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 Microbiolog...
Published by
Jitendar P Vij
Jaypee Brothers Medical Publishers (P) Ltd
Corporate Office
4838/24 Ansari Road, Daryaganj, Ne...
PREFACE
PracticalManualofMedicalMicrobiologyisaimedtohelptheteachers
and students to conduct practical/demonstration class...
CODE OF PROFESSIONAL CONDUCT FOR
MEDICAL LABORATORY PERSONNEL
1. Place the well-being and service of the sick above your o...
CONTENTS
1. LaboratorySafety ............................ 1
2. FirstAid ..................................10
3. Hand Washi...
30. Urease Test ............................... 122
31. CitrateUtilisationTest ....................... 124
32. Voges–Prosk...
Laboratory Safety 1
Laboratory safety is a vital component of functioning of any
laboratory.Safetyproceduresandprecautions...
2 Practical Manual of Medical Microbiology
BSL-3: In addition to BSL-2, it has special laboratory clothing,
controlledacce...
Laboratory Safety 3
Figure 1.1: Hazard warning symbols
Figure 1.2: Biological safety cabinets
4 Practical Manual of Medical Microbiology
• Plugpipetteswithcotton.
• Avoidrapidmixingofliquidsbyalternatesuctionandexpul...
Laboratory Safety 5
• Placeusedneedle-syringeunitsdirectlyintoapuncture-resistant
container and decontaminate before disas...
6 Practical Manual of Medical Microbiology
and heavy risk are being carried out. Only authorized visitors
shall enter the ...
Laboratory Safety 7
Clothing
• Allemployeesandvisitorsinmicrobiologicallaboratoriesshall
wear laboratory clothing and labo...
8 Practical Manual of Medical Microbiology
Decontaminationofworksurfaces
• Floodthetotalspillageareaincludingthebrokencont...
Laboratory Safety 9
• Donotwanderaboutthelaboratory;uncontrolledactivitiescause:
– Accidents
– Distractothers
– Promote co...
Knowledgeoffirstaidcanhelptoreducesufferingandconsequences
of serious accidents. In some situations, first aid can be life...
First Aid 11
10. Hydrocortisone cream (H)
11. Acetaminophen and ibuprofen
12. Tweeters
13. Sharp scissors
14. Safety pins
...
INTRODUCTION
“Soap and common sense can prevent 80% of Nosocomial of
infections”. This fact points to the importance of so...
Hand Washing 13
towels are not available. Alcohol would not be effective for soiled
hands which require prior washing.
In ...
The international system of units has been developed and agreed
internationally in the interest of world health. It overco...
Units 15
Sl.No. SI derived units Symbol Quantity measured
1. Square metre m2
area
2. Cubic metre m3
volume
3. Metre per se...
16 Practical Manual of Medical Microbiology
Prefix Symbol Function Multiplied by
Deca da 101
10
Hecta h 102
100
Kilo K 103...
INTRODUCTION
Microscope was invented by Antony Van Leuwenhoek (1632-1723).
He was a Dutch lens maker and was the first per...
18 Practical Manual of Medical Microbiology
Microscope that is suitable for the study of microorganisms is
the light compo...
Microscope 19
b. Limb: It forms the arm which bears the illuminating parts, stage
andtheobservationtube.Insomemicroscopest...
20 Practical Manual of Medical Microbiology
In some new microscopes focusing is done by moving the stage
(movable stage) b...
Microscope 21
10 × – 15.98 mm
40 × – 4.31 mm
100 × – 1.81 mm (This gap is filled with cedar wood oil)
The eyepiece/ocular ...
22 Practical Manual of Medical Microbiology
Electric Bulb
Insteadofmirrorinnewermodelstherewillbeanelectricbulbwhich
will ...
Microscope 23
For Oil-immersion Examination:
1. Raise the condenser completely.
2. Open the iris diaphragm
3. Use plane mi...
INTRODUCTION
The measurement of objects using a calibrated eye piece scale
(micrometer)ismicrometry.Thisisusedtomeasurethe...
Micrometry 25
Calibration of Eye Piece Micrometer
Theeyepiecemicrometerwillrequirecalibrationforeachobjective
of the micro...
26 Practical Manual of Medical Microbiology
6. Measure the distance between ‘0 point and where the alignment
occurs. The m...
Sterilisation is defined as the destruction or removal of all
microorganisms and their spores.
Disinfection is the destruc...
28 Practical Manual of Medical Microbiology
The containers having clinical material are subjected to heat
treatment in the...
Sterilisation 29
Operating Instructions
• Ensure that there is sufficient water inside the chamber.
• Load the autoclave a...
30 Practical Manual of Medical Microbiology
• Allow the material to cool before these are handled (usually agar
bottles ta...
Sterilisation 31
Operating Instructions
• Bring the jacket of the autoclave to operating temperature.
• Load the chamber, ...
32 Practical Manual of Medical Microbiology
Operating Instructions
• Arrange the material to be sterilized loosely and eve...
Sterilisation 33
The synopsis of a few commonly-used disinfectants is given in
Table 7.1.
Preferred methods of sterilizati...
34 Practical Manual of Medical Microbiology
Table 7.2: Preferred methods of sterilization for common-use articles
Autoclav...
Sterilisation 35
Storage
Prior to disposal, all biohazardous waste should be maintained
and stored separately from the gen...
36 Practical Manual of Medical Microbiology
containers, labelled as disinfected waste and disposed of in an
approved facil...
INTRODUCTION
This experiment is mainly used to study the motility of bacteria. It is
also useful to study the shape, relat...
38 Practical Manual of Medical Microbiology
Procedure (Fig. 8.3)
• Take a clean cover slip and apply a little of Vaseline ...
Hanging Drop Preparation 39
• Focus the drop under low power objective so that the edge of the
drop is exactly in the cent...
40 Practical Manual of Medical Microbiology
3. Oxygen tension is more towards the edge of the drop and hence
aerobes tend ...
A proper fixed smear is essential for many staining procedures like
Gram’s staining and Acid fast staining. A good smear w...
42 Practical Manual of Medical Microbiology
Procedure
Step 1—Making of smears
Step 2—Drying of smears
Step 3—Fixation of s...
• Emulsify the colony in the saline and spread evenly on the
slide in a circular manner.
2. Pus: Spread the purulent mater...
44 Practical Manual of Medical Microbiology
Note: Afterpassingtheslidethroughtheflamethreetimes,itshould
bepossibletotouch...
INTRODUCTION
This is a simple staining procedure, both background and object
will stain blue.
Requirements
Methylene blue ...
46 Practical Manual of Medical Microbiology
Uses
The stain is used to make out clearly the morphology of the
organisms, e....
INTRODUCTION
This is a negative staining, useful for demonstration of capsule of
bacteria and yeasts. The capsule will not...
48 Practical Manual of Medical Microbiology
employed for diagnosis especially for Cryptococcus neoformans and
Pneumococcus...
INTRODUCTION
In 1884 Hans Christian Gram described this method of staining,
which is the most important stain in routine b...
50 Practical Manual of Medical Microbiology
or Bunsen burner by holding the slide with the thumb and index
finger. (DON’T ...
Observation (Fig. 12.2)
• Grampositiveorganismstainsviolet.
• Gram negative organism stains pink.
COCCI
Grampositivecocci ...
52 Practical Manual of Medical Microbiology
crystalvioletandiodine.Thosethatresistdecolourisationremain
violetincolouranda...
Mycobacteria have the power of retaining certain stain even when
they are decolorised by mineral acids. This is known as “...
54 Practical Manual of Medical Microbiology
2. “Fixation of the smear”-fix the smear by gently passing the slide
over the ...
Ziehl-Neelsen’s Stain (Acid Fast Stain) 55
bacilli are both acid and alcohol fast, Smegma bacilli is acid fast but
not alc...
INTRODUCTION
Some bacteria possess granules in the cytoplasm these granules are
known by different names like metachromati...
Albert's Stain 57
Requirement
1. Albert’sStain
Toludine blue - 1.5 gm
Malachite green - 2 gm
Glacialaceticacid - 10 ml
Eth...
58 Practical Manual of Medical Microbiology
Figure 14.1: Albert stained smear of corynebacterium
(For colour version, see ...
INTRODUCTION
This stain is a modification of Romanowsky stain. It is very useful
forthedemonstrationofprotozoainbloodfilms...
60 Practical Manual of Medical Microbiology
• Using a pipette add double the volume of distilled water/buffer
to the slide...
Leishman’s Stain 61
Observation (Fig. 15.1)
RBC : Red
W B C : Blue
Malarialparasite : Inside RBC with blue cytoplasm and r...
NEW GLASSWARES
New glass wares may contain resistant spores which may be present
inpackingmaterialandnewglasswarestendtogi...
Figure 16.1: Laboratory wares (plastic/glass)
Preparation and Cleaning of Glassware 63
64 Practical Manual of Medical Microbiology
Figure 16.2: Laboratory wares (plastic/glass)
Chromic Acid Cleaning
This clean...
Figure 16.3: Preparation of glass Pasteur pipette
Figure 16.4: Plastic Pasteur pipettes
Preparation and Cleaning of Glassw...
66 Practical Manual of Medical Microbiology
• DryinoveniftheglasswareisnotusedforaccurateVolumetric
purposes.
Cleaning of ...
Microorganisms are sensitive to the varying pH of the external
environment. Where there is an optimum pH for the growth an...
68 Practical Manual of Medical Microbiology
Table 17.1: Indicative dyes and pH range
Indicator Range of pH Colour change
T...
The role of suitable quality culture media for cultivation of
microorganisms cannot be over emphasized. The success of iso...
70 Practical Manual of Medical Microbiology
growth-promoting qualities are enriched media, e.g. blood agar,
chocolate agar...
Bacteriological Media 71
The pH determination can be conveniently done with the use of
pH indicator papers.
Adjust the pH ...
72 Practical Manual of Medical Microbiology
Figure 18.1: Nutrient broth
(For colour version, see Plate 3)
Figure 18.2: Nut...
Bacteriological Media 73
Figure 18.3: Blood agar (For colour version, see Plate 4)
Figure 18.4: Colony morphology
74 Practical Manual of Medical Microbiology
Blood Agar (Fig. 18.3)
Nutrient agar 100 ml
Sheep blood (defibrinated) 10 ml
M...
Bacteriological Media 75
Buffered Glycerol Saline
Glycerol 300 ml
Sodium chloride 4.2 gm
Disodium hydrogen phosphate 10.0 ...
76 Practical Manual of Medical Microbiology
Glycerolated blood tellurite mixture
Sterile defibrinated sheep blood 14 ml
St...
When inoculating or seeding culture media an aseptic (sterile)
technique must be used to:
• To prevent contamination of cu...
78 Practical Manual of Medical Microbiology
Figure 19.1: Aseptic inoculation of culture medium
Figure 19.2: Bunsen burner
Inoculation of Culture Media 79
• Make slide preparations from specimens after inoculating the
culture media.
• Decontamin...
80 Practical Manual of Medical Microbiology
1. Cut a piece of wire about 125 mm in length and thickness (swg)
26 or 27, Wi...
Inoculation of Culture Media 81
To inoculate a plate, apply the inoculum to a small area of the
plate (‘the well’) using a...
