies. 2 Tubes growing bacterial colonies are discarded. Different kinds of media are available to grow and isolate different types of fungi. PROCEDURE 1 Choose and take two appropriate sterile medium tubes and label RT (room temperature) and 37°C. and exudate from lesions. II Reagents and lab wares Glassware for preparation of media. tubes for aliquots of media. 3 Incubate RT tube at room temperature and 37°C tubes at 37°C temperature. 71-1) is the most frequently used media in a diagnostic mycology laboratory (Box 71-1). List of different media used for culture of fungi are summarized in the table 71-1. potato glucose agar. trypticase soy agar and urea agar. Sabouraud’s dextrose agar with antibiotics It consists of 20 gm of glucose/dextrose. KEY FACTS 1 2 3 4 Choose appropriate medium based upon clinical history of the patient. FURTHER READINGS 1 Collier L.0. Sahm DF and Weissfeld AS. It contains 40 mg of chloramphenicol and 40 mg of cycloheximide (actidione).214 Cultivation of Fungi Table 71-1 List of media used for fungal culture Sabouraud’s dextrose agar (SDA) Sabouraud’s dextrose agar with antibiotics Ascospore medium Bennett’s agar for Nocardia Blood agar base Casein agar base for identification of Trichophyton species Casein agar for Nocardia Casitone medium of Howell and Pine for Actinomyces Carbohydrate broth for Candida Czapek – Dox medium 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 BOX 71-1 SABOURAUD’S DEXTROSE AGAR Sabouraud’s dextrose agar It consists of 40 gm of glucose / dextrose. St. VIVA 1 What are the different types of media used in fungal culture? 2 What are the most commonly used media in laboratory for routine diagnosis of fungal infections? Ans. 9th Edition. Always inoculate in duplicate tubes and incubate one at room temperature and another at 37°C. FIGURE 71-1 SDA with Candida albicans colonies. Most media used for fungal culture are sterilized by autoclaving at 15 lbs for 15 mins.5 – 6. The media that are prepared can be tested to see for its proper working by inoculating standard strains of appropriate fungi and incubating them both at 37°C and at room temperature. Interprint. Sterilised by autoclaving. Topley and Wilson’s Microbiology and Microbial Infections. 3 How do you sterilize different media used for culturing fungi? Ans. 4 How do you test for the proper working of different media? Ans. Volume 4. 711. Mycology. 2000.Dox medium. A Text Book of Medical Mycology. Sussman M. 10 gm of neopeptone and 35 gm of agar in 1000 ml of distilled water. 3 Jagadish Chander. pH is 7. 10 gm of neopeptone and 20 gm of agar in 1000 ml of distilled water. Media should be sterile. pp. Sabouraud’s dextrose agar (SDA) is the most frequently used media in a diagnostic mycology laboratory The other commonly used media include carbohydrate broth.0. (The CV Mosby Company. Bailey and Scott’s Diagnostic Microbiology. pH should be appropriate. . Balows A. gelatin medium. 2 Forbes BA. Arnold publishers. Czapek. Louis) 2002. 11 th ed. pH is 5. The appearance of purple coloured Gram positive bacteria and pink coloured Gram negative bacteria in the control smear indicates proper staining technique and stained test smear is compared with it. 7 Rinse the smear again gently under tap water. REQUIREMENTS I Equipments Compound light microscope. II Reagents and lab wares Bunsen flame. 95% ethanol (decolourising agent). 9 Rinse the smear again gently under tap water and air dry it. 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 end of the slide test smear is made. 11 Record the observations in the note book. PRINCIPLE The Gram reaction is dependent on the permeability of the fungal cell wall. 3 Rinse the smear gently under tap water. 8 Cover the smear with dilute carbol fuchsin for 30 seconds to 1 minute. 2 Cover the smear with the methyl violet. PROCEDURE I Preparation of fungal smear: 1 Take clean. spread the cell suspension into a thin area. 4 Allow the smear to air dry.Textbook of Practical Microbiology 215 LESSON 72 INTRODUCTION Gram’s Staining for Fungi LEARNING OBJECTIVES After completing this practical you will be able to: 1 Stain fungal smears by Gram’s staining method. Fungal material appears Gram positive. Gram’s iodine (mordant). 5 Rinse the smear again gently under tap water. 10 Observe the smear first under low power (10x) objective. 6 Decolourise the smear with 95% alcohol for 15 to 20 seconds. and grease free glass slides for making the smears. The slide with control and test smears is stained by Gram’s staining. QUALITY CONTROL On the same slide. and quickly passing the smear over the flame of Bunsen burner two to three times. and then under oil immersion (100x) objective. Sabouraud’s dextrose agar and place on the clean slide with a bacteriological loop. albicans culture on . methyl violet (basic dye). Allow it to stand for one minute. 4 Cover the smear with Gram’s iodine and allow it to stand for one minute. 2 Take one or two loopful of C. to the dye-iodine complex like those of bacteria (refer chapter 7). and 1% safranine or dilute carbol fuchsin (counter stain). III Specimen Candida albicans culture on Sabouraud’s dextrose agar. II Staining Procedure 1 Heat fixes the smear by passing the slide 2–3 times gently over the flame with the smear side up. loop wire. 3 Then with circular movement of the loop. 5 Heat fix the smear while holding the slide at one end. Gram stain was devised by Christian Gram in 1884 as a method of staining bacteria in tissues. . 2 Forbes BA. 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. Volume 4. Topley and Wilson’s Microbiology and Microbial Infections. Balows A. 2000. C. Louis) 2002. Mycology. albicans (Fig. 3 Jagadish Chander. St. 11 th ed. Sussman M. RESULTS AND INTERPRETATION The stained smear contains Gram positive yeast . Arnold publishers. KEY FACTS 1 Gram staining is a differential stain. Sahm DF and Weissfeld AS. 9th Edition. pp. 711. A Text Book of Medical Mycology. 72-1). Bailey and Scott’s Diagnostic Microbiology. Interprint. FURTHER READINGS 1 Collier L. (The CV Mosby Company. x 1000. FIGURE 72-1 Gram stained smear of Candida albicans.216 Gram’s Staining for Fungi OBSERVATION Presence of Gram positive budding yeast cells. 2 Demonstrate fungi and fungal elements under the microscope in LPCB wet mount preparation. In case of scotch tape preparation. .05 gram cotton blue. LPCB wet mount of both Scotch tape preparation and tease mount preparations will be described. and 3 Slide culture preparation. chapter 75) is the most advantageous in that. 3 With a needle. and 40 ml of glycerol to 20 ml of distilled water. PRINCIPLE Identification of filamentous fungi is made by their characteristic microscopic morphology such as shape. Tease mount preparation. Remove a small portion of the colony and the supporting agar at a point between the centre and periphery and place it in the drop of LPCB. 20 ml of lactic acid. Fungal infections can be diagnosed by their demonstration in clinical specimens. Slide culture (Ref. Add 0. III Specimen Rhizopous culture on Sabouraud’s dextrose agar. Tease mount preparation 1 Place a drop of LPCB on a clean glass slide.Textbook of Practical Microbiology 217 LESSON 73 INTRODUCTION Lactophenol Cotton Blue (LPCB) Wet Mount II Reagents and lab wares Glass Petri dishes. tease the fungal culture first and spread in the LPCB. slide. the fungus as it grows on the medium can be visualized. 4 Examine the preparation under 10x and 40x of a light microscope. 2. lactophenol cotton blue (LPCB) stain. Dissolve the ingredients by heating the container in a hot water bath. straight/bent wire and needle. place one drop of LPCB. There are three different preparations of LPCB mounts as mentioned below: 1 Scotch tape preparation. Preparation of Lactophenol cotton blue stain: Weigh and add 20 gm of phenol crystals. the fungal elements are disturbed but finer details of the fungus can be visualized. LPCB staining wet mount is the most commonly used method adopted in a mycology laboratory to identify filamentous fungi. PROCEDURE Scotch tape preparation 1 On a clean glass slide. LEARNING OBJECTIVES After completing this practical you will be able to: 1 Prepare lactophenol cotton blue (LPCB) wet mount of fungal colony. usually 30 mins. size. In this chapter. 2 Touch the adhesive side of the tape of transparent scotch tapes on the surface of the colony at a point intermediate between its centre and periphery. cover slip. the fungal elements are undisturbed whereas in tease mount preparation. 4 Examine microscopically after giving sufficient time for the structures to take up the stain. 3 Fix the adhesive side of the tape over an area on the glass slide containing the LPCB. arrangement of spores and hyphae. 2. REQUIREMENTS I Equipments Microscope. 711. x 400. 4 What is the other use of LPCB apart from examination of fungi? Ans. 2000. LPCB is also used in wet mount of stool for intestinal parasites. 11 th ed.218 Lactophenol Cotton Blue (LPCB) Wet Mount QUALITY CONTROL Preparation of lactophenol cotton blue should be done by heating the ingredients in a waterbath. OBSERVATIONS 1 The stained preparation should be observed under 10x or 40x. b Phenol: Acts as disinfectant. This should be thoroughly differentiated and the fungus identified. 9th Edition. 2 Fungi appear as dark blue stained mycelium (Fig. pp. KEY FACTS 1 The fungal culture should be first is teased well and then spread in LPCB for better results. Fast growing fungi in case of slide culture preparation will give satisfactory results in 24–48 hr. Sahm DF and Weissfeld AS. types of morphological structures including hyphae and spores. Mycology. Louis) 2002. Bailey and Scott’s Diagnostic Microbiology. . d Glycerol: Hygroscopic agent. VIVA 1 What is lactophenol cotton blue and how is it prepared? 2 What are three different methods of wet mounts used and their advantages in a lactophenol cotton blue preparation? 3 What are the functions of each component of lactophenol cotton blue stain? Ans. A Text Book of Medical Mycology. RESULTS AND INTERPRETATIONS The fungal element grown should be observed and results interpreted depending on the morphology of the hyphae and the spores. St. Interprint. 3 Jagadish Chander. for presence of mould. Topley and Wilson’s Microbiology and Microbial Infections. Sussman M. a Lactic acid: Helps in preserving the morphology of the fungal element. Balows A. (The CV Mosby Company. Different fungi under LPCB wet mount will show different FIGURE 73-1 LPCB mount of fungi. 73-1). Arnold publishers. 2 Forbes BA. It prevents drying. c Cotton blue: Stains the fungal elements. FURTHER READINGS 1 Collier L. Volume 4. etc. 3 Cover the smear with the cover slip. 4 Leave it for 5–10 min. .) which can be clearly seen in a KOH wet mount. septate. granules. Bunsen flame and 10% KOH. Different fungi will have different morphological forms (yeasts. respiratory specimen. corneal scrapings. skin scrapings. needle. 5 Examine the slide under low (10x) and high power (40x) magnifications 6 Examine the slide for 15–20 min. The potassium hydroxide (KOH) wet mount preparation is very useful for the presumptive diagnosis of the type of fungal infection. slide. Interpretation of results should be done by critical analysis of the type. III Specimen Pus from draining sinuses. and aseptate hyphae. cells with pseudo hyphae. 2 Emulsification of specimen should be homogenous in KOH solution. REQUIREMENTS RESULTS AND INTERPRETATIONS I Equipments Microscope. PROCEDURE 1 Emulsify the specimen in a drop of 10% KOH on a microscopic slide with the help of a loop.Textbook of Practical Microbiology 219 LESSON 74 INTRODUCTION Potassium Hydroxide Wet Mount LEARNING OBJECTIVES After completing this practical you will be able to: 1 Prepare potassium hydroxide (KOH) wet mount of clinical specimens. The procedure also helps in the selection of appropriate culture media for the isolation of etiological fungal agent. straight/bent wire. OBSERVATIONS Shining fungal elements shall be observed in microscopy of the clinical specimens. aspirate from nasal sinuses. material from external ear. 2 Demonstrate the presence of fungal elements in the given clinical specimen by KOH wet mount preparation. hair. etc. 2 Apply gentle heat by passing the slide over a Bunsen flame for 3–4 times. size and color of fungal elements which will be different for different fungi. QUALITY CONTROL 1 10% KOH should be prepared at the right concentration. budding. for demonstration of shining fungal elements. nail scrapings. PRINCIPLE The KOH clears out the background scales or cell membranes that may be confused with fungal hyphal elements in microscopy of clinical specimens. Gentle heating also accelerates clearing of artifacts. II Reagents and lab wares Glass Petri dishes. cover slip. 2 Forbes BA. Volume 4. 232. Topley and Wilson’s Microbiology and Microbial Infections. 2 The KOH should be prepared at the right concentration (10%). Balows A. Interprint. 2000. 711. VIVA 1 What percentage of KOH should be used? 2 What is the function of KOH? 3 How do you interpret results of a KOH wet mount? FURTHER READINGS 1 Collier L. . A Text Book of Medical Mycology. 3 Emulsification of specimen should be homogenous. St. 3 Jagadish Chander. Sahm DF and Weissfeld AS. Mycology.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. Arnold publishers. (The CV Mosby Company. pp. pp. Louis) 2002. 11 th ed. Bailey and Scott’s Diagnostic Microbiology. Sussman M. 9th Edition. in cerebrospinal fluid (CSF) is a very useful procedure for diagnosis of meningitis caused by C. 5 Blot dry the excess fluid. 6 Examine the slide under low (10x) and high power (40x) magnifications Capsule is a protective layer found around some bacteria and some fungi like C. OBSERVATIONS The India ink preparation is observed under microscope and noted for presence of clear halo around yeast cells (Fig. An early diagnosis will help for prompt treatment of the condition. the background appear black. preparations with such appearance can be confirmed to have yeast cells with capsules and the organism may be identified as C. Place a cover slip over the preparation. 2 Demonstrate the presence of capsulated yeast. PROCEDURE 1 2 3 4 Put a drop of CSF on the microscopic slide. 2 Care should be taken not to trap air bubbles which will mimic capsules of yeast cells. taking care not to trap air bubbles in the preparation.Textbook of Practical Microbiology 221 LESSON 75 INTRODUCTION India Ink Preparation LEARNING OBJECTIVES After completing this practical you will be able to: 1 Prepare India ink wet mount of cerebrospinal fluid (CSF). loop wire and 0. II Reagents and lab wares Microscopic slide. Cryptococcus neoformans in the CSF. neoformans. Emulsify the specimen with India ink on the slide. x 400. neoformans. . PRINCIPLE India ink is used as a negative stain preparation. 75-1). RESULTS AND INTERPRETATION Since India ink stains the background and leaves a clear halo around the cells. cover slip.5% India ink in distilled water. FIGURE 75-1 India ink preparation showing Cryptococcus neoformans with capsule. and the unstained capsule of C. especially in an emergency conditions. Hence demonstration of capsule by India ink preparation. When used in wet mount preparation of the CSF. glassware. REQUIREMENTS I Equipments Microscope.5%. neoformans. III Specimen Cerebrospinal fluid (CSF). neoformans appears as a white halo around the yeast cells in microscopy. QUALITY CONTROL 1 India ink preparation in distilled water should be made exactly to 0. Put a drop of India ink to the CSF on the microscopic slide. Arnold publishers. Sussman M. trapped between the cover slip and slide. 2 Forbes BA. neoformans in CSF is significant in emergency mycology laboratory for prompt administration of antifungal agents. A Text Book of Medical Mycology. . 9th Edition. 3 How to interpret results of India ink preparation? Ans. Interprint. Topley and Wilson’s Microbiology and Microbial Infections. The India ink preparation should be critically interpreted for the presence of a clear capsule around the yeast cell. (The CV Mosby Company. St. 11 th ed. 711. 2000. Louis) 2002. Volume 4. India ink preparation is one of the best methods to demonstrate the presence of capsule in case of capsulated yeasts. The demonstration of capsulated yeast like C. VIVA 1 What is the percentage of India ink used? 2 What is the advantage of India ink preparation and how is it useful in emergency mycology laboratory? Ans. Mycology.222 Indian Ink Wet Mount Preparation KEY FACTS A clear distinction should be made between capsules and air bubbles. pp. Bailey and Scott’s Diagnostic Microbiology. FURTHER READINGS 1 Collier L. This should not be confused with any air bubble that might be present. 3 Jagadish Chander. Balows A. Sahm DF and Weissfeld AS. but not to excess. 1 glass cover slip on top of the filter paper. The aerial hyphae including the conidiophores will be seen to grow along the undersurface of the cover slip PRINCIPLE Slide culture is a very useful technique in identification of the type of fungi. 3 With a probe. the tips of which have been flamed. stained and observed under a microscope for identification of the fungi. when incubated on a suitable growth medium. (b) Place a V-shaped glass rod on the filter paper. Several of these setups should be kept ready on hand. 6 Incubate the slide culture at room temperature. III Specimen Fragments of mould to be cultured is used as the specimen. (d) Wrap the preparation in Kraft 20 paper for sterilizing in hot air oven. The fungal element that is to be identified will produce characteristic hyphae and spores. This can be visualized undisturbed using this technique. place an agar block in the centre of the slide in the slide culture set up. 8 When growth appears peneath the cover slip. 4 With forceps.Textbook of Practical Microbiology 223 LESSON 76 INTRODUCTION Slide Culture LEARNING OBJECTIVES After completing this practical you will be able to: 1 Perform slide culture of the fungal preparation. if present. place one piece of filter paper slightly smaller in diameter than the Petri dish. 2 Sterile test tube with rimless mouth and an inside diameter of approximately 15 mm. The mould that is to be cultured is inoculated onto a small piece of an agar below a cover slip. An entire fungal colony can be demonstrated within a short period of time with the use of minimum materials. 76-1). (a) Into a Petri dish. or with a spatula. Slide culture is a very useful technique to study undisturbed morphological details of fungi. thoroughly moisten. REQUIREMENTS I Equipments 1 Slide culture set (Fig. 2 Mount the cover slip after the growth of fungus. 9 Place the slide on the microscope stage and examine. (c) Place a 1 by 3 inch glass slide and a 22 mm square. place the cover slip on the agar block. The cover slip after incubation is lifted. . 7 Remove the slide culture from the Petri dish and dry the bottom of the slide with a tissue. PROCEDURE 1 From the Petri dish containing Sabouraud’s agar cut out one square cm block of agar for each slide culture to be inoculated. Take a slide place a drop of LPCB. 3 Standard laboratory glassware. particularly relationship between reproductive structures like conidia. 5 With a pipette. three to four fragments of the mold to be cultured. No. 2 With the flat side of a sterile bacteriological loop. inoculate around the periphery of the agar block. II Reagents Sterile distilled water and Petri dish containing Sabouraud’s agar (or other medium of choice) to a depth of 2 mm. 3 Demonstrate fungal morphology without disturbing the aerial hyphae and conidiophores. The whole setup is kept in a Petri dish with moisture. the filter paper with sterile distilled water. place the cover slip removed from the block on the LPCB. 232. 3 What are the main advantages of slide culture? Ans. VIVA 1 What are the requirements for slide culture? Ans. pp. 1 by 3 inch glass slide. (The CV Mosby Company. Sabouraud’s agar (or other medium of choice) to a depth of 2 mm and standard laboratory glassware and. Balows A. Topley and Wilson’s Microbiology and Microbial Infections. 3 Jagadish Chander. Louis) 2002. staining characters etc. is needed before a slide culture shows growth of aerial hyphae. Sussman M. The requirements for slide culture include Petri dish. Bailey and Scott’s Diagnostic Microbiology. 2 When is slide culture used in routine diagnosis of fungal infections? Ans. 22 mm square. V shaped glass rod. FIGURE 76-1 Slide culture set. RESULTS AND INTERPRETATION Small spore bearing fungi make beautiful permanent mounts. if present.. Slide culture is a convenient method to demonstrate an entire colony without disturbing the aerial hyphae and conidiophores of fungi such as Aspergillus species. . With the type of hyphae. Some large spore bearing organisms like Microsporum gypsum do not stain as well. Mycology. Arnold publishers. No. the final interpretation of the fungal type can be made. Sahm DF and Weissfeld AS. OBSERVATIONS Usually a minimum of 48 hr. Volume 4. 3 Sterility should be maintained to the maximum. arrangement of conidiophores. A Text Book of Medical Mycology. 711. Penicillium species etc. pp. sterile distilled water. Kraft 20 paper. 2 Distilled water to be used should be checked for sterility .Slide culture can be used in routine diagnosis of fungal infections when examination of the entire fungal colony is required without disturbing the aerial hyphae and conidiophores. the culture may be examined after 48 hours incubation and as frequently thereafter as necessary. 2 Forbes BA. piece of filter paper slightly smaller in diameter than the Petri dish.224 Slide Culture QUALITY CONTROL 1 All the materials should be sterilized and checked for sterility before use. St. 11 th ed. 15 mm. 3 Components of the medium used should be adjusted according to standard procedure. 1 glass coverslip. KEY FACTS 1 The Petri dish chamber should be always moist. 2 Agar used should support growth of the suspected fungus. FURTHER READINGS 1 Collier L. Thus. 2000. Interprint. 9th Edition. Slide culture is a technique used to study an entire fungal colony within a short period of time with the use of minimum materials. sterile test tube with rimless mouth and an inside diameter of approx. However. albicans and C. urine. 6 Incubate all the tubes at 37°C for a maximum of 1½ hrs. 2 Add 0.stellatoidea and rarely C. . II Reagents and glass wares Standard laboratory glassware. albicans single colony by using a sterile loop. and mix it with serum in the test tube 3. Candida species can cause a wide variety of opportunistic infections. take a half of C. foetal calf or rabbit serum. PRINCIPLE Germ tube is an initial hypha from a sprouting conidia. 3 Take a half of a single colony to be tested by using a sterile loop. and test tubes 12×75 mm. The suspension is then incubated at 37°C for a minimum of 1½–2 hours. 4 Control organisms should be tested individually for production of germ tubes. nail. 7 Place one drop of suspension from tube 1. and 3 onto 3 different slides and place cover slips over the drops. 24 hour culture of known strains of C. the whole field under the cover slip is examined for any cell showing production of germ tube (Fig. in case of immunocompromised individuals. 77-1).Textbook of Practical Microbiology 225 LESSON 77 INTRODUCTION Germ Tube Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Demonstrate the production of germ tube by Candida species. and mix it with serum in the test tube 2. after which a drop is examined under the microscope for the germ tube. etc. fetal calf or human serum with a small quantity of growth of Candida species. It is very widely used in diagnostic laboratories because of its reproducibility. albicans isolated from the patients with antifungal drugs or patients with cancer do not produce germ tubes. human. These may be isolated from respiratory secretions. tropicalis. some strains of C. vaginal secretions. skin. REQUIREMENTS I Equipments Microscope. spore or yeast. QUALITY CONTROL 1 Positive control for germ tubes: C. 4 Similarly. parapsilosis single colony by using a sterile loop. albicans. 5 Similarly. of serum to each of the test tube. parapsilosis. PROCEDURE 1 Take three test tubes and label as 1. albicans. Formation of germ tube can be demonstrated by inoculating rabbit. III Specimens 24 hour culture of suspected fungal colony on Sabouraud’s dextrose agar to be tested.5ml. C. Germ tube test is a very simple and efficient test to distinguish pathogenic Candida from non-pathogenic ones. 2. 3 Human or rabbit serum should be checked for contamination prior to use. gastric washings. OBSERVATIONS Under the microscope. 2 Negative control for germ tubes: C. 8 Examine the slide under low (10x) and high power (40x) magnifications. and certain other situations (Box 77-1). Germ tubes are produced by C. 2 and 3. At times. Under normal conditions Candida species are not pathogenic. parapsilosis colony on Sabouraud’s dextrose agar. take a half of C. and mix with serum in the test tube 1. stool. Candida species are usually found as normal flora of the oral cavity and gastrointestinal tract of man. Tube 2 contains C. The drop from tube 1 should be read and compared with these controls. BOX 77-1 PREDISPOSING FACTORS FOR CANDIDIASIS Immunosuppression Long term antibiotic therapy Use of oral contraceptives Pregnancy Premature birth Obesity Diabetes mellitus Immunocompromised status Table 77-1 Differences between germ tubes and pseudohyphae Germ tubes No constriction at the site of attachment. 9th Edition. 711. RESULTS AND INTERPRETATION Tube 2 will show production of germ tube and Tube 3 will not. Pseudohyphae Constriction at the site of attachment. Septate and not necessarily with parallel sides. VIVA 1 What is a germ tube and how is it significant in routine identification of fungi? 