Part 1 book “Practical manual of medical microbiology” has contents: Laboratory safety, first aid, hand washing, units, microscope, micrometry, sterilisation, hanging drop preparation, preparation and fixation of smears, methylene blue staining, indian ink staining, gram’s stain,… and other contents.
Practical Manual of Medical Microbiology (For Medical, Dental and Paramedical Students) Practical Manual of Medical Microbiology (For Medical, Dental and Paramedical Students) CP Prince MSc (Medical Microbiology), PhD, FAGE Lecturer Department of Microbiology Mother Theresa Institute of Health Sciences (A Government of Puducherry Institution) Puducherry, India ® JAYPEE BROTHERS MEDICAL PUBLISHERS (P) LTD ————————————————————————————— New Delhi • Ahmedabad • Bengaluru • Chennai • Hyderabad Kochi • Kolkata • Lucknow • Mumbai • Nagpur • St Louis (USA) Published by Jitendar P Vij Jaypee Brothers Medical Publishers (P) Ltd Corporate Office 4838/24 Ansari Road, Daryaganj, New Delhi - 110 002, India, +91-11-43574357 (30 lines) Registered Office B-3 EMCA House, 23/23B Ansari Road, Daryaganj, New Delhi 110 002, India Phones: +91-11-23272143, +91-11-23272703, +91-11-23282021, +91-11-23245672, Rel: +91-11-32558559 Fax: +91-11-23276490, +91-11-23245683 e-mail: jaypee@jaypeebrothers.com, Website: 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1-A Indian Mirror Street, Wellington Square Kolkata 700 013 Phones: +91-33-22651926, +91-33-22276404, +91-33-22276415 Rel: +91-33-32901926, Fax: +91-33-22656075, e-mail: kolkata@jaypeebrothers.com • Lekhraj Market III, B-2, Sector-4, Faizabad Road, Indira Nagar Lucknow 226 016 Phones: +91-522-3040553, +91-522-3040554 e-mail: lucknow@jaypeebrothers.com • 106 Amit Industrial Estate, 61 Dr SS Rao Road, Near MGM Hospital, Parel Mumbai 400012 Phones: +91-22-24124863, +91-22-24104532, Rel: +91-22-32926896 Fax: +91-22-24160828 e-mail: mumbai@jaypeebrothers.com • “KAMALPUSHPA” 38, Reshimbag, Opp Mohota Science College, Umred Road Nagpur 440 009 (MS) Phone: Rel: +91-712-3245220, Fax: +91-712-2704275 e-mail: nagpur@jaypeebrothers.com USA Office 1745, Pheasant Run Drive, Maryland Heights (Missouri), MO 63043, USA Ph: 001-636-6279734 e-mail: jaypee@jaypeebrothers.com, anjulav@jaypeebrothers.com Practical Manual of Medical Microbiology © 2009, Jaypee Brothers Medical Publishers All rights reserved No part of this publication should be reproduced, stored in a retrieval system, or transmitted in any form or by any means: electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the author and the publisher This book has been published in good faith that the material provided by author is original Every effort is made to ensure accuracy of material, but the publisher, printer and author will not be held responsible for any inadvertent error(s) In case of any dispute, all legal matters are to be settled under Delhi jurisdiction only First Edition: 2009 ISBN 978-81-8448-637-7 Typeset at JPBMP typesetting unit Printed at Ajanta Offset PREFACE Practical Manual of Medical Microbiology is aimed to help the teachers and students to conduct practical/demonstration classes and to solve the difficulty of maintaining the practical record book A sincere effort is made to provide brief knowledge on the principles and procedures of common laboratory experiments The figures and photographs especially the line diagrams illustrated in this book will be useful to perform various experiments and write the observations and reports The list of spotters and identification points given in the last chapter will help the students to excel in their practical examinations The content of the book covers the syllabus requirements of many Universities and other regulatory bodies like Medical, Dental and Nursing Councils The WHO recommended test procedures and quality assurance programmes which are mentioned in this book These procedures and programmes may be helpful to standardise and streamline the experiments in newly established medical institutions all over the world This book is tailored to the need of students of MBBS, BDS, BSc (MLT), BSc (Microbiology), DMLT and other paramedical courses and those who work in the field of microbiology needing short and concise information I am extremely grateful to Dr V Balu, Dean, Mother Theresa Institute of Health Sciences (MTIHS), Dr V Gopal, Principal, College of Pharmacy, MTIHS and Dr Helen PS Mannuel, former DirectorProfessor, Madras Medical College for their constant encouragement and support I owe special thanks and gratitude to my colleagues and family for their support and help Readers’ suggestions and comments will help for further improvement of the book in future editions CP Prince CODE OF PROFESSIONAL CONDUCT FOR MEDICAL LABORATORY PERSONNEL Place the well-being and service of the sick above your own interests Be loyal to your medical