82 Practical Manual of Medical Microbiology
Inoculation of Stab Media (deeps)
Use a Sterile straight wire to inoculate a s...
Inoculation of Culture Media 83
metabolism. The length of time of incubation depends on how long
an organism takes to deve...
Depending on oxygen requirement, miroorganisms can be classified
into4groups:
1. OBLIGATE AEROBES
Thosebacteriawhichrequir...
Anaerobic Cultivation 85
Ex.: a. Staphylococci.
b. Esch.coli.
4. MICROAEROPHILIC ORGANISMS
Thosethatgrowjustinthepresenceo...
86 Practical Manual of Medical Microbiology
themediawithinoculatedspecimenisinvertedoverthefilter
paper and the edge is se...
Anaerobic Cultivation 87
McIntosh and Filde's Anaerobic Jar
This jar is used to provide complete anaerobic environment for...
BACTERIAL PATHOGENS AND RELATED DISEASES
Organism Morphology Important diseases
Gram Positive Cocci
Staphylococcus Cocci i...
Important Bacterial Pathogens and the Diseases 89
Contd...
Viridans Streptococci Cocci in chains Bacterial endocarditis,
b...
90 Practical Manual of Medical Microbiology
Contd...
Clostridium tetani Non-motile, long Tetanus
(Drum-stick bacillus) thi...
Important Bacterial Pathogens and the Diseases 91
Contd...
Yersinia Motile Acute mesenteric
pseudotuberculosis pleomorphic...
92 Practical Manual of Medical Microbiology
Contd...
Shigella species Non-motile rods Bacillary dysentery
Salmonella speci...
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CP Prince - Practical Manual of Medical Microbiology
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CP Prince - Practical Manual of Medical Microbiology
CP Prince - Practical Manual of Medical Microbiology
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CP Prince - Practical Manual of Medical Microbiology

  1. 1. Practical Manual of Medical Microbiology (For Medical, Dental and Paramedical Students)
  2. 2. 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) ®
  3. 3. 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: www.jaypeebrothers.com Branches • 2/B, Akruti Society, Jodhpur Gam Road Satellite Ahmedabad 380 015 Phones: +91-79-26926233, Rel: +91-79-32988717 Fax: +91-79-26927094 e-mail: ahmedabad@jaypeebrothers.com • 202 Batavia Chambers, 8 Kumara Krupa Road, Kumara Park East Bengaluru 560 001 Phones: +91-80-22285971, +91-80-22382956, +91-80-22372664 Rel: +91-80-32714073, Fax: +91-80-22281761 e-mail: bangalore@jaypeebrothers.com • 282 IIIrd Floor, Khaleel Shirazi Estate, Fountain Plaza, Pantheon Road Chennai 600 008 Phones: +91-44-28193265, +91-44-28194897, Rel: +91-44-32972089 Fax: +91-44-28193231 e-mail: chennai@jaypeebrothers.com • 4-2-1067/1-3, 1st Floor, Balaji Building, Ramkote Cross Road Hyderabad 500 095 Phones: +91-40-66610020, +91-40-24758498, Rel:+91-40-32940929 Fax:+91-40-24758499 e-mail: hyderabad@jaypeebrothers.com • No. 41/3098, B & B1, Kuruvi Building, St. Vincent Road Kochi 682 018, Kerala Phones: +91-484-4036109, +91-484-2395739, +91-484-2395740 e-mail: kochi@jaypeebrothers.com • 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
  4. 4. PREFACE PracticalManualofMedicalMicrobiologyisaimedtohelptheteachers and students to conduct practical/demonstration classes and to solvethedifficultyofmaintainingthepracticalrecordbook. A sincere effort is made to provide brief knowledge on the principles and procedures of common laboratory experiments. The figuresandphotographsespeciallythelinediagramsillustratedin this book will be useful to perform various experiments and write theobservationsandreports. Thelistofspottersandidentification points given in the last chapter will help the students to excel in theirpracticalexaminations. Thecontentofthebookcoversthesyllabusrequirementsofmany 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 institutionsallovertheworld. 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 conciseinformation. I am extremely grateful to Dr V Balu, Dean, Mother Theresa InstituteofHealthSciences(MTIHS),DrVGopal,Principal,College of Pharmacy, MTIHS and Dr Helen PS Mannuel, former Director- Professor,MadrasMedicalCollegefortheirconstantencouragement 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
  5. 5. CODE OF PROFESSIONAL CONDUCT FOR MEDICAL LABORATORY PERSONNEL 1. Place the well-being and service of the sick above your own interests. 2. Beloyaltoyourmedicallaboratoryprofessionbymaintaining high standards of work and striving to improve your professional skills and knowledge. 3. Work scientifically and with complete honesty. 4. Do not misuse your professional skills or knowledge for personalgain. 5. Never take anything from your place of work that does not belongtoyou. 6. Do not disclose to a patient or any unauthorised person the resultsofyourinvestigations. 7. Treatwithstrictconfidentialityanypersonalinformationthat you may learn about a patient. 8. Respect and work in harmony with the other members of yourhospitalstafforhealthcentreteam. 9. Be,atalltimes,courteous,patient,andconsideratetothesick andtheirrelatives. 10. Promotehealthcareandthepreventionandcontrolofdisease. 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.
  6. 6. CONTENTS 1. LaboratorySafety ............................ 1 2. FirstAid ..................................10 3. Hand Washing .............................12 4. Units ....................................14 5. Microscope ................................17 6. Micrometry ................................24 7. Sterilisation ................................27 8. Hanging Drop Preparation .....................37 9. Preparation and Fixation of Smears ...............41 10. Methylene Blue Staining .......................45 11. Indian Ink Staining ..........................47 12. Gram’s Stain ...............................49 13. Ziehl-Neelsen’sStain(AcidFastStain) ............53 14. Albert’sStain ..............................56 15. Leishman’s Stain ............................59 16. Preparation and Cleaning of Glassware ............62 17. pH in Microbiology ..........................67 18. BacteriologicalMedia .........................69 19. Inoculation of Culture Media ...................77 20. Anaerobic Cultivation ........................84 21. Important Bacterial Pathogens and the Diseases ......88 22. CollectionofClinicalMaterials ..................95 forMicrobiologicalInvestigations 23. BiochemicalTestsandIdentificationofBacteria ..... 106 24. O/F Test (Hugh and Leifson’s Test) .............. 108 25. CatalaseTest .............................. 110 26. Oxidase Test .............................. 112 27. Sugar Fermentation Test ...................... 115 28. Nitrate Reduction Test ....................... 118 29. Hydrogen Sulphide Production Test ............. 120
  7. 7. 30. Urease Test ............................... 122 31. CitrateUtilisationTest ....................... 124 32. Voges–Proskauer Test (VP Test) ................ 126 33. Methyl Red Test (MR Test) .................... 128 34. IndoleTest ............................... 130 35. BileSolubilityTest .......................... 133 36. Coagulase Test ............................ 135 37. AntimicrobialSusceptibilityTesting ............. 138 38. BrucellaAgglutinationTest ................... 148 39. Anti-Streptolysin O (ASO) Test ................. 150 40. CRP Screen Latex Agglutination Slide Test......... 152 41. VDRL Test ............................... 156 42. Treponema Pallidum Haemagglutination Assay .... 159 (TPHA) 43. Widal Test ............................... 162 44. Enzyme Linked Immunosorbent Assay (ELISA) ..... 165 45. Experimental Animals ....................... 172 46. Potassium Hydroxide Wet Mount ............... 175 47. Lactophenol Cotton Blue Mount ................ 177 48. Culture of Fungi ........................... 179 49. Fungal Slide Culture (Riddle’s Method) ........... 182 50. IdentificationofFungalIsolates ................ 184 51. Germ Tube Test ............................ 195 52. DiagnosisofVirusInfections .................. 197 53. Lab Diagnosis of Malaria ..................... 201 54. Parasitological Examination of Faeces ............ 210 55. Medical Entomology ........................ 220 56. Bacteriological Examination of Water ............ 233 57. Microbiology of Milk ........................ 239 58. Lab Diagnosis of Tuberculosis ................. 241 59. UrinaryTractInfection ....................... 247 60. Spotters ................................. 253 Index .................................... 267 x Practical Manual of Medical Microbiology
  8. 8. Laboratory Safety 1 Laboratory safety is a vital component of functioning of any laboratory.Safetyproceduresandprecautionstobefollowedinthe 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,laboratorybenchesorintheair)fromentering 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.Nospecialrequirementasregardscontainmentequipment. Biosafety Level-2 (BSL-2): In addition to the use of standard microbiological practice, laboratory coats, decontamination of infectiouswastes,limitedaccess,protectiveglovesanddisplayof biohazard sign and partial containment equipment are the requirementsforthislevel. Most peripheral and intermediate laboratories need BSL-1 or BSL-2laboratoryfacilities. Laboratory Safety 1 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
  9. 9. 2 Practical Manual of Medical Microbiology BSL-3: In addition to BSL-2, it has special laboratory clothing, controlledaccesstolaboratoryandpartialcontainmentequipment. 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) • Laboratoryshouldbedesignedinsuchawaythatitcanbeeasily cleaned. • Laboratory contains a sink for washing. • Laboratory tops are impervious to water but resistant to acids, alkalisandorganicsolvents. • Anautoclavetodecontaminateinfectiousmaterialisavailable. • Illuminationisadequateforalllaboratoryactivities. • Storagespaceisadequate. • Preventivemeasuresagainstlaboratoryinfections These are aimed to protect workers, patients and cultures. Following steps are suggested: • Performadequatesterilizationbeforewashingordisposingwaste. • Provide receptacle for contaminated glassware. • Providesafetyhood. • Ensure that tissues are handled and disposed of properly. • Promote regular hand washing and cleaning of bench tops. • Ensureuseofgloves. • Provide mechanical pipetting devices. • Protect patients from laboratory personnel with skin or upper respiratorytractinfections. • Providespecialdisposalcontainersforneedlesandlancets. Pipetting Pipettingandsuctioninghavebeenidentifiedasthesignificantand consistent causes of occupational infections. Various important precautionsthatmustbetakenwhilepipettingare: • Developpipettingtechniquesthatreducethepotentialforcreating aerosols.
  10. 10. Laboratory Safety 3 Figure 1.1: Hazard warning symbols Figure 1.2: Biological safety cabinets
  11. 11. 4 Practical Manual of Medical Microbiology • Plugpipetteswithcotton. • Avoidrapidmixingofliquidsbyalternatesuctionandexpulsion. • Donotforciblyexpelmaterialfromapipette. • Donotbubbleairthroughliquidswithapipette. • Preferpipettesthatdonotrequireexpulsionoflastdropofliquid. • Drop material having pathogenic organisms as close as possible tothefluidoragarlevel. • Place contaminated pipettes in a container having suitable disinfectantforcompleteimmersion. Avarietyofpipettesareavailable.Selectionshoulddependupon the ease of operation and the type of work to be performed. Hypodermic Syringes and Needles Accidents involving the use of syringes and needles while drawing bloodfrompatientsorperformingexperimentsonlaboratoryanimals are among the most common causes of occupational infections in laboratoriesandhealthcarefacilities.Theyaccountforalmost25% of the laboratory-acquired infections that occur by accidents. The practices which are recommended for hypodermic needle and syringesare: • Avoid quick and unnecessary movements of the hand holding thesyringe. • Examineglasssyringesforchipsandcracks,andexamineneedles forbarbsandplugs. • Use needle locking (Luer Lock type) syringes only and be sure thatneedleislockedsecurely. • Wearsurgicalorothergloves. • Fillsyringescarefullytominimizeairbubblesandfrothing. • Expelexcessair,liquidandbubblesverticallyintoacottonpledget moistenedwithsuitabledisinfectant. • Do not use syringe to forcefully expel infectious fluid into an openvialformixing.Mixingwithasyringeisappropriateonlyif thetipoftheneedleisheldbelowthesurfaceofthefluidinthe tube. • Do not bend, shear, recap or remove the needle from syringe by hand.