2 Why is germ tube test important in a mycology laboratory? 3 What are the advantages of a germ tube test? FURTHER READINGS 1 Collier L. albicans isolated from the patients with antifungal drugs or patients with cancer do not produce germ tubes. FIGURE 77-1 Germ tube test. Bailey and Scott’s Diagnostic Microbiology. Topley and Wilson’s Microbiology and Microbial Infections. This should be differentiated from pseudohyphae (Table 77-1). pp. some strains of C.stellatoidea and rarely C. x 400. tropicalis. . albicans and few other species which are pathogenic. Arnold publishers. Volume 4. Louis) 2002. albicans. Sahm DF and Weissfeld AS. Non-septate with parallel sides. A Text Book of Medical Mycology. Rabbit. 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. KEY FACTS 1 2 3 4 Germ tube is a useful test to identify C.226 Germ Tube Test Germ tubes are seen as long tube like projections extending from yeast cells. Interprint. 11 th ed. (The CV Mosby Company. foetal calf or human serum can be used for demonstrating germ tube formation. Sussman M. At times. C. tropicalis may show germ tube formation after 3 hours with a constriction at the base of the germ tube. albicans and hence shows germ tube production while tube 3 does not show production of germ tube since it contains C. 3 Jagadish Chander. St. 5 C. 2 Forbes BA. Balows A. parapsilosis. 2000. Germ tubes are produced by C. Mycology. The un inoculated medium is colour less. Ammonia reacts in solution to form ammonium carbonate. Some fungi produce the enzyme urease that hydrolyses the urea releasing ammonia into the medium. leading to an increase in the pH. An un inoculated medium is incubated along with the test to compare the colour change. REQUIREMENTS I Equipments Incubator. 4 Observe any change of colour in the inoculated medium. fungi from culture tube. PROCEDURE 1 Pick up one colony of C.Textbook of Practical Microbiology 227 LESSON 78 INTRODUCTION Urease Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Demonstrate production of the enzyme urease by the fungus Cryptococcus neoformans. III Specimen C. When positive. Negative control: Candida albicans and Trichophyton rubrum (Urease negative fungi). which is alkaline. OBSERVATION Examine after 2 days of incubation. the enzyme produced by the fungi. . after incubation. PRINCIPLE Urea is a diamide of carbonic acid. medium shows growth of the colony and the color of the test medium as well as positive culture medium changes to purple pink. the colour of the medium changes to purple pink. thus indicating the presence of urease activity. and Christensen’s urea agar slope. No colour change is seen in negative culture medium. In a positive test. Urease. QUALITY CONTROL Positive control: Trichophyton mentagrophytes (urease positive fungi). 3 Incubate the tube at 37°C for 2 days. II Reagents and lab wares Inoculating wire. hydrolyses urea and releases ammonia and carbon dioxide. RESULTS AND INTERPRETATION Positive reaction is detected within 1 to 2 days of incubation. Bunsen flame. neoformans. Ammonia in turn produces a change in the pH of the medium that can be detected by the colour change in the indicator dye. The test is considered negative if no colour change of the test medium is observed. 2 Inoculate Christensen’s urea agar slope with these fungal colonies. The test should not be considered negative till after 2 days of incubation. Phenol red that is incorporated in the medium changes its colour from yellow to red in alkaline pH. This test can be used to differentiate different groups of fungi. neoformans culture. test tubes. 9th Edition. (The CV Mosby Company. thus indicating the presence of urease activity. Bailey and Scott’s Diagnostic Microbiology. VIVA 1 What is the medium used in urease test? 2 What is the principle of urease test? 3 Give examples of urease positive fungi? FURTHER READINGS 1 Collier L. St. 5 Observe the growth of inoculum irrespective of change in colour. Sahm DF and Weissfeld AS. . 2 Phenol red that is incorporated in the medium changes its color from yellow to red in alkaline pH. Louis) 2002. Interprint. 11 th ed. Topley and Wilson’s Microbiology and Microbial Infections. Sussman M.228 Urease Test KEY FACTS 1 Certain fungi possess the enzyme urease that hydrolyzes urea releasing ammonia into the medium. 2 Forbes BA. Mycology. A Text Book of Medical Mycology. 2000. Volume 4. pp. Arnold publishers. 3 Jagadish Chander. 3 Always check the sterility of the slants before inoculation. Balows A. 711. 4 An un inoculated medium must be incubated along with the test. filter paper discs. 4 Remove the excess fluid and allow the surface of the agar to dry. PRINCIPLE Yeast and yeast-like fungi are identified by the pattern of carbohydrate assimilation. utilization of carbohydrate is used as a definitive diagnosis for yeast or yeast-like fungi. and filter paper discs containing different carbohydrates III Specimens Pure growth of test fungi on Sabouraud’s dextrose agar (SDA) medium. sterile saline. After incubation for appropriate time. PROCEDURE 1 Pipette 2. 5 With sterile forceps place the carbohydrate disc onto the surface of agar in such a way that at least 30 mm space is present between each disc. . growth of the fungi around the discs is observed and the carbohydrate utilization pattern is assessed.0 ml. QUALITY CONTROL 1 Carbohydrate discs and media should be checked using standard control strains as follows: Candida albicans ATCC 14053 C. 2 With a sterile loop pick up few isolated colonies of the organism from the SDA plate and emulsify to a turbidity equal to McFarland 4 units. it is considered negative for assimilation of that carbohydrate.Textbook of Practical Microbiology 229 LESSON 79 INTRODUCTION Carbohydrate Assimilation Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Find out the pattern of assimilation of carbohydrates by yeast and yeast-like fungi. guilliermondii ATCC 6260 C. Hence in this test. 7 At the end of the incubation period observe the plate for growth around the disc. McFarland standard. Similarly. if the fungus does not grow in the proximity of a particular carbohydrate disc. pseudotropicalis ATCC 4135 2 Sterility of glassware and media should be ensured. yeast nitrogen base agar (Table 79-1). of sterile saline into a test tube. and that organism assimilates that particular carbohydrate in the disc then it is considered positive. They are inoculated on the carbohydrate-free yeast nitrogen base agar on which different filter paper discs containing various carbohydrates are placed. 3 Cover the surface of yeast nitrogen base agar with the suspension of the yeast cells. Yeast and yeast-like fungi use carbohydrates as sources of energy. II Reagents and lab wares Standard laboratory glassware and equipments. RESULTS AND INTERPRETATION Growth of the fungi around each carbohydrate disc is observed and results are interpreted in such a way that if the fungus has grown touching the edges of the disc. REQUIREMENTS I Equipments Incubator. growth around the discs is observed and the carbohydrate utilization pattern is assessed. OBSERVATION After incubation. 6 Incubate the plate at 30°C or 37°C for 24–48 hours. pp. 232. pp. Sussman M. Louis) 2002. Arnold publishers. 9th Edition. 10 ml. The basic salt solution is prepared and 10 ml. 3 Growth of fungi near carbohydrate discs should be confirmed after thorough examination. Ans. of this solution is added to 90 ml of 2% molten agar and poured in plates. (The CV Mosby Company. 2 How do you interpret carbohydrate assimilation test result? FURTHER READINGS 1 Collier L. Balows A. Volume 4.0 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 Preparation All the above ingradients are to be sterilized by filtration and dispersed aseptically. 2000. VIVA 1 Describe how carbohydrate medium is prepared. Bailey and Scott’s Diagnostic Microbiology. Interprint.230 Carbohydrate Assimilation Test Table 79-1 Preparation of Yeast Nitrogen Base Composition Boric acid – 500 µgm Copper sulphate – 40 µgm Potassium iodide – 100 µgm Ferric chloride – 200 µgm 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. St. KEY FACTS 1 Turbidity using the organism should be done in such a way that it is equal to McFarland Standard 4 units. 11 th ed. 2 Sterility should be maintained for medium and carbohydrate discs. A Text Book of Medical Mycology. 711. 2 Forbes BA. of the above solution to 90ml of 2% molten agar and pour into the plates. 3 Jagadish Chander. Add. Sahm DF and Weissfeld AS. Topley and Wilson’s Microbiology and Microbial Infections. . Mycology. In such cases. 3 Inoculate each sugar tube with 0. galactose. fill 5–6 ml IBM just above the Durham’s tube and sterilize at 121°C for 15 min. 0.. 3 Quality of medium used should be checked by growing known standard strains of fungi. Note: Do not screw caps on tubes tightly. Fermentation of carbohydrate in the medium produces a color change and bubbles which can be used as a marker to find out the type of yeast in culture.2 ml (5 drops) of culture suspension. PROCEDURE PRINCIPLE Sometimes. Preparation of indicator broth medium: The indicator broth medium (IBM) contains peptone.10 ml and distilled water.3 ml of 20% filter sterilized carbohydrate solution (glucose. bromocresol purple (0. Different carbohydrates. 1. and indicator broth medium (IBM). Durham’s tube is placed in an inverted position in screw capped test tubes. II Reagents Standard laboratory glassware and equipments. Candida kefyr: Glucose. 2 Sterility of medium used should be confirmed before use. This is an indicator of fermentation.0 gm. Aseptically. 4 Incubate the tubes at 30°C or 37°C for 2-10 days. carbohydrate fermentation tests are used as a supplement. McFarland standard. lactose. sucrose. sucrose. maltose. maltose positive. 2 With a sterile loop pick up few isolated colonies of the fungus from the SDA plate and emulsify to a turbidity equal to McFarland 4 units. and trehalose are the sugars used in carbohydrate fermentation tests. beef extract. 5 Observe the presence of air bubbles in Durham’s tubes.) are added to the medium. . a definite identification of the suspected fungi cannot be made using carbohydrate assimilation test.0 ml.. maltose.5 gm. III Specimen Pure growth of test fungus on Sabouraud’s dextrose agar (SDA) medium. etc. Yeasts and yeast-like fungi use carbohydrates as sources of energy. REQUIREMENTS I Equipments Incubator.4%). 0. Candida albicans: Glucose. 90. Glucose. and bubbles will be produced in the Durham’s tube.Textbook of Practical Microbiology 231 LESSON 80 INTRODUCTION Carbohydrate Fermentation Test LEARNING OBJECTIVES After completing this practical you will be able to: 1 Demonstrate the ability of different yeast and yeast-like fungi to ferment various carbohydrates. Fermentation of carbohydrates produces a visible color change in the medium. sodium chloride. 0.5 gm. In each tube. 1 Take tubes with indicator broth medium with sugars. lactose. QUALITY CONTROL 1 Quality of sterile glassware must be checked for proper sterility. sucrose positive. 2000. VIVA 1 2 3 4 What are the different carbohydrates used in carbohydrate fermentation test? What percentage of carbohydrate should be incorporated in carbohydrate medium? How do you interpret carbohydrate fermentation test? What are the advantages of carbohydrate fermentation test over carbohydrate assimilation test? FURTHER READINGS 1 Collier L. 3 Jagadish Chander. 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. Topley and Wilson’s Microbiology and Microbial Infections. 2 Forbes BA. Sussman M. . RESULTS AND INTERPRETATION Presence of bubbles or drop in the fluid level in Durham’s tube indicates fermentation of sugars. the Durham’s tubes are observed for presence of any gas bubbles. Arnold publishers. Interprint. Balows A. 711. St. (The CV Mosby Company. Bailey and Scott’s Diagnostic Microbiology.232 Carbohydrate Fermentation Test OBSERVATIONS After incubation. pp. Louis) 2002. 9th Edition. Mycology. 11 th ed. Volume 4. 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. Sahm DF and Weissfeld AS. A Text Book of Medical Mycology. glass slides. the fungal element can be stained and observed under a microscope and presumptive identification can be done. sterile cotton swab. conidia. III Specimen Fungal culture grown on SDA’s medium. OBSERVATION The wet mount preparation should be visualized under low power (10x) and high power (40x) objectives and the morphology observed. hyphae. etc. RESULTS AND INTERPRETATION By microscopic observation. Further. Hence it is necessary to know different fungi causing infections in man and methods to identify them. II Reagents and lab wares Standard laboratory glassware. QUALITY CONTROL Quality of sterility of medium should be checked before use.Textbook of Practical Microbiology 233 LESSON 81 INTRODUCTION Identification of Common Fungi LEARNING OBJECTIVES After completing this practical you will be able to: 1 Know important characteristic morphological features of different fungi. hard lens. Bunsen burner. cover slips. lactophenol cotton blue stain. Colony morphology Rapidly growing white colored fungus swarms over entire plate showing aerial mycelium cottony. SDA medium. glass marking pencil. Molds Rhizopus Mucor Alternaria Fusarium Aspergillus Penicillium Cladosporium Cephalosporium Trichophyton Epidermophyton Yeast Torulopsis Candida REQUIREMENTS I Equipments BOD incubator and dissecting microscope. hence identification of the fungi is a very important approach in patient management. . Fungi cause varied infections in man. the fungi can be cultivated. PROCEDURE Lactophenol cotton blue wet mount preparation of the fungus (refer chapter 73). mold. PRINCIPLE In a mycology laboratory. 2 Identify a given fungus. can be made out and used in classifying the type of fungus isolated. characters of the yeast. and its colonial characteristics will throw some light on the type of fungus. Rhizopus A Common laboratory contaminant. Microscopy Conidia multi celled. Colony morphology Grayish green or black colonies with grey edges and swarming over entire plate with grayish white aerial hyphae. Colony morphology Woolly white colonies may change to pink. Vesicle Club or flask shaped. Conidiophore Smooth. Conidia Globose. Mycelium non-septate. Almost sixteen different species cause human disease. Fusarium Usually found in soil. Aspergillus fumigatus Colonies Colonies are blue-green. More than 170 species are known. Microscopy Conidia multi celled. Colony morphology Resembles colony of Rhizopus. and color of ascospore and conidia. 81-2). uniseriate. compact. surface powdery or granular. Sterigmata Produced on upper half of the vesicle. Aspergillus Usually infect plants and animals. green. FIGURE 81-1 SDA with colonies of Fusarium species. appears green in color and crowded. purple or yellow (Fig. Axis of sterigmata is roughly parallel to that of conidiophore and conidia produced in chains on sterigmata. They are: Aspergillus fumigatus Aspergillus flavus Aspergillus niger Aspergillus oryzae Aspergillus amstelodami Aspergillus verscicolor Aspergillus terreus Aspergillus nidulans Aspergillus candidus Aspergillus ustus Aspergillus carnens Aspergillus arenaceus Aspergillus clavatus Aspergillus caseillus Aspergillus restricutus The characteristics used to identify them are: A) Colour and shape of conidial head. sporangiophores straight and terminate in black sporangium containing sporangiospores and columella. Alternaria Usually found on plants. attached to conidiophores arising from a septate hypha. pear-shaped and attached to single conidiophores arising from septate mycelium. Conidial head Columnar. root like hyphae (rhizoids) penetrating the medium. 81-1). velvety. and F) Size and shape. oval or crescent shaped. non-septate mycelium giving rise to single sporangiophores and terminate in globular sporangium containing columella and sporangiospores. . colorless and sometimes brown. terminate into club or flask shaped green. sometimes radially folded (Fig. B) Number of sterigmata/phialids. C) Shape of vesicles. E) Presence and absence of cleistothecia. D) Colour of conidiophore. Mucor Common food contaminant. Microscopy Oval spores. green and echinulate.234 Identification of Common Fungi Microscopy Spores are oval. branched and gives rise to brush like appearance. Vesicle Globose. loosely columnar. Sterigmata Monoseriate and biseriate sterigmata seen in same strain. Vesicle Globose. Aspergillus niger Aspergillus flavus Colonies White. thick smooth walls. Colonies Rapidly growing. subglobose or elliptical. producing sterigmata. initially globose. FIGURE 81-3 SDA with colonies of Aspergillus niger. Conidia echinulate and appears yellow green. Conidiophore hyaline. Penicillium species Colonies Initially white and fluffy. yellowish green and velvety colony (Fig. produced by small and large vesicles respectively. covered with black spores (Fig. Hyphae Septate. Conidia Conidia globose. hyaline. concave under surface. brownish sterigmata produced in two series. sub globose or elliptical and sterigmata cover entire surface or at least three fourth. conidiophore hyaline and brownish near the vesicle. FIGURE 81-4 SDA with colonies of Aspergillus flavus. with radial folds. Conidial head Radiate.Textbook of Practical Microbiology 235 FIGURE 81-2 SDA with colonies of Aspergillus fumigatus. later turns to green and blue green. Conidial head large black to brownish. septate primary sterigmata and short secondary sterigmata. become radiate then splits into divergent spore columns. Conidia Globose. Conidiophore Variable in size. fluffy. Phialids Flask shaped. echinulate. . Conidiophores Long with branching phialids. thick walled and roughened. reverse buff colored. 81-4). 81-3). septa 2–3. Erect. port wine or deep violet colored. Epidermophyton floccosum Colony Velvety or powdery surface. Cephalosporium Used in antibiotic production Colony Rapidly growing compact. spherical or oval in shape. bean shaped. wrinkled loose pigment present. Microscopy Conidia develop at the end of complex conidiophores arising from brown septate mycelium. white glabrous heaped up colony. Macroconidia String-like. . rubrum may be present. Macroconidia Generally not present. thick walled. Microconidia Not produced. septate. No pigment produced on reverse. sometimes button like with velvety texture. Microscopy Single celled conical or elliptical conidia held together in clusters at the tips of conidiophores. with gray or rose colored aerial hyphae. Colony Small. Swollen antler-like hyphae produced rarely. reverse of colony yellow tan. Thick walled structures resembling macroconidia of T. flat or heaped up. heaped. producing chains of conidia. Chlamydospores Produced. obverse and reverse should be kept in mind. Chains of chlamydospores produced. pyriform. Cladosporium Usually found on dead and decaying plants. greenish black and powdery. cottony. racquet hyphae and nodular bodies present. septation. size and color are very important for identifying the fungus. moist colonies. with characteristic rat tail appearance. color. Trichophyton violaceum Colony Slow growth. Macroconidia Clavate. Trichophyton verrucosum Colony Slow grower.236 Identification of Common Fungi Sterigmata Long with tapering end. rarely produced. characteristics clusters of twos and threes. KEY FACTS 1 Characters of aerial hyphae. rarely produced. conidial shape. unbranched conidiophores arise from septate mycelium. Microconidia Tear-shaped. Variants with yellow or grey white colonies. Microconidia Generally not present. may produce chains of chlamydospores. rugal folds seen. smooth. 2 Colony characters. Conidia Long chains of conidia. surface waxy and glabrous. surface folded in radiating furrows. etc. 711. Sussman M. The colonies produced by many pathogenic fungi differ greatly from each other. 2000. A Text Book of Medical Mycology. Moreover different fungi produce a wide variety of morphological structures like conidiophores. . Arnold publishers. Different fungi have their own predilection to grow on different media depending upon the exact nutritional requirement.Textbook of Practical Microbiology 237 VIVA 1 What are the common fungi that cause disease in humans? Ans. microconidia. 4 How is the colony morphology of fungi important in diagnosing fungal infections? Ans. Hence this helps for a preliminary identification of the type of fungus FURTHER READINGS 1 Collier L. which on staining can be visualized and the fungi identified. 3 Jagadish Chander. conidia. 3 What are different types of spores or conidia produced by different molds? Ans. Bailey and Scott’s Diagnostic Microbiology. sporangiophores. Volume 4. 2 Forbes BA. Louis) 2002. septate or aseptate hyphae. Mycology. Topley and Wilson’s Microbiology and Microbial Infections. sporangia. St. like chlamydospores. 11 th ed.. Different molds produce different types of spores and conidia. pp. 9th Edition. etc 2 How are culture and staining of fungal elements useful in characterizing fungi? Ans.. ascospores. Interprint. Sahm DF and Weissfeld AS. The common fungi that cause human infections include pathogenic species of Curvularia Rhizopus Mucor Alternaria Fusarium Aspergillus Penicillium Cladosporium Cephalosporium Trichophyton Epidermophyton Torulopsis Candida Absidia Cryptococcus Histoplasma Sporothrix Blastomyces. macroconidia etc. (The CV Mosby Company. Moreover production of pigments by these colonies also vary greatly. Balows A. 238 . Textbook of Practical Microbiology UNIT 239 XI Virology Lesson 82 Cultivation of Viruses in the Cell lines Embryonated Egg Lesson 83 Cultivation of Viruses in . II Reagents and lab wares Sterile glassware. REQUIREMENTS I Equipments Inverted microscope (Fig. 2 Count the cells with the medium in a hemocytometer for appropriate splitting.Vero (Vervet monkey kidney cell line). incubator (Fig. and d) interference. membrane filter. Some viruses however do not produce any cytopathic effect (e. McCoy (Human synovial carcinoma cell line). sodium bicarbonate (NaHCO3). Contamination of cell lines should be prevented and even cross contamination among cell lines should be avoided. Hep 2 (Human epithelioma of larynx cell line).240 LESSON 82 INTRODUCTION Cultivation of Viruses in the Cell lines LEARNING OBJECTIVES After completing this practical you will be able to: 1 Know and understand the common cell lines used for routine maintenance of viruses in a virology laboratory. 3 Inoculate the cells into sterile flasks or tubes for viral inoculation. human amnion cells. EDTA trypsin mixture. foetal calf serum (FCS). discard jar. minimum essential medium (MEM). HeLa (Human carcinoma of cervix cell line). PROCEDURE 1 Discard the trypsin versene mixture and add a small amount of MEM with 10% FCS to the monolayer of cells. virus inoculum. b) immunofluorescence. III Specimen Suspected virus infected specimen like the cerebrospinal fluid (CSF). b) biochemical studies of viral replication and c) production of viral antigens and vaccines. Many viruses kill the infected viral cells in which they grow and bring about detectable changes in morphology of the cells. Now a days monolayer cell cultures are mostly used in diagnostic and research work in viral diseases. vials.g. pre-sterilized tissue culture plasticware (Fig.. The growth of viruses in the cell lines can be known by a) cytopathic effects. PRINCIPLE Viruses infect healthy cells grown in the laboratory. These changes are collectively known as cytopathic effects. Monolayer of a cell culture in a culture flask is treated with trypsin or versene to disperse cells. Depending upon the number of divisions which a cell line undergoes in-vitro before dying. The cell cultures are used for a) isolation of viruses from clinical specimens for diagnosis of viral diseases. rectal swab. the cell lines have been classified as primary. syringes. Eagle’s. rubella virus). . sterile double distilled water. 82-1). 82-2). The most commonly used cell culture systems in most laboratories include chick embryo fibroblast cells. c) haemagglutination and haemadsorption. and throat swab. The most important precaution to be taken during maintenance of cell lines is sterility. Cell culture is the most widely used system for cultivation of viruses since it is more convenient method compared to the other methods like egg inoculation and animal inoculation. spirit and sodium hypochlorite. stool. they show pathological changes and the viruses can be harvested from the cells for further tests. 82-3). diploid and continuous cell lines. Pasteur pipettes and measuring pipettes. and W1-38 (Human embryonic lung cell line). When susceptible cells are used for inoculation of viruses. Viruses are intracellular pathogens which are dependent on host cell machinery for their multiplication and growth. haemocytometer and biological safety cabinet. Rhesus monkey kidney cells. Vero cells. Multinucleated giant cell formation. 6 Inoculate the monolayer of cells with virus using sterile Pasture pipette. Table 82-1 The cell lines and indications for the viruses and cytopathic effects they produce Type of viruses Adenovirus Herpes simplex virus Varicella zoster virus Cytomegalovirus Enterovirus Susceptible cell line Primary human embryonic kidney cells. and incubate at 37°C. Giant cell formation. Viruses are known to produce cytopathic effects are identified by observing the same in the infected cell lines. Rapid rounding of cells progressing to complete cell destruction. RD cells. Syncytia formation. HeLa cells. the flasks are observed for confluency and healthy monolayer of cells and virus infected cells are classified. Human kidney cells. 