laboratory profession by maintaining high standards of work and striving to improve your professional skills and knowledge Work scientifically and with complete honesty Do not misuse your professional skills or knowledge for personal gain Never take anything from your place of work that does not belong to you Do not disclose to a patient or any unauthorised person the results of your investigations Treat with strict confidentiality any personal information that you may learn about a patient Respect and work in harmony with the other members of your hospital staff or health centre team Be, at all times, courteous, patient, and considerate to the sick and their relatives 10 Promote health care and the prevention and control of disease 11 Follow safety procedures and know how to apply First Aid 12 Do not drink alcohol during laboratory working hours or when on emergency stand-by 13 Use equipment and laboratory ware correctly and with care 14 Do not waste reagents or other laboratory supplies 15 Fulfill reliably and completely the terms and conditions of your employment CONTENTS 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Laboratory Safety First Aid 10 Hand Washing 12 Units 14 Microscope 17 Micrometry 24 Sterilisation 27 Hanging Drop Preparation 37 Preparation and Fixation of Smears 41 Methylene Blue Staining 45 Indian Ink Staining 47 Gram’s Stain 49 Ziehl-Neelsen’s Stain (Acid Fast Stain) 53 Albert’s Stain 56 Leishman’s Stain 59 Preparation and Cleaning of Glassware 62 pH in Microbiology 67 Bacteriological Media 69 Inoculation of Culture Media 77 Anaerobic Cultivation 84 Important Bacterial Pathogens and the Diseases 88 Collection of Clinical Materials 95 for Microbiological Investigations Biochemical Tests and Identification of Bacteria 106 O/F Test (Hugh and Leifson’s Test) 108 Catalase Test 110 Oxidase Test 112 Sugar Fermentation Test 115 Nitrate Reduction Test 118 Hydrogen Sulphide Production Test 120 x Practical Manual of Medical Microbiology 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Urease Test 122 Citrate Utilisation Test 124 Voges–Proskauer Test (VP Test) 126 Methyl Red Test (MR Test) 128 Indole Test 130 Bile Solubility Test 133 Coagulase Test 135 Antimicrobial Susceptibility Testing 138 Brucella Agglutination Test 148 Anti-Streptolysin O (ASO) Test 150 CRP Screen Latex Agglutination Slide Test 152 VDRL Test 156 Treponema Pallidum Haemagglutination Assay 159 (TPHA) Widal Test 162 Enzyme Linked Immunosorbent Assay (ELISA) 165 Experimental Animals 172 Potassium Hydroxide Wet Mount 175 Lactophenol Cotton Blue Mount 177 Culture of Fungi 179 Fungal Slide Culture (Riddle’s Method) 182 Identification of Fungal Isolates 184 Germ Tube Test 195 Diagnosis of Virus Infections 197 Lab Diagnosis of Malaria 201 Parasitological Examination of Faeces 210 Medical Entomology 220 Bacteriological Examination of Water 233 Microbiology of Milk 239 Lab Diagnosis of Tuberculosis 241 Urinary Tract Infection 247 Spotters 253 Index 267 Laboratory Safety 1 Laboratory Safety ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ Laboratory safety is a vital component of functioning of any laboratory Safety procedures and precautions to be followed in the Microbiology laboratory should be designed to: • Restrict microorganisms present in specimens or cultures to the vessels in which they are contained • Prevent environmental microorganisms (normally present on hand, hair, clothing, laboratory benches or in the air) from entering specimens or cultures and interfering with the results of the studies Laboratory Biosafety Levels Four Biosafety levels have been recommended based on the type of microbes you are working with Biosafety Level -1 (BSL-1): Adherence to standard microbiological practices No special requirement as regards containment equipment Biosafety Level-2 (BSL-2): In addition to the use of standard microbiological practice, laboratory coats, decontamination of infectious wastes, limited access, protective gloves and display of biohazard sign and partial containment equipment are the requirements for this level Most peripheral and intermediate laboratories need BSL-1 or BSL-2 laboratory facilities Practical Manual of Medical Microbiology BSL-3: In addition to BSL-2, it has special laboratory clothing, controlled access to laboratory and partial containment equipment BSL-4: BSL-3 plus entrance through change room where laboratory clothing is put on, shower on exit, all wastes are decontaminated before exit from the facility It requires maximum containment equipment Laboratory facilities in BSL-2 (Figs 1.1 and 1.2) • Laboratory should be designed in such a way that it can be easily cleaned • Laboratory contains a sink for washing • Laboratory tops are impervious to water but resistant to acids, alkalis and organic solvents • An autoclave to decontaminate infectious material is