  12. 12. Laboratory Safety 5 • Placeusedneedle-syringeunitsdirectlyintoapuncture-resistant container and decontaminate before disassembly, reuse or disposal. Opening Containers Theopeningofvials,flasks,petridishes,culturetubes,embryonated eggs,andothercontainersofpotentiallyinfectiousmaterialsposes potential but subtle risks of creating droplets, aerosols or contamination of the skin or the immediate work area. The most common opening activity in most health care laboratories is the removal of stoppers from containers of clinical materials. It is imperative that specimens should be received and opened only by personnelwhoareknowledgeableaboutoccupationalinfectionrisks. Variousprecautionsthatcanbetakeninthisregardare: • Open containers with clinical specimens in well-lighted and designatedareasonly. • Wear a laboratory coat and suitable gloves. • Ifpossible,useaplastic-backedabsorbentpapertowelto: – Facilitateclean-up – Reduce generation of aerosols • Specimens which are leaking or broken may be opened only in safetycabinets. Tubescontainingbacterialculturesshouldbehandledwithcare. Vigorousshakingofliquidculturescreatesaheavyaerosol.Whena sealedampoulecontainingalyophilizedorliquidcultureisopened, an aerosol may be created. Ampoules should be opened in a safety cabinet(Fig.1.3). Laboratory Access • As far as possible children and pregnant women visitors should notenterthemicrobiologicallaboratories. • Appropriate signs should be located at points of access to laboratoryareasdirectingallvisitorstoareceptionistorreceiving officeforaccessprocedures. • The universal biohazard symbol shall be displayed at specific laboratories in which manipulations of organisms with moderate
  13. 13. 6 Practical Manual of Medical Microbiology and heavy risk are being carried out. Only authorized visitors shall enter the laboratory showing universal biohazard sign (Fig.1.4).Doorsdisplayingbiohazardsymbolshallnotbepropped open, but shall remain closed when in use. . Figure 1.4: Universal biohazard sign Figure 1.3: Biological safety cabinet
  14. 14. Laboratory Safety 7 Clothing • Allemployeesandvisitorsinmicrobiologicallaboratoriesshall wear laboratory clothing and laboratory shoes or shoe covers. • Disposableglovesshallbewornwhereverradiological,chemical, carcinogenicmaterialsorviruspreparationsofmoderatetohigh riskarehandled. • Laboratory clothing including shoes shall not be worn outside theworkarea. Accidents in Laboratory Inthemicrobiologicallaboratory,infectionsposethemostfrequent risk. The important pathogens are: HepatitisBvirus, Shigellaspp. HIV, Salmonellaspp.includingStyphi Brucellaspp. Bacillusanthracis LeptospiresYersiniapestis Mycobacteriaspp. Histoplasma Accidents and Spills Theorderofprioritiesisasfollows: • Protectionofpersonnel • Confinement of contamination • Decontamination of personnel • Decontamination of area involved Decontamination of skin: The area is washed thoroughly with soap and water. Detergents or abrasive materials must not be used and care must be taken not to damage the skin. Decontaminationofcutseyes:Theseareirrigatedwithwatertaking caretopreventthespreadofcontaminationfromoneareatoanother. Decontamination of clothing: Contaminated garments should be removed immediately and placed in a container. They should not be removed from the spill location until contamination has been monitored.
  15. 15. 8 Practical Manual of Medical Microbiology Decontaminationofworksurfaces • Floodthetotalspillageareaincludingthebrokencontainerwith disinfectant. • Leave undisturbed for 10 minutes. • Mop with cotton wool or absorbent paper. • Wear disposable gloves, apron and goggles. • Ifadustpanandbrushorforcepshavebeenusedthesetoorequire disinfection. • For blood or viruses, hypochlorites (10 gm/L) are used. • Donotusehypochoritesolutionincentrifuges. • Use activated gluteraldehyde (20 gm/L) on surfaces for viral decontamination. • Place all potentially contaminated materials in a separate containerandretainuntilmonitored. • Restricttheentrytosuchanareauntilcontaminationmonitoring hasbeencarriedout. Management of Laboratory Accidents An adequately equipped first-aid box should be kept in the laboratoryinaplacethatisknownandaccessibletoallmembersof staff. The box must be clearly marked and preferably be made of metal or plastic to prevent from damage by pests. A medical officer should be consulted regarding the contents of the box. A first-aid chartgivingtheimmediatetreatmentofcuts,burns,poisoning,shock and collapse, should be prepared and displayed in the laboratory. General Laboratory Directions for Safety The salient general laboratory directions which must be obeyed by allare: • Long hair should be bound back neatly away from shoulders. • Do not wear any jewellery to laboratory sessions. • Keepfingers,pencils,bacteriologicalloops,etc.outofyourmouth. • Do not smoke in the laboratory. • Donotlicklabelswithtongue(usetapwater). • Do not drink from laboratory glassware.
  16. 16. Laboratory Safety 9 • Donotwanderaboutthelaboratory;uncontrolledactivitiescause: – Accidents – Distractothers – Promote contamination • Do not place contaminated pipettes on the bench top. • Donotdiscardcontaminatedcultures,glassware,pipettes,tubes or slides in wastepaper basket or garbage can. • Avoiddispersalofinfectiousmaterials. • Operate centrifuges, homogenizer and shakers safely. • Immunize the laboratory workers against vaccine-preventable diseasessuchashepatitisB,meningococcalmeningitis,rabies,etc.
  17. 17. Knowledgeoffirstaidcanhelptoreducesufferingandconsequences of serious accidents. In some situations, first aid can be life saving. All laboratory workers should receive a basic practical training in First Aid, with particular attention being paid to the types of accidents which may occur in the laboratory. First Aid Box An adequately equipped first aid box should be kept in the laboratory. The box should be clearly marked with a red cross. The commonitemskeptinaFirstAidBoxaregivenbelow. Thelaboratory incharge should regularly check the items; replace the missing items or medicines that may have expired. 1. First aid manual 2. Sterile gauze 3. Adhesive tape 4. Adhesive bandages in several sizes 5. Elastic bandage 6. Antiseptic wipes 7. Soap 8. Antibiotic cream (triple antibiotic ointment) 9. Antiseptic solution (like hydrogen peroxide) First Aid 2 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
  18. 18. First Aid 11 10. Hydrocortisone cream (H) 11. Acetaminophen and ibuprofen 12. Tweeters 13. Sharp scissors 14. Safety pins 15. Disposable instant cold packs. 16. Calamine lotion 17. Alcohol wipes and ethyl alcohol 18. Thermometer 19. Plastic gloves (at least two pairs) 20. Flash light and extra batteries 21. Mouth piece for administering CPR 22. List of emergency phone numbers 23. Blanket (stored nearby)
  19. 19. INTRODUCTION “Soap and common sense can prevent 80% of Nosocomial of infections”. This fact points to the importance of soap and hand washing in the control of infectious diseases. Hand hygiene has been described as the single most effective means of preventing spread of infections. The aim of hand washing is to remove transient microorganisms (not commensal organisms) as soon as possible following acquisition fromcontactwithpossiblesources,orimmediatelybeforeperforming invasive procedures and touching susceptible patients or susceptible sites. Method Proper hand washing can easily achieve by thorough hand washing technique with soap and water using mechanical friction for 10-15 seconds followed by drying. Surgical hand wash involves washing both hands and fore arms using a defined technique for at least 2 minutes (Fig. 3.1). In order to disinfect clean hands, alcohol rub can be applied following washing. Alternatively, alcohol based product can be used which are shown to be very effective when running water, soap and Hand Washing 3 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
  20. 20. Hand Washing 13 towels are not available. Alcohol would not be effective for soiled hands which require prior washing. In hospitals and laboratories hand washing facilities should be conveniently located in sufficient numbers. The facility should include sink of adequate size with elbow or sensor operated tap and wall mounted liquid hand washing agent like soap or disinfectant and paper towel unit. Waste bin with foot-operated lid should be provided along side. Commonly used disinfectant solutions are chlorhexidine, povidone iodine and commercially available antiseptic solutions like Dettol or Savlon. Figure 3.1: Hand washing procedure
  21. 21. The international system of units has been developed and agreed internationally in the interest of world health. It overcomes language barriers, enabling an exchange of health information with in a country and between nations. SI units (Systeme International d’ Unites) are commonly used. There are seven basic SI units, meter, kilogram, second, mole, ampere, Kelvin and candela. All other units are derived from these seven base units. Some SI derived units have been given special names. Sl.No. SI base units Symbol Quantity measured 1. Metre m length 2. Kilogram kg mass 3. Second S time 4. Mole mol amount of substance 5. Ampere A Electric current 6. Kelvin K temperature 7. Candela cd luminous intensity. Units 4 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
  22. 22. Units 15 Sl.No. SI derived units Symbol Quantity measured 1. Square metre m2 area 2. Cubic metre m3 volume 3. Metre per second m/s speed Sl.No. Named derived Symbol Quantity measured unit 1. Hertz Hz frequency 2. Joule J energy, quantity of heat 3. Newton N Force 4. Pascal Pa Pressure 5. Watt W Power 6. Volt V Electric potential difference 7. Degree o C Celsius temperature SI Unit Prefixes To enable the measurement of larger or smaller units, SI system also includes a set of prefixes. The use of a prefix makes a unit larger or smaller. Example: if the prefix milli is put in front of the metere (millimeter) this indicates that the unit should be divided by a thousand (10–3 ) Prefix Symbol Function Divided by Deci d 10–1 10 Centi C 10–2 100 Milli M 10–3 1000 Micro u 10–6 1000 000 Nano n 10–9 1000 000 000 Pico P 10–12 1000 000 000 000 Femto f 10–15 1000 000 000 000 000
  23. 23. 16 Practical Manual of Medical Microbiology Prefix Symbol Function Multiplied by Deca da 101 10 Hecta h 102 100 Kilo K 103 1000 Mega M 106 1000 000 Giga G 109 1000 000 000 Tera T 1012 1000 000 000 000 Peta P 1015 1000 000 000 000 000
  24. 24. INTRODUCTION Microscope was invented by Antony Van Leuwenhoek (1632-1723). He was a Dutch lens maker and was the first person to observe Bacteria. Microscope is an essential optical instrument of Microbiology laboratory. It consists of combination of lenses which will give a magnified image of minute objects or micro organisms like Bacteria, Fungi, and Protozoa. Types of Microscopes 1. Simple microscope 2. Compound microscope 3. Ultraviolet microscope 4. Fluorescent microscope 5. Polarizing microscope 6. Dark ground microscope 7. Phase contrast microscope 8. Inverted microscope 9. Electron microscope Microscope 5 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
  25. 25. 18 Practical Manual of Medical Microbiology Microscope that is suitable for the study of microorganisms is the light compound Microscope. This consists of two converging lenses fixed at the ends of a brass tube. The lens which is nearer to the object is called “Objective” and the lens which is close to the eye is called Eyepiece or “Ocular”. The final image can be observed through the ocular. The objective magnifies the specimen to a definite amount and produces a real, inverted intermediate image of the object, which lies within the principle focus of the eyepiece. The eyepiece further magnifies the image formed by the objective so that the image seen by the eye has a magnification, equal to the product of magnification of the two systems. The individual magnification of objectives and eyepiece are engraved on each part. The final image seen is thus inverted, magnified and virtual. Parts of Compound Microscope Based on the manufacturer the parts and adjustments may slightly vary from each other. Microscopes with fixed stage and mechanical stage are available. Microscope with mechanical stage is more convenient for microbiological studies (Fig. 5.1). I Mechanical parts Base (Foot) Limb(Arm) Stage Adjustment knobs Revolving nose piece II Magnifying parts Objective Ocular III Illuminating parts Sub stage condenser Iris diaphragm Filter holder Mirror Electric bulb MECHANICAL PARTS a. Base: It forms the stand or foot of the microscope, often horse shoe shaped to give the stability. The mirror or electric bulb illuminator is attached to the base.