7 Observe for the cytopathic effect (CPE) 7 days after inoculation. and interference FIGURE 82-1 Incubator. which is also confluent. 2 Susceptible cells to be selected for the appropriate virus. Hep 2 cells. for viral inoculation. FIGURE 82-1 Inverted microscope. Paramyxovirus . Primary human and monkey kidney cells. HEp 2 cells. QUALITY CONTROL 1 Sterility precautions should be taken perfectly. Rounding and swelling of cells.Textbook of Practical Microbiology 241 4 Fill the new flasks with MEM and incubate in horizontal position. Cytopathic effect Rounding and clustering of swollen cells. Human fibroblast cells. haemagglutination and haemadsorption. OBSERVATIONS After incubation. Non-cytopathogenic viruses are identified by other methods like immunofluorescence. LLC-MK2. RESULTS AND INTERPRETATION The cell lines are observed for any cytological alterations that are diagnostic of viral infections (Table 82-1). 5 Select a healthy monolayer. FIGURE 82-3 Cell culture bottle. Lippincott Williams and Wilkins. Volume 1. 9th Edition. Hence precautions for sterility should be meticulously followed. 3 Zuckerman AJ. Principles and Practice of Clinical Virology. Pattison JR. Topley and Wilson’s. John Willey and sons Ltd. Sussman M. 2001. VIVA 1 Why is cell culture method. 2 Knipe DM. 5th Edition. 4th Edition. 2 Susceptible cells should be selected for the appropriate viral inoculation. Field’s Virology. . 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. Griffiths PD and Schoub BD. 2004. Balows A. Virology. Howley PM. Banatvala JE.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. Microbiology and Microbial Infections. Arnold publishers. The eggs are used because they are inexpensive. and also for yellow fever (17D strain) and rabies (Flury strain) vaccines. Since eggs lack a well developed defence mechanism of their own. formation of pocks. The eggs are candled to determine the position of the embryo and its viability. PRINCIPLE Usually 8-11 days old chick embryos are used. easily available and much simpler to handle than animals. Pocks produced by different viruses vary in their morphology. egg. or hemagglutination. gloves. allantoic cavity and chorioallantoic membrane (CAM). pencils. Viral growth is recognized by death of embryo. III Specimen Virus isolate or specimen suspected to contain virus. Nevertheless. melted paraffin wax and 70% ethanol. Amniotic sac is used for the primary isolation of influenza virus. Prior to 1950s the technique of propagation of viruses in embryonated eggs was popular because of non-availability of cell culture techniques during those times. they do not interfere with growth of viruses. . Any of the viruses or specimen suspected to contain viruses are inoculated.1 ml of 10-3 dilution of NDV in GLB). Dead embryos are promptly discarded. 2 Drill the egg shell. After inoculation.Textbook of Practical Microbiology 243 LESSON 83 INTRODUCTION Cultivation of Viruses in Embryonated Egg LEARNING OBJECTIVES After completing this practical you will be able to: 1 Know the routes of inoculation of eggs for propagation of viruses. the embryonated eggs are still used for the isolation of avian viruses. or haemagglutination property of embryonic fluid. II Reagents and lab wares Syringes (1 tuberculin syringe preloaded with 0. 3 Inoculate the specimen in the embryonated egg through appropriate route. These produce characteristic visible lesions such as pocks. These eggs are inoculated by one of the following routes: yolk sac. candling is important. candling lamp and hole puncher. amniotic sac. Age of the egg chosen depends on route of inoculation described since various membranes and their contents vary in size as embryo matures. certain bacteria (Chlamydia and Rickettsia) and parasite (Toxoplasma gondii). However. Note: Age of the embryonated egg is chosen depending on the route of inoculation since various membranes and their contents vary in size as embryo matures (Table 83-1). Pasteur pipette with bulb. many viruses fail to grow on primary inoculation into eggs. pocks. viral growth in the egg is recognized by death of embryo. eggs are incubated for 2-9 days. screw capped sterile vials. CAM is mainly used for growing pox viruses. green or brown color indicates contamination. The viruses can grow in different compartments of the egg . influenza viruses and also for vaccine production. healthy embryo has an orange color with evident vasculature. Allantoic cavity is mainly employed for harvesting influenza virus for vaccine production . When candled. 4 After 2–5 days post injection. sterile forceps. Since viruses need living tissues to replicate. Black. Dead embryo shows clear yellow with no vasculature. gauze. PROCEDURE 1 Disinfect the egg shell. REQUIREMENTS I Equipments Egg holders. Yolk sac is mainly used for culture of some viruses. Mumps virus. Higher yield of viruses cannot be expected from dying or dead embryo since viruses require healthy living tissues for their multiplication. Table 83-1 The routes of inoculation of the egg and the viruses isolated Route of inoculation Chorio allantoic membrane. OBSERVATIONS The inoculated part of embryonated egg is observed for changes due to viral infection. green or brown color indicates contamination. candling of eggs is important. 2 Since viruses need living tissues to replicate. When candled. Yolk sac. Herpes simplex virus. Isolation. Parainfluenza viruses. FIGURE 83-1 CAM showing pocks. Vaccine titration. Isolation. vaccine production. 9-12 days. 83-1). Amniotic sac. Flavi viruses. 3 Sterile precautions should be taken throughout the procedure. Influenza A. Isolation. black. 2 Age of the embryonated egg is chosen depending on the route of inoculation since various membranes and their contents vary in size as embryo matures. candling is important. KEY FACTS 1 Usually 8-11 days old chick embryos are used for cultivation of viruses. 4 Would you expect a higher yield of virus from dying or dead embryo. haemagglutination. Vaccinia virus. RESULTS AND INTERPRETATION Viral growth in the inoculated egg is recognized by death of the embryo. healthy embryo has an orange color with evident vasculature. pock formation in CAM (Fig. Mumps virus. Influenza B. Hybrid vaccine production. Use Isolation. Isolation and typing. Virus Variola virus. Isolation. dead embryo show clear yellow colour with no vasculature. . Influenza A virus. Allantoic sac. Age of embryo 10-14 days. VIVA 1 Why is the age of the embryo an important factor in virus inoculation? 2 What are some important parameters to be noted before passaging viruses in embryonated eggs? 3 What is the purpose of candling? Ans. Since viruses need living tissues to replicate. 6-8 days. etc. the eggs are candled to determine the position of the embryo and its viability.244 Cultivation of Viruses in Embryonated Egg QUALITY CONTROL 1 Before inoculation. HA antigen preparation. 10-12 days. why? Ans. Topley and Wilson’s.Textbook of Practical Microbiology 245 FURTHER READINGS 1 Collier L. Principles and Practice of Clinical Virology. . 3 Zuckerman AJ. Howley PM. Virology. Lippincott Williams and Wilkins. Pattison JR. Banatvala JE. Microbiology and Microbial Infections. 9th Edition. Griffiths PD and Schoub BD. John Willey and sons Ltd. 2 Knipe DM. Field’s Virology. 2004. 4th Edition. Sussman M. 2001. Balows A. 5th Edition. Arnold publishers. Volume 1. 246 . Milk and Air Lesson 84 Microbiology of Water Lesson 85 Microbiology of Milk Lesson 86 Microbiology of Air .Textbook of Practical Microbiology UNIT 247 XII Microbiology of Water. 248 LESSON 84 INTRODUCTION Microbiology of Water LEARNING OBJECTIVES After completing this practical you will be able to: 1 Know various methods used for testing potable water. Acinetobacter. colourless. Faecal or thermotolerant coliforms These satisfy the criteria for coliforms but have the additional property of the ability to grow at a higher temperature 44°C. etc. However. In order to include anaerogenic bacteria and those which are nonlactose fermenters. and Clostridium perfringens. Clostridium perfringens The presence of this organism in water in the absence of other indicators of contamination of water implies remote or intermittent fecal pollution. The presence of E. Microscopically it should be free from pathogenic organisms. Serratia. Giardia species and . odourless and without disagreeable taste. whose presence indicates the contamination of water with fecal matter. These are: 1 2 3 4 5 Coliforms. in the presence of azide and 40% bile. Faecal Escherichia coli. When the concentration of free residual chlorine is at least 0. these organisms are present in large quantities in the environment. Drinking water is acceptable and fit for drinking when it is clear. Fecal (thermotolerant) coliforms. The total coliform count is widely regarded as the most reliable indicator of potable water quality. 2 Test the quality of the potable water for drinking. Viruses in water are destroyed by chlorination. Faecal streptococci These are catalase negative. Coliforms Coliforms are defined as 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.5 mg per litre. because. faecium) . hydrolyse aesculin and can grow at 45°C. Alcaligenes. Such organisms which can survive 60°C for 30 min and can grow at 10°C at pH 9. it has been modified as the members of the Enterobacteriaeceae capable of growing galactosidase at 37°C that normally posses. Water gets contaminated by pathogens which are introduced into water by sewage pollution. coli is considered as the contamination of water with feces of human or animal origin.5% of sodium chloride (NaCl) belong to the genus Enterococcus (Enterococcus faecalis and E. Chromobacterium. Gram positive cocci present in the intestinal tract of man and animals. These saprophytes are harmless.6 and in 6. These organisms have the Lancefield group D antigen. Faecal Escherichia coli These are defined as thermotolerant coliforms which ferment lactose (or) mannitol at 44°C with the production of acid and gas within 24 hours and also form indole from tryptophan at 44°C. Flavobacterium. Faecal Streptococci. Natural sources of water usually contain some saprophytic bacteria. such as Pseudomonas. the presence of coliforms not necessarily indicate faecal or sewage contamination. There are some indicator organisms. A wide varieties of diseases are transmitted by contaminated water. protozoa such as Entamoeba histolytica . for a minimum contact period of 30 minutes at pH below 8 and a turbidity of 1 nephelometric turbidity unit or less. The agar count at 22°C gives an indication of the amount of decomposing organic matter in the water available for bacterial nutrition. etc. the more likely is the water to be contaminated with parasitic and potentially pathogenic organisms. All the bacteria present are retained on its surface.Textbook of Practical Microbiology 249 Balantidium coli may be present in the drinking water. Further confirmation of the presence of E. 5 grams. Examination for Cl. II Reagents MacConkey broth.5grams in 100 ml of distilled water. When collecting water samples from lakes or streams the bottle is opened at a depth of about 30 cm with its mouth facing the current. 20 grams. Colonies on the surface of the membrane are counted. Plate count This consists of inoculating the nutrient agar with water to be tested and incubating the agar aerobically in parallel at 37°C for 1–2 days and at 22°C for 3 days. 2 Detection of coliform bacteria and E. . and vegetables. subcultures are made from all the bottles showing acid and gas to fresh tubes of single strength MacConkey medium already warmed to 37°C. b Differential coliform test. Vibrio. Collection of water samples Water is collected in heat sterilised bottles containing a sufficient volume of sodium thiosulphate to neutralise the bactericidal effect of any chlorine or chloramine in the water. the bottle is stoppered.01 grams and final pH (at 25°C) is adjusted at 7. Detection of faecal streptococci Subcultures are made into tubes containing 5 ml of glucose azide broth from the positive bottles in the above test.3 ± 0. After 18 hours of incubation the presumptive coliform counts and detection of E. water is allowed to run to waste for 2–3 min. brilliant green bile broth Preparation of MacConkey broth: This is prepared by mixing pancreatic digest of gelatin. 50 ml and 10 ml of the media are dispensed into screw capped bottles with inverted Durham’s tubes and are sterilized by autoclaving at 15 lbs (121°C for 15 minutes). Preparation of double strength medium: It is prepared by suspending 35 grams in 500 ml of distilled water and heated to dissolve the medium completely. autoclave and water bath. At least 100 ml of water to be tested is collected in each bottle. This test is usually employed to find out whether the coliform bacilli detected in the presumptive test are E. bile salt. and 5 Test for pathogenic bacteria. because of the presumption that the reactions are due to coliforms organisms. results are read off the probability tables. 5 ml quantities of the medium are dispensed in screw capped bottles with Detection of coliform bacteria and E. before collecting it into the bottle. After collecting the water in the bottle. Preparation of single strength medium: It is prepared by suspending 3. stormy fermentation occurs. Streptococcus faecalis if present produces acid in the medium within 18 hours at 45°C. In this chapter Presumptive coliform test and diffrential coliform test will be discussed. For collection from tap. lactose. Differential coliform test This is called Eijkman test. coli. corresponding special media are used. 0. 3 Detection of faecal streptococci. 4 Examination of Cl.coli Presumptive coliform test – Multiple tube technique The test is called presumptive. perfringens. REQUIREMENTS I Equipments Bacteriological incubator. and sent to the laboratory as quickly as possible within 6 hours keeping it in a cool container and protecting it from light. Though these are non-pathogenic the greater the amount of organic matter present. Coliforms are not the reliable indicator of protozoal contamination. The count is made by adding varying quantities of water (0. PRINCIPLE The following tests can be done for bacteriological analysis of water: 1 Plate count. The agar count at 37°C is a more important index of dangerous pollution. Examination for pathognic bactrial cells test For other special pathogens like Salmonella. bromocresol purple. Membrane filtration method A measured volume of water is filtered through a Millipore filter. Those showing gas in Durham’s tubes contain E. They are incubated at 44°C for 24 hours. c Membrane filtration method. After the usual presumptive test. Acid and gas formation indicate the growth of coliform bacilli. From the number of positive tubes obtained. Those which grow 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. coli can be directly made. After incubation number of the colonies formed in the agar are counted. soil.2. coli or not.1 ml–50 ml) to bile salt lactose peptone water with an indicator for acidity. coli. 10 grams. if positive. perfringens Water is incubated in litmus milk medium at 37°C for 5 days. This is then placed on suitable media and incubated at the appropriate temperatures. a Presumptive coliform count: multiple tube technique. coli is done by testing for indole production and citrate utilization tests. Grades of the quality of drinking water supplied as determined by results of periodic Escherichia coli and coliform counts is listed in the table 84-1. coli or any coliform present in consecutive samples. of tubes giving positive reaction. 0. coli. for Differential coliform counts: No. Table 84-1 Grades of the Quality of Drinking Water Supplies Determined by results of Periodic Escherichia coli and Coliform counts Quality of supply Results from routine samples Coliform counts / 100 ml E. lactose. 5 Inoculate 1 ml of water into 5ml single strength medium (5 bottles). of bottles showing acid and gas is counted and compared with the MacGrady’s table. III Specimen Water specimen to be tested. for Presumptive coliform count No. 2 Satisfactory. Result is expressed as. PROCEDURE 1 Collect 500 ml of water in a sterile bottle. In any sample.coliforms most probable no (MPN)/ 100ml. Presence of gas in brilliant green bile broth and indole production at 44°C is indicative of the presence of E. 3 Inoculate 50 ml of water into 50 ml double strength media (1 bottle) 4 Inoculate 10 ml of water into 10 ml double strength medium (5 bottles). coli count / 100 ml 0 1–3 0 0 Tolerance 1 Excellent.2. of tubes giving positive reaction 50 ml 10 ml 1 ml 1 2 2 Result: Coliforms most probable no 10/100 ml. both gas productions in BGBB and Indole test at 44°C is compared with MacGrady’s table. Differential coliform count The no of tubes showing positive results i. For interpretation refer MacGrady’s table.0133 gm and adjusting the final pH (at 25°C) to 7.g. 7 Check for acid and gas after 24 hours and 48 hours. 4–9 0 10 coliforms or 1 or more E. 6 Incubate all the bottles at 37°C for 48 hours. RESULTS AND INTERPRETATION Presumptive coliform count The no. 10 grams. 50 ml 10 ml 1 ml 1 1 1 Results: Escherichia coli MPN —— 5 /100 ml Report: Unsatisfactory. 8 Any positives are inoculated into brilliant green bile broth and peptone water. The media is distributed in 5ml volumes in test tubes with inverted Durham’s tubes and sterilized by autoclaving at 15 1bs pressure (121°C) for 15 minutes. 4 Unsatisfactory. ox gall.e. Provided that coliforms do not occur in Consecutive samples or in more than 5% of samples. 2 Inoculate water sample immediately into the MacConkey broth medium. . 20 grams and brilliant green. QUALITY CONTROL Sterility of media and strength of media should be properly checked. or presence of any coliform organisms in more than 5% of routine samples. Preparation of brilliant green bile broth (BGBB): This is prepared by mixing peptic digest of animal tissue. 3 Intermediate. 9 Incubate at 44°C in a water bath overnight. In all samples.2 ± 0. Eg. e. OBSERVATIONS Observe for the presence of gas and production of indole. 10 grams.250 Microbiology of Water inverted Durham’s tubes and are sterilized by autoclaving at 15 lbs (121°C for 15 minutes). 4 grams of the medium is suspended in 100 ml distilled water and mixed well. Result is expressed a Escherichia coli Most probable No (MPN) / 100 ml. viral hepatitis. Typhoid fever. pp. Paniker. colourless. Practical Medical Microbiology. 2 Mention the routine laboratory tests done for the analysis of water. cysticercosis. 5 Eijkman test is usually employed to find out whether the coliform bacilli detected in the presumptive test are E.Textbook of Practical Microbiology 251 KEY FACTS 1 Drinking water is acceptable and fit for drinking when it is clear. coli or not. Microscopically it should be free from pathogenic organisms. 3 They are indicator of faecal contamination.K. 921. C. VIVA 1 What are the common water-borne infections transmitted by contaminated water? Ans. 4 Coliforms are not the reliable indicator of protozoal contamination. Churchill Livingstone. and odourless and without disagreeable taste. Orient Longman. 2 Mackie and McCartney. 1996. cholera. 2005. 14th Edition. . etc. 3 Which are the organisms which indicate fecal contamination of water? FURTHER READINGS 1 Ananthanarayanan. giardiasis. amoebiasis. R. 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. pp 603 – 609. poliomyelitis. 7th Edition. Ananthanarayanan and Paniker’s Textbook Of Microbiology. For un refrigerated milk there is a consistent relationship between the bacterial count and the dye reduction time. PRINCIPLE Time taken for bacterial dehydrogenases to reduce methylene blue dye and decolourise it. 3 Aseptically transfer 10ml of sample of milk with a pipette to a sterile test tube. This method will be described in this chapter. and Examination for specific pathogens. invert it once or twice to mix the contents. 2 Mix thoroughly the milk sample. sterile pipettes. is taken as an indicator of the . Turbidity test. REQUIREMENTS I Equipments Water bath. The test should not be done if the atmospheric shade temperature exceeds 21°C. Test for coliform bacilli.000 solution of methylene blue by dissolving a standard methylene blue tablet in 200ml cold sterile glass-distilled water and making upto 800ml with more of such water. The routine bacteriological examination of milk to detect bacterial contaminations can be done by the following methods: 1 2 3 4 5 6 Viable count. or from milk handlers such as dairy workers. from the environment. streptococcal and staphylococcal infections. 4 Add 1ml of methylene blue solution by a 1ml sterile pipette. methylene blue test is a rapid. sterile rubber stopper. inexpensive and most widely used test for testing the milk. Note: This solution can be stored in dark in a refrigerator and can be used for 2months. Methylene blue reduction test. simple. Human infections may be caused by the ingestion of animal milk which contains microorganisms derived either from the animal (faeces). 6 Place these test tubes at 37°C in a thermostatically controlled water bath for 30 min. A wide number of bacteria are found in contaminated milk (Table 85-1). 5 Put a sterile rubber stopper to the test tube. number of viable bacteria in the milk. brucellosis. salmonellosis and Q fever. Certain precautions are taken to prevent contamination. They include tuberculosis. Phosphatase test.252 LESSON 85 INTRODUCTION Microbiology of Milk LEARNING OBJECTIVES After completing this practical you will be able to: 1 Test the quality of the un refrigerated milk by methylene blue test. III Specimen Milk to be tested. The infections that can be transmitted from infected animals to humans through contaminated milk are many. Of these tests.00. PROCEDURE The time of reduction is affected by the temperature at which the milk is held before testing. methylene blue tablets and distilled water. 1 Prepare a 1 in 3. II Reagents and glass wares Sterile test tubes. R. 1996. . Ananthanarayanan and Paniker’s Textbook Of Microbiology. Churchill Livingstone. 7th Edition. Because. 2 Mackie and McCartney. Add 10 ml milk to 1 ml of tap water. B. KEY FACTS 1 Tuberculosis. 2 What is the principle of methylene blue test? 3 List the routine tests used for the bacteriological examination of milk? FURTHER READING 1 Ananthanarayanan. 2 Methylene blue test is rapid. to inactivate its reducing system. the milk fails the test. brucellosis. Orient Longman.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. The time for complete decolourisation need to be recorded. 2005. 921. The methylene blue reduction test should not be done if the atmospheric shade temperature exceeds 21°C. Paniker. QUALITY CONTROL With each test the following milk specimens are incubated as controls: A. Table 85-1 List of bacteria that can be found in contaminated milk 1 2 3 4 5 6 7 8 9 10 Streptococcus lactis Streptococcus faecalis Achromobacter Clostridium perfringens Clostridium butyricum Bacillus subtilis Bacillus cereus Proteus vulgaris Staphylococcus aureus Gaffkya tetragena OBSERVATIONS After incubation. VIVA 1 List common infections transmitted by contaminated milk.K. Practical Medical Microbiology. 14th Edition. C. streptococcal and staphylococcal infections. salmonellosis and Q fever are some of the infections transmitted by contaminated milk. Add 1ml methylene blue to 10ml milk that has been held at 100°C for 3 min. inexpensive and most widely used test for testing the microbial contamination of the milk. pp 603 – 609. untreated milk is often considered satisfactory if it fails to decolourize the dye in 30 minutes. compare the test mixture with control A to see whether there is any decolourization in the former and with control B to see whether decolourisation is complete. pp. simple. RESULTS AND INTERPRETATION If the dye is wholly decolourised or decolourised within 5mm of the milk surface. 3 The time of reduction is affected by the temperature at which the milk is held before testing. It is considered as contaminated milk. 2 Remove cover of the Petri dish in its chosen position for the measured period of time. 4 Count the colonies. For example. streptococcal and staphylococcal infections. OBSERVATIONS Observe the colonies grown on the medium after incubation.254 LESSON 86 INTRODUCTION Microbiology of Air LEARNING OBJECTIVES After completing this practical you will be able to identify: 1 Bacteria present in the air. petri dishes containing an agar medium of known surface area are left open for a measured period of time. 2 Organisms present in the air by settle plate method. The number of bacteria in the air at any given time is dependent on the number of persons present. etc. in the premises where safe working depends on the bacterial content in air being kept at a very low level and premises where certain foods or pharmaceutical materials are prepared. 