available • Illumination is adequate for all laboratory activities • Storage space is adequate • Preventive measures against laboratory infections These are aimed to protect workers, patients and cultures Following steps are suggested: • Perform adequate sterilization before washing or disposing waste • Provide receptacle for contaminated glassware • Provide safety hood • Ensure that tissues are handled and disposed of properly • Promote regular hand washing and cleaning of bench tops • Ensure use of gloves • Provide mechanical pipetting devices • Protect patients from laboratory personnel with skin or upper respiratory tract infections • Provide special disposal containers for needles and lancets Pipetting Pipetting and suctioning have been identified as the significant and consistent causes of occupational infections Various important precautions that must be taken while pipetting are: • Develop pipetting techniques that reduce the potential for creating aerosols Collection of Clinical Materials for Microbiological Investigations 103 Other specimens: Materials like splenic puncture, liver puncture, pus from liver abscess, CSF, urine rectal swabs and blood may also be sent for parasitological examination Note: Stool for culture must be sent in a sterile bottle G SPECIMENS FOR VIROLOGICAL INVESTIGATION The diagnostic virology is entirely dependent on the correct selection of clinical specimens and their collection and transport to the laboratory under appropriate conditions Specimen should be collected early during the course of the illness as successful isolation may be less in the later stages All specimens should be colleted in sterile containers Each specimen should be accompanied with requisition, from containing patient details like name, age sex, ward and hospital number with brief history of nature of specimen, and date of collection and examination required Most viruses are unstable above 40 oC, hence specimens should be sent immediately, preferably kept in chilled packet in ice to the laboratory For isolation of certain viruses the specimens should be collected in a transport medium The common transport medium used for virological specimens is Hank’s balanced salt solution + 10% bovine albumin + Sodium carbonate + 50 units of penicillin + 50 mg of Sterptomycin The Specimens that can be transported in this medium are swabs, biopsy and autopsy materials The seriological tests can be conducted where the isolation of viruses is impractical, and also to find out significance of the isolation of certain viruses Specimens for serological tests should not be frozen, but should reach the laboratory within 24 hours of collection For serological procedure two samples of clotted blood should be sent once during the acute illness and another 14 days after the 1st sample They should be sent in sterile screw capped bottles a Diseases of central nervous system Faeces: The specimen should be collected from the acute stage upto IInd week 5-10 grams of Faeces should be collected in a 104 Practical Manual of Medical Microbiology sterile screw capped bottle The contents should fill only 3rd of the bottle Freezing is not necessary as certain viruses are stable at normal room temperature CSF: ml collected during the acute stage of the illness The specimen should be chilled by keeping the container in ice cubes and sent to the laboratory Throat swabs: The specimen should be collected during the first few days of illness After swabing it is immediately broken into a sterile screw capped bottle containing ml of transport medium Neuropsy specimens: Several grams of the brain substance can be sent to the laboratory in the frozen state Blood: ml of heparinised blood should be collected during first few days of suspected infection Saliva: It is collected for suspected rabies cases Paired sera are collected for serological tests b Disease of the Skin Macules or papules should be scrapped with scalpel Smears should be made on several glass slides Vesicular fluid should be collected into capillary tubes These should be kept in screw capped bottles After removing the vesicular fluid scrapings from the base of the vesicles should be taken and smeared on slides for microscopic examination The specimens may be dangerous and proper care should be taken while handling them c Disease of the Eye Sterile swabs with wooden stick should be used After collecting swabs from the diseased part of the eye, the tip of the swabs should be cut and the swab should be kept in a screw capped bottle containing ml of transport medium; at the same time several smears can be prepared on glass slides d Diseases of the Respiratory Tract The specimens collected in the respiratory viral infections are mouth garglings, Throat swabs and nasopharynegeal swabs They should Collection of Clinical Materials for Microbiological