  26. 26. Microscope 19 b. Limb: It forms the arm which bears the illuminating parts, stage andtheobservationtube.Insomemicroscopesthelimbisattached to the foot by a hinged joint so that the microscope can be set at a comfortable angle for the observer. c. Stage: The stage is a plat form which accommodates a glass slide on which the object to be examined is mounted. It has an aperture in its centre to permit light to reach the object. The stage can be of 2 types: i. A fixed stage on which the object is fixed by clips. ii. The mechanical stage holds the slide secure and allows the specimen to be moved smoothly backwards, forwards or sideways. Sometimes a scale is fitted to two sides of the stage to show the extent of the movement. This is called the Vernier Scale and it is useful to trace a part of the blood film or sputum smearthatyouneedtore-examineorshowtoyoursupervisor. d. Adjustment knobs: They are used to focus the object two types of knobs will be present (1) Coarse adjustment for initial adjustment and (2) Fine adjustment for getting a clear image. Figure 5.1: Compound microscope Eye piece Limb Coarse adjustment Fine adjustment Body tube Body Nose piece Objective Stage Condenser Mirror Base
  27. 27. 20 Practical Manual of Medical Microbiology In some new microscopes focusing is done by moving the stage (movable stage) by coarse and fine adjustments. In older versions, the stage is fixed and focusing is done by moving the Body tube by coarse and fine adjustment knobs. e. Revolving nose piece: A number of objective lenses of different magnifications are screwed to the nosepiece of the microscope, which can be revolved to increase or decrease the magnification of the specimen being examined by selecting the objective lens. MAGNIFYING PARTS This consists of eyepieces and objectives. They are kept separated in a graduated tube. Objectives are referred to by their magnifying power, which is marked on the side. The microscope commonly used in student laboratories will have the following objectives (Fig. 5.2). Figure 5.2: Objectives of microscope 10 × (Low power objective) 40 × (High power objective) 100 × (Oil immersion objective) As the magnification differs between objectives, so does the working distance. The working distance is the distance between the front lens of the objective and the specimen on the stage (when the specimen is in focus). The higher the magnifying power of the objective the shorter is the working distance. Working distances for the standard objectives are likely to be indicated as follows.
  28. 28. Microscope 21 10 × – 15.98 mm 40 × – 4.31 mm 100 × – 1.81 mm (This gap is filled with cedar wood oil) The eyepiece/ocular commonly used will have a magnification of 10 ×. Oculars with 5 ×, 6 ×, 15 × and 20 × are also available. ILLUMINATING PARTS Sub-stage Condensers It is made up of a system of convex lenses, which focus light from the illuminating source on the place of the object. The height of the condenser can be varied by a rack and pinion mechanism. Lowering of condenser diminishes illumination whereas raising the condenser increases the illumination. While using oil immersion objective, the condenser is completely raised as it requires more light. When the other objectives are used, it is to be lowered suitably. Iris Diaphragm Immediately below the condenser and incorporated in the same mount is the sub stage iris diaphragm operated by a small lever which protrudes to one side. Opening or closing of this iris diaphragm controls the amount of light reaching the object. Iris diaphragm is opened widely when the oil-immersion objective is used as it requires maximum light and closed partially when the other objectives are in use. Filter Holder Just below the iris diaphragm is ring shaped filter holder designated to carry circular, blue colored glass filters required to reduce the excessive red or yellow component of some light sources. Mirror Fitted to the base, below the condenser is a Plano- concave mirror. This is the illuminating source. It helps to reflect the light to the sub stage condenser. The flat surface (Plane mirror) is used whenever the oil immersion objective is employed. The concave mirror is employed with low and high power objectives.
  29. 29. 22 Practical Manual of Medical Microbiology Electric Bulb Insteadofmirrorinnewermodelstherewillbeanelectricbulbwhich will act as source of illumination. It can also be replaced by mirror. The intensity of light can also be adjusted by the regulator. MAGNIFICATION It is defined as the degree of enlargement of the image of the object achieved by the microscope. The total magnification achieved by various objectives are given in the Table 5.1. Table 5.1: Total magnification Objective Ocular Mirror Oil Condenser Iris diaphragm Magnification 10 × 10 × Concave No Low Closed 10 × 10 = 100 times 40 × 10 × Concave No Middle Half Open 40 × 10 = 400 times 100 × 10 × Plane Yes Raised Open 100 × 10 = 1000 times Adjustment of Microscope Adjust the various parts as follows. For unstained preparation: 1. Lower the condenser 2. Close the iris diaphragm (Fig. 5.3) 3. Use concave mirror 4. Focus under low power and then turn to high power. For Stained preparations: 1. Lower the condenser 2. Use concave mirror 3. Adjust the iris diaphragm to give an even illumination of field (Fig. 5.3). 4. Focus under low power and then turn to high power, if required. Figure 5.3: Adjustments of diaphragm
  30. 30. Microscope 23 For Oil-immersion Examination: 1. Raise the condenser completely. 2. Open the iris diaphragm 3. Use plane mirror 4. Place a drop of cedar wood oil on the object and focus under oil immersion lens. Rack oil immersion objective down till its tip dips in the oil. Using the fine adjustment focus the object. 5. After this, remove the oil from the objective and the object with lens cleaning paper. 6. Leave low power objective in position till further use. Note: Cedar wood Oil has same refractive index as that of glass, Addition of oil in the gap between objective and object prevents refraction of light rays in order to get a bright image of the object. Care of Microscope • Clean the microscope with a clean soft cloth • The Objectives and oculars must be cleaned with lens paper • Alcohols should not be used as it dissolves the cement, which binds the lenses. • Direct sunlight on to the mirror should be avoided. • For prolonged work, artificial light of particular wavelengths are advisable. • When not used, protect it from dust and damages.
  31. 31. INTRODUCTION The measurement of objects using a calibrated eye piece scale (micrometer)ismicrometry.Thisisusedtomeasurethesizeofobjects observed under microscope. Measurement of size is useful for the identificationofmicroorganismespeciallycystandOvaofParasites. Requirement 1. Eyepiece micrometer: A suitable line scale for the eye piece micrometerisdividedinto50divisions. 2. StageMicrometer:AsuitableScaleforthestagemicrometerisone that measures 2 mm in length with each large division measuring 0.1 mm (100 μm) and each small division measuring 0.01 mm (10μm)(Fig.6.1). Method 1. Caliberationofeyepiecemicrometer 2. Measuring an object with calibrated eye piece micrometer. Micrometry 6 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
  32. 32. Micrometry 25 Calibration of Eye Piece Micrometer Theeyepiecemicrometerwillrequirecalibrationforeachobjective of the microscope at which measurements will be required. For parasitology, the scale should be calibrated for the 40 × objectiveandforBacteriologyitisusefultocalibrateforthe100× objective. A table can be prepared for each objective giving measurement1to50divisionsoftheeyepieceScale. 1. Remove the normal eyepiece and insert the eye piece micrometer inthetubeofthemicroscope. 2. Placethestagemicrometerslideonthestageofthemicroscope. 3. Focusthestagemicrometerscaleusingtherequiredobjective. 4. Adjustthefielduntilthe0lineoftheeyepieceScalealignsexactly withthe‘0’lineofthestage. 5. Look along the Scales and note where a division of the eye piece scalealignsexactlywithadivisionofstagescale. Figure 6.1: Micrometry: Stage micrometer scale (upper) and ocular micrometer (lower)
  33. 33. 26 Practical Manual of Medical Microbiology 6. Measure the distance between ‘0 point and where the alignment occurs. The measurements Calibration Scale are 0.1mm to 2.0 mm each small division measures 0.01mm. 7. Count the number of divisions of the eye piece Scale covered between the ‘0’ point and where the alignment occurs. 8. Calculate the measurement of 1 of the divisions of the eye piece Scalein1μm. Example: Distance measured = 0.2 mm Number of divisions = 27 1 division measures = 0.2 27 = 0.0074 mm To Convert mm to μm = 0.0074 × 1000 = 7.4 μm Measuring the Object • Remove the stage micrometer • Placetheobjectslide • Witheyepiecemicrometermeasuredivisionscoveredfortheobject (e.g.Cystorova) • Referthepreparedtablefortheobjectivebeingusedtoobtainthe measurement of size.