3 Incubate the plates aerobically for 24 hours at 37°C. anthrax. Settle plate method and slit sampler method are the two methods used for routine bacteriological examination of the air. Settle plate method will be described in this chapter. the monitoring of density of microbial pathogens in the air is a priority. pour it into plates and dry off any surface moisture. II Reagents and lab wares These include Petri dishes containing nutrient agar media for the growth of organisms. QUALITY CONTROL The Petri dish agar plates should remain open for a specified and adequate time. and then replace its lid. The list of bacteria commonly found in the air are summarized in the table 86-1. Demonstration and estimation of the number of bacteria carrying particles in air may be required in certain situations. PRINCIPLE In the settle plate method. The plates are incubated and a count of the colonies formed shows the number of settled particles that contained bacteria capable of growth on the medium. Large bacteria carrying dust particles settle on to the medium. preferably with the use of a plate microscope. PROCEDURE 1 Prepare nutrient agar media. the amount of their body movements and the amount of disturbance of their clothing. RESULTS AND INTERPRETATION Growth rate on the medium in a given time indicates the bacterial load in a given area. Table86-1 List of bacteria commonly found in air 1 2 3 4 5 6 7 Staphylococcus aureus Streptococcus pyogenes Mycobacterium tuberculosis Pseudomonas aeruginosa Bacillus anthracis Bacillus subtilis Proteus vulgaris REQUIREMENTS I Equipments Incubator and plate microscope. tuberculosis. . The infections that can be transmitted through air include wound infections. 2 What are the methods used for the detection of microorganisms in air? FURTHER READINGS 1 Ananthanarayanan. Churchill Livingstone.K. Orient Longman. 1996. Ananthanarayanan and Paniker’s Textbook Of Microbiology. 2 Mackie and McCartney. C. the amount of their body movements and the amount of disturbance of their clothing. 921. Paniker. pp 603 – 609. Practical Medical Microbiology. 7th Edition. . etc. 2 Settle plate method and slit sampler method are the two methods used for routine bacteriological examination of the air.Textbook of Practical Microbiology 255 KEY FACTS 1 The number of bacteria in the air at any given time is dependent on the number of persons present. 2005. VIVA 1 List the infections transmitted by air. R. 14th Edition. pp. 256 . Textbook of Practical Microbiology UNIT 257 XIII Animal Experiments Lesson 87 Intravenous Inoculation into Mice Tail Vein Vein of Rabbit Lesson 89 Animals and their uses in the Laboratory Lesson 88 Collection of Blood from the Marginal Ear . 0. 3 Inject carefully the material (approx. 1 ml syringe. 1 Hold the syringe and needle in such a way that animal should be horizontal to the tail and vein. III Specimen Laboratory bred mouse and culture material to be tested. PRINCIPLE Mouse is a very suitable laboratory animal for different types of pathogens. Cryptosporidium parvum. Bunsen flame. (Box 87-1).2 ml) into the tail vein. 3 Lift the mouse by holding the tail halfway. . Animal preparation for injection 1 Remove the selected animal from the cage. 4 Remove the needle. 2 Fill the syringe with the culture to be injected. 6 Apply little xylol over the area of the vein. cotton. The suckling mouse is used for isolation and cultivation of viruses like Coxsackie A and B viruses and arboviruses. colour dye and other standard laboratory glassware. After inoculation of the animal with appropriate specimens. 5 Wipe clean the tail with spirit or antiseptic soaked cotton. II Reagents and lab wares Discarding jar. 26G needle.258 LESSON 87 INTRODUCTION Intravenous Inoculation into Mice Tail Vein LEARNING OBJECTIVES After completing this practical you will be able to: 1 Inoculate the appropriate culture suspension into the tail vein of mouse. Anaesthetic agent. 2 Keep the animal on a rough surface such as over a mouse cage or a wire. Injection of material REQUIREMENTS I Equipments Mice cage. then place plain sterile cotton over the prick and hold it firmly until bleeding stops. the changes can then be observed in the inoculated mouse and result interpreted. Note: The air is to be expelled by pushing the needle into the cotton inserted into a sterile tube and the volume to be adjusted 3 The needle should be recapped carefully and placed ready for use. etc. 4 Insert the tail through the mesh of a wire basket and hold the animal in such a way that the animal is inside the wire mesh and tail outside. and antiseptic. Mouse is one of the most commonly used lab animal for different animal experiments. culture tube. and also for testing enterotoxins. The adult mouse is used for isolation and cultivation of organisms such as Mycobacterium leprae. 5 Keep back the animal in the cage after labeling with colored dye. Note: It should be ensured that the needle is inside the vein by withdrawing some blood.1 ml and 0. 2 Then insert the needle into the vein midway between base and tip of tail. and allow it to act for a minute so that the vein will be prominent. PROCEDURE Procedure for loading the syringe for injection 1 Open the culture tube near the flame. Toxoplasma gondii. before inoculation should be tested for any infection or other physiological changes. 2 The mouse.Textbook of Practical Microbiology 259 6 Clean the table after performing the experiment and then dispose off the needle into a discarding jar.5 to 1. Cryptococcus neoformans. Rickettsia species. Francisella tularensis. OBSERVATIONS The inoculated mouse should be observed and handled with care every day and any change in its health condition should be monitored. Bordetella pertussis. 3 For pathogenicity and virulence testing of (a) (b) (c) (d) (e) (f) (g) (h) Streptococcus pneumoniae. Nocardia asteroides. Herpes simplex virus. RESULTS AND INTERPRETATION Depending on the type and size of inoculation. Leishmania spices. Bacillus anthracis. Trypanosoma brucei. Adult mice 1 For isolation and cultivation of (a) (b) (c) (d) Mycobacterium leprae – foot pad. the animal will show changes and typical characteristic health manifestations. QUALITY CONTROL 1 The mouse has to be marked well before use for appropriate selection of animal. . BOX 87-1 USES OF MICE IN LABORATORY Suckling mice Less than 48 hours old weighing 0. Rabies virus.tail.0 gm. Toxoplasma gondii – intraperitoneal. Plasmodium berghei . 2 For isolation and cultivation of coxsackie A and B virus. Chlamydia psittaci. 1 For testing enterotoxins of enteropathogenic bacteria. 2 For isolation of the causative agents namely (a) (b) (c) (d) (e) (f) (g) Borrelia recurrentis.intestinal. These are to be noted and interpreted. Cryptosporidium species . and arbovirus. Histoplasma capsulatum. Listeria monocytogenes. (The CV Mosby Company. 14th Edition. Churchill Livingstone. 1996. The mouse should be handled only with gloved hands. pp. VIVA 1 What are the common uses of mice as laboratory animal? 2 What care should be taken during handling the mouse? Ans.260 Intravenous Inoculation into Mice Tail Vein KEY FACTS 1 The mouse to be inoculated should be first checked thoroughly for proper health conditions. After inoculation. Louis) 2002. Its health should be monitored regularly. the mouse should be kept in an individual cage with proper labeling. FURTHER READINGS 1 Forbes BA. It should be handled gently and the handler should thoroughly disinfect his hands after the inoculation. 2 Mackie and McCartney. Practical Medical Microbiology. The animal should be fed and watered well. St. 3 What post inoculation care should be given for mice? Ans. Bailey and Scott’s Diagnostic Microbiology. Sahm DF and Weissfeld AS. . 11 th ed. 921. 2 The area for inoculation should be disinfected before inoculation. 8 Clean the ear with a sterile swab and return the rabbit to its cage. 2 Shave the hair from the rear margin of an ear. Smear the skin over the marginal ear vein with petroleum jelly to delay clotting. 7 After collection release pressure on the vein. Streptococcus pneumoniae. etc. and for testing of enterotoxins of enteropathogenic pathogens. RESULTS AND INTERPRETATION Repeated samples of blood upto 50 ml can be collected at 2-4 week intervals. before inoculation should be tested for any infection or other physiological changes. Rabbit is one of the most commonly used lab animal for different experiments. Adult rabbits are used for the isolation of Listeria monocytogenes .5. test tube. 3 Grasp the ear near its base so that the venous return is impeded and the vein raised. 2 The rabbit. Bunsen flame. OBSERVATIONS The inoculated rabbit should be observed and handled with care every day and any change in its health condition should be monitored. Uses of rabbits are summarized in the box 88-1. . Infant rabbits are used for the propagation of Vibrio cholerae . 6 Collect the blood in a clean sterile test tube. These antibodies can be obtained by drawing the blood from marginal ear vein of the rabbit. Note: The cut should penetrate the skin and vein but not be so deep as to sever the vein. PRINCIPLE Rabbit is widely employed for raising and production of antibodies against specific antigens. When the flow begins to recede. cover the cut with a small piece of cotton and press firmly. wiping the cut with cotton will restart it. III Specimen Laboratory bred adult healthy rabbit weighing 1. 4 With a scalpel blade make a diagonal cut across the vein. REQUIREMENTS I Laboratory wares Jar. Blood samples can also be collected from central artery of the ear or directly from the heart by cardiac puncture. LEARNING OBJECTIVES After completing this practical you will be able to: 1 Collect the blood from the ear marginal vein of rabbit.3 Kg.Textbook of Practical Microbiology 261 LESSON 88 INTRODUCTION Collection of Blood from the Marginal Ear Vein of Rabbit PROCEDURE 1 Restrain the rabbit by gently wrapping it in a blanket. Antigen is injected into rabbit and antibodies are produced. 5 Blood will then start to flow. and petroleum jelly. QUALITY CONTROL 1 The rabbit has to be marked well before use for ease in identification. and sterile scalpel. colour dye. II Reagents Antiseptic. cotton. The ear should not be released at this time. . (e) Increasing virulence of rabies virus and vaccinia virus. Practical Medical Microbiology. Adult rabbit 1. Other uses (a) Differentiating M. Sahm DF and Weissfeld AS.262 Collection of Blood from the Marginal Ear Vein of Rabbit BOX 88-1 USE OF RABBITS IN LABORATORY Infant rabbit 1. 1996. 2 Sudden movements or noise should not be made during the procedure. For testing virulence and pathogenicity of (a) Listeria monocytogenes. (f) Candida albicans. (g) To raise antibodies to antigen. In enteropathogenic organisms. (b) Sereny’s test. (c) Rabbit ileal loop test (enterotoxin testing). (h) For serum as source of complement for cytotoxic assays. 2. 921. testing of enterotoxins can be done. 11 th ed. (d) Mycobacterium tuberculosis. Louis) 2002. (b) Streptococcus pneumoniae. St. (The CV Mosby Company. bovis. the ear should not be released immediately. 2 Mackie and McCartney. KEY FACTS 1 After cutting the ear and vein. pp. 14th Edition. Churchill Livingstone. Animal model for Vibrio cholerae. (e) Herpes simplex virus. Bailey and Scott’s Diagnostic Microbiology. 3 Blood samples can also be collected from central artery of the ear or directly from the heart by cardiac puncture. (c) Nocardia spp. (f) For collecting blood for antistreptolysin O test. VIVA 1 What are the other routes for the collection of blood from rabbit? FURTHER READINGS 1 Forbes BA. (d) Maintenance of Treponema pallidum. tuberculosis and M. 2. intramuscular. then the animal should be disinfected and incinerated with proper care. 4 Take care of animal to prevent it from other infections. rabbit. age of the animal. 5 Test results should be monitored and recorded regularly. intraperitoneal. They are used for a wide range of applications and form an integral part of research (Box 89-1). etc. PRINCIPLE The principle behind using of laboratory animals are that when an experimental material is inoculated into a laboratory animal. intracranial. etc. intraperitoneal. guinea pigs. intradermal. masks. etc. exotoxin and enterotoxin.Textbook of Practical Microbiology 263 LESSON 89 INTRODUCTION Animals and their uses in the Laboratory LEARNING OBJECTIVES After completing this practical you will be able to: 1 Know the commonly used laboratory animals (mice. . Laboratory animals have been used for ages. Animals such as these should be properly taken care of. etc. After inoculation following care of the animals should be taken: 1 Physical properties of the animal should be monitored regularly. it shows pathological changes which can be later studied. The routes of inoculation in various laboratory animals include subcutaneous. intra-abdominal. REQUIREMENTS I Equipments Wire cage. hamsters. intracerebral. intradermal. bacterial. physiological condition of the animal. in laboratories for different purposes. intramuscular. 89-3). Different animals and their usage are listed in the table 89-1. monkeys. rabbits. spirit/antiseptic and soaked cotton. PROCEDURE 1 Different routes of inoculation are followed for different types of inoculum and lab animals. personnel protective equipment. wire mesh. pyrogen. III Specimen Specimen to be inoculated may be toxins including endotoxin. Animals such as mouse (Fig. intravenous. intrathecal. 2 Know the uses of these animals in the laboratory. The information that are to be recorded before and after inoculation of a lab animal include the number of the animal. and should be in accordance to the governing rule for animal usage in laboratory. 3 Make sure that the animal is taking normal amount of food and water as before to inoculation. These different routes are selected depending on the nature of the inoculum and it is important because different organisms have different tissue tropism. 89-1). 89-2) and guinea pig (Fig. hood with Bunsen flame. 2 Commonly used routes of inoculation include intravenous. rabbit (Fig. nature of the specimen. guinea pig and other animals) for animal experiments. subcutaneous. fungal or viral culture. 2 Health of the animal should be checked regularly. These animals include mice. date of inoculation of the specimen and route of inoculation. The animal if dies due to the effect of the specimen inoculated. II Reagents and animals Syringe with needle. It should also include the changes noted in animals on a daily basis and the date of death of the animal if the inoculated specimen proves fatal. intra-nasal. type of inoculation etc. To study immune response to an antigen. QUALITY CONTROL 1 The laboratory animal’s health and physical condition should be checked before using them. To study changes in the condition of animals by inoculating test substances. To study hypersensitivity by producing reactions due to introduction of allergens. BOX 89-1 USES OF LABORATORY ANIMALS To test endotoxin and exotoxin production by various bacteria. RESULTS AND INTERPRETATION Depending upon the changes that the animal shows.264 Animals and their uses in the Laboratory 3 Whichever are the lab animal used and whichever route of inoculation be the case the following general procedure can be adopted. 4 If box is unavailable. and is comfortable. 5 Guinea pigs. To produce complements. hamsters can also be held using a cloth wrapped around them. OBSERVATIONS The animal is marked or tagged with all necessary information like the number. To produce monoclonal and polyclonal antibodies against select antigens. 2 The animal should be labeled properly before and after inoculation. FIGURE 89-1 Mouse. the mouse is held tightly in one hand with the tail running between two fingers. b In case of mice. the animal is kept inside a rabbit holding box. FIGURE 89-3 Guinea pig. FIGURE 89-2 Rabbit. and kept in a place away from other animals. . rabbit or guinea pig. 7 The area of prick can be later wiped with dry cotton and after monitoring the animal for any adverse reaction. it can be left in its cage. date of inoculation. route of administration. To use the blood/serum of animals. To test and producte vaccines. keep the rabbit on a towel and wrap it completely leaving the head outside in such a way that the animal does not move. To test for pyrogens. the results of the test shall be interpreted. a The animal be it mouse. they are properly labeled first before use. c In case of rabbit. 6 The syringe is loaded with the appropriate material and gently introduced into the marked area of inoculation. (c) Clostridium perfringens. (d) Clostridium botulinum. (b) Leishmania species. 2 For isolation of (a) Leptospira species. (c) Clostridium tetani. 2 For RBC for serological tests. (c) Yersinia pestis. (e) Sereny’s test. (b) Entamoeba histolytica. (d) Leptospira icterohaemorrhagiae. 6 Laboratory cultivation of hepatitis A virus and Plasmodium falciparum. (e) Brucella abortus.Textbook of Practical Microbiology 265 Table 89-1 Laboratory animals and their usage Monkeys Monkeys are primates and are extensively used in laboratories. (g) Toxoplasma gondii. lesions in spleen and liver). (b) Mycobacterium bovis. 3 Pathogenicity testing of (a) Listeria monocytogenes. (f) Chlamydia species. tuberculosis (spleen enlargement. Guinea pig Uses 1 For the isolation of (a) Mycobacterium tuberculosis. (c) Pathogenicity testing of M. 5 Testing hemagglutinating property of measles virus. 3 For testing virulence of Entamoeba histolytica. Uses 1 To raise antiserum. Adult hamster 1 For pathogenicity testing of (a) Leishmania donovani. 2 For toxigenicity testing of (a) Corynebacterium diphtheriae. (b) Bacillus anthracis. (d) Entamoeba histolytica. 3 Other uses (a) Maintenance of virulence of Entamoeba histolytica. (b) Clostridium perfringens. Since they are genetically evolved animals. (d) Pyrogen testing. (e) Cryptococcus neoformans. they are considered very suitable for lab testing. 4 Testing pathogenicity of polio virus. (b) Drug efficiency testing against Leishmania donovani. (b) For the evaluation of bronchodilator compounds. Hamsters Uses Infant hamsters 1 To test tumorigenicity of adeno viruses. 4 Other uses (a) For the preparation of complement. (c) Toxoplasma gondii. (c) Bacillus anthracis. 7 Animal model for influenza virus. . Bailey and Scott’s Diagnostic Microbiology. 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. 2 The lab animal’s health and physiological condition should be checked before and after inoculation. 2 Mackie and McCartney. 921. pp. and should be in accordance to the governing rule of CPSEA for animal usage in laboratory. Louis) 2002. St. (The CV Mosby Company. 14th Edition. Practical Medical Microbiology. Churchill Livingstone.266 Animals and their uses in the Laboratory KEY FACTS 1 Animals should be properly taken care of. . Sahm DF and Weissfeld AS. 11 th ed. 1996. Textbook of Practical Microbiology UNIT 267 XIV Medical Entomology Lesson 90 Identification of Common Insects . Characteristic of unspotted wings and white stripes on a black body are present. d). Head has a long needle-like structure named proboscis. glass slides. Identifying features of Anopheles 1. Head is semi-globular in shape with a pair of large compound eyes. Antennae are bushy in male. Palpi is as along as proboscis. Dark (blackish) or white scales on the wing veins. bearing a pair of wings dorsally and three pairs of legs ventrally. a pair of palpi.268 LESSON 90 INTRODUCTION Identification of Common Insects LEARNING OBJECTIVES After completing this practical you will be able to: 1 Know important identifying features of common insects. Tip of abdomen is pointed. glass marking pencil. These are day bitters. Diseases transmitted 1. c). Other characteristic features are: a). All the general features of mosquitoes as mentioned above. So they are called as tiger mosquitoes. 6. etc. Insects are responsible for transmitting a wide variety of infections to humans. c). abdomen are at an angle of 45° to the resting surface. a pair of antennae or feelers. 1. Other characteristic features are: a). When at rest. MOSQUITOES General features 1. 2. 3. II Specimen Insect to be identified. cover slips. 2. Abdomen has 10 segments. Dengue haemorrhagic fever. All the general features of mosquitoes as mentioned above. Palpi are short in female. sterile cotton swab. each situated on either side of proboscis. Dengue. e). 2. body shows a hunch back. b). 2. 5. . 4. thorax. d). Thorax is large and rounded. Wings are characterized by a fringe of scales on the posterior border. Diseases transmitted Malaria REQUIREMENTS Identifying features of Aedes I Equipments and lab wares Standard dissecting microscope. b). head. Hence it is essential to know different insects causing infections in man and methods to identify them. Legs are striped or banded in nature. The middle lobe of tri-lobed salivary gland is short. When anopheles is at rest. PRINCIPLE The insects can be identified by observing the killed insect under a dissecting microscope and observing their morphological features for specific identification. Trombicula akamush is the vector in Japan. 8 Dorsal surface has backwardly pointed spines. smaller than mosquito. Poliomyelitis. Has 4 pairs of legs. Helminthic infections. 4 Thorax is marked with 2 to 4 dark longitudinal stripes. TROMBICULID MITE It is also known as scrub typhus mite. The second longitudinal vein on the wing branches twice. 2 Greyish brown having dark conspicuous eye and hairy body. 5 Yellow fever. 6 Legs and body are covered with numerous short and stiff hairs called Tenent hairs which secrete a sticky substance. 7 Only females suck blood at night. Conjunctivitis. Diseases transmitted by sand fly are summarized in the box 90-1. 3 West Nile fever. 4 Thorax bears a pair of wings and three pairs of legs. Anthrax. 7 Abdomen is segmented and shows dark and light markings. slender. Identifying features 1 2 3 4 5 It is 0. 2 These mosquitoes bite about midnight. Gastroenteritis. SAND FLY General features 1 2-4mm in length. for sucking liquid food. Body is hairy. visible to naked eye. Dysentery. ITCH MITE The itch mite or Sarcoptes scabies var hominis belongs to the class Arachnida. Diseases transmitted 1 Bancroftian filariasis. Front legs have suckers at the end. 9 They do not fly. Diseases transmitted 1 2 3 4 5 6 7 8 9 10 11 12 Typhoid and paratyphoid fever. In males. 6 Males are smaller than the females. 3 Head bears a pair of antennae. a pair of large compound eyes and a refractive proboscis. Has 2 pairs of legs in front and 2 pairs behind. the first 2 pairs of legs possess suckers. 5 Wings are upright and lanceolate shaped. hairy antennae and a proboscis with 16 segments. except the tip of abdomen is blunt. 6 Legs are longer and hairy. 2 Body is divided in to head. 2 Japanese encephalitis. 4 Rift valley fever. Trachoma. 2 Forage mite and house dust mite cause allergic respiratory distress. and abdomen. Body is constricted between the 3rd and 4th pairs of legs. the fourth pair of legs also have suckers. Norwegian itch of man. 5 Each leg is provided with a pair of pads which enable the fly to walk on highly polished surfaces. 3 Head contains a pair of long. they only hop about up to 3 feet. Diarrhea. and the hind legs end in long bristles. Yaws. Trombicula dilensis is the vector in India. Cholera. Diseases transmitted Rickettesia orientails (Orientia tsutsugamushi). Amoebiasis. Eyes of the male are close together and those of the female are set apart widely. 8 Males have three pairs of clappers on the last abdominal segment. Diseases transmitted 1 Scabies (seven year itch). Body is tortoise shaped.Textbook of Practical Microbiology 269 3 Chikungunya fever. Covered dorsally and ventrally with numerous feathered hairs.5mm in size. Identifying features 1 2 3 4 Adult mites are 1-2mm in length and are red in colour. first branching takes place in the middle of the wing. Identifying features of Culex 1 Similar to that of Aedes aegypti. It bears a pair of wing and three pairs of legs. which is characteristic of the genus Musca. HOUSE FLIES Identifying features 1 It is mouse gray in colour. rounded above and flattened below. giving a figure of eight appearance. thorax. . 7 In females. 3 Trench fever or 5 day fever caused by Bartonella quintana. and abdomen. 4 Coxal segments lack spurs. 15 Life Span is 3 years. 8 They do not have wings. abdomen is bilobed. whereas in female it is found in small portion. each having 5 segments. 3 Spiracular openings lies behind the 3rd pair of coxal segments. thorax and abdomen is not clear. 11 Only one nymphal stage is present. c Pubic louse – Phthirus pubis. 10 Males have coiled genitalia. 6 Thorax has 6 legs. which is an adaptation to move freely in hairs and feathers. 14 Cannot resist starvation for a long time. b Two pairs of antennae. 5 Pulvilli are absent. . Dorsal surface is covered by a chitinous shield. 9 They can withstand starvation for one year. b Body louse – Pediculus corporis. 7 Abdomen has 9 segments. 6 More than one nymphal stage is present. 7 Mouth parts are conspicuously blood sucking and pointed downwards. Males have scutum on the entire dorsal surface. Contains 4 pairs of legs but no antennae. Wingless ectoparasite and flattened dorsoventrally. Grayish or reddish after blood meal. Diseases transmitted by hard ticks are summarized in the box 90-2. 5 Demarcation into head. d Five pairs of legs which are swimming appendages. Head is narrower than thorax and has a pair of eyes. 10 Life span is 16 – 21 years. 5th pair is vestigeal. 1-8mm in size. 12 Takes blood meal at both day and night time. 11 Females have a spermatheca at the posterior end and Genal combs are useful for species identification. 2 Oval in shape. Female dies after laying eggs. thorax. 10 Pulvilli are present. In females. it is pointed in males. RAT FLEA Identifying features 1 Light to dark brown in colour. 2 Epidemic relapsing fever caused by Borrelia recurrentis. 4 Contains a hard chitinous exoskeleton and backwardly directed strong bristles. the last segment has penis. SOFT TICK Identifying features All features of hard ticks except 1 Scutum is absent 2 Capitulum lies ventrally. c Three pairs of jaws (1 pair mandibles and 1 pair maxillae). mouth parts are not visible from above. 7 It has anteriorly placed capitulum called head. 7 Take blood meals only at night. 