Investigations 105 be collected in the first days after the illness After taking the specimens they should be placed in screw capped bottles containing ml of transport medium and sent to the laboratory H MATERIALS FOR STERILITY TESTS Suture materials, gauze pieces, cotton, catgut, can be sent in sterile bottle or test tubes Spore paper is supplied by the Microbiology laboratory This should be kept in the centre of the articles to be sterilized before autoclaving The same to be sent to the laboratory to know whether the sterilization is proper or not Once in every 15 days, swabs may be collected from theatre, equipment and sent to the laboratory for testing anaerobic spore bearing organisms 23 Biochemical Tests and Identification of Bacteria ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ After the successful cultivation of bacteria in the laboratory, the next step is identification of the genus and species of the cultivated organism For the identification individual bacteria various methods are available They include staining techniques, biochemical tests, serological tests, animal pathogenecity tests and nucleic acid techniques Among these, biochemical tests are very useful for the routine identification of pathogenic bacteria The Validity of the identification of an unknown bacterial culture by its reaction in a range of biochemical tests depends on the use of a pure culture of the bacteria for inoculation of the test media Single, well separated colonies grown on a non-selective culture medium should be used as inoculum for the tests Common Biochemical Test Used to Differentiate Bacteria Catalase test Oxidase test Sugar fermentation test O/F test Citrate utilisation test Biochemical Tests and Identification of Bacteria 107 10 11 12 13 14 Bile solubility tests Indole production test Coagulase test Voges-Proskauer test Methyl red test Urease test Nitrate reduction test Coagulase test H2S production test 24 O/F Test (Hugh and Leifson's Test) ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ This test is used to distinguish between aerobic and anaerobic break down of Carbohydrate by Bacteria A semisolid medium containing glucose and pH indicator is used for the test if acid is produced only at the surface of the medium, where conditions are aerobic, the attack on the sugar is oxidative if acid is found throughout the tube, including the lower layers where conditions are anaerobic the breakdown is fermentative (Fig 24.1) Requirements Medium Peptone - gm NaCl - gm K2HPO4 - 0.3 gm Bromothymol blue (1% aqueous solution) : ml Agar - gm Water - litre The pH is adjusted to 7.1 before adding bromothymol blue Autoclave the medium O/F Test (Hugh and Leifson's Test) 109 Figure 24.1: O/F test The medium is then tubed to a depth of about cm Straight wire Sterile melted petroleum jelly liquid culture (test organism) Method • Two tubes of medium are taken • Inoculate the medium by stabbing • One tube is promptly sealed with a layer of sterile melted petroleum jelly to a depth of 5-10 mm • One tube without sealing • Incubate both tubes Results Open tube Sealed tube Oxidation (e.g : Acinetobacter) Fermentation (e.g : Esch.coli) No action on Carbohydrate (e.g : Alcaligenes faecalis) Yellow Yellow Green Yellow Green Green Note: This medium can also be used for detecting gas production and motility 25 Catalase Test ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ PRINCIPLE This test demonstrates the presence of catalase, an enzyme that catalyses the release of oxygen from hydrogen peroxide Hydrogen peroxide is a by-product of aerobic respiration and is a toxic substance for bacterial cell Aerobic bacteria produce catalase to breakdown hydrogen perioxide into water and oxygen This can be demonstrated in vitro by catalase test Requirements Hydrogen peroxide solution (10 vol) Platinum wire loop or Plastic loop or sealed capillary tube Pure growth bacteria Method • Take H2O2 solution in a clean test tube • Pick up culture to be tested from a nutrient agar slope with a clean sterile platinum loop or sealed cappillary tube • Insert the wire loop into H2O2 Solution Catalase Test 111 Result • Immediate Production of gas bubbles from the surface of the solid culture material – Positive e.g Staphylococcus spp • No bubbles – Negative e.g Streptococci spp Note: A false positive reaction may be obtained if the culture medium contains catalase (e.g Blood agar) or if an iron wire loop is used 26 Oxidase Test ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ PRINCIPLE This test depends on the presence in bacteria of certain oxidases that will catalyse the transport of electrons between electron donors in the bacteria and a redox dye – tetra methyl-p- phenylene-diamine The dye is reduced to a deep purple colour This test is very useful in screening of Neisseria, Aeromonas, Vibrio, Campylobacter and