  34. 34. Sterilisation is defined as the destruction or removal of all microorganisms and their spores. Disinfection is the destruction of many microorganisms but not usually the bacterial spores. Sterilization is usually achieved with the help of heat whereas chemical agents are employed to effect disinfection. Sterilisation and disinfection are part of the daily routine of microbiological laboratories and constitute a vital activity which ensures that cultures, containers, media and equipment are treated in such a way that only the inoculated organisms will grow while all others will be eliminated. Sterilisation by Heat This can be achieved by autoclaving, by exposing articles to dry heat in hot air ovens or boiling. Autoclave Autoclaves can sterilise anything that can withstand a temperature of 121o Cfor30minutes.Apressurecookerusedinhomesforcooking purposes can also be used as a makeshift autoclave (Fig. 7.1). Sterilisation 7 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
  35. 35. 28 Practical Manual of Medical Microbiology The containers having clinical material are subjected to heat treatment in the autoclave after which these are emptied and washed and put back into service. Only autoclaves designed for laboratory work and capable of dealing with a mixed load should be used. Porous load and bottle fluid sterilisers are rarely satisfactory for laboratory work. There are two varieties of laboratory autoclaves. 1. Pressure cooker type 2. Gravity displacement models with automatic air and condensate discharge. Pressure-cooker type Laboratory Autoclaves The most common type is a device for boiling water under pressure. It has a vertical metal chamber with a strong metal lid which can be fastened down and sealed with a rubber gasket. An air and steam discharge tap, pressure gauge and a safety valve are fitted in the lid. Waterinthebottomoftheautoclaveisheatedbyexternalgasburners, an electric immersion heater or a steam coil (Fig. 7.2). Figure 7.1: Steam circulation in a double jacketed autoclave
  36. 36. Sterilisation 29 Operating Instructions • Ensure that there is sufficient water inside the chamber. • Load the autoclave and fasten the lid keeping the discharge tap open. • Adjust the safety valve to the required temperature and turn the heat on. • Allow the mixture of air and steam to pass out freely till all air has been discharged. • Close the air discharge tap and let the steam pressure rise within the chamber till it attains a temperature of 121o C (1.5 kg/cm2 ). • Hold on the pressure for 15 minutes. • Turn off the heat and let the autoclave cool. • Slowly open the air and steam discharge taps after the pressure gauge has reached zero. Figure 7.2: Vertical autoclave
  37. 37. 30 Practical Manual of Medical Microbiology • Allow the material to cool before these are handled (usually agar bottles take hours before these become safe to handle). Autoclave with air discharge by gravity displacement These are usually rectangular in shape and arranged horizontally. These autoclaves have a jacket around the chamber (Figs 7.1 and 7.3). Figure 7.3: Horizontal autoclave Figure 7.4: Candle filter
  38. 38. Sterilisation 31 Operating Instructions • Bring the jacket of the autoclave to operating temperature. • Load the chamber, close the door and open the steam valve so that steam can freely enter the top of the chamber. Air and condensate shall automatically flow out through the drain at the bottom (Fig. 7.4). • When the drain thermometer reaches the required temperature, allow further period for the load to reach that temperature (this has to be determined initially and periodically for each autoclave). • Continue the autoclave cycle for the holding period. • Close the steam valve and let the autoclave cool till a temperature of 80o C is reached. • Gradually and softly open the autoclave enabling the steam to escape and allow the load to cool further. Hot Air Oven A hot air oven is electrically operated and should be equipped with afantoensureuniformtemperatureinside.Therequiredtemperature for sterilization is generally 160o C for one hour (Fig. 7.5). Figure 7.5: Hot air oven
  39. 39. 32 Practical Manual of Medical Microbiology Operating Instructions • Arrange the material to be sterilized loosely and evenly on the racks of the oven allowing free circulation of air and thereby even heating of the load. • Do not pack the load tightly since air is a poor conductor of heat. • Switch on the power supply and control the temperature of the oven by adjusting the thermostat. • Note the time when the desired temperature is reached (heating- up time). • Hold the load in the oven at this temperature for a definite period of time (holding period). This is usually 60 minutes at 160o C. • Do not overheat since it would char the cotton plugs and paper wrappings. Autoclaves and hot air ovens can be used for disinfection of infectious waste before it is discarded. In addition, waste can be disposed of by boiling in detergent or by burial. Boiling In the absence of an autoclave, most specimen containers can be boiled in water having detergents to decontaminate. This process kills the vegetative bacteria but fails to destroy the spores and certain viruses. The easiest way to get best results is to add washing powder or sodium carbonate crystals, 60 grams to one litre of water in a big container and boil specimen containers in it for a minimum of 30 minutes. Disinfection Disinfection can be undertaken either chemically or by boiling. Boiling is an effective method to disinfect equipment, e.g. needles and syringes, if autoclaving facilities are not available. Equipment which has already been cleaned should be boiled for 20 minutes. Chemical disinfection is used for heat-sensitive equipment that is damaged at high temperatures. Commonly-used chemical disinfec- tants include chlorine releasing compounds; ethyl and isopropyl alcohol, quaternary ammonium compounds and gluteraldehyde.
  40. 40. Sterilisation 33 The synopsis of a few commonly-used disinfectants is given in Table 7.1. Preferred methods of sterilization for common articles are given in Table 7.2. Decontamination of some of the commonly reusable equipment has been briefly presented in Table 7.3. Table 7.1: Disinfectants and their mode of application* Target Disinfectant Strength to use Application Time of exposure (disinfectant/ material V/V) Skin Ethanol 70% Direct 2 minutes Iodine 1% Direct 2 minutes Povidone 1% Direct 2 minutes iodine Quaternary Direct 2 minutes ammonium comp Blood Cresol (pH 9) 5% 2:1 6 hours Ca hypochlorite 1% 2:1 6 hours Urine Cresol (pH 9) 5% 1:1 4 hours Sputum Cresol (pH 9) 5% 1:1 4 hours Faeces Cresol (pH 9) 5% 2:1 6 hours Hypochlorite 1% 3:1 6 hours (Na/Ca) Ca hydroxide 20% 2:1 6 hours Work Lysol 5% Direct 4 hours benches Cresol 1% Direct 4 hours Hypochlorite 5% Direct 4 hours Chloramine-T Direct 4 hours Glassware Hypochlorite 1% Direct 4 hours Lab Hypochlorite 0.1% Direct 4 hours instruments Isopropanol 70% Direct 4 hours * Based upon: Basics of quality assurance: WHO/EMRO, 1992, page 162 Biohazard Waste Management Waste is defined as any solid, liquid or gaseous material that is no longer used and will either be recycled, disposed of or stored in anticipation of treatment and/or disposal.
  41. 41. 34 Practical Manual of Medical Microbiology Table 7.2: Preferred methods of sterilization for common-use articles Autoclaving Hot air oven Animal cages Glass ware Sugar tubes Beakers Lab. coats Beakers Cotton Petridish Filters Pipette Instruments Slides Culture media Glass syringes Test tubes Powders Rubber Wood Gloves, stopper, tubing Tongue depressor, applicator Glass Slides, syringes, test tubes Enamel metal trays Wire baskets Table 7.3: Disinfection of specific equipment Container/material Method of choice Alternative method of for decontamination decontamination Reusable stool Autoclaving 121o C Fill the jar having stool with container for 30 minutes 5% solution of phenol and keep for 24hours Empty into lavatory* Empty into lavatory* Reusable containers Autoclaving Boiling in detergent of CSF, pus, sputum Urine bottles (after Autoclaving Fill with 2% phenol or emptying in lavatory*) 1% bleach, leave for 4 hours, clean with detergent Blood containers Autoclaving Soak overnight in strong disinfectant (5% cresol; 1% Ca hypochlorite, 1:2 V/V) Glass microscope Autoclaving Soak overnight in 5% phenol slides** * If the lavatory is connected to a septic tank, phenol or other antiseptics should not be put into the lavatory. ** Glass microscope slides which have been used for the diagnosis of tuberculosis should be discarded after keeping them soaked in detergent overnight.
  42. 42. Sterilisation 35 Storage Prior to disposal, all biohazardous waste should be maintained and stored separately from the general waste stream and from other hazardous wastes. The containers used to store biohazardous waste should be leak-proof, clearly labelled with a red or orange universal biohazard symbol and sealed tightly when transported. In certain cases,itmaybenecessarytodouble-bagthewastetopreventleakage. Any biohazardous sharps, such as infectious needles and scalpels, must be placed in containers that are puncture-resistant, leak-proof on all sides and the bottom, and close-able. These containers can then be placed in a standard biohazard bag. Disposal Options There are three main disposal options: Render the waste noninfectious by autoclaving and dispose it in the general waste stream. If autoclaving is not possible, deconta- minatewithchemicaldisinfectantsorbyboilingfor20minutesbefore disposal. On-site incineration, if possible. Transportation of locally-generated waste to a distant appropriate facility. Incineration is the preferred disposal option. Not only does this method render the waste noninfectious but it also changes the form and shape of the waste. Sterilization is an effective method for decontaminating waste, but it does not alter the appearance of the waste. Steam sterilization in an autoclave at a temperature of 121o C for at least 15 minutes destroys all forms of microbial life, including high numbers of bacterial spores. This type of complete sterilization can also be accomplished using dry heat which requires a temperature of 160-170o C for 2-4 hours. However, it must be ensured that heat comes in contact with the material to be rendered sterile. Therefore, bottles containing liquid material should have loosened caps or cotton plug caps to allow for steam and heat exchange within the bottle. Biohazard bags containing waste should be tied loosely. Once sterilized, biohazardous waste should be sealed in appropriate
  43. 43. 36 Practical Manual of Medical Microbiology containers, labelled as disinfected waste and disposed of in an approved facility. Biological waste should be clearly labelled prior to disposal and complete records should be maintained. Burial It is not a recommended decontaminating process. However, it does preventtheinfectiousmaterialfrombecomingareservoirofinfection if properly buried. It requires digging a pit of almost 5 meters depth and 2 meters width and having a tightly fitted heavy lid on top. Disposable containers with clinical material are thrown daily into it and the lid is replaced immediately after throwing the specimens. Once a week, the refuse is covered with a layer of quicklime. If quicklime is not available, the refuse is covered with almost 10 cm thick layer of dried leaves once a week.
  44. 44. INTRODUCTION This experiment is mainly used to study the motility of bacteria. It is also useful to study the shape, relative size and arrangement of bacteria, protozoan parasites, sperm motility and stool examination. The term hanging drop is self explanatory, after the preparation the drop hangs from the coverslip. The hanging drop provides enough space for the movement of Bacteria Unlike wet mount where a thin film is available for movement. Requirements 1. Young broth culture 2. Clean cavity slide (depression slide). cavity slide is slide with central depression or concavity (Figs 8.1 and 8.2). 3. Clean cover slip 4. Vaseline 5. Spirit lamp 6. Wire loop 7. Compound microscope. Hanging Drop Preparation 8 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
  45. 45. 38 Practical Manual of Medical Microbiology Procedure (Fig. 8.3) • Take a clean cover slip and apply a little of Vaseline to the corners. • Place a drop of broth culture on the centre of the cover slip using a sterile wire loop. • Invert a clean cavity slide gently over the cover slip, the concavity facing the drop (i.e. downwards) See that the drop does not touch the slide. • Quickly and carefully, turn the slide over. So that the coverslip is upper most and the drop is suspending from the coverslip. • Lower the substage condenser to reduce the light.  Partially close the iris diaphragm if necessary. Figure 8.1: Cavity slides Figure 8.2: Cavity slide with hanging drop
  46. 46. Hanging Drop Preparation 39 • Focus the drop under low power objective so that the edge of the drop is exactly in the centre of the microscopic field. • Turn to high power objective and focus the edge of the drop. • Study the motility, shape, relative size and arrangement of the organisms. Observation (Fig. 8.4) Motile and non motile organisms may be observed. Figure 8.3: Hanging drop preparation Figure 8.4: Edge of the drop with motile bacilli Notes The edge of the drop is focussed because of the following reasons. 1. The contrast or optical aberration due to the different refractive indices (Water and glass) is less at the edge of the drop. 2. The organisms move in a horizontal direction at the edge of the drop, contrary to the vertical movement seen at the centre of the drop.