8 Remain ectoparasite only during their short feeding time. 8 Spiracular openings are situated behind the basal segments of the fourth pair of legs. CYCLOPS Identifying features 1 2 3 4 5 Pear-shaped. which helps them to leap up to 10-15cms.5 to 1mm in length. In India relapsing fever is transmitted by Ornithodorus tholozani. 9 Spurs are present in coxal segments which assist in classification of ticks. and oval in shape. 8 The louse are of 3 different types: a Head louse – Pediculus capitus. Varies from 0. Cannot distinctly be separated into head. 1 2 3 4 5 6 3 Laterally compressed. but legs are highly developed. Body shows anterior cephalothorax and posterior abdomen. Identifying features 1 2 3 4 5 Measures 2-4mm in length. Dorsoventrally flattened.270 Identification of Common Insects HARD TICK Identifying features Sac-like body varying in size from 2mm to 10mm. which projects forward beyond the scutum. Cephalothorax has a Pigmented eye. Semitransparent. Diseases transmitted 1 Louse borne epidemic typhus caused by Rickettsia prowazaki. called scutum. Diseases transmitted Ornithodorus species is the vector for tick borne relapsing fever caused by Borrelia duttoni. 13 Remain ectoparasite on host for long time. LOUSE It is also called as Pediculus humanus. 9 Abdomen is bulky and has 10 segments. Ornithodorus lahorensis and Argas persicus. Antenna in the larval stages is short having 3 segments while in adult it has 5 segments. 6 Head has simple eyes and short paired antennules. 1996. Species Dermacentor. Ixodes. variabilis.Textbook of Practical Microbiology 271 6 Abdomen has 5 segments. Mesocyclops act as intermediate host for Dracunculosis medinensis. 2 Oriental sore . They can be seen as small moving specks in water. Lutzomyia columbiana. Diseases transmitted 1 Dracunculiasis. First intermediate host for Diphyllobothrium latum. BOX 90-2 DISEASES TRANSMITTED BY HARD TICKS Diseases 1 Tick paralysis. (The CV Mosby Company. 9 Rocky mountain Spotted fever. Dermacentor andersoni. What are the identifying features of Anopheles mosquitoes? What are the diseases transmitted by house fly? FURTHER READINGS 1 Forbes BA. Ixodes scapularis. 5 Russian spring summer encephalitis. 10 Boutonneuse fever. Sahm DF and Weissfeld AS. Practical Medical Microbiology. andersoni. The last segment has two feathered filaments. Rhipicephalus sanguineus. Hence called as water fleas. 2 Colorado tick fever. 3 Kyasanur forest disease. Dermacentor marginatum. Dermacentor. Haemophysalis. Lutzomyia peruensis. 3 They also act as vectors for Gnathostoma spinigerum and Gnathostoma hispidium. 8 Lyme disease. 6 Tick borne encephalitis. Churchill Livingstone. (Bartonellosis. 7 Swims with characteristic jerky movements in water. Haemophysalis spinigera. . 4 Oroya fever . BOX 90-1 DISEASES TRANSMITTED BY SAND FLY Diseases 1 Kala azar. D. 14th Edition. Ixodes. Dermacentor marginatus. St. 2 Diphyllobothriasis. 2 Mackie and McCartney. 11 th ed. pp. Hyalomma marginatum. 7 Osmk hemorraghic fever. 3 Sand fly fever. Carrion’s disease) Species Phlebotomus argentipes. In females the first abdominal segment has got 2 ovisacs on both sides to carry the eggs. D. 4 Crimean – congo hemorrhagic fever. Bailey and Scott’s Diagnostic Microbiology. Lutzomyia verrucarum. Haemophysalis concinna. 921. VIVA 1 2 3 4 List the diseases transmitted by mosquitoes. List the identifying features of cyclops. Phlebotomus sergenti. Ixodes persculatuis. Louis) 2002. Phlebotomus papatasi and Sergentomyia spp. 272 . Textbook of Practical Microbiology UNIT 273 XV Common Viva Spots Lesson 91 Identification of Common Viva Spots . Culture media with growth. Biochemical reactions. and Microscopic slides. FIGURE 91-1 Nutrient agar. . Preparation Prepared by adding agar in nutrient broth and sterilized by autoclaving. etc. Agar : 2–3%. Uses 1 2 3 4 For growing of non fastidious bacteria. Sheep blood : 5–10%. FIGURE 91-2 Blood agar. Concentrated agar (3% and more) prevents swarming of bacteria such as Proteus vulgaris.274 LESSON 91 1 2 3 4 5 6 7 Identification of Common Viva Spots LEARNING OBJECTIVES After completing this practical you will be able to identify the following common viva spots given in Microbiology examination: Culture media. Specimens of parasites. Glass wares. Instruments. Clostridium tetani. Composition Nutrient agar.2–0. Modifications 1 Semi-solid agar if concentration of agar is 0.5%. The colour of the medium is red like that of blood (Fig. 91-1). Preparation of blood agar. Composition Nutrient broth : Peptone water. 91-2). Determination of antibiotic sensitivity. CULTURE MEDIA NUTRIENT AGAR BLOOD AGAR Also referred as ‘Agar’ (Fig. Meat extract : 1%. The blood agar is an enriched medium as well as an indicator medium. 2 Concentrated agar if agar concentration is 2–6%. horse. 91-3). 1 litre. etc. If it is acid. 20 g. etc. 5 g. The medium should be a distinct reddish brown colour. Uses FIGURE 91-4 Mac Conkey agar. Pour plates. commercial Water Agar Neutral red solution. etc) blood may be used. Composition Nutrient agar. Add the agar and dissolve it in the steamer or autoclave. This is a useful medium for cultivation of enteric bacteria (Fig.). Haemophilus.5. Chocolate agar is an enriched medium (Fig. The agar is melted and cooled it in water bath at 75°C. 3. Preparation It is prepared by heating 10% sterile blood in sterile nutrient agar. Adjust pH to 7. FIGURE 91-5 Loeffler’s serum slope. 2 Used in preparation of the potassium tellurite blood agar. Heat in autoclave with free steam (100°C) for 1 hr. MACCONKEY AGAR MacConkey agar is a differential or indicator medium. then at 115°C for 15 min. rabbit.Textbook of Practical Microbiology 275 Preparation Prepared by adding sterile blood to sterile nutrient agar that has been melted and cooled to 55°C. Either human or animal (sheep. 91-4). Add lactose and neutral red which should be well shaken before use and mix. Shigella. Composition 1 2 3 4 5 Peptone Sodium taurocholate. 2 percent in 50 percent ethanol 6 Lactose 10% aqueous solution. The blood agar is sterilized by autoclaving. 1 For growing enteric bacteria. 1 For growth of most of the pathogenic bacteria. Concentration of blood vary from 5% to 50% for special purposes. . 2 To differentiate lactose fermenter (LF) from non-lactose fermenter (NLF) colonies (Salmonella. it assumes rose-pink colour. CHOCOLATE AGAR The medium is so called because it is chocolate in colour. If necessary.5 ml. The medium is sterilized by autoclaving. This is used for culture and isolation of Corynebacterium diphtheriae (Fig. sheep). 10% is most usual concentration used. Uses FIGURE 91-3 Chocolate agar. LOEFFLER’S SERUM SLOPE Loeffler’s serum slope is an enriched medium. Uses For growing fastidious bacteria such as Neisseria. : : : : : 20 g. Heated 10% sterile blood (horse. clear by filtration. blood is added and the medium continues to remain at 75°C till the blood becomes chocolate brown in colour. Preparation Dissolve peptone and taurocholate (bile salt) in water by heating. Pneumococcus. 91-5). Preparation Malachite green solution: Prepare 2% solution of malachite green in sterile water with sterile precautions by dissolving the dye in incubator for 1–2 hr. diphtheriae. 2. Uses 1 For growing Mycobacterium tuberculosis (takes 3 to 6 weeks to grow). Mix the complete medium.6 g. 500 ml. distribute it in 5 ml volumess in sterile McCartney bottles and screw the caps tightly on. Dissolve mineral salts by heating. 6 Malachite green solution. The green colour of the medium is due to the presence of malachite green (Fig.6 g. 3 Glycerol : 12 ml. 5 Egg. FIGURE 91-7 Robertson’s cooked meat medium. 600 ml. particularly of Clostridium species. Add glucose broth to the serum with sterile precautions and distribute in test tubes or in 2–5 ml amounts in sterile conditions to fill up nearly one fourth of the bottles. ROBERTSON COOKED MEAT (RCM) BROTH Robertson cooked meat (RCM) broth is a selective medium used for culture of anaerobic bacteria (Fig. MgSO4 : 0. Uses 1 Loeffler’s serum slope is used especially for cultivation of C.25 g. Preparation of medium Mineral salt solution : Malachite green solution : Beaten egg (20 to 22 hen’s eggs depending on size) : Preparation Dissolve glucose in nutrient broth and autoclave at 115°C for 20 min. 2 For antibiotic sensitively testing of M. 2 Asparagine : 3.24 g. 1. 2 It is also used to show proteolytic properties. tuberculosis. 1 litre. 20 ml. 500 ml. LOWENSTEIN-JENSEN (LJ) MEDIUM Lowenstein-Jensen (LJ) medium is an enriched medium used for growing Mycobacterium species. Magnesium sulphate. 4 Water : 600 ml. Composition 1 Mineral salt solution Potassium dihydrogen phospphate anhydrous KH2 PO4 : 2. Lay the bottles horizontally in the inspissator and heat at 75°–80°C for 1 hr. After sterilization the pH of FIGURE 91-6 Lowenstein Jenson’s medium. Magnesium citrate : 0. 91-6). 1 g.5 g. The medium is sterilised by autoclaving at 121°C for 20 min. .4 g. Autoclave at 121°C for 25 min to sterilize. This is to solidify the medium. Composition 1 2 3 4 5 Cooked meat (fresh bullock heart) Peptone Sodium chloride Water Liquid from cooked meat : : : : : 500 g. Preparation Place the meat in each 1 oz bottle to a depth of one inch and cover with about 10 ml broth. This medium is sterilized by inspissation. 91-7). (RCM ) broth.276 Identification of Common Viva Spots Composition 1 Sterile ox. 100 ml. producing luxuriant growth in 6–12 hr. sheep. horse serum 2 Nutrient broth 3 Glucose : : : 300 ml. 5 and filter. It also acts as reducing agent and used for shake cultures of anaerobes (Fig. 91-10). FIGURE 91-8 Sabouraud’s dextrose agar (SDA). 2 For preservation of stock cultures of anaerobic bacteria. Filter through cotton gauze and adjust to pH 5. 91-9). For making hanging drop preparation of bacteria. adjust pH to 7. Preparation Dissolve the ingredients in warm water. This medium is sterilized by autoclaving. Uses 1 For growing anaerobic bacteria.Textbook of Practical Microbiology 277 broth over meat is 7. Preparation Dissolve the ingredients in the steamer or autoclave. Autoclave at 115°C for 15 min. 2 The agar medium is suitable for the primary isolation of fungi from clinical material. Dissolve in stock bottles or in tubes. For preparation of sugar media. SABOURAUD’S DEXTROSE AGAR (SDA) Sabouraud’s dextrose agar (SDA) is used for culture of fungi. 91-8). 5 g. 20 g. Low pH and high sugar content of this medium make it particularly selective for fungi as against bacterial contaminants (Fig. For growing bacteria for antibiotic susceptibility testing. Distribute as required and autoclave at 121°C for 15 min.4. Composition Peptone : Sodium chloride (NaCl) : Water : 10 g. PEPTONE WATER Peptone water is used as the basis for sugar fermentation media since broth may contain a small amount of sugar derived from FIGURE 91-10 Glucose broth. 10 g.5. meat and it is essential that basal medium to which various carbohydrates added for fermentation tests should be free from natural sugars (Fig.4–7. Uses 1 2 3 4 5 For growing pathogenic bacteria. GLUCOSE BROTH Glucose added to nutrient media promotes luxuriant growth of many organisms. 3 Fungi can take weeks to grow but yeasts grow in 24 hours to 48 hours on this medium. Composition Glucose Peptone Agar Water : : : : 40 g. To test the formation of indole. If test tubes are used the surface of the medium may be covered with a layer of sterile liquid paraffin 1cm deep but this is not essential. 1 L. . 1 L. FIGURE 91-9 Peptone water. Uses 1 For growing fungi. Variants are nonflagellate and non motile.5%). discrete. 2 The colonies are opaque convex with a shining surface and may be pigmented white (var. In young culture they may be filamentous as longs as (80 µm). 91-11). Confluent growth appears like oil paint (Fig. Salient features 1 Seen as b-hemolytic colonies on blood agar with golden yellow pigment. They possess more than one type of fimbriae. 1–3 µm long and 0. FIGURE 91-11 Streptococcus pyogenes on blood agar. 5 Seen as b-hemolytic colonies in blood agar plates. 7 It is catalase negative. PROTEUS SPP. This sterile glucose solution is then added to the sterile basal medium. Salient features 1 Blood agar colonies 0. albus) yellow. after 24 hrs incubation circular. This test differentiates it from Staphylococcus. CULTURE MEDIA WITH GROWTH STREPTOCOCCUS PYOGENES ON BLOOD AGAR FIGURE 91-12 Staphylococcus aureus on nutrient agar. are Gram negative. 4 S. coccobacilli. showing a-hemolysis on fresh blood agar plates (Fig. Preparation Prepare 20% of glucose solution separately.6 µm wide. and for their antibiotic susceptibility testing. aureus is catalase positive which differentiates it from streptococci. 2 Virulent strains isolated from lesions give a matt type of colony whereas avirulent strains produce glossy colonies. Peptone water. Continuous swarming or discontinuous swarming are a series of concentric circles of growth around point of inoculation (Fig. 4 Str. low convex disks. golden yellow or golden (var. Add a drop of phosphoric acid to endure pH not more than 7.5–1 mm in diameter. 3 Gram staining shows as Gram positive cocci in clusters. semi-transparent. (0. 91-12). Swarming is seen in blood agar plates and the growth may eventually appear either as a uniform growth / film over the whole plate. . aureus). 91-13). 3 They are motile by peritrichous flagella. 6 Gram staining shows Gram positive cocci in chains.278 Identification of Common Viva Spots Composition 1 2 3 4 Nutrient broth. Meat extract Glucose STAPHYLOCOCCUS AUREUS ON NUTRIENT AGAR 1%. Uses 1 Used for growing fastidious organisms such as Streptococcus pyogenes and Enterococci. FIGURE 91-13 Proteus species on blood agar. ON BLOOD AGAR Salient features 1 Group of cells at the edge of developing micro colony migrate to an uninoculated area of the medium and present as swarming. 5 It is coagulase positive.0 and autoclave at 115°C for 20 mins. 2 Proteus spp. 3 A mucoid colony type is also encountered and corresponds in virulence to matt type. pyogenes is the commonest organism causing sore throat. non capsulate. 2 It is an aerobe and facultative anaerobe. non motile bacilli. 91-17).0 µm × 0. 4 They are urease positive.5–3.Textbook of Practical Microbiology 279 4 The two common species are Proteus vulgaris and Proteus mirabilis. non sporing. urease negative. 1–2 µm long and 0. Gram negative bacillus. FIGURE 91-16 Klebsiella species on MacConkey agar. 5 They cause urinary tract infections. CORYNEBACTERIUM DIPHTHERIAE ON POTASSIUM TELLURITE AGAR Salient features 1 Potassium tellurite agar is used as selective medium for C. . non capsulated bacillus measuring 1–3 µm × 0. 4 It is oxidase positive. etc. 2 Ps. diphtheriae (Fig.5 µm arranged singly.4–0. and indole negative. 4 They are indole positive. 5 PPA and urease tests are positive. though mucoid strains may sometime occur and usually motile by one or two polar flagella.5–0. 5 It is one of the common causes of nosocomial infections. 5 They cause urinary tract infections and diarrhoea. 3 It is a Gram negative. 1. 3 It is non sporing.7 µm. 91-15). Most of the strains are motile by peritrichate flagella. 2 C. mucoid colonies on MacConkey agar (Fig. FIGURE 91-15 Escherichia coli on MacConkey agar. 91-16). 91-14). 2 They are Gram negative. ESCHERICHIA COLI ON MACCONKEY AGAR Salient features 1 Escherichia coli shows lactose fermenting. 3 Freshly isolated strains possess a well defined polysaccharide capsule.8 µm wide with parallel or bulging sides and slightly pointed or rounded ends. diphtheriae produces black coloured colonies in potassium tellurite agar. in pairs or short chains. pneumonia. FIGURE 91-14 Pseudomonas aeruginosa on nutrient agar. aeruginosa is a strict aerobe. non-mucoid colonies on MacConkey agar (Fig. KLEBSIELLA SPP ON MACCONKEY AGAR Salient features 1 Klebsiella spp produces lactose fermenting. PSEUDOMONAS AERUGINOSA ON NUTRIENT AGAR Salient features 1 On nutrient agar growth of the organism shows green diffusible pigment (Fig. slender. FIGURE 91-17 Corynebacterium diphtheriae on potassium tellurite agar. 280 Identification of Common Viva Spots 3 S. aureus is another bacteria which produces black colonies on this medium 4 Other media used for C. diphtheriae are Loeffler’s serum slope, and blood agar containing fresh, lysed or heated blood. BIOCHEMICAL REACTIONS CARBOHYDRATE FERMENTATION TESTS Salient features 1 It is used to determine the ability of an organism to ferment a particular carbohydrate to produce acid or acid and gas (Fig. 91-20). 2 A large variety of sugars are used as follws: Pentoses Hexoses Disaccharides : : : Arabinose, xylose, rhamnose. Glucose, fructose, mannose, sorbose, galactose. Sucrose, maltose, lactose, trehalose, cellobiose. Raffinose. Starch, insulin, dextrin, glycogen. Glycerol, erythritol, adonitol, mannitol, dulcitol, sorbitol, inositol. Salicin, aesculin. MYCOBACTERIUM TUBERCULOSIS ON LÖWENSTEIN JENSEN (LJ) MEDIUM Salient features 1 M. tuberculosis takes 3–6 weeks to grow on the LJ medium on incubation at 37°C (Fig. 91-18). 2 Cultures should not be discarded as negative until they have been observed for 12 weeks. 3 The colonies are identified by their rough, tough and buff coloured eugonic growth. Trisaccharides : Polysaccharides : Sugar alcohols : Glycosides : FIGURE 91-18 Mycobacterium tuberculosis on Lowenstein- Jensen’s medium. CANDIDA ALBICANS ON SABOURAUD’S DEXTROSE AGAR (SDA) Salient features 1 Candida albicans species grow well on SDA at 25–37°C (Fig. 91-19). 2 Cream coloured, smooth pasty colonies appear in 1–2 days. 3 Lacto-phenol cotton blue preparation and Gram stained smears showing budding yeast cells and pseudohyphae. 4 C. albicans can be differentiated from other species by germ tube test, sugar fermentation and sugar assimilation reactions 3 A suitable indicator that will change colour only as a result of formation of acids during fermentation of sugar is used in the test. A small inverted tube (Durham’s fermentation tube) is placed in each culture tube to detect gas. Andrade’s indicator is the indicator used. Pink colour of the solution is due to acid production by fermentation of carbohydrates. Air bubble is formed in the Durham’s tube if gas is produced. 4 The test is done by inoculating a drop or loopful of the culture. The inoculated tubes are incubated, aerobically at 37°C for required period, and colour change and gas formation noticed. Fermentation reactions are observed usually after a period of 24 hours of incubation. 5 These tests are used to identify some Gram negative bacilli such as E. coli, Klebsiella species, Salmonella species, Proteus species etc. + FIGURE 91-19 Candida albicans on Sabourauds dextrose agar. + + – FIGURE 91-20 Carbohydrate fermentation test. Textbook of Practical Microbiology 281 GLUCOSE WITH DURHAM’S TUBE Salient features 1 It is used to determine the ability of an organism to ferment a particular carbohydrate to produce acid or acid and gas (Fig. 91-21). 2 All members of the family Enterobacteriaceae are glucose fermenters with or without gas production. 3 This test is used to identify some Gram negative bacilli such as E. coli, Klebsiella species, Salmonella species, Proteus species, etc. 4 Examples of indole positive bacteria are E. coli, Proteus vulgaris, Edwardsiella species, etc. 5 Kovac’s reagent consists of amyl iosoamyl alcohol, 150ml; p-dimethyl – amino benzaldehyde; 10g and conc. HCl; 50ml. UREASE TEST Salient features 1 This test determines the ability of an organism to produce an enzyme urease which splits urea to ammonia (Fig. 91-23). 2 The occurrence of this enzyme, urease can be tested for alkali (NH3) production by means of suitable pH indicator. 3 Christensen’s urease medium is an example of the medium which contains phenol red as an indicator. Ammonia produced from urea makes the medium alkaline and phenol red changes to pink / red in colour. 4 Development of pink colour indicates positive test while the persistence of the pale yellow colour indicates the negative test. 5 Examples of urease producing bacteria are Klebsiella species, Proteus species, Yersinia enterocolitica, Helicobacter pylori,etc. – + FIGURE 91-21 Glucose with Durham’s tube. INDOLE TEST Salient features 1 The indole test demonstrates the ability of certain bacteria to decompose the amino acid tryptophan to indole which accumulates in the medium. The production of indole is then tested for by a colorimetric reaction with p-dimethyl amino benzaldehyde (Fig. 91-22). 2 The test is performed by inoculating the peptone water medium with bacterium to be tested and incubated for 24-48 hours at 37°C. Then 0.5ml Kovac’s reagent is added to the medium and shaked gently. 3 Formation of a red coloured ring near the surface of medium indicates positive test. The presence of yellow coloured ring near surface of medium indicates negative test. NEGATIVE POSITIVE FIGURE 91-23 Urease test. CITRATE UTILIZATION TEST Salient features 1 This is a test for the ability of an organism to utilize citrate as the sole carbon and energy source for growth and an ammonium salt as the sole source of nitrogen (Fig. 91-24). 2 Koser’s liquid citrate medium or Simmon’s citrate agar may be used. Bromothymol blue is the indicator in Simmon’s citrate medium. 3 Positive Koser’s citrate medium is indicated by the formation of a turbidity i.e. growth. No turbidity indicates negative growth. 4 In Simmon’s citrate medium positive test is indicated by formation of blue colour and streak of growth. Original green colour and no growth indicates negative growth. 5 Examples of citrate positive bacteria are Klebsiella species, Salmonella species except Salmonella Typhi, Citrobacter species, etc. + FIGURE 91 -22 Indole test. 282 Identification of Common Viva Spots NEGATIVE POSITIVE FIGURE 91-26 Triple sugar iron agar. FIGURE 91-24 Citrate utilization test. PHENYL PYRUVIC ACID TEST (PPA) Salient features 1 It is used to determine the ability of an organism to deaminate phenyl pyruvic acid (Fig. 91-25). 2 Positive PPA test is indicated by a green colour and negative PPA test is indicated by no colour change. Examples of PPA positive bacteria are Proteus species, Providencia species, and Morganella species. 4 Yellow colour formation occurs with fermentation of carbohydrates, while bubbles in butt show the formation of gas during fermentation process. 5 When H2S is produced by the bacteria, the medium shows blackish discolouration. 6 Combinations of TSI reactions: K/A (red/yellow) A/A (yellow/yellow) Glucose only fermented. Glucose, and lactose or sucrose fermented or both fermented. K/K (red / red) : Neither glucose, lactose nor sucrose fermented. Note: K – Alkaline, A- Acidic. : : SPECIMENS OF PARASITE ASCARIS LUMBRICOIDES ADULT WORM Salient features NEGA TIVE FIGURE 91-25 Phenyl pyruvic acid test. 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). 2 Route of infection of the organism is by ingestion of the food or water contaminated with eggs. 3 Embryonated egg is the infective form of the parasite. TRIPLE SUGAR IRON (TSI) AGAR Salient features 1 TSI agar is used to determine the ability of the bacterium to break down specific carbohydrates incorporated in a growth medium, with or without the production of gas, along with the production of hydrogen sulphide (Fig. 91-26). 2 Three carbohydrates i.e., glucose, lactose and sucrose are present in the TSI agar. It also contains ferric salts for detection of H2S production. 3 The medium in a test tube has a butt and slant. Phenol red is the indicator. FIGURE 91 -27 Ascaris lumbricoides adult worm. Textbook of Practical Microbiology 283 HYDATID CYST Salient features 1 Caused by Echinococcus granulosus. 2 Cyst wall consists of two layers (i.e.) ectocyst and endocyst (Fig. 91-28). 3 Hydatid fluid which is present in cyst is used as antigen in immunodiagnostic tests. 4 Man is the intermediate host and dog is the definitive host. 5 Infective agent is the egg, which is present in dog’s faces. 6 Infection is acquired by ingestion of the food or water contaminated eggs. BIJOU BOTTLE Sterilised by hot air oven or autoclave (Fig. 91-30). Uses 1 Specimen collection like CSF, blood, urine, ascitic fluid, etc. 2 For preparation of media e.g. LJ medium, Loeffers, serum slope, etc. 3 For preparation of urease medium. FIGURE 91-30 Bijou bottle. FIGURE 91-28 Hydatid cyst. GLASS SYRINGE GLASS WARES UNIVERSAL CONTAINER Universal containers (Fig. 91-29) are sterilised by autoclave . Glass syringes (Fig. 91-31) are sterilised by hot air oven. Uses 1 For collection of blood by venepuncture. 2 To collect body fluids, pus, etc. 3 To collect blood from animals (sheep, rabbit) which may be used for preparation of blood agar. 4 For injecting medicine to patients. Uses 1 Specimen collection. 2 For measuring and mixing purposes. FIGURE 91-31 Glass syringe. TUBERCULIN SYRINGE These are of two types: Glass or plastic syringe, and graduated 1ml syringe (Fig. 91-32). a) Glass syringes are sterilized by hot air oven. b) Plastic syringes are sterilized by gamma radiations. FIGURE 91-29 Universal containers. 284 Identification of Common Viva Spots FIGURE 91-32 Tuberculin syringe. FIGURE 91-34 Graduated pipette. Uses 1 2 3 4 5 6 Lepromin test. Tuberculin test. BCG vaccination. Insulin injection. Tetanus toxoid injection. To give intradermal and other sub cutaneous injections; and to inject small amount of test material into animals. PASTEUR PIPETTE Pasteir pipettes (Fig. 91-35) are sterilised by hot air oven. Uses 1 Used for delivering solutions or reagent in test tubes, containers etc., during various procedures. 