Pseudomonas, which give positive reactions and for excluding the enterobacteriaceae which give negative reactions Requirements 1% solution of tetramethyl – P-phenylene diamine dihydrochloride Whatman’s no.1 filter paper Glass rod/platinum wire loop Methods The test can be performed by three methods: Plate method Oxidase Test 113 Dry filter paper method Wet filter paper method Plate Method • Cultures are made on suitable solid culture medium (e.g.: nutrient agar) • A freshly prepared 1% solution of tetramethyl–p-phenylenediamine dihydrochloride is poured on to the plate • Decant the solution Results • Colonies rapidly develop a purple colour–Oxidase positive • Colonies not change their colour–Oxidase negative Dry filter paper method • Strips of Whatman’s no.1 filter paper are soaked in a freshly prepared solution of tetramethyl–p-phenylene-diamine dihydrochloride • After draining for about 30 seconds the strips are freeze dried and stored in a dark bottle tightly sealed with a screw cap The papers will have a light purple tint • A strip is laid in a petridish and moistend with sterile distilled water • The colony to be tested is picked up with a platinum wire loop and smeared over the paper Results Intense deep-purple colour on the paper develops within 5-10 seconds positive oxidase Absence of colourisation or colouration after 60 seconds negative oxidase (Fig 26.1) Wet Filter Paper Method • Strips of Whatman’s no.1 filter paper are soaked in a freshly prepared solution of oxidase reagent 114 Practical Manual of Medical Microbiology Figure 26.1: Oxidase test (For colour version, see Plate 7) • Place the paper in a petridish • Smear the paper with the culture to be tested with a platinum wire loop • Result same as for the dry filter paper method Example Oxidase Positive Bacteria • Pseudomonas • Neisseria • Vibrio Example: Oxidase Negative • Esch coli • Salmonella • Shigella 27 Sugar Fermentation Test ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ PRINCIPLE Many bacteria are able to ferment a variety of carbohydrates or related compouds They are broken down to acids like lactic acid, pyruvic acid, or in to gases like CO2 or hydrogen The fermentation range of a particular bacterial species is usually constant By performing a battery of sugar fermentation tests we can identify a species In a routine laboratory various carbohydrates like glucose, lactose, maltose, inulin, salicin, esculin, mannitol, sucrose, etc are used If the organism ferments a given carbohydrate the pH of the medium turns into acidic and this can be detected by using a suitable pH indicator Gas production can be detected by using an inverted Durham’s tube in the medium (Fig 27.1) Requirments Medium Sugar - Peptone water/Serum peptone water/Serum agar - Glucose/Fructose/Mannose/Galactose/ Sucrose/Maltose/ Lactose/Salicin, etc Suitable indicators - Andrade’s indicator - Bromocresol purple 116 Practical Manual of Medical Microbiology Figure 27.1: Sugar fermentation test (phenol red indicator) showing negative, acid formation, and acid and gas formation (For colour version, see Plate 7) - Phenol red - Bromothymol blue Inverted Durham’s tube Method Peptone water fermentation test (common method) Serum peptone water fermentation test This media are suitable for more exacting organism like Corynebacterium diphtheriae Serum agar fermentation test: This media are recommended for organisms such as Meningococci and Gonococci that grow poorly in liquid media The commonest nutrient medium for fermentation tests is peptone water The nutrient medium is prepared and indicater added • Each carbohydrate is incorporated in to peptone water to a concentration of 1% • Add pH indicator (Commonly used indicator is Andrade’s indicator It is made by adding NaOH mol/litre to a 0.5% solution of acid fuchsin until the colour just becomes yellow It is used at a a final concentration of 0.005% in the medium It turns dark reddish pink at about pH 5.5 (Fig 27.2) Sugar Fermentation Test 117 Figure 27.2: Sugar fermentation test (Andrade’s indicator) showing negative reaction and acid formation • The Medium is sterlized after distributing into sugar tubes with a small inverted glass tube called Durham’s tube • Before inoculation, confirm the absence of bubbles gas from Durham’s tube in liquid media • Inoculate the sugar media with a drop or loopful of liquid culture • Incubate at 37 oC for 24 hours Result Pink colour with gas bubble in the Durham’s tube Carbohydrate fermented with acid and gas (+) (e.g Esch.coli, Klebsiella) Pink colour with no gas bubbles in the Durham’s tube Carbohydrate is fermented without gas production (+) (e.g Salmonella Shigella, Vibro) No color change or yellow colour Carbohydrate not fermented (-) and no gas in Durham’s tube e.g Pseudomonas, Alkaligenes ... 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