  47. 47. 40 Practical Manual of Medical Microbiology 3. Oxygen tension is more towards the edge of the drop and hence aerobes tend to accumulate at the edge of the drop. Bacterial Motility Bacteria exhibits true motility and false motility. When a bacterium changes its position and moves in different directions, we can consider it as true motivity. False mobility is mainly due to Brownian movement, i.e. the organisms exhibit an oscillatory movement with- out change of position, due to the bombardment of water molecules True motility is different types. 1. Darting (missile like), e.g. Vibrio, Pseudomonas 2. Tumbling motility, e.g. Salmonella, Esch.coli. Examples of Motile Bacilli • Salmonella • Vibrio cholerae • Pseudomonas. • Proteus. Examples of Non-motile Bacilli • Klebsiella • Shigella • Clostridium welchii • Haemophilus influenzae. Motility of Anaerobic Bacteria Motility of anaerobic bacteria can be checked by inoculating them into a semisolid medium. Motile anaerobes are seen to spread at the deep layers of the medium where oxygen content is less or neglibile. Another microscopic method used for anaerobic motility observation is closed Capillary tube method. Liquid growth from a liquid anaerobic culture medium (Thioglycollate broth) is taken in a Capillary tube, close the ends of cappillary tube by heating, observe the capillary tube under microscope.
  48. 48. A proper fixed smear is essential for many staining procedures like Gram’s staining and Acid fast staining. A good smear will provide reliable information if they are stained systematically, labelled properly, spread evenly and fixed with care. Note • It is recommended to prepare only one smear on a single slide. • Slides with positive AFB Smears should always be discarded and never reused. • Precaution should be taken when handling infectious material. • The Smears should be spread evenly covering an area of about 15-20 mm diameter on a slide. Requirements 1. Clean, clear, grease free glass slide 2. Wire loop 3. Burner or spirit lamp (Fig. 9.1) 4. Sterile normal saline or distilled water 5. Glass marking pencil 6. 70% methanol or Ethanol (for Alcohol fixation of M. tuberculosis) 7. Absolute alcohol (for alcohol fixation other Bacteria) Preparation and Fixation of Smears 9 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
  49. 49. 42 Practical Manual of Medical Microbiology Procedure Step 1—Making of smears Step 2—Drying of smears Step 3—Fixation of smears Step 4—Labelling of slides Making of Smears Smears can be made out of various clinical specimens and bacterial cultures. 1. Bacterial cultures: Smear can be prepared by the following procedure. • Sterilise the wire loop by showing it in the flame, make it red hot and allow to cool. • Place one or two drops sterile saline on a clean slide. • Using the sterile wire loop pick up a small portion of the isolated Bacterial colony. Figure 9.1: Spirit lamp
  50. 50. • Emulsify the colony in the saline and spread evenly on the slide in a circular manner. 2. Pus: Spread the purulent material thinly using a sterile wire loop. The flame sterilized loop must be allowed to cool before it is used. 3. Sputum: Use a piece of clean stick to transfer and spread purulent and caseous material on a slide. Dip the stick in a disinfectant solution before discarding. 4. Swab: Roll the swab on a slide. Rolling the swab prevents damage to the pus cells. 5. Faeces: Use a piece of clean stick to transfer pus and mucus to a slide. Decontaminate the stick before discarding. 6. CSF: Make on evenly spread smear of a drop of purulent CSF or the sediment from a centrifuged sample on a slide using a sterile wire loop. (AFB is difficult to detect in CSF. The chances of finding Bacteria are increased if the CSF it centrifuged for 20-30 min and several drops of sediment are used). Drying of Smears After making smears, the slides should be kept in a safe place to air dry. Protect the slide from flies and dust. If the smears can not be stained immediately, they should be Fixed and stored in a covered box. Fixation of Smears Thepurposeoffixationistopreservemicroorganisms,andtoprevent smears being washed from slides during staining. Smears are fixed by heat, alcohol or by other chemicals. Heat fixation: The air dried smear is fixed by passing the slide (Smeared slide up) rapidly over the burner by holding the slide with the thumb and index finger (Don’t over heat). The slide is them allowed to cool (Fig. 9.2). The aim of this fixing is to coagulate the aluminous material whereby the film adheres better to the glass slide and thus may not be detached in the subsequent staining process. Heat fixation is commonly used for Gram’s staining. Preparation and Fixation of Smears 43
  51. 51. 44 Practical Manual of Medical Microbiology Note: Afterpassingtheslidethroughtheflamethreetimes,itshould bepossibletotouchtheslideonthebackofyourhandwithout the hand feeling uncomfortably hot. The disadvantages of heat fixation are : 1. It damages leucocytes and 2. M. tuberculosis is not killed by this method. Alcohol fixation: This fixation is more bactericidal than heat fixation, it kills M. tuberculosis. Another advantage of alcohol fixation is, it doesnotproducedamagetothepuscells,andtheyarewellpreserved. Alcohol fixation is recommended for fixing smears containing instracellular GonococcusandMeningococcus. methodofalcoholfixing smear is as follows. 1. Allow the smear to air-dry completely. 2. Keep the slide on a staining rack. 3. Add one or two drops of alcohol on the smear. 4. Leave the alcohol on the smear for 2 minutes to dry the alcohol on the smear. Example other chemical fixations: • 40 gm/l potassium permagnate is recommended for fixing smears containing Anthrax bacilli. • Formaldehyde Vapour. For smear containing Mycobacterium. Labeling of Slides Using a grease pencil mark the area of smear. This will be useful while focusing under microscope. Label the slide with date, name and numbers. Figure 9.2: Heat fixed smear
  52. 52. INTRODUCTION This is a simple staining procedure, both background and object will stain blue. Requirements Methylene blue 0.3 gm Distilled water 100 ml Dissolve the dye in water. Filter through a filter paper. Staining Method Make a smear on a glass slide, dry in air and fix by passing it over the flame of a burner 3-4 times. Stain for one minute by pouring methylene blue solution over the smear. Wash with water, blot dry and examine under the oil immersion of light microscope (Fig. 10.1). Methylene Blue Staining 10 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
  53. 53. 46 Practical Manual of Medical Microbiology Uses The stain is used to make out clearly the morphology of the organisms, e.g. Yersinia pestis in exudate, Haemophilus influenzae in CSF and Gonococci in urethral pus. Figure 10.1: Methylene blue staining of yeast cells
  54. 54. INTRODUCTION This is a negative staining, useful for demonstration of capsule of bacteria and yeasts. The capsule will not take up the stain and the remaining areas of the field will be darkly stained. Staining Procedure • Place a loopful of India ink on the side of a clean slide. • A small portion of the solid culture is suspended in saline on the slide near the ink and then emulsified in the drop of ink, or else, mix a loopful of liquid culture of specimens like CSF with the ink. • Place a clean cover slip over the preparation avoiding air bubbles. • Press down, or blot gently with a filter paper strip to get a thin, even film. • Examine under dry objectives followed by oil immersion. Observation To demonstrate the capsule which is seen as an unstained halo around the organisms distributed in a black background. This is Indian Ink Staining 11 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
  55. 55. 48 Practical Manual of Medical Microbiology employed for diagnosis especially for Cryptococcus neoformans and Pneumococcus (Fig. 11.1). Figure 11.1: Indian ink preparation of Pneumococcus
  56. 56. INTRODUCTION In 1884 Hans Christian Gram described this method of staining, which is the most important stain in routine bacteriology. It is differentialstainusedfortheidentificationofBacteria. Requirement a. Crystalviolet Composition: Crystal violet and distilledwater. b. Gram’s iodine Act as mordant. Composition – Iodine, potassium iodideanddistilledwater c. Absolute alcohol/acetone Decolouriser. d. Dilute carbol Fuchsin Counterstain. Carbolfuchsinanddistilledwater. Procedure (Fig. 12.1) 1. Preparationoffilm–Athinuniformsmearisabsolutelyessential. 2. Fixation of the smear – The smear is dried in air and fixed by passingtheslide(Smearedslideup)rapidlyoverthespiritlamp Gram's Stain 12 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
  57. 57. 50 Practical Manual of Medical Microbiology or Bunsen burner by holding the slide with the thumb and index finger. (DON’T OVER HEAT). The slide is then allowed to cool. Theobjectofthisfixingistocoagulatethealbuminousmaterial whereby the film adheres better to the glass and thus may not be detached in the subsequent processes. A film should never be heated over not be detached in the subsequent processes. A film shouldneverbeheatedovertheflameuntilitisthoroughlydried up. 3. Cover the smear with crystal violet solution and allow to remain ontheslidefor1minute. 4. Discardthecyrstalvioletstainandholdtheslideatasteepand wash off the residual stain with an excess of iodine solution. Cover the smear with fresh iodine solution and leave it for 1 minute. 5. Rinse the smear with absolute alcohol and continue application until no more colour appears to flow from the preparation (about 15-20 seconds).if acetone is used for decolorisation, wash immediately. 6. Wash with water. 7. Coverthesmearwithdilutecarbolfuchsinandallowittoremain for1minute. 8. Rinse with water and dry in air or between blotting paper. 9. Then examine under oil immersion. Figure 12.1: Gram’s stained smear with GPC, GPB, GNC, and GNB (For colour version, see Plate 1)
  58. 58. Observation (Fig. 12.2) • Grampositiveorganismstainsviolet. • Gram negative organism stains pink. COCCI Grampositivecocci -Staphylococcusarrangedinclusters -Sterptococcusinchains. -Pneumococciislanceolate,diplococciand Capsulated. Gramnegativecocci -Neisseria-beanshapeddiplococci. BACILLI Grampositivebacilli: Corynebacterium diphtheriae, Clostridium spp. Gramnegativebacilli: Salmonella,Shigella,Proteusand Pseudomonas. Comments Gramstaindividesthebacteriaintotwocatergories,dependingupon whether they can be decolorised with alcohol after staining with Figure 12.2: Gram’s staining steps (For colour version, see Plate 1) Gram's Stain 51
  59. 59. 52 Practical Manual of Medical Microbiology crystalvioletandiodine.Thosethatresistdecolourisationremain violetincolourandaredesignatedasGrampositive,andthosethat aredecolourisedandtakeupthecounterstainsuchasdilutecarbol fuchsin appear pink and are termed as Gram negative. The suggested reasonsforthisdifferentiationintotwocategoriesare: 1. Gram positive organisms have a more acidic protoplasm (pH 2.0) than the Gram negative organisms (pH 5.0), there by having greateraffinityforbasicdyes. 2. Cellwalltheory—Afterstainingwithcrystalvioletandiodine,a dye iodine complex is formed within the cell. This complex is insoluble in water but moderately soluble and dissolvable in alcohol.Undertheactionofdecoloriserthedyeandiodinediffuse freelyoutinGramnegativecellbutnotfromGrampositivecell, presumably because the latter surface is less permiable to the decoloriseroritsiodinesolute. 3. Cyto plasmic theory—Gram positivity depends upon the integrity of cellular structure and the presence in the cell of a specific magnesium ribonucleate complex. Thus Gram positive bacteria becomeGramnegativeiftheyarerupturedmechanicallyorifthis ribonucleateisremoved.