2 For mixing the constituents for reactions. Example: RBC s coated with specific antigen to perform IHA test for detection of antibodies. PETRI DISH Petri dishes (Fig. 91-33) are sterilised by hot air oven. Uses 1 For preparation of culture media such as nutrient agar, blood agar, MacConkey agar, etc. 2 For counting colonies as in pour plate method. FIGURE 91-35 Pasteur pipette. SWAB TUBE Swab tubes (Fig. 91-36) are sterilized by hot air oven. Unplugged swab tubes are contaminated. Presterilized disposable swabs are commercially prepared. These are sterilized by gamma radiations. FIGURE 91-33 Petri dish. GRADUATED PIPETTE These are of 2 types: measuring pipette (Fig. 91-34) and delivery pipette. Sterilised by hot air oven. Uses 1 For measuring quantity of fluid used in serological tests or other tests. 2 For delivering the exact required volume. FIGURE 91-36 Swab tube. Textbook of Practical Microbiology 285 Uses 1 For collection of specimens from various sites e.g. throat, cervix, local lesions, etc. 2 For streaking in case of lawn culture of bacterial growth as in antibiotic sensitivity testing HOT AIR OVEN Salient features 1 Hot air oven (Fig. 91-39) uses the principle of dry heat sterilization (holding temperature at 160°C for 60 min) method. 2 It is used for sterilization of glass wares such as glass syringes, Petri dish, flasks, pipettes, test tubes, etc. NIH SWAB This is a swab used for collection of perianal scrapings. Uses 1 Specially used for collection of specimen from perianal region for demonstration of eggs of Enterobius vermicularis (Fig. 91-37). FIGURE 91-37 Egg of Enterobius vermicularis, x 400. FIGURE 91-39 Hot air oven. INSTRUMENTS INCUBATOR Salient features 1 Incubators (Fig. 91-38) are used for incubating culture plates 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. AUTOCLAVE Salient features 1 Autoclave (Fig. 91-40) uses the principle of moist heat sterilization (121°C at 15 lbs pressure for period of 15 minutes) method. 2 It is used for sterilization of culture media, rubber material, gloves, gowns, dressing, test tubes, etc. FIGURE 91-38 Incubator. FIGURE 91-40 Autoclave. Examples E. 2 Gram positive cocci occur either in pairs.286 Identification of Common Viva Spots MICROSCOPY SLIDES GRAM POSITIVE COCCI Salient features 1 In Gram stained smear. Arranged in chains. FIGURE 91-43 Neisseria gonorrhoeaein a Gram’s stained smear. etc. 1 In Gram stained smear. FIGURE 91-44 Gram negative bacilliin a Gram’s stained smear. pneumoniae can be typically demonstrated by negative staining with India ink. Arranged in groups of eight. capsule can be seen as unstained structure surrounding the cell. 2 They usually do not have any typical arrangement 3 In capsulated organisms. Arranged in pairs. Neisseria gonorrhoea appear as pink coloured Gram negative diplococci with adjacent sides concave typically kidney shaped (Fig. coli. 2 Found predominantly within the polymorphonuclear cells. STREPTOCOCCUS PNEUMONIAE Salient features 1 In Gram stained smear. 2 Capsule is seen as a clear halo around the diplococci. Proteus species. x 1000. chains or clusters. Examples S. Klebsiella species. 91-44). the Gram positive cocci appear violet in colour (Fig. 91-41). the Gram negative bacilli appear red in colour (Fig. . 91-43). GRAM NEGATIVE BACILLI Salient features FIGURE 91-41 Gram positive cocci. NEISSERIA GONORRHOEAE Salient features 1 In Gram stained smear. x 1000. x 1000. but some may be seen outside. aureus Streptococcus pneumoniae Micrococci Sarcina species Streptococcus pyogenes : : : : : Arranged in clusters. 91-42). FIGURE 91-42 Sterptococcus pneumoniae in a Gram’s stained smear. pneumoniae appear as Gram positive lanceolate shaped cocci arranged in pairs. x 1000. Str. Arranged in tetrads. 3 Capsule of Str. surrounded by capsule (Fig. 2 Seen as black spirals against yellowish brown background FIGURE 91-49 Triponema pallidum in a Levaditi stained smear. VIBRIO CHOLERAE Salient features 1 Gram stained smear shows pink coloured comma shaped. . CLOSTRIDIUM PERFRINGENS Salient features 1 Gram stained films of necrotic muscle tissue shows Gram positive bacillus of 4-6 µm x 1 µm with straight. 2 Vibrios are seen arranged on parallel rows fish in stream appearance. x 1000. 2 Ends of the bacilli are truncated. 91-49). x 1000. x 1000. 91-45). often concave and somewhat swollen giving the chain of bacilli a bamboo stick appearance. BACILLUS ANTHRACIS Salient features 1 In a Gram stained smear. 91-46). 2 Pus cells are absent or scanty. Bacillus anthracis appears as short chains of thick purple coloured Gram positive rods arranged end to end surrounded by capsule (Fig. x 1000.Textbook of Practical Microbiology 287 HAEMOPHILUS INFLUENZAE Salient features 1 Gram stained smear shows pink coloured Gram negative pleomorphic bacilli of Haemophilus influenzae (Fig. 91-48). TREPONEMA PALLIDUM Salient features FIGURE 91-46 Vibrio cholerae in a Gram’s stained smear. FIGURE 91-45 Haemophilus influenzaein a Gram’s stained smear. FIGURE 91-48 Clostridium perfringens in a Gram’s stained smear. FIGURE 91-47 Bacillus anthracis in a Gram’s stained smear. 2 Coccobacillary forms of the bacteria can also be seen. 1 Treponema pallidum in a smear stained by Levaditi stain appear as thin delicate spirochaetes of 10 µm long with about 10 regular spirals which are sharp and angular at regular intervals of about 1 µm (Fig. x 1000. parallel sides and rounded or truncated ends without spores occurring singly or in chains (Fig. 91-47). curved Gram negative bacilli with rounded or slightly pointed ends (Fig. hence it does not satisfy Koch’s postulates for a pathogenic bacteria. tuberculosis causes tuberculosis. M. diphtheriae can be seen as early as 6-8 hours after incubation. on which colony of C. 6 Löwenstein Jensen (LJ) medium is the most frequently used medium to grow M. 6 No artificial cell-free media is available for growth of M. FIGURE 91-51 Ziehl Neelsen staining for Mycobacterium tuberculosis. 7 It can be grown in foot pads of 9 banded armadillo. diphtheriae causes diphtheria. x 1000. diphtheriae. 2 Negri bodies are usually found within the nerve cells of the hippocampus and cerebellar region. 3 M.NEELSEN STAINING FOR MYCOBACTERIUM LEPRAE Salient features 1 It is a special stain for demonstrating acid fastness of Mycobacterium leprae (Fig. NEGRI BODIES Salient features 1 Negri bodies are 3 . leprae resist decolourisation with 5% sulphuric acid. leprae. 2 The bacteria appears as green coloured bacilli with bluish black metachromatic granules. leprae are seen as pink coloured acid fast bacilli arranged as parallel rows of bacilli giving a ‘cigar bundle’ or ‘globi’ appearance against a blue background. 4 M. 91-53). 91-51). leprae causes leprosy. 6 Loeffler’s serum slope is an egg based medium .288 Identification of Common Viva Spots ALBERT STAINING Salient features 1 It is a special stain for demonstrating metachromatic granules of C. 4 M. x 1000. FIGURE 91-52 Ziehl Neelsen staining for Mycobacterium leprae. 5 M. tuberculosis.NEELSEN STAINING FOR MYCOBACTERIUM TUBERCULOSIS Salient features 1 It is a special stain for demonstrating acid fastness of M. x 1000. tuberculosis appear as approx. 91-50).27 µm size. tuberculosis to grow in this medium. diphtheriae (Fig. leprae is only acid fast but not alcohol fast. M. FIGURE 91-50 Alberts stained smear of Corynebacterium diphtheriae. 3 Green colour of the bacilli is due to malachite green and bluish black granules due to toluidine blue reagents of Albert’s stain. oval. intra cytoplasmic inclusion body which appear as round . 4 C. 5 M. 3 M. tuberculosis is both acid and alcohol fast. arranged in Chinese letter pattern (V or L pattern). 2 20% sulphuric acid is used for ZN stain. 91-52). 5 Potassium tellurite blood agar is the selective medium for C. 3 These are usually demonstrated in the impression smears of . 1-3 µm size slender pink coloured acid fast bacilli with curved ends against a blue background. ZIEHL . 2 5% sulphuric acid is used for ZN stain. ZIEHL . tuberculosis (Fig. 7 It takes 3-6 weeks for M. tuberculosis resist decolourisation with 20% sulphuric acid. The bacteria forms black colour on this medium after 36-48 hrs of incubation at 37°C. purplish pink structures with characteristic basophilic inner granules (Fig. with numerous acidophilic nuclear and cytoplasmic inclusions in Giemsa stained smears. 2 These giant cells show multinucleate syncytium formation. 4 All ages of erythrocytes are infected. x 1000. 2 P. . FIGURE 91.55 Molluscum bodies.54 Multinucleate giant cells-measles.53 Negri bodies. Ring stages are found within both normal sized young and older RBCs. 4 It is a Giemsa stained thin blood smear. PLASMODIUM VIVAX RING STAGE Salient features 1 Plasmodium vivax is the causative agent of vivax malaria (Fig. falciparum is the causative agent of falciparum malaria and causes cerebral malaria (Fig. 91-55). 2 Molluscum bodies are composed of large nucleus of virus particles. MOLLUSCUM BODIES Salient features 1 Section of the lesion shows large (20 µm to 30 µm) sized eosinophilic hyaline inclusion bodies with nuclei displaced to the margin (Fig. x 1000. 5 The erythrocyte which is invaded by the parasite is not enlarged 6 P. 91-57). 4 Negri bodies can also be demonstrated by Mann’s and Giemsa stain. PLASMODIUM FALCIPARUM RING STAGE FIGURE 91. x 1000. 3 Multiple ring forms are found within a single RBC. Salient features 1 In a Giemsa stained thin blood smear.56 Plasmodium vivax ring stage in a Giemsa stained blood smear. falciparum shows frequent drug resistance to chloroquine. FIGURE 91. FIGURE 91. embedded in protein matrix. 2 Female Anopheles mosquito transmits the disease and is the definitive host 3 Man is the intermediate host. 91-54). 91-56). The cytoplasm portion of ring opposite the nucleus is thickened. MULTINUCLEATE GIANT CELLS: MEASLES Salient features 1 These multinucleate giant cells are produced by measles virus in Hela cell lines in tissue culture (Fig. 5 Presence of basophilic inner granules within the Negri bodies helps to differentiate canine distemper which lacks it. 5 Young erythrocytes are predominantly infected and they are enlarged. along with accole forms. the ring stage appear as a blue stained ring of cytoplasm with a red chromatin dot. ring stage appears as blue ring of cytoplasm surrounding a central vacuole with red chromatin dot at centre.Textbook of Practical Microbiology 289 the rabid dog brain stained by Seller’s technique (basic fuschin and methylene blue in methanol). x (1000. Cytoplasm is purple and nucleus is small. 2 Female gametocyte are typically crescent (banana) shaped with rounded or pointed ends. Cytoplasm stains deep blue and nucleus is compact. 2 Female gametocyte (macrogametocyte) is spherical and large than the male. FIGURE 91. in a Giemsa stained blood smear. x 1000. x 1000. FIGURE 91. 91-58). 2 Tachyzoites appear as 3-7 µm. Gomori methenamine silver. 91-61). x 1000. Some LD bodies are found outside macrophages.58 Plasmodium vivax female gametocyte in a Giemsa stained blood smear. and Wright-Giemsa stain. donovani shows frequent drug resistance to pentavalent antimonials. 3 Tachyzoites can be stained with periodic acid schiff (PAS). 91-60). TOXOPLASMA GONDII Salient features 1 The typical active multiplying tachyzoites is an important diagnostic form of Toxoplasma gondii (Fig. 3 LD bodies. Cytoplasm stains light blue or pale blue and nucleus is large (Fig. 4 L.57 Plasmodium falciparum ring stage in a Giemsa stained blood smear.290 Identification of Common Viva Spots MALE FEMALE FIGURE 91. otherwise known as amastigote stage of L. broader and shorter. . PLASMODIUM FALCIPARUM MALE AND FEMALE GAMETOCYTES Salient features 1 Male gametocyte (microgamete) is sickle shaped. FIGURE 91. donovani is found mostly inside the macrophages.60 LD bodies in a Giemsa stained blood smear. PLASMODIUM VIVAX MALE AND FEMALE GAMETOCYTES Salient features 1 Male gametocyte (microgametocyte) is spherical and smaller than the female. LD BODIES Salient features 1 It is a Giemsa stained thin blood smear showing a red coloured nucleus and pale blue coloured cytoplasm multiple rings in one red blood cell.59 Plasmodium falciparum male and female gametocytes. oval to crescent shaped structures with pointed anterior end and rounded posterior end. 2 Leishmania donovani is the causative agent of visceral leishmaniasis or kala-azar (Fig. 91-59). Cytoplasm stains light blue and nucleus is diffuse (Fig. hematoxylin and eosin. x 1000. CYST OF GIARDIA INTESTINALIS Salient features 1 Iodine wet mount of stool showing iodine stained brown oval cyst of Giardia intestinalis (Fig. 3 No nuclei are present in the tail end.The cyst is separated from the cyst wall by a clear space. etc. 4 The quadrinucleate cyst is the infective form of the parasite. FIGURE 91. x 1000. gondii which appear as 40mm to 50mm spherical structure surrounded by eosinophilic.61 Toxoplasma gondii tachyzoites in Wrisht-Giemsa stained blood smear. FIGURE 91. 6 Man is the definitive host. FIGURE 91. weakly PAS positive cyst wall. 2 Cyst contains four nuclei. histolytica and E. Culex and Aedes mosquito transmit the disease and are the intermediate host. slow growing trophozoites known as bradyzoites. 2 Cyst contains 4 nuclei. FIGURE 91. 4 The infection is transmitted by ingestion of water and food contaminated with cysts. . etc.dispar are morphologically similar. 4 Wuchereria bancrofti is the causative agent of lymphatic filariasis (Fig. 5 The infection is transmitted by ingestion of water and food contaminated with cysts.Textbook of Practical Microbiology 291 4 Tissue cyst is the resting form of T. 2 The body of microfilaria shows few nuclei in the body and more distinct tail is tapering and pointed. forming a dividing line within the cyst wall. 91-64). each nucleus has a central karyosome. x 400. 91-63). x 400. 5 Anopheles. and an axostyle which lie diagonally.63 Cyst of Entamoeba histolytica / dispar. WUCHERERIA BANCROFTI MICROFILARIA Salient features 1 It is a Giemsa stained thin blood smear showing the sheathed microfilaria.64 Cyst of Giardia intestinalis. CYST OF ENTAMOEBA HISTOLYTICA/ DISPAR Salient features 1 Iodine wet mount of stool showing quadrinucleate cyst of Entamoeba histolytica/dispar (Fig. malabsorption . amoebic liver abscess.62 Wuchereria bancrofti microfilaria in a Giemsa stained blood smear. 3 The cysts of E. x 1000. 3 The cyst is the infective form of the parasite. It contains hundred of strongly PAS positive. 91-62). 5 It is the causative agent of diarrhoea. 6 It is the causative agent of amoebic dysentery. 292 Identification of Common Viva Spots EGG OF ROUND WORM Salient features 1 Bile stained egg of Ascaris lumbricoides in the saline wet mount of stool (Fig. 91-69). nana infection is transmitted by faeco -oral transmission. 2 Egg contains oncosphere with three pairs of hooklets. 3 Egg is the infective form of the parasite. x 100. 2 Embryonated egg is the infective form of the parasite. EGG OF HYMENOLEPIS NANA FIGURE 91-65 Egg of round worm. vermicularis infection. 4 Hook worm infection is transmitted by filariform larva piercing the intact skin. 3 Filariform larva is the infective form of the parasite. americanus are morphologically similar. 5 It is the causative agent of microcytic hypochromic anemia and tropical pulmonary eosinophilia. 4 It is the causative agent of intestinal ascariasis. 3 A. lumbricoides infection is transmitted by faeco -oral transmission. x 400. 4 It is the causative agent of pruritus ani in children. FIGURE 91-67 Egg of Enterobius vermicularis. 5 Auto infection is characteristic of E. 91-66). 91-68). x 400. 2 The eggs of A. duodenale and N. FIGURE 91-68 Egg of Hymenolepis nana. trichiura egg is barrel – shaped with mucous plug at each pole. 4 It is the causative agent of hymenolepiasis EGG OF HOOK WORM Salient features 1 Saline wet mount of stool showing non. vermicularis infection is transmitted by faeco-oral transmission. EGGS OF TRICHURIS TRICHIURA Salient features 1 Saline wet mount of stool showing bile stained egg of Trichuris trichiura (Fig. 2 Egg is the infective form of the parasite. 2 T. Salient features 1 Saline wet mount of stool showing non-bile stained egg of Hymenolepis nana (Fig. FIGURE 91-66 Egg of Hook worm. x 400. 91-65). 91-67). . 3 Egg is the infective form of the parasite.bile stained egg of hook worms: Ancylostoma duodenale and Necator americanus (Fig. EGG OF ENTEROBIUS VERMICULARIS Salient features 1 Saline wet mount of stool showing plano-convex and nonbile stained egg of Enterobius vermicularis (Fig. 3 H. etc. 3 E. albicans from other Candida species 3 The test is performed by incubating Candida in patients serum at 37°C for 2hours 4 Germ tube production is due to the formation of pseudohyphae by the fungus. Parija SC. pp.69 Egg of Trichuris trichura. C. 2006. 2 It is used to identify and differentiate C.71 Germ tube test. Louis) 2002. vaginitis. 4 It is the causative agent of gastrointestinal infections. FIGURE 91. Parija SC. (The CV Mosby Company. 14th Edition. Practical Medical Microbiology. Bailey and Scott’s Diagnostic Microbiology. Mackie and McCartney. Sahm DF and Weissfeld AS. . 3nd Edition. 1996. onychonychia etc. 3 They cause opportunistic infection especially in patients with HIV. albicans appear purple in colour (Fig.Textbook of Practical Microbiology 293 3 T. x 1000. FIGURE 91.. All India Publishers and Distributors. Nepal. GERM TUBE TEST Salient features 1 The test is also called Reynold – Braude phenomenon (Fig. in immunocompetent patients. 921. 4 They cause oral thrush. 91-71). x 400. FURTHER READINGS 1 2 3 4 Forbes BA. Churchill Livingstone. 11 th ed. St. x 400. CANDIDA ALBICANS Salient features 1 It is a yeast-like fungus.70 Candida albicans in a Gram’s stained smear. Textbook of Medical Parasitology. trichiura infection is transmitted by faeco-oral transmission. Dharan. 91-70) FIGURE 91. Stool Microscopy. BPKIHS. Heavy infection may complicate as appendicitis. 2 In Gram stained smear. 1998. 294 . 291 Aerotolerant anaerobes 52 Aesculin 173 Agar 274 Agar dilution method 97. 244 Amoebiasis 269 Amoebic antigens 130 Amoebic dysentery 291 Amoebic liver abscess 291 Anaerobic bacilli 54 . 33. 176. 268. 32.Textbook of Practical Microbiology 295 Index A Abbe condenser 4 ABO system 110 Acetoin 78 Acetylcholinesterase 140 Acid fast staining method 27 Acid-alcohol 27 Acid-fast staining 27 Learning Objectives 27 Introduction 27 Principle 27 Requirements 27 Equipments 27 Reagents and glass wares 27 Preparation of strong carbol fuchsin 27 Preparation of 20% sulphuric acid 27 Preparation of 95% alcohol 27 Preparation of acid-alcohol decolouriser 28 Specimen 28 Procedure 28 Quality Control 28 Observation 28 Results and Interpretation 28 Key Facts 29 Viva 30 Acid-fast Staining of Stool Smears 198 Learning Objectives 198 Introduction 198 Principle 198 Requirements 198 Equipments 198 Reagents and lab wares 198 Specimen 198 Procedure 198 Quality Control 198 Observations 199 Results and Interpretation 199 Key Facts 199 Viva 200 Adult hamster 265 Adult mice 259 Adult rabbit 261. 288 Albert’s staining 31 Learning Objectives 31 Introduction 31 Principle 31 Requirements 31 Equipments 31 Reagents and glass wares 31 Preparation of Albert’s stain I 31 Preparation of Albert’s stain II 31 Specimen 31 Procedure 31 Quality Control 32 Observation 32 Results and Interpretation 32 Viva 32 Key Facts 33 Alcohol 27. 131 Amniotic sac 243. 262 Aedes aegypti 269. 175. 98 Learning Objectives 97 Introduction 97 Principle 97 Requirements 97 Equipments 97 Reagents and lab wares 97 Preparation of stock solutions of antibiotics 97 Specimens 97 Preparation of suspension of bacteria 97 Procedure 97 Test procedure 98 Quality Control 98 Observations 98 Results and Interpretation 98 Key Facts 98 Viva 99 Agarose 130 Agglutination 108. 140 Allantoic cavity 243 Allantoic sac 244 Allergic respiratory distress 269 Alsever’s solution 121 Amido black 126. 117 Albert’s stain 31. 181 Alkaline phosphatase 138. 60 Alkaline peptone water 46. 165. 94. 125 Biotin-avidin ELISA 141 Bismuth sulfite agar 86 Blastomyces dermatitides 6 Blocking solution 139 Blood agar 44. 163. 60 Antiseptics 60 Anti-Streptolysin O (ASLO) Test 121 Learning Objectives 121 Introduction 121 Principle 121 Requirements 121 Equipments 121 Reagents and lab wares 121 Specimen 121 Procedure 121 Serum dilution 121 Test procedure 121 Quality Control 121 Observations 121 Results and Interpretation 122 Key Facts 122 Viva 122 Antony Von Leeuwenhoek 2 Argas persicus 270 Arginine dihydrolase test 185 Armadillo 288 Asbestos filters 56 Ascaris lumbricoides 206. 171 Auxotrophs 144 Axenic culture medium 208. 45. 161. 184.296 Index Auramine O 30 Autoclave 285 Autolysis 169. 249 Bancroftian filariasis 269 Barbitone buffer 126 Bartonella quintana 270 Basal media 45. 99. 94. 101 Balantidium coli 6. 46. 292 ASLO 122 Aspergillus flavus 235 Aspergillus fumigatus 234 Aspergillus niger 235 Aspergillus species 6 B Babes Ernst granules 31 Babesia 204 Babesia species 203 Bacilli 6 Bacillus anthracis 6. 274 Blood group antigens 110 . 287 Bacitracin test 172 Bacterial Agglutination Test 108 Learning Objectives 108 Introduction 108 Principle 108 Requirements 108 Reagents and glass wares 108 Specimen 108 Procedure 108 Quality Control 109 Observations 109 Results and Interpretation 109 Positive agglutination 109 Negative agglutination 109 Auto agglutination 109 Key Facts 109 Viva 109 Bacterial agglutination tests 109 Bacterial conjugation 155 Learning Objectives 155 Introduction 155 Hfr strains 155 F’ factors and sexduction 155 Principle 155 Requirements 155 Equipments 155 Reagents and glass wares 155 Specimen 155 Procedure 155 Quality Control 156 Observations 156 Results and Interpretation 156 Key Points to Remember 156 Viva 157 Bacterial endospores 38 Bacterial plasmids 145 Bactericidal drugs 93. 162. 101 Bacteriostatic drug 93. 209 Anaerobic bacteria 277 Anaerobic cocci 54 Ancylostoma duodenale 292 Animals and their uses in the laboratory 263 Learning Objectives 263 Introduction 263 Principle 263 Requirements 263 Equipments 263 Reagents and animals 263 Specimen 263 Procedure 263 Quality Control 264 Observations 264 Results and Interpretation 264 Key Facts 266 Viva 266 Anopheles 268. 94. 46 Basic dye 215 Basic stains 20 Bicarbonate buffer 50 Bijou bottle 283 Bile aesculin test 173 Bile solubility test 169 Biological false positive (BFP) reactions 123. 164. 100 Antiseptic 59 Antiseptics and Disinfectants 59 Learning Objectives 59 Introduction 59 Principle 59 Requirements 59 Equipments 59 Reagents and media 59 Specimen 59 Procedure 59 Quality Control 59 Observation 59 Results and Interpretation 59 Key Facts 60 Viva 60 antiseptics 59. 289 291 Anthrax 269 Antibiotic resistance 157 Antibiotic sensitivity 92 Antibiotic susceptibility testing 278 Antigen 133 Antimicrobial agent 93. Atkins and Munch-Peterson) test 173 Candida albicans 6. 57 Brownian movement 12 Brucellosis 253 Buffered distilled water 201 Buffered methylene blue 191 C CAMP (Christie.Textbook of Practical Microbiology Blood group antisera 110 Blood grouping 110 Learning Objectives 110 Introduction 110 Principle 110 Requirements 110 Reagents and lab wares 110 Specimen 110 Procedure 110 Quality Control 110 Observations 110 Results and Interpretation 110 Key Facts 111 Viva 111 Blood parasites 201 Body louse 270 Boeck and Drbohlav’s medium 208 Borrelia duttoni 270 Borrelia recurrentis 270 Bound coagulase 68. 281 Broth dilution agar method 101 Broth dilution method 100 Learning Objectives 100 Introduction 100 Principle 100 Requirements 100 Equipments 100 Reagents and lab wares 100 Preparation of stock solutions of antibiotics 100 Specimens 100 Preparation of suspension of bacteria 100 Procedure 100 Quality Control 100 Observations 101 Results and Interpretation 101 Key Facts 101 Viva 101 Browne’s tube 56. 286 Capsule Staining 3 4 Learning Objectives 34 Introduction 34 Principle 34 Requirements 34 Equipments 34 Reagents and glass wares 34 Specimen 34 Procedure 34 For positive staining of smears 34 For negative staining of smears 35 Quality Control 35 Observation 35 Observation of positive staining method 35 Observation of negative staining method 35 Results and Interpretation 35 Positive staining method 35 Negative staining method 35 Key Facts 35 Viva 36 Carbohydrate assimilation 229 Carbohydrate Assimilation Test 229. 69 Boutonneuse fever 271 Box 1-1 Terminology 5 Box 1-2 Size of Different Organisms 6 Box 12-1 List of most common capsulated organisms that can be demonstrated by negative staining 41 Box 17-1 List of Anaerobic Bacilli and Cocci 54 Box 18-1 Pasteurisation 58 Box 19-1 Commonly used disinfectants and their mechanism of actions 60 Box 2-1 Principle of Dark Ground Microscopy 8 Box 20-1 Uses of catalase Test 63 Box 20-2 Catalase Test for Mycobacteria 63 Box 22-1 Free Coagulase 69 Box 29-1 List of media used for detecting production of hydrogen sulphide 86 Box 31-1 Glossary of terms 93 Box 4-1 Demonstrating Motility of Anaerobic Bacteria 12 Box 43-1 VDRL-ELISA 125 Box 43-2 Biological False Positive Reactions of VDRL Test 125 Box 47-1 Reverse Passive Haemagglutination test 133 Box 49-1 1st. 2nd and 3rd Generation ELISA 140 Box 49-2 Dot ELISA 140 Box 5-1 Terminology 17 Box 50-1 Role of Plasmids in drug resistance 146 Box 51-1 Advantages and Disadvantages of Page 150 Box 52-1 Point Mutations and Large Scale Mutations 153 Box 52-2 Mechanisms of Mutation 154 Box 52-3 The Importance of Mutation 154 Box 53-1 Mechanisms of DNA Transfer 156 Box 54-1 Beneficial Effects of Normal Flora 161 Box 6-1 Simple stains and their uses in microbiology laboratory 21 Box 61-1 Identification of Escherichia Coli 179 Box 61-2 Identification of Klebsiella species 179 Box 62-1 Identification of Vibrio Cholerae 182 Box 63-1 Identification of Pseudomonasaeruginosa 185 Box 67-1 Acid Fast Parasites and Parasitic Components 199 Box 68-1 The Parasites found in the Peripheral Blood Smear 203 Box 69-1 Advantages and Disadvantages of the Concentration Methods 207 Box 71-1 Sabouraud’s Dextrose Agar 214 Box 77-1 Predisposing Factors for Candidiasis 226 Box 8-1 Different Modifications of Acid Fast Stain and their uses 29 Box 8-2 Frequently examined specimens for the Detection of Mycobacterium Tuberculosis 29 Box 87-1 Uses of Mice in Laboratory 259 Box 88-1 Use of Rabbits in Laboratory 262 Box 89-1 Use of Laboratory Animals 264 Box 9-1 Rapid staining by direct fluorescent antibody method 32 Box 90-1 Diseases Transmitted by Sand Fly 271 297 Box 90-2 Diseases Transmitted by Hard Ticks 271 Box 7-1 Modifications of Gram’s staining 25 Box 7-2 Uses of Gram’s Staining 25 Brain heart infusion agar 46 Bright field microscopy 2 Brilliant green bile broth 249 Bromothymol blue 81. 