  60. 60. Mycobacteria have the power of retaining certain stain even when they are decolorised by mineral acids. This is known as “Acid fastness”. This acid fastness appears to be due of presence of lipid (Mycolic acid) and integrity of the cell wall. The improvement of acid fast staining was done by Ziehl and Neelsen. It is a differential stain used to differentiate bacteria into acid fast and non-acid fast. Requirements a. Strong carbol fuchsin-composition: Basic fuchsin, absolute alcohol, phenol and water. b. Sulphuric acid 20% solution. c. Absolute alcohol (Used only if the genitourinary specimens are processed) d. Methylene blue-composition: Methylene blue in alcohol and potassium hydroxide and water. Procedure 1. Preparation of smear: A portion of the mucopurulent material is taken on a slide and a smear is made and dried. Ziehl-Neelsen’s Stain (Acid Fast Stain) 13 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
  61. 61. 54 Practical Manual of Medical Microbiology 2. “Fixation of the smear”-fix the smear by gently passing the slide over the flame. 3. Flood the slide with strong carbol fuchsin and heat until steam rises. Allow the preparation to stain for 5 to 7 minutes. Heat being applied at intervals until steam rises and stops it and continue in this way for 5-7 minutes (never heat too much to produce boiling and charring. The stain must not be allowed to evaporate and dry on the slide, if necessary, pour more carbol fuchsin on the slide. 4. Wash in running tap water. 5. Decolourise the smear with sulphuric acid (20%) by dipping the slide in the acid solution in a wide mouth jar. Decolourisation should be continued till the film becomes faintly pink. Decolourisation must be done in stages and generally requires about 10 minutes. 6. Wash in running tap water. 7. Counter stain the smear with methylene blue for 1-2 minutes. 8. Wash in running tap water. 9. Blot dry and examine with the oil immersion objective. Observation (Fig. 13.1) • Acid fast bacilli – Pink • Tissue and other organisms – Blue • Mycobacteria are acid fast bacilli and most of the other bacteria are non-acid fast. • Nocardia is another bacterium which shows acid fastness. Notes Acidfastbacteriaareverydifficulttostain.Henceheatingisnecessary while staining which act as a mordant and makes the tough lipid cell wall of the bacterium permeable to the dye. In cases of genitourinary specimens the absolute alcohol or 3% acid alcohol can be used as decolouriser. In case of suspected lepra bacilli, only 5% sulphuric acid is to be used as decolouriser because it is less acid fast. Most important acid fast bacteria of medical importance are Tubercle bacilli (Human, bovine, atypical) lepra bacilli and smegma bacilli, M. smegmatis is not pathogenic to human beings. Tubercle
  62. 62. Ziehl-Neelsen’s Stain (Acid Fast Stain) 55 bacilli are both acid and alcohol fast, Smegma bacilli is acid fast but not alcohol fast. Lepra bacilli are acid fast to a lesser degree and alcohol fast. Some of the suspected tuberculosis cases may fail to show acid fast bacteria by ordinary Z- N stain methods. In such cases other methods like concentration techniques and direct flourescent staining may be helpful. In concentration technique the specimen is digested suitably with alkali at neutral pH and the digested material is centrifuged. The smear made from the deposit are stained by Z-N-stain and observed. Figure 13.1: Z-N stained sputum smear with AFB (pink) (For colour version, see Plate 2)
  63. 63. INTRODUCTION Some bacteria possess granules in the cytoplasm these granules are known by different names like metachromatic granules or volutin granules or Babes-Ernst granules or polar bodies. Demonstration of thesegranulesisusefulinidentificationoftheBacterialSpecies. TheseGranulesaremadeupofglycogen,Starch,lipidsandpoly- metaphosphates. The granules consisting of polymetaphosphate are called voluntin granules or Babes-Ernst granules. Babes-Ernst granules are predominant in Corynebacterium diphtheriae. Fordemonstrationofthevolutingranulesthreespecialstaining methodsareavailabletheyareAlbertsstaining,Neisser’sstaining and Ponder’s staining. Albert’sstainingisthecommonstainingprocedureintheroutine microbiology laboratories. This staining method is useful for the identificationofCorynebacteriumdiphtheriae. Thegranulestakeupadifferentstainingcolourfromthatofthe stain itself and hence the name metachromatic granules. Usually each bacillus contains 2 to 6 such granules, mainly in the poles of the bacilli. These are reserve food materials and appear more prominent during starvation. Albert’sStain 14 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
  64. 64. Albert's Stain 57 Requirement 1. Albert’sStain Toludine blue - 1.5 gm Malachite green - 2 gm Glacialaceticacid - 10 ml Ethyl alcohol (95%) - 20 ml Distilledwater - 1litre Dissolve the dyes in the alcohol and add to the water and acetic acid.Allowtostandforonedayandthenfilter. 2. Albert’sIodine Iodine - 6 gm Potassium Iodide - 9 gm Distilledwater - 900 ml Method • Prepareasmear,allowtodryand fixwithgentleheat. • Cover the smear with Albert’s stain and allow to act for 3-5 minutes. • Discard Albert’s stain and hold the slide at a steep slope and wash off the residual stains with an excess of Albert’s Iodine SolutionCovertheSmearwithfreshAlbert’siodinesolutionand allowtoactfor1minute. • Wash with water, blot dry. • Observe under oil immersion objective. Observation (Fig. 14.1) • Granules bluish black, the protoplasm green • Granules mainly seen in the poles. • Other organisms usually pale green. • ThediptheriabacillishowtypicalarrangementofletterslikeL,V,N orlikeChineseletters.
  65. 65. 58 Practical Manual of Medical Microbiology Figure 14.1: Albert stained smear of corynebacterium (For colour version, see Plate 2)
  66. 66. INTRODUCTION This stain is a modification of Romanowsky stain. It is very useful forthedemonstrationofprotozoainbloodfilmslikeMalariaparasite. Requirements 1. Leishman staining solution 2. Distilledwaterorbuffersolution Leishman’s Stain is prepared by taking 0.15 gm of Leishman’s powder and dissolving the powder in 100 ml methanol. Distilled water should be neither acid nor alkaline. Any slight variations from neutrality may alter considerably the colour of granules in white blood corpuscles and give rise to supposed pathologicalappearancesincellswhicharereallynormal.Thiscan beovercomebyusingabuffersolution. Method • Make thin film of blood smear. • Pourtheundilutedstainontounfixedfilmandallowittoactfor 1min. Leishman’s Stain 15 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
  67. 67. 60 Practical Manual of Medical Microbiology • Using a pipette add double the volume of distilled water/buffer to the slide mixing the fluids by alternately sucking them up in the pipette and expelling them. Allow the diluted stain to react for12min. • Floodtheslidegentlywithdistilledwater. • Remove the excess water and blot dry. Note Methylalcoholpresentinthestainactsasafixativeandfixesthe filmtotheslide. Figure 15.1: Leishman stained blood smear (For colour version, see Plate 3)
  68. 68. Leishman’s Stain 61 Observation (Fig. 15.1) RBC : Red W B C : Blue Malarialparasite : Inside RBC with blue cytoplasm and ruby red nucleus.
  69. 69. NEW GLASSWARES New glass wares may contain resistant spores which may be present inpackingmaterialandnewglasswarestendtogiveofffreealkali which may be sufficient to interfere with the growth in certain organisms. To overcome these problems the following cleaning methodsshouldbefollowed(Figs16.1to16.4). • Place in 1% HCl overnight • Wash with tap water • Wash in distilled or deionised water • Autoclave. Used Glasswares • Reusable glass ware should be autoclaved before cleaning. • The discarded cultures and their containers are then placed in a hotdetergentsolutioncleanwithasuitablebrush. • Rinse with deionized water • Drain and dried in a hot air oven • Drysteriliseat160o Cfor3hours. Preparation and Cleaning of Glassware 16 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
  70. 70. Figure 16.1: Laboratory wares (plastic/glass) Preparation and Cleaning of Glassware 63
  71. 71. 64 Practical Manual of Medical Microbiology Figure 16.2: Laboratory wares (plastic/glass) Chromic Acid Cleaning This cleaning of glassware is mainly for biochemical work. • Remove any grease with petroleum. Wash with warm tap water. • Place in dichromate Sulphuric acid cleaning solution for 12-24 hours. • Remove and wash by density in hot tap water at least four times andindistilledwatertwice.
  72. 72. Figure 16.3: Preparation of glass Pasteur pipette Figure 16.4: Plastic Pasteur pipettes Preparation and Cleaning of Glassware 65
  73. 73. 66 Practical Manual of Medical Microbiology • DryinoveniftheglasswareisnotusedforaccurateVolumetric purposes. Cleaning of Pipettes • If contaminated with infected material, place the used pipette intodisinfectantsolutionandleaveuntilconvenienttowash. • Rinseintapwater. • Ifnecessary,keepovernightindetergentordichromate–Sulphuric cleaningfluid. • Wash with tap water followed by deionized water. • The top end of the pipette is plugged with cotton-wool. Press it entirelywithintheendofthepipettesothattherearenoprotruding strandsofcottontopreventclosefittingofarubberteatorother pipettefillingdevicewhichmaylaterbeattachedtooperatethe pipette. • To sterlise the pipettes, Pack them in aluminium or copper cylinders.Placeinahotairovenat160o Cfor3hours. Note: Accurately caliberated volumetric glassware should never be heated in an oven, since the expansion and contraction of the glass makes the graduations inaccurate.
  74. 74. Microorganisms are sensitive to the varying pH of the external environment. Where there is an optimum pH for the growth and multiplication the microorganisms will survive and flourish. So when micro organisms are cultivated in the laboratory it should be noted that the medium should have an optimum pH. Media should be adjusted as far as possible to the pH optimal for the growth of the organism concerned. Most pathogenic bacteria have a fairly restricted PH range and grow best around pH 7.3, i.e. at a slightly alkaline reaction. Methods Used for Measurement of pH a. The pH meter: The accurate method of measuring pH is with a pH meter. It is easy and quick to use. Care must be taken in its maintenance. b. pH indicator dyes: Indicator dyes are substances that will change in colour with variations in the pHof the solution in which they are dissolved. Examples of indicative dyes and pH range are given in the Table 17.1. pH in Microbiology 17 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
  75. 75. 68 Practical Manual of Medical Microbiology Table 17.1: Indicative dyes and pH range Indicator Range of pH Colour change Thymol blue 1.2-2.8 Red to yellow Bromophenol blue 2.8-4.6 Yellow to violet Bromocresol green 3.6-5.2 Yellow to blue Methyl red 4.4-6.2 Red to yellow Litmus 4.5-8.3 Red to yellow Bromocresol purple 5.2-6.8 Yellow to violet Bromothymol blue 6.0-7.6 Yellow to blue Neutral red 6.8-8.0 Red to yellow Phenol red 6.8-8.4 Yellow to purple pink Cresol red 7.2- 88 Yellow to violet red Thymol blue 8.0-9.6 Yellow to blue Phenol phthalein 8.3-10.0 Colourless to red Thymol phthalein 9.3-10.5 Colourless to blue pH Indicator Papers The simplest method of determining the pH of a solution is use of commercially available pH indicator papers. These papers are impregnated with an indicator that gives a change of colour over a specific or general range of pH. The Paper can simply be dipped in the solution to be tested or a drop the solution can be withdrawn by a wire crop or pasteur pipette are placed on the paper. The resulting colour is compared with the chart supplied with the papers. Comparater and Capillator Methods These methods are not available now so not used.