12 Capsular antigens 36 Capsulated bacteria 36 Capsule 36. 215. 230 Learning Objectives 229 Introduction 229 Principle 229 Requirements 229 Equipments 229 . 293 Candida albicans on Sabourauds dextrose agar 280 Candida species 225 Candle jar 51 Candling 244 Capillary tube method 11. 244 Christensen’s urea agar 71. 64 Catalase negative bacteria 63 Catalase positive bacteria 63 Catalase test 62. 81 Learning Objectives 80 Introduction 80 Principle 80 Requirements 80 Equipments 80 Reagents and lab wares 80 Specimen 80 Procedure 80 Quality Control 81 Positive control 81 Negative control 81 Observations 81 Results and Interpretation 81 Key Facts 81 Viva 81 Citrate utilization test 185. 251 Collection of Blood from the Marginal Ear Vein of Rabbit 261 Learning Objectives 261 Reagents and lab wares 229 Specimens 229 Procedure 229 Quality Control 229 Observation 229 Results and Interpretation 229 Key Facts 230 Viva 230 Carbohydrate Fermentation Test 231. 280 Learning Objectives 231 Introduction 231 Principle 231 Requirements 231 Equipments 231 Reagents 231 Preparation of indicator broth medium 231 Specimen 231 Procedure 231 Quality Control 231 Observations 232 Results and Interpretation 232 Key Facts 232 Viva 232 Carbohydrate fermentation tests 231 Carbol fuchsin 27. 178.298 Index CIEP test 130. 167 Learning Objectives 68 Introduction 68 Principle 68 Bound coagulase 68 Free coagulase 68 Requirements 68 Reagents and lab wares 68 Specimen 68 Procedure 68 Slide test 68 Tube test 68 Quality Control 69 Positive control 69 Negative control 69 Observation 69 Results and Interpretation 69 Key Facts 69 Viva 70 Coccidian parasites 198 Coccidioides immitis 6 Cold agglutination test 120 Coliforms 248. 115 Learning Objectives 114 Introduction 114 Principle 114 Requirements 114 Reagents and lab wares 114 Specimen 114 Procedure 114 Quality Control 114 Observations 114 Results and Interpretation 114 Key Facts 115 Viva 115 CO 2 incubator 160 Coagulase 68 Coagulase negative bacteria 69 Coagulase negative staphylococci 168 Coagulase positive bacteria 69 Coagulase reacting factor (CRF) 69 Coagulase test 68. 287 Co-agglutination test 114. 185 Learning Objectives 62 Introduction 62 Principle 62 Requirements 62 Reagents and glass wares 62 Specimen 62 Procedure 62 Slide method 62 Tube method 62 Quality Control 63 Positive control 63 Negative control 63 Observations 63 Slide method 63 Tube method 63 Results and Interpretation 63 Key Facts 64 Viva 64 Catalyst 53 Cell culture 240. 161. 163. 269 Chorioallantoic membrane (CAM) 243. 282 Cladosporium 236 CLED medium 46. 180 Clostridium perfringens 248. 281. 242 Cell lines 240 Cephalosporium 236 Cerebral malaria 289 Cetrimide agar 46. 198 Carbonate buffer 139 Carbonic acid 71 Cardiolipin antigen 123. 131 Citrate negative bacteria 81 Citrate positive bacteria 81 Citrate sulfide agar 86 Citrate utilisation test 80. 227. 232. 275 Cholera 181. 166. 281 Chromoblastomycosis 6 . 125 Cassette ELISA 141 Catalase 62. 185 Chick cell agglutination 181 Chick Martin test 60 Chick RBCs agglutination 182 Chikungunya fever 269 Chlorhexidine 60 Chlorination 248 Chocolate agar 44. 149 Counter-current Immunoelectrophoresis Test 130 299 Learning Objectives 130 Introduction 130 Principle 130 Requirements 130 Equipment 130 Reagents and glass wares 130 Specimen 130 Preparation of Veronal buffer 0. 275 Corynebacterium diphtheriae on potassium tellurite agar 279 Counter-current immunoelectrophoresis (CIEP) 130. 207 Concentration of Stool for Parasites 205 Learning Objectives 205 Introduction 205 Principle 205 Requirements 205 Equipments 205 Reagents and lab wares 205 Specimen 205 Procedure 206 Saturated salt solution flotation method 206 Formalin-ether sedimentation method 206 Quality Control 206 Observations 206 Results and Interpretation 206 Key Facts 207 Viva 207 Condenser 4 Conjugation 155. 157 Conjunctivitis 269 Coomassie staining 149 Corynebacterium diphtheriae 31.075 M (pH 8. 291 Cultivation of Fungi 213 Learning Objectives 213 Introduction 213 Principle 213 Requirements 213 Equipments 213 Reagents and lab wares 213 Specimen 213 Procedure 213 Quality Control 213 Observations 213 Results and Interpretation 213 Key Facts 214 Viva 214 Cultivation of Viruses in the Cell lines 240 Learning Objectives 240 Introduction 240 Principle 240 Requirements 240 Equipments 240 Reagents and lab wares 240 Specimen 240 Procedure 240 Quality Control 241 Observations 241 Results and Interpretation 241 Key Facts 242 Viva 242 Cultivation of Viruses in Embryonated Egg 243 Learning Objectives 243 Introduction 243 Principle 243 Requirements 243 Equipments 243 Reagents and lab wares 243 Specimen 243 Procedure 243 Quality Control 244 Observations 244 Results and Interpretation 244 Key Facts 244 Viva 244 Culture media 274 Culture of anaerobic bacteria 53 Learning Objectives 53 Introduction 53 .Textbook of Practical Microbiology Introduction 261 Principle 261 Requirements 261 Laboratory wares 261 Reagents 261 Specimen 261 Procedure 261 Quality Control 261 Observations 261 Results and Interpretation 261 Key Facts 262 Viva 262 Colorado tick fever 271 Competitive ELISA 138.congo hemorrhagic fever 271 Cryptococcal antigen 130 Cryptococcus neoformans 6. 258 Culex 269. 200. 199.6) 130 Preparation of agarose 130 Procedure 130 Quality Control 131 Observations 131 Results and Interpretation 131 Key Facts 131 Viva 131 Coxsackie A and B viruses 258 Craigie’s tube method 11 Crimean . 141 Compound Microscope 3 Learning Objectives 3 Introduction 3 Parts of the compound microscope 3 Microscope stand 3 Main tube 3 Body and arm 3 Substage 3 Foot 3 Stage 3 Microscope optics 4 Mechanical adjustment of a microscope 4 Coarse and fine focusing adjustments 4 Condenser adjustment 4 The light source 4 Principle 4 Magnification 4 Principle involved in the magnification of the object 4 Importance of numerical aperture 5 Requirements 5 Equipment 5 Reagents 5 Specimen 5 Procedure 5 Observations 5 Results and Interpretation 5 Key Facts 6 Viva 6 Compound light microscope 9 Concentration of stool 205. 175. 221 Cryptosporidium parvum 198. 141 Learning Objectives 138 Introduction 138 Principle 138 The sandwich ELISA for antigen 138 The indirect ELISA for antibodies 138 Competitive ELISA for antibodies 138 Indirect ELISA to Detect Antibodies 139 Requirements 139 Equipments 139 Reagents and glass wares 139 Carbonate buffer (pH 9. 149 Electron microscopes 2 Electrophoresis 148 Embryonated eggs 243 Endospores 37 Enriched media 45. 291 Differential coliform test 249. 196. 215 Dengue haemorrhagic fever 268 Deoxycholate citrate agar (DCA) medium 44. 292 Egg of Hymenolepis nana 292 Egg of round worm 292 Egg of Trichuris trichiura 190. 193. 251 Electro-immuno transfer blot (EITB) 150 Electroimmunodiffusion 127. 135. 11 Learning Objectives 7 Introduction 7 Principle 7 Requirements 7 Equipments 7 Reagents 7 Specimen 7 Procedure 7 Observations 7 Results and Interpretation 7 Key Facts 8 Viva 8 Decolourising agent 26. 196. 60 Disinfection 59. 250 Dysentery 269 Principle 53 Requirements 53 Equipments 53 Reagents and media 53 Specimen 53 Procedure 53 Quality Control 54 Observations 54 Results and Interpretation 54 Viva 54 Key Facts 55 Culture of Stool for Entamoeba histolytica 208 Learning Objectives 208 Introduction 208 Principle 208 Requirements 208 Equipments 208 Reagents and lab wares 208 Specimen 208 Procedure 209 Quality Control 209 Observations 209 Results and Interpretation 209 Key Facts 209 Viva 209 Cutaneous mycoses 212 Cyclops 270 Cyclospora cayetanensis 195.0 139 Substrate 139 Specimen 139 Procedure 139 Quality Control 139 Observations 139 Results and Interpretation 139 Sandwich ELISA to Detect Antigen 139 Requirements 139 Equipments 139 Reagents and glass wares 139 Specimen 139 Procedure 139 Quality Control 140 Observations 140 Results and Interpretation 140 Key Facts 141 Viva 141 D D’ Antonie’s iodine 192 Dark ground microscopy 2. 181 Dettol 60 Diarrhea 269. 206. 136 Direct plate technique 65 Disc diffusion test 94 Disinfectants 59. 196. 140 Double diffusion method 126 Dracunculiasis 271 Dracunculus medinesis 6 Draughtsman’s colonies 169 Drbohlav’s Locke-egg-serum (LES) medium 208 Dry heat sterilization 285 Durham’s tube 231.300 Index Dobell’s iodine 192 Dot ELISA 139. 193. 46. 248 Enteric fever 116. 208. 196. 198. 250 Differential media 46 Differential stain 21. 193. 291 Cyst of Giardia intestinalis 190. 292 Eggs of Taenia saginata 199 Ehrlich’s reagent 74. 241 E E-test 103 Earthenware candles 56 Echinococcus granulosus 283 EDTA 69 Egg of Ascaris lumbricoides 190. 26 Diffusion tests 97 Dilute carbol fuchsin 215 Diphtheroids 163 Diphyllobothriasis 271 Diphyllobothrium latum 271 Direct fluorescent antibody test 32. 193. 60 DNAase test 167 Dobell and O’Connor’s iodine 192 . 200 Cyst of Entamoeba coli 190193. 206. 75 Eijkman test 249. 199. 291 Cytopathic effect (CPE) 240.1M) pH 5. 275 Enrichment media 46 Entamoeba histolytica 8. 196 Egg of Enterobius vermicularis 292 Egg of hook worm 206. 27. 206 Cyst of Entamoeba histolytica/dispar 190.6) 139 Washing buffer (PBS-Tween 20) 139 Conjugate 139 Citric acid phosphate buffer (0. 7. 86. 118 Enterobius vermicularis 292 Enzyme-linked immunosorbent assay 138. 218 Gnathostoma hispidium 271 Gnathostoma spinigerum 271 Goat antimouse immunoglobulin 139 Gomori methenamine silver 290 Gram negative cocci 24 Gram positive bacilli 24 Gram positive bacteria 24 Gram positive cell wall 26 Gram positive cocci 24. 265 Hanging drop preparation 11. 136 Fluorescent staining 30 Foetal calf serum (FCS) 240 Forage mite 269 Formalin 205 Formalin-ether sedimentation method 205. 286 Gram variable bacteria 26 Gram’s iodine 215 Gram’s iodine 23 Gram’s stain 26. 37. 264. 215 Gram’s Staining for Fungi 215 Learning Objectives 215 Introduction 215 Principle 215 Requirements 215 Equipments 215 Reagents and lab wares 215 Specimen 215 Procedure 215 Preparation of fungal smear 215 Staining Procedure 215 Quality Control 215 Observation 216 Results and Interpretation 216 Key Facts 216 Grams staining 23 Learning Objectives 23 Introduction 23 Principle 23 Requirements 23 Equipments 23 Reagents and glass wares 23 Preparation of methyl violet stain 23 Preparation of Gram’s iodine 23 Preparation of 1% safranine 23 Specimen 24 Preparation of bacterial smear 24 Preparation of bacterial smear 24 Procedure 24 Quality Control 24 Observation 24 Results and Interpretation 24 Key Facts 26 Viva 26 Group A streptococci 172 Group B streptococci 172 Group D streptococci 172 Guinea pig 263. 162. 164. 226 Giardia species 248 Giemsa stains 201. 181 Learning Objectives 11 Introduction 11 Principle 11 Requirements 11 Equipments 11 Reagents and glass wares 11 . 161. 226. 203 Glucose 6-phosphate dehydrogenase 140 Glucose broth 277 H H2S producing bacteria 86 Haemoparasites 201 Haemophilus influenzae 287 Hamsters 263. 265 301 F F factor 155 Facultative anaerobes 52. 207 Free coagulase 68 Fungal elements 219 Fusarium 234 G Gas gangrene 38 Gaspak system 53 Gel diffusion test 127 Gelatin hydrolysis test 176 Germ tube test 225. 54 Faecal Escherichia coli 248 Faecal Streptococci 248 Falciparum malaria 289 Filariform larva 292 Floatation method 207 Fluorescence microscopy 2 Fluorescent microscope 135. 293 Learning Objectives 225 Introduction 225 Principle 225 Requirements 225 Equipments 225 Reagents and glass wares 225 Specimens 225 Procedure 225 Quality Control 225 Observations 225 Results and Interpretation 226 Key Facts 226 Viva 226 Germ tubes 225.Textbook of Practical Microbiology Enzymes 138 Epidemic relapsing fever 270 Epidemic typhus 270 Epidermophyton floccosum 236 Epsilometer test (E-test) 102 Learning Objectives 102 Introduction 102 Principle 102 Requirements 102 Reagents and lab wares 102 Specimens 102 Procedure 102 Opening an E-test package 102 Application of strips 102 Quality Control 103 Observations 103 Results and Interpretation 103 Key Facts 103 Viva 103 Escherichia coli 178 Escherichia coli on MacConkey agar 279 Ether 205 Ethidium bromide solution 145 Eye pieces 4 Glucose phosphate medium 82 Glucose with Durham’s tube 281 Glutaraldehyde 60 Glycerol 195. 206. 302 Index Fusarium 234 Colony morphology 234 Microscopy 234 Aspergillus fumigatus 234 Aspergillus niger 235 Aspergillus flavus 235 Penicillium species 235 Conidiophores 235 Phialids 235 Cladosporium 236 Colony 236 Microscopy 236 Cephalosporium 236 Colony 236 Microscopy 236 Trichophyton verrucosum 236 Colony 236 Trichophyton violaceum 236 Colony 236 Epidermophyton floccosum 236 Colony 236 Viva 237 Identification of Common Insects 268 Learning Objectives 268 Introduction 268 Principle 268 Requirements 268 Equipments and lab wares 268 Specimen 268 Mosquitoes 268 General features 268 Identifying features of Anopheles 268 Diseases transmitted 268 Identifying features of Aedes 268 Diseases transmitted 268 Identifying features of Culex 269 Diseases transmitted 269 Sand Fly 269 General features 269 House Flies 269 Identifying features 269 Diseases transmitted 269 Itch Mite 269 Identifying features 269 Diseases transmitted 269 Trombiculid Mite 269 Identifying features 269 Diseases transmitted 269 Hard Tick 270 Identifying features 270 Soft Tick 270 Identifying features 270 Diseases transmitted 270 Rat Flea 270 Identifying features 270 Louse 270 Identifying features 270 Diseases transmitted 270 Cyclops 270 Identifying features 270 Viva 271 Identification of Common Viva Spots 274 Learning Objectives 274 Culture Media 274 Nutrient Agar 274 Composition 274 Preparation 274 Modifications 274 Specimen 11 Procedure 11 Quality Control 11 Observations 12 Results and Interpretation 12 Key Facts 13 Viva 13 Hard ticks 270 Head louse 270 Heat stable catalase test 63 Hektoen enteric agar 86 HeLa 240 Hemolysis on blood agar 167 Hep 2 240 Hepatitis B antigen 130 Heterophile agglutination tests 107 Histoplasma capsulatum 6 Holder method 58 Hook worm egg 190. 132 Hydatid cyst 283 Hydatid fluid 283 Hydrogen Sulfide Test 85 Learning Objectives 85 Introduction 85 Principle 85 Requirements 85 Equipments 85 Reagents and lab wares 85 Specimen 85 Procedure 85 Quality Control 86 Positive control 86 Negative control 86 Observations 86 Results and Interpretation 86 Viva 86 Key Facts 87 Hymenolepis nana 6. 285 House dust mite 269 House flies 269 Hungate procedure of anaerobiosis 54 Hybridization probes 144 Hydatid antigens 130. 196 Horseradish peroxidase 138. 292 I Identification of Common Fungi 233 Learning Objectives 233 Introduction 233 Principle 233 Requirements 233 Equipments 233 Reagents and lab wares 233 Specimen 233 Procedure 233 Quality Control 233 Observation 233 Results and Interpretation 233 Rhizopus 233 Colony morphology 233 Microscopy 234 Mucor 234 Colony morphology 234 Microscopy 234 Alternaria 234 Colony morphology 234 Microscopy 234 . 140 Hot air oven 58. 193. 275. 284. 278. 285 Blood Agar 274 Composition 274 Preparation 275 Chocolate Agar 275 Composition 275 Preparation 275 MacConkey Agar 275 Composition 275 Preparation 275 Loeffler’s Serum Slope 275 Composition 276 Preparation 276 Lowenstein-Jensen (LJ) Medium 276 Composition 276 Preparation 276 Robertson Cooked Meat (RCM) Broth 276 Composition 276 Preparation 276 Sabouraud’s Dextrose Agar (SDA) 277 Composition 277 Preparation 277 Peptone water 277 Composition 277 Preparation 277 Glucose Broth 277 Composition 278 Preparation 278 Culture Media with Growth 278 Streptococcus pyogenes on Blood Agar 278 Salient features 278 Staphylococcus aureus on Nutrient Agar 278 Salient features 278 Proteus spp. on MacConkey Agar 279 Salient features 279 Corynebacterium diphtheriae on Potassium Tellurite Agar 279 Salient features 279 Mycobacterium tuberculosis on Lowenstein Jensen (LJ) Medium 280 Salient features 280 Candida albicans on Sabouraud’s Dextrose Agar (SDA) 280 Salient features 280 Biochemical Reactions 280 Carbohydrate Fermentation Tests 280 Salient features 280 Glucose with Durham’s Tube 281 Salient features 281 Indole Test 281 Salient features 281 Urease Test 281 Salient features 281 Citrate Utilization Test 281 Salient features 281 Phenyl Pyruvic Acid Test (PPA) 282 Salient features 282 Triple Sugar Iron (TSI) Agar 282 Salient features 282 Specimens of Parasite 282 Ascaris lumbricoides Adult Worm 282 Salient features 282 Hydatid Cyst 283 Salient features 283 Glass Wares 283 Universal Container 283 Bijou Bottle 283 Tuberculin Syringe 283 Graduated Pipette 284 Pasteur Pipette 284 NIH Swab 285 Incubator 285 Salient features 285 Hot Air Oven 285 Salient features 285 Autoclave 285 Salient features 285 Microscopy Slides 286 Gram Positive Cocci 286 Salient features 286 Streptococcus pneumoniae 286 Salient features 286 Neisseria gonorrhoeae 286 Salient features 286 Gram Negative Bacilli 286 Salient features 286 Haemophilus influenzae 287 Salient features 287 Vibrio cholerae 287 Salient features 287 Bacillus anthracis 287 Salient features 287 Clostridium perfringens 287 Salient features 287 Treponema pallidum 287 Salient features 287 Albert staining 288 Salient features 288 Ziehl-Neelsen Staining for Mycobacterium tuberculosis 288 Salient features 288 Ziehl-Neelsen Staining for Mycobacterium leprae 288 Salient features 288 Negri Bodies 288 Salient features 288 Multinucleate Giant Cells Measles 289 Salient features 289 Molluscum Bodies 289 Salient features 289 Plasmodium vivax Ring Stage 289 Salient features 289 Plasmodium falciparum Ring Stage 289 Salient features 289 Plasmodium vivax Male and Female Gametocytes 290 Salient features 290 Plasmodium falciparum Male and Female Gametocytes 290 Salient features 290 LD Bodies 290 Salient features 290 Toxoplasma gondii 290 Salient features 290 Wuchereria bancrofti Microfilaria 291 Salient features 291 Cyst of Entamoeba histolytica/dispar 291 Salient features 291 Cyst of Giardia intestinalis 291 Salient features 291 Egg of Round Worm 292 Salient features 292 Egg of Hook Worm 292 Salient features 292 Egg of Enterobius vermicularis 292 Salient features 292 Egg of Hymenolepis nana 292 303 . 277. on Blood Agar 278 Salient features 278 Pseudomonas aeruginosa on Nutrient Agar 279 Salient features 279 Escherichia coli on MacConkey Agar 279 Salient features 279 Klebsiella spp. 283.Textbook of Practical Microbiology Uses 274. 276. 286 India ink preparation 35. Coli and Klebsiella spp. 221. 134. 221.304 Index Learning Objectives 181 Introduction 181 Specimens 181 Tests for Identification of Vibrio Cholerae 181 Direct examination 181 Hanging drop preparation 181 Culture 181 Biochemical tests 181 Antibiotic susceptibility testing 181 Key Facts 182 Viva 183 Identification of b -haemolytic Streptococci 172 Learning Objectives 172 Introduction 172 Specimen 172 Tests for Identification of Streptococcus pyogenes 172 Direct examination 172 Culture 172 Bacitracin sensitivity test 172 CAMP (Christie. 178 Direct examination 178 Gram’s stain 178 Culture 178 Biochemical tests 178 Antibiotics susceptibility testing 178 Key Facts 180 Viva 180 Identification of Pseudomonas aeruginosa 184 Learning Objectives 184 Introduction 184 Specimens 184 Tests for Identification of Pseudomonas Aeruginosa 184 Direct examination 184 Culture 184 Oxidase test 184 Biochemical tests 184 Antibiotic susceptibility testing 184 Key Facts 185 Viva 185 Identification of Staphylococcus aureus 166 Learning Objectives 166 Introduction 166 Specimen 166 Tests for the Identification of Staphylococcus aureus 166 Direct examination 166 Culture 166 Coagulase test 166 Deoxyribonuclease test 166 Mannitol salt agar 166 Novobiocin sensitivity 167 Key Facts 168 Viva 168 Identification of Streptococcus pneumoniae 169 Learning Objectives 169 Introduction 169 Specimen 169 Tests for the Identification of Streptococcus pneumoniae 169 Direct examination 169 Culture 169 Bile solubility test 169 Optochin test 169 Inulin fermentation 170 Key Facts 170 Viva 171 Identification of Vibrio cholerae 181 . 212. 135 Learning Objectives 128 Introduction 128 Principle 128 Requirements 128 Equipments 128 Reagents and glass wares 128 Specimen 128 Procedure 128 Quality Control 129 Observations 129 Results and Interpretation 129 Key Facts 129 Viva 129 Immunoelectrophoresis 128. 222. 284 Illuminating source 4 Illumination 5 Immunodiffusion 127 Immunoelectrophoresis test 107. 41. 129 Immunofluorescence Test 135 Learning Objectives 135 Introduction 135 Principle 135 Requirements 135 Equipments 135 Reagents and lab wares 135 Specimen 135 Procedure 135 Quality Control 136 Observations 136 Results and Interpretation 136 Key Facts 136 Viva 136 In-use test 60 Incineration 58 Incomplete antibodies 108 Incubators 285 India ink 34. Atkins and Munch-Peterson) test 173 Bile aesculin test 173 Viva 173 Key Facts 174 IHA test 133. 128. 222 Learning Objectives 221 Introduction 221 Principle 221 Requirements 221 Equipments 221 Reagents and lab wares 221 Salient features 292 Eggs of Trichuris trichiura 292 Salient features 292 Candida albicans 293 Salient features 293 Germ Tube Test 293 Salient features 293 Identification of Corynebacterium diphtheriae 175 Learning Objectives 175 Introduction 175 Specimen 175 Tests for the identification of Corynebacterium diphtheriae 175 Direct examination 175 Albert’s stain 175 Culture 175 Biochemical tests 175 Key Facts 176 Viva 176 Identification of Lactose Fermenting Enterobacteriaceae 178 Learning Objectives 178 Introduction 178 Specimens 178 Tests for Identification of E. 194 Iodine Wet Mount of Stool 192 Learning Objectives 192 Introduction 192 Principle 192 Requirements 192 Equipments 192 Reagents and glass wares 192 Specimen 192 Procedure 192 Quality Control 192 Observations 193 Results and Interpretation 193 Key Facts 194 Viva 194 Iodine wet mount preparation 189. 16 For pour plate method 15.Textbook of Practical Microbiology Specimen 221 Procedure 221 Quality Control 221 Observations 221 Results and Interpretation 221 Key Facts 222 Viva 222 India-ink method 40 Indicator broth medium (IBM) 231 Indicator medium 275 Indirect ELISA 141 Indirect haemagglutination test 132 Learning Objectives 132 Introduction 132 Principle 132 Requirements 132 Lab wares 132 Reagents 132 Specimen 133 Procedure 133 Sensitisation of chick RBCs with OSD of the antigen 133 Performance of the IHA test 133 Quality Control 133 Observations 133 Results and Interpretation 133 Key Facts 133 Viva 134 Indirect paper strip procedure 65 Indole negative bacteria 74. 281 Learning Objectives 74 Introduction 74 Principle 74 Requirements 74 Equipments 74 Reagents and lab wares 74 Specimen 74 Procedure 74 Quality Control 74 Positive control 74 Negative control 74 Observation 74 Results and Interpretation 74 Key Facts 75 Viva 75 Infant hamsters 265 Infant rabbit 262 Insects 268 Inspissation 58 Intestinal coccidian parasites 199. 192. 75 Indole positive bacteria 74. 200 Intravenous Inoculation into Mice Tail Vein 258 Learning Objectives 258 Introduction 258 Principle 258 Requirements 258 Equipments 258 Reagents and glass wares 258 Specimen 258 Procedure 258 Procedure for loading the syringe for injection 258 Animal preparation for injection 258 Injection of material 258 Quality Control 259 Observations 259 Results and Interpretation 259 Key Facts 260 Viva 260 305 Inulin fermentation 170 Iodine wet mount 191. 185. 16 Quality Control 15 Observations 15 Results and Interpretation 16 Viva 16 . 75 Indole test 74. 16 For spread plate method 15. 192 Isoantibodies 111 Isolation of Antibiotic Resistant Mutant 152 Learning Objectives 152 Introduction 152 The importance of mutation 152 Principle 152 Requirements 152 Equipments 152 Reagents and lab wares 152 Specimen 152 Procedure 152 Quality Control 153 Observations 153 Results and Interpretation 153 Key Facts 154 Viva 154 Isolation of Plasmids 145 Learning Objectives 145 Introduction 145 Principle 145 Requirements 145 Equipments 145 Reagents and lab wares 145 Preparation of ethidium bromide stock solution 145 Preparation of ethidium bromide working solution 145 Preparation of Luria-Bertani medium (LB medium) 145 Specimen 145 Procedure 145 Extraction of plasmid 145 Electrophoresis on agarose gel 146 Quality Control 146 Observations 146 Results and Interpretation 146 Key Facts 146 Viva 146 Isolation of Pure Cultures 14 Learning Objectives 14 Introduction 14 Principle 14 Requirements 14 Equipment and labwares 14 Reagents 14 Specimen 15 Procedure 15 For streak plate method 15. 199 Itch mite 269 J Japanese encephalitis 269 Jaswant Singh Bhattacharjee stain 203 Jensen’s Gram method for smears 25 K K antigen 36 Köhler illumination 3. 217 Lactose fermenting Enterobacteriaceae 178 Lane’s saturated salt solution floatation method 205 Latex agglutination test 112. 81 Koser’s liquid citrate medium 281 Kovac’s reagent 74. 280 Louse 270 LPCB Wet Mount of Stool 195 Learning Objectives 195 Introduction 195 Principle 195 Requirements 195 Equipments 195 Reagents and glass wares 195 Specimen 195 Procedure 195 Quality Control 196 Observations 196 Results and Interpretation 196 Key Facts 197 Viva 197 LPCB wet mount preparation 196 Lugol’s’ iodine 192 Luria-Bertani medium 145 Lyme disease 271 Lymphatic filariasis 291 Lysine decarboxylation test 185 Lysine iron agar 86 Lysol 60 Key Facts 17 Isospora belli 198. 185 Koch’s postulates 288 KOH wet mount preparation 219. 86. 86 Leishman stains 201. 113 .5 McFarland standard 92 Specimens 92 Preparation of suspension of bacteria 92 Procedure 92 Quality Control 93 Observations 93 Results and Interpretation 93 Key Facts 93 Viva 94 Kirby-Bauer’s chart 93 Klebsiella species 178 Klebsiella species on MacConkey agar 279 Kligler’s iron agar median 85. 6 Kala azar 271 Kinyoun’s modification of acid-fast stain 29 Kirby-Bauer disc diffusion method 92 Kirby-Bauer method 92. 275.306 Index Learning Objectives 112 Introduction 112 Principle 112 Requirements 112 Reagents and lab wares 112 Specimen 112 Procedure 112 Quality Control 112 Observations 112 Results and Interpretation 113 Key Facts 113 Viva 113 LD bodies 290 Lead acetate agar 85. 75. 276. 203 Leishman’s 201 Leishman’s stain 201 Leishman’s Staining of Peripheral Blood Smears 201 Learning Objectives 201 Introduction 201 Principle 201 Requirements 201 Equipments 201 Reagents and lab wares 201 Preparation of EDTA anticoagulated blood 201 Preparation of Leishman’s stain 201 Specimen 202 Procedure 202 Preparation of thin blood smear 202 Preparation of thick blood smear 202 Preparation of combined thick and thin films 202 Leishman’s staining 202 Quality Control 202 Observations 202 Results and Interpretation 203 Key Facts 203 Viva 204 Leishmania donovani 290 Lepromin test 284 Levaditi stain 287 Light microscopy 2 Listeria monocytogenes 261 Locke’s solution 208 Loeffler’s methylene blue 21. 28 Loeffler’s serum slope 175. 281 Krebs cycle 80 Kyasanur forest disease 271 L Löwenstein-Jensen medium 44 Laboratory animals 263 Lactophenol cotton blue (LPCB) stain 195. 27. 96 Learning Objectives 92 Introduction 92 Principle 92 Requirements 92 Equipments 92 Reagents and lab wares 92 Preparation of 0. 217 Lactophenol Cotton Blue (LPCB) Wet Mount of Fungi 217 Learning Objectives 217 Introduction 217 Principle 217 Requirements 217 Equipments 217 Reagents and lab wares 217 Specimen 217 Procedure 217 Scotch tape preparation 217 Tease mount preparation 217 Quality Control 218 Observations 218 Results and Interpretation 218 Key Facts 218 Viva 218 Lactophenol cotton blue (LPCB) wet mount 212. 220 Kopeloff and Beerman’s Gram method 25 Koser’s citrate 80. 32. 215 Methylene blue (counter stain) 198 Methylene blue reduction test 252 Methylene blue test 252 Mice 263 Microbiology of Air 254 Learning Objectives 254 Introduction 254 Principle 254 Requirements 254 Equipments 254 Reagents and lab wares 254 Procedure 254 Quality Control 254 Observations 254 Results and Interpretation 254 Key Facts 255 Viva 255 Microbiology of Water 248 Learning Objectives 248 Introduction 248 Coliforms 248 Faecal or thermotolerant coliforms 248 Faecal Escherichia coli 248 Faecal streptococci 248 Clostridium perfringens 248 Collection of water samples 249 Principle 249 Plate count 249 Detection of coliform bacteria 249 Presumptive coliform test – Multiple tube technique 249 Differential coliform test 249 Membrane filtration method 249 Detection of faecal streptococci 249 Examination for Cl. 184.Textbook of Practical Microbiology 307 M MacConkey agar 45. 178. 167 Mc Fadyean reaction 21 McFadyean reaction 35 McFarland standard 92. 288 Methyl alcohol 201 Methyl red test 76 Learning Objectives 76 Introduction 76 Principle 76 Requirements 76 Equipments 76 Reagents and lab wares 76 Specimen 76 Procedure 76 Quality Control 77 Positive control 77 Negative control 77 Observation 77 Results and Interpretation 77 Key Facts 77 Viva 77 Methyl violet stain 23. 46. perfringens 249 Requirements 249 Equipments 249 Reagents 249 Preparation of MacConkey broth 249 Preparation of double strength medium 249 Preparation of single strength medium 249 Preparation of brilliant green bile broth 250 Specimen 250 Procedure 250 Quality Control 250 Observations 250 Results and Interpretation 250 Differential coliform count 250 Key Facts 251 Viva 251 Microbiology of Milk 252 Learning Objectives 252 Introduction 252 Principle 252 Requirements 252 Equipments 252 Reagents and glass wares 252 Specimen 252 Procedure 252 Quality Control 253 Observations 253 Results and Interpretation 253 Key Facts 253 Viva 253 Microcapsule 34 . 95 McIntosh and Fildes jar 53 Measurement of Microorganisms 9 Learning Objectives 9 Introduction 9 Principle 9 Requirements 9 Equipments 9 Reagents 9 Specimen 9 Procedure 9 Quality Control 10 Observations 10 Results and Interpretation 10 Key Facts 10 Viva 10 Media for Routine Cultivation of Bacteria 44 Learning Objectives 44 Introduction 44 Principle 44 Basal media 44 Enriched medium 44 Enrichment media 44 Selective media 44 Differential media 44 Requirements 45 Equipments 45 Reagents and media 45 Specimen 45 Procedure 45 Quality Control 45 Observations 45 Results and Interpretation 46 Key Facts 46 Viva 46 Membrane filtration method 249 Mesocyclops 271 Metachromatic granules 31. 176. 275 MacConkey broth 249 Macleod’s potassium tellurite agar media 175 Macrocapsule 34 Macrogametocyte 290 Magnesium sulphate floatation method 206 Magnification 6 Malabsorption 291 Malachite green solution 276 Malachite green stain 37 Malaria 268 Male gametocyte 290 Mannitol salt agar 166. 46. 184. 185 Learning Objectives 88 Introduction 88 Principle 88 Requirements 88 Equipments 88 Reagents and lab wares 88 Specimen 88 Procedure 88 Quality Control 88 O O/129 reagent 183 Obligate aerobes 52 . 100. 33.308 Index Positive control 88 Negative control 88 Observation 88 Results and Interpretation 88 Key Facts 89 Viva 89 Nitrates 89 Non agglutinable vibrios 183 Non motile bacteria 12 Non-bile stained eggs 193 Non-fermenters 184 Nonsense mutations 153 Normal flora 160 Normal Microbial Flora of the Mouth 160 Learning Objectives 160 Introduction 160 Principle 160 Requirements 160 Equipments 160 Reagents and lab wares 160 Specimen 160 Procedure 160 Quality Control 160 Observations 160 Results and Interpretation 161 Key Facts 161 Viva 161 Normal Microbial Flora of the Skin 164 Learning Objectives 164 Introduction 164 Principle 164 Requirements 164 Equipments 164 Reagents 164 Specimen 164 Procedure 164 Observations 164 Results and Interpretation 164 Viva 164 Key Facts 165 Normal Microbial Flora of the Throat 162 Learning Objectives 162 Introduction 162 Principle 162 Requirements 162 Equipments 162 Reagents and lab wares 162 Specimen 162 Procedure 162 Observations 162 Results and Interpretations 162 Key Facts 163 Viva 163 Normal flora in the throat 162 Normal flora on the skin 164 Normal microbial flora 163 Northern blotting 149 Norwegian itch of man 269 Nosocomial fungal infections 212 Novobiocin sensitivity 167 Novobiocin-sensitive staphylococci 168 Numerical aperture 5. 288 Mycobacterium tuberculosis 28. 77 Mucor 234 Multinucleate giant cells 289 Mutations 152. 89. 40 NIH Swab 285 Nitrate agar slant 88 Nitrate broth 89 Nitrate reduction test 88. 101 Minimum inhibitory concentration (MIC) 97. 276 Mycobacterium tuberculosis on Lowenstein. 265 Monoclonal antibodies 139 Mordant 24.Jensen’s medium 280 N N-N tetramethyl para-phenylene diamine hydrochloride 65 NCCLS QC ranges 103 NCCLS table 98 Necator americanus 292 Negative staining 40. 260. 41 Learning Objectives 40 Introduction 40 Principle 40 Requirements 40 Equipments 40 Reagents and glass wares 40 Specimen 40 Procedure 40 Quality Control 40 Observations 41 Results and Interpretation 41 Key Facts 41 Viva 41 Negri bodies 288 Neisser’s stain 31. 274 Nutrient broth 274 Microfilaria of Brugia malayi 203 Microfilaria of Loa loa 203 Microfilaria of Mansonella perstans 203 Microfilaria of Wuchereria bancrofti 203 Miliary tuberculosis 29 Minimum bactericidal concentration (MBC) 100. 176 Neisseria gonorrhoea 286 Neutral red 180 Neutralization test 107 Nigrosin staining 34. 215 Mosquitoes 268 Motile bacteria 12 Mouse 258. 98. 101. 154 Mycobacterium leprae 258. 102 Missense mutations 153 Modified acid fast staining 199 Modified acid fast staining of faeces 198 Modified Ziehl-Neelsen stain 37 Moist heat sterilization 285 Molluscum bodies 289 Monkeys 263. 264 Mouse virulence 170 MR negative bacteria 77 MR positive bacteria 77 MR test 76. 6 Nutrient agar 45. 274. 277 Periodic acid schiff (PAS) 290 Permanent staining of blood smear 201 PH requirement of bacteria 49 pH Requirement for Growth of Bacteria 49 Learning Objectives 49 Introduction 49 Principle 49 Requirements 49 Equipments 49 Reagents 49 Specimen 49 Procedure 49 Quality Control 49 Observations 49 Results and Interpretation 49 Key Facts 50 Viva 50 Phase-contrast microscope 2. 149 Specimen 149 Procedure 149 Quality Control 149 Observations 149 Results and Interpretation 149 Viva 150 Key Facts 151 Polychrome methylene blue 20. 281 P-phenylene diamine dihydrochloride 67 Paracoccidioides brasiliensis 6 Parvo virus 6 Passive agglutination tests 106 Pasteur pipette 284 Pasteurisation 56. 38 Phenol 60. 146. 281. 74. 185 Learning Objectives 65 Introduction 65 Principle 65 Requirements 65 Reagents and glass wares 65 Specimen 65 Procedure 65 Direct plate technique 65 Indirect filter paper strip procedure 65 Quality Control 66 Positive control 66 Negative control 66 Observations 66 Direct plate technique 66 Indirect filter paper strip procedure 66 Results and Interpretation 66 Viva 66 Key Facts 67 Oxygen Requirement for Growth of Bacteria 51 Learning Objectives 51 Introduction 51 Principle 51 Aerobes 51 Microaerophiles 51 Obligate anaerobes 51 Aerotolerant anaerobes 51 Facultative anaerobes 51 Requirements 51 Equipments 51 Reagents and media 51 Specimen 51 Procedure 52 Quality Control 52 Observations 52 Results and Interpretation 52 Key Facts 52 Viva 52 Penicillium species 235 Peptone water 46. 153. 149 Destaining solution: 1000ml. 21 309 P P-dimethyl amino benzaldehyde 75. 66. 282 Phenyl pyruvic acid test 282 Phosphatase test 252 Phosphate buffer 50 Phosphate buffer saline 132 Phthirus pubis 270 Physiological saline 189 Pikes medium 46 Plasmids 145. 157 Plasmodium 204 Plasmodium falciparum 203 Plasmodium Falciparum male and female Gametocytes 290 Plasmodium Falciparum Ring stage 289 Plasmodium malariae 203 Plasmodium ovale 203 Plasmodium vivax 203 Plasmodium Vivax male and female Gametocytes 290 Plasmodium vivax ring stage 289 Pneumococcal antigen 114 Point mutations 152.Textbook of Practical Microbiology Obligate anaerobes 52. 58 Paul-Bunnel test 120 Pediculus capitus 270 Pediculus corporis 270 . 154 Polar bodies 31 Polar flagellum 11 Poliomyelitis 269 Polyacrylamide Gel Electrophoresis 148 Learning Objectives 148 Introduction 148 Principle 148 Requirements 148 Equipments 148 Reagents and lab wares 148 Stock solutions 148 Working solutions 148 Separating gel buffer (4x) 148 Stacking gel buffer (4x) 148 10% Ammonium persulfate (APS) 148 Electrophoresis/Running Buffer (1x) 149 Sample buffer 149 Staining solution: 1000ml. 53 Ocular micrometer 9 Onychonychia 293 Optimum sensitizing dose (OSD) of the antigen 132 Optochin ( ethyl hydrocupreine hydrochloride) 169 Optochin sensitivity 170 Optochin test 169 Oral thrush 293 Oriental sore 271 Orientia tsutsugamushi 269 Ornithodorus lahorensis 270 Ornithodorus species 270 Ornithodorus tholozani 270 Oroya fever 271 Osmk hemorraghic fever 271 Oxidase negative bacteria 66 Oxidase positive bacteria 66 Oxidase reagent 65 Oxidase test 65. 218 Phenol red 82. 33 Pulmonary tuberculosis 29 Pure culture 14. 204 Prozone reaction 108 Pseudomonas aeruginosa 184 Pseudomonas aeruginosa on nutrient agar 279 Pubic louse 270 Puch’s stain 31. 204 Russian spring summer encephalitis 271 Polymetaphosphate 175 Polymyxin-B (50U) sensitivity 181. 263. 46 Seller’s technique 289 Semi quantitative catalase test 63 Sereny’s test 262 Q Q fever 253 Quellung reaction 170 R Rabbit 261. 209 Ponder’s stains 176 Positive staining technique 35 Potable water 248 Potassium hydroxide (KOH) wet mount 212. 233 Rhodamine 135 Rickettesia orientails 269 Rickettsia prowazaki 270 Rickettsial infection 119 Rideal Walker test 60 Rift valley fever 269 Robertson cooked meat (RCM) 53. 206. 279. 120 Proteus species on blood agar 278 Proteus vulgaris 279 Prototrophs 144 Protozoal cysts 195. 69 Radial immuno diffusion 126 Radial Immunodiffusion Test 126 Learning Objectives 126 Introduction 126 Principle 126 Requirements 126 Equipments 126 Reagents and glass wares 126 Specimen 126 Procedure 126 Preparation of antibody containing gels 126 Calibration of reference graph 126 . 264 Rabbit plasma 68. 17 Pyocyanin 184 Pyruvic acid 78 S Sabouraud’s dextrose agar (SDA) 164. 279 Polyxenic culture medium 208. 225. 219 Potassium Hydroxide Wet Mount of Fungi 219 Learning Objectives 219 Introduction 219 Principle 219 Requirements 219 Equipments 219 Reagents and lab wares 219 Specimen 219 Procedure 219 Quality Control 219 Observations 219 Results and Interpretations 219 Key Facts 220 Viva 220 Potassium nitrate broth (KNO3 ) 88 Potassium tellurite blood agar 275. 250 Protein-A ELISA 141 Proteus mirabilis 279 Proteus OX 19. 288 Pour plate inoculation procedure 14 Pour plate method 14 Pox virus 6 PPA test 282 Precipitation tests 107 Preston and Morrell’s Gram method 25 Presumptive coliform count 249. 213. 214.310 Index Testing unknown serum samples 127 Quality Control 127 Observations 127 Results and Interpretation 127 Key Facts 127 Viva 127 Rat Flea 270 Reaginic antibodies 123 Recombinant DNA technology 144 Relapsing fever 270 Resolution 5. 207 Scabies 269 Schaeffer Fulton method 37. 276 Robertson’s cooked meat (RCM) medium 52 Robinson’s medium 208 Rocky mountain Spotted fever 271 Romanowsky’s stains 201. 133 Reynold – Braude phenomenon 293 Rheumatoid factor (RF) 112 Rhinosporidium seeberi 6 Rhizopus 217. OX 2 and OX K antigens 119. 6 Resolving power 5 Restriction endonucleases 144 Reverse passive haemagglutination test 132. 38 Scolices of Echinococcus granulosus 199 Scotch tape preparation 217 Scrub typhus mite 269 SDS-PAGE 150 Sedimentation method 207 Selenite F broth 45. 182 Polysaccharide capsule 36. 277 Safranine 23 Safranine stain 37 Saline Wet Mount of Stool 189 Learning Objectives 189 Introduction 189 Principle 189 Requirements 189 Equipments 189 Reagents and glass wares 189 Specimen 189 Procedure 189 Quality Control 189 Observations 189 Results and Interpretation 190 Key Facts 191 Viva 191 Saline wet mount preparation of faeces 189 Salmonella-Shigella agar 86 Sand Fly 269 Sand fly fever 271 Sandwich ELISA 141 Sarcocystis hominis 199 Sarcoptes scabies var hominis 269 Saturated salt solution flotation method 205. 215. 16. 229. 205 Protozoal infections 201. 150 Sodium hypochlorite 60 Soft Tick 270 Southern blotting 149 Specific soluble substance 36 Spore of Clostridium tetani 56 Spore Staining 37 Learning Objectives 37 Introduction 37 Principle 37 Requirements 37 Equipments 37 Reagents and lab wares 37 Preparation of malachite green stain 37 Preparation of safranine stain 37 Specimen 38 Procedure 38 Quality Control 38 Observations 38 Results and Interpretation 38 Viva 38 Key Facts 38 Spores of B. 278 Streptococcus pyogenes on blood agar 278 Streptolysin O 121 Streptolysin S 121. 122 Streptomycin-resistant mutants 152 String test 183 Stuart’s broth 72 Subcutaneous mycoses 212 Suckling mouse 259. 261 Streptococcus pyogenes 172. 281 Simmon’s citrate medium 80. 170. 224 Slide Culture for Fungi 223 Learning Objectives 223 Introduction 223 Principle 223 Requirements 223 Equipments 223 Reagents 223 Specimen 223 Procedure 223 Quality Control 224 Observations 224 Results and Interpretation 224 Key Facts 224 Viva 224 Slide culture preparation 217 Slide culture set 223 Slide flocculation test 123 Slide test 68 Slit sampler method 254. 206 Significant bacteriuria 25 Simmon’s citrate agar 280. 258 Sulphur indole motility medium 86 Superficial mycoses 212 Swarming 11 Systemic mycoses 212 311 T TAB vaccine 117 Table 1-1 Types of condensers 4 Table 13-1 List of different type of media commonly used forisolation of bacteria 46 Table 14-1 Examples of bacteria showing different temperaturesfor their growth 48 . 60 Sterilization of Commonly Used Culture Media 56 Learning Objectives 56 Introduction 56 Principle 56 Sterilization by heat 56 Sterilization by filtration 56 Requirements 56 Equipments 56 Reagents 56 Specimen 56 Procedure 56 Quality Control 57 Autoclave 57 Hot air oven 57 Filters 57 Observations 57 Results and Interpretation 57 Key Facts 58 Viva 58 Sterilization of culture media 56 Stokes Method 95. 255 Sheather’s sucrose floatation method 200. 108 Slide coagulase test 70 Slide culture 223. stearothermophilus 57 Spores of Bacillus stearothermophilus 56 Spores of Microsporidia 199 Sporothrix schenckeii 6 Spread plate inoculation procedure 14 Standard test of syphilis 125 Staphylococcus aureus 6. 278 Staphylococcus aureus on nutrient agar 278 Sterile Mueller Hinton broth 97 Sterilisation 56.polyacrylamide gel electrophoresis (SDSPAGE) 148. 96 Learning Objectives 95 Introduction 95 Principle 95 Requirements 95 Equipments 95 Reagents and lab wares 95 Specimen 95 Procedure 95 Quality Control 95 Observations 96 Results and Interpretation 96 Key Facts 96 Viva 96 Streak plate inoculation procedure 14 Streptococcus MG agglutination 120 Streptococcus pneumoniae 169.Textbook of Practical Microbiology Settle plate method 254. 166. 255 Sodium dodecyl sulfate . 81 Simple stain 21 Simple staining 20 Learning Objectives 20 Introduction 20 Principle 20 Requirements 20 Equipments 20 Reagents and glass wares 20 Preparation of Loeffler’s methylene blue stain 20 Preparation of polychrome methylene blue 20 Specimen 20 Procedure 20 Quality Control 20 Observations 20 Results and Interpretation 21 Key Facts 21 Viva 21 Sintered glass filter 56 Slide agglutination tests 106. 228 Urease negative bacteria 72 Urease negative fungi 227 Urease positive bacteria 72 Urease positive fungi 227 . 72. 202. 282 Tube agglutination tests 106 Tube coagulase test 69. 156 Transformation 156 Transversions 154 Trench fever 270 Treponema pallidum 287 Trichinella spiralis 6 Trichomonas vaginalis 8 Trichophyton verrucosum 236 Trichophyton violaceum 236 Triple sugar iron (TSI) agar 82 Triple sugar iron agar (TSI) test 82 Learning Objectives 82 Introduction 82 Principle 82 Requirements 82 Equipments 82 Reagents and lab wares 82 Specimen 82 Procedure 83 Quality Control 83 Observations 83 Results and Interpretation 83 Viva 83 Key Facts 84 Trombicula akamush 269 Trombicula dilensis 269 Trombiculid Mite 269 Tryptone broth 74 TSI agar 82. cholerae biotype El Tor 182 Table 65-1 List of bile-stained and non-bile stained eggs 193 Table 69-1 Other floatation methods of concentration stool 206 Table 7-1 List of Gram positive and Gram negative bacteria 24 Table 71-1 List of media used for fungal culture 214 Table 77-1 Differences between germ tubes and pseudohyphae 226 Table 79-1 Preparation of Yeast Nitrogen Base 230 Table 8-1 List of acid-fast structures 29 Table 82-1 The cell lines and indications for the viruses andcytopathic effects they produce 241 Table 83-1 The routes of inoculation of the egg and the virusesisolated 244 Table 84-1 Grades of the Quality of Drinking Water Supplies Determined by results of Periodic Escherichia coli andColiform counts 250 Table 85-1 List of bacteria that can be found in contaminatedmilk 253 Table 86-1 List of bacteria commonly found in air 254 Table 89-1 Laboratory animals and their usage 265 Table Applications of various tests used in a microbiologylaboratory 106 Tachyzoites 290 Taenia saginata 6 Taenia soluim 6 Tautomerization 154 TCBS 181 Tease mount preparation 217 Temperature Requirement for Growth of Bacteria 47 Learning Objectives 47 Introduction 47 Principle 47 Psychrophilic bacteria 47 Mesophilic bacteria 47 Thermophilic bacteria 47 Requirements 47 Equipments 47 Reagents and media 47 U Universal Container 283 Universal donors 110 Urea 71. 86. 70 Tube dilution method 100 Tuberculin test 284 Tuberculous meningitis 29 Turbidity test 252 Tyndallization 58 Table 16-1 Examples of bacteria grouped depending on theirrequirement of oxygen 52 Table 18-1 Different methods of sterilization of varioussubstances 58 Table 19-1 List of commonly used disinfectants and antiseptics 60 Table 20-1 Catalase positive and negative bacteria 63 Table 21-1 List of oxidase positive and negative bacteria 66 Table 22-1 List of coagulase positive bacteria 69 Table 23-1 Urease producing bacteria and fungi 72 Table 24-1 List of indole positive and negative bacteria 75 Table 25-1 List of MR positive and negative bacteria 77 Table 26-1 VP positive and negative bacteria 79 Table 27-1 Differences between Simmon’s citrate and Koser’scitrate 80 Table 27-2 List of citrate positive and negative bacteria 81 Table 29-1 List of H2S positive bacteria 86 Table 32-1 Comparison of Kirby-Bauer and Stokes methods 96 Table 34-1 Preparation of stock dilutions of the antibiotic stocksolutions 101 Table 4-1 Motile and non-motile bacteria 12 Table 43-1 Advantages and disadvantages of VDRL test 124 Table 44-1 Uses of gel diffusion tests 127 Table 48-1 Advantages and disadvantages of the immunofluorescencetest 136 Table 49-1 Enzymes and substrates used in the ELISA 140 Table 49-2 Advantages and disadvantages of ELISA test 140 Table 51-1 List of other types of electrophoresis in gels 149 Table 51-2 Calculation for X% separating or stacking gel 150 Table 52-1 Different types of mutations and their role inmicrobial infections 153 Table 53-1 Differences between F+ strain and Hfr strain 156 Table 57-1 Laboratory tests for differentiation of staphylococcalspecies 167 Table 58-1 Differences between Streptococcus pneumoniae and Streptococcus mitis 170 Table 62-1 Differences between V. 258. 204 Thin blood films 201. 227 Urea hydrolysis test 176 Urease 71. 204 Thioglycollate broth 52 Thioglycollate broth culture 53 Tick borne encephalitis 271 Tick paralysis 271 Ticks 270 Toluidine blue 31 Total coliform count 248 Toxoplasma gondii 199. 85. 290 Trachoma 269 Transcription 144. 227.312 Index Specimen 47 Procedure 47 Quality Control 47 Observations 48 Results and Interpretation 48 Key Facts 48 Viva 48 Test for coliform bacilli 252 Test for pathogenic bacteria 249 Tetramethyl paraphenylene diamine dihydrochloride 160 Thick blood smear 201. cholerae biotypeclassical and V. 84. 202. 206 Widal test 116. 72. 227. 125 Learning Objectives 123 Introduction 123 Principle 123 Requirements 123 Equipments 123 Reagents and glass wares 123 Preparation of buffered saline solution 123 Preparation of un buffered saline solution 123 Specimen 123 Procedure 123 Preparation of serum 123 Preparation of antigen emulsion 124 Maturation of the antigen 124 Qualitative serum test 124 Quantitative serum test 124 Quality control 124 Observations 124 Results and Interpretation 124 Qualitative serum test 124 Quantitative serum test 124 Key Facts 125 Viva 125 VDRL-ELISA 123. 287 Vibriostatic (O/129) agent 182 Voges-Proskauer (VP) test 182 Voges-Proskauer Test 78 Learning Objectives 78 Introduction 78 Principle 78 Requirements 78 X Xylose lysine deoxycholate agar 86 Y Yaws 269 Yeast-like fungi 229 Yellow fever 269 Yolk sac 243 Yolk sac 244 . 125 Veronal buffer 130 Viable count 252 Vibrio cholerae 181.Textbook of Practical Microbiology Urease producing bacteria 72 Urease producing fungi 72 Urease test 71. 279 Learning Objectives 227 Introduction 227 Principle 227 Requirements 227 Equipments 227 Reagents and lab wares 227 Specimen 227 Procedure 227 Quality Control 227 Observations 227 Results and Interpretation 227 Key Facts 228 Viva 228 Equipments 78 Reagents and lab wares 78 Specimen 78 Procedure 78 Quality Control 79 Positive control 79 Negative control 79 Observations 79 Results and Interpretation 79 Key Facts 79 Viva 79 Volutin granules 31 VP broth 78 VP negative bacteria 79 VP positive bacteria 79 VP test 79 313 W Water fleas 271 Weil-Felix test 119 Learning Objectives 119 Introduction 119 Principle 119 Requirements 119 Equipments 119 Reagents and glass wares 119 Specimen 119 Procedure 119 Preparation of master dilution of the serum 119 Performance of the test 119 Quality Control 120 Observations 120 Results and Interpretation 120 Key Facts 120 Viva 120 West Nile fever 269 Western blot 150 Wet mount preparation 191. 281 Learning Objectives 71 Introduction 71 Principle 71 Requirements 71 Equipments 71 Reagents and glass wares 71 Specimen 71 Procedure 71 Quality Control 72 Positive control 72 Negative control 72 Observation 72 Results and Interpretation 72 Key Facts 72 Viva 72 Urease Test for fungi 227. 228. 117 Learning Objectives 116 Introduction 116 Principle 116 Requirements 116 Equipments 116 Reagents and glass wares 116 Specimen 116 Procedure 116 Preparation of master dilution of the serum 116 Performance of the test 116 Quality Control 117 Observations 117 Results and Interpretation 117 Viva 117 Key Facts 118 Wright’s stain 201 Wuchereria bancrofti 291 Wuchereria bancrofti microfilaria 291 V VDRL test 123. 185. 37 .314 Index Zinc dusts 89 Zinc sulphate floatation method 206 Zone reactions 124. 125 Zygomycetes 6 Z Zephiran 60 Ziehl Neelsen staining 288 Ziehl-Neelsen acid-fast staining 27 Ziehl-Neelsen stain 29. to help students to perform various practicals. and to learn and apply the knowledge of practical microbiology in clinical medicine Subhash Chandra Parija is Director-Professor and Head of Microbiology at JIPMER. Every effort has been made to incorporate all aspects of practical microbiology. Rs Ahuja Publishers New Delhi . A sincere effort is made to provide the essential underlying principles of practical microbiology.1 / Running Head 13 TEXTBOOK OF PRACTICAL MICROBIOLOGY The intent of the book is to provide recent information and explain in detail the routine diagnostic methods performed in a Microbiology laboratory. Pondicherry.