  76. 76. The role of suitable quality culture media for cultivation of microorganisms cannot be over emphasized. The success of isolation of aetiological agents depends on the quality of the medium. Only in exceptional cases, can an organism be identified on the basis of its morphological characteristics alone. Types of Media Bacteriological media can be broadly sub-divided into four categories. 1. Ordinary Culture Media These are routinely employed in a laboratory, e.g. nutrient broth, nutrient agar, infusion broth and lysate media. 2. Enriched Media Certain organisms do not grow on ordinary nutrient media. They requiregrowth-promotingingredientssuchasblood,glucose,serum, egg, etc. The media containing ingredients which enhance their Bacteriological Media 18 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
  77. 77. 70 Practical Manual of Medical Microbiology growth-promoting qualities are enriched media, e.g. blood agar, chocolate agar and Loeffler medium. 3. Enrichment Media Enrichment media are liquid media containing chemical constituents which inhibit some normal flora and allow pathogens which may be present in very small number in the specimen, to grow unhampered and thus enriching them. Isolated colonies of these organisms may be obtained by subculturing onto solid media. An example of enrichment media is selenite broth used for primary isolation of enteric bacteria. 4. Differential and Selective Media Differential media have got some chemical constituents which characterize different bacteria by their special colonial appearances in the culture, e.g. MacConkey agar contains lactose as a substrate and neutral red as an indicator. Bacteria fermenting lactose produce acid and this will change the colour of the indicator and thus the colonies will turn red. The red lactose fermenting colonies can be differentiated from the pale non-lactose fermenting colonies. Selective media will selectively permit the growth of pathogens and inhibit the commensals. In addition, it may differentiate the pathogen from commensals that grow by the colour and opacity of the colonies, e.g. blood tellurite medium for C. diphtheriae. In addition, transport media are also frequently used to sustain the viability of organisms when a clinical specimen is to be transported from the periphery to laboratory. The transport medium prevents the outgrowth of contaminants during transit and sustains the pathogen. Cary and Blair and Stuart media are two examples of this group of media. Preparation of Media and Checking of pH Presently, a wide range of culture media are available commercially in the form of dehydrated media. These media are simply reconstituted by weighing the required quantities and by adding distilled water, as per the manufacturer’s instructions.
  78. 78. Bacteriological Media 71 The pH determination can be conveniently done with the use of pH indicator papers. Adjust the pH using NaOH (1 Normal) and HCl (1 Normal) Check the pH of the medium once again before use. The quantity of agar given in the formulae of media may have to be changed depending upon the quality of agar used. The concentration varies from batch to batch and should be such that will produce a sufficiently firm surface on solidification. This can be tested by streaking with inoculating wire. In some laboratories media are prepared by individual measurement of ingredients and then mixing the same (Figs 18.1 to 18.4). Nutrient Broth (Fig. 18.1) • Meat extract 10.0 gm • Peptone 10.0 gm • Sodium chloride 5.0 gm • Distilled water 1000 ml Mix the ingredients and dissolve them by heating in a steamer. When cool, adjust the pH to 7.5-7.6. Nutrient Agar (Fig. 18.2) To the ingredients as in nutrient broth, add 15 gm agar per litre. Dissolve the agar in nutrient broth and sterilize by autoclaving at 121o C for 15 minutes. Prepare plates and slopes as required. Glucose Broth • Nutrient broth 900 ml • Glucose (10% solution) 100 ml • Dissolve 9 gm glucose in distilled water and sterilize by tyndallisation. • Add l00 ml of the glucose solution to 900 ml of sterile nutrient broth. • Dispense 60 ml each in 100 ml pre-sterilized culture bottles. • Sterilize by open steaming at l00o C for one hour.
  79. 79. 72 Practical Manual of Medical Microbiology Figure 18.1: Nutrient broth (For colour version, see Plate 3) Figure 18.2: Nutrient agar with bacterial colonies (For colour version, see Plate 4)
  80. 80. Bacteriological Media 73 Figure 18.3: Blood agar (For colour version, see Plate 4) Figure 18.4: Colony morphology
  81. 81. 74 Practical Manual of Medical Microbiology Blood Agar (Fig. 18.3) Nutrient agar 100 ml Sheep blood (defibrinated) 10 ml Melt the sterile nutrient agar by steaming, cool to 45o C. Add required amount of sheep blood aseptically with constant shaking. Mix the blood with molten nutrient agar thoroughly but gently, avoiding froth formation. Immediately pour into petri dishes or test tubes and allow to set. Chocolate Agar The ingredients are essentially the same as in blood agar. Melt the sterile nutrient agar by steaming and cool to about 75o C. Add blood to the molten nutrient agar and allow to remain at 75o C after gently mixing till it is chocolate brown in colour. Pour in Petri dishes or test tubes for slopes as desired. XLD Agar Xylose 3.5 gm 1–lysine 5.0 gm Lactose 7.5 gm Sucrose 7.5 gm Sodium chloride 5.0 gm Yeast extract 3.0 gm Sodium desoxycholate 2.5 gm Sodium thiosulphate 6.8 gm Ferric ammonium citrate 0.8 gm Phenol red 0.08 gm Agar agar 15.0 gm Water 1000 ml Weigh the ingredients into a flask and add distilled water. Mix the contents well and steam it for 15 minutes (do not autoclave). Cool to 56o C and pour in plates.
  82. 82. Bacteriological Media 75 Buffered Glycerol Saline Glycerol 300 ml Sodium chloride 4.2 gm Disodium hydrogen phosphate 10.0 gm Na2 HPO4 Anhydrous 15.0 gm Phenol red aqueous solution 0.02 per cent 15.0 ml Water 700 ml Dissolve NaCl in water and add glycerol. Add disodium hydrogen phosphate to dissolve. Add phenol red and adjust pH to 8.4. Distribute 6 ml in universal containers (screw -capped bottles of 30 ml capacity). Autoclave at 115o C for 15 minutes. Loeffler Serum Medium Nutrient broth 100 ml Serum (sheep or horse or ox) 300 ml Glucose 1.0 gm Dissolve glucose in nutrient broth and sterilize at 121o C for 15 minutes. Add serum aseptically. Mix thoroughly but gently, avoiding froth formation. Distribute in sterile test tubes or quarter ounce screw-cap bottles. Inspissate the medium in a slanting position in a water inspissator at 82o C for two hours. In the absence of an inspissator, the medium may be coagulated by standing over the top of a steam sterilizer for 6-7 minutes. Blood Tellurite Agar Agar base Meat extract 5.0 gm Peptone 10.0 gm Sodium chloride 5.0 gm Agar 25.0 gm Water 1000 ml Dissolve the ingredients and adjust the pH to 7.6. Distribute in 100 ml quantities in a bottle and autoclave at 121o C for 15 minutes.
  83. 83. 76 Practical Manual of Medical Microbiology Glycerolated blood tellurite mixture Sterile defibrinated sheep blood 14 ml Sterile glycerol 6 ml Sterile potassium tellurite solution (1% in water) 4 ml Sterilise the glycerol in hot air oven at 160o C for 60 minutes and the tellurite solution by autoclaving at 115o C for 20 minutes. Mix the ingredients in a sterile flask, incubate for 1-2 hours. at 37o C, then refrigerate.Haemolysisiscompleteafter24hours.Themixturekeeps well in a refrigerator. One per cent solution of good quality tellurite is sufficient but 2% of some batches may be required. Preparation of complete medium Glycerolated blood tellurite mixture 24 ml Agar base 100 ml Melt the agar, cool to 45o C, add blood and tellurite and pour in sterile petri dishes.
  84. 84. When inoculating or seeding culture media an aseptic (sterile) technique must be used to: • To prevent contamination of cultures and specimen. • Topreventinfectionofthelaboratoryworkerandtheenvironment. Aseptic Technique (Fig. 19.1) • Flame sterilize wire loops, straight wires, and metal forceps before and after use whenever possible, use a hooded Bunsen burner (Fig. 19.2). Note: To prevent the release of aerosols, wire loops should be well made. Aerosols can also be released when spreading inoculation media containing air bubbles. • Flame the necks of specimen bottles, culture bottles, and tubes after removing and before replacing caps, bungs, or plugs. • When inoculating, do not let the tops or caps of bottles and tubes touch an unsterile surface. This can be avoided by holding the top or cap in the hand. • Always use racks to hold tubes and bottles containing specimens or culture media. Inoculation of Culture Media 19 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
  85. 85. 78 Practical Manual of Medical Microbiology Figure 19.1: Aseptic inoculation of culture medium Figure 19.2: Bunsen burner
  86. 86. Inoculation of Culture Media 79 • Make slide preparations from specimens after inoculating the culture media. • Decontaminate the work bench before starting the day’s work and after finishing. • Use a safety cabinet when working with hazardous pathogens. • Wear protective clothing, wash the hands after handling infected material, and never mouth-pipette, eat, drink, or smoke in the laboratory. Making a Wire Loop Loops must be made correctly to ensure inocula are well spread, and to prevent the release of aerosols from long and springy loops or loops that are not completely closed. The length of wire from the loop to the loop holder should be about 50 mm. The method of making a wire loop is as follows (Fig. 19.3): Figure 19.3: Preparation of wire loop
  87. 87. 80 Practical Manual of Medical Microbiology 1. Cut a piece of wire about 125 mm in length and thickness (swg) 26 or 27, Wind it around a loop holder. 2. Using a pair of scissors, cut off one arm of the wire leaving the loop and about 50 mm of wire. Bend the loop back to make it central using a pair of forceps. 3. Insert the wire in a loop holder. Make sure the loop is completely closed. Note: When sterilising a wire loop, hold it in the blue part of a Bunsen burner flame allow the loop to cool before using it. Inoculation of Media in Petri Dishes (Fig. 19.5) The technique used to inoculate media in petri dishes (plates) must provide single colonies for identification and to see whether a culture is pure or mixed, i.e. consisting of a single type of organism or several different organisms. A pathogen must be isolated in pure culture before it can be identified and antimicrobial sensitivity tested. The inoculation of media in petri dishes is referred to as ‘plating out’ or ‘looping out’. It is not necessary to use whole plates of media. Considerable savings can be made by using a half or even a third of a plate (especially if the medium is a selective one). The area of medium used must be sufficient to give separate colonies (Fig. 19.4). Before inoculating a plate of culture medium, the surface must be dried first otherwise single colonies will not be formed. Usually 30- 40 minutes incubation at 37o C is adequate. Figure 19.4: Incubator (For colour version, see Plate 5)
  88. 88. Inoculation of Culture Media 81 To inoculate a plate, apply the inoculum to a small area of the plate (‘the well’) using a sterile wire loop or swab of the specimen held with sterile forceps. Flame sterilize the loop. When cool (or using a second sterile loop), spread and thin out the inoculum. This will ensure single colony growth. Inoculation of Slopes To inoculate slopes such as Dorset egg medium or Loeffler serum, use a sterile straight wire to streak the inoculum down the centre of the slope and then spread the inoculum in a zig-zag pattern. To inoculate a slope and butt medium, such as Kigler iron agar, use a sterile straight wire to stab into the butt first and then use the same wire to streak the slope in a zig-zag pattern. Figure 19.5: Inoculation of media
  89. 89. 82 Practical Manual of Medical Microbiology Inoculation of Stab Media (deeps) Use a Sterile straight wire to inoculate a stab medium, for example mannitol motility medium. Stab through the centre of the medium taking care to withdraw the wire along the line of inoculum without making further stab lines. Inoculation of Fluid Media Broths and other fluid media are inoculated using a sterile wire loop, straight wire, or Pasteur pipette depending on whether the inoculum is colonial growth or a fluid culture or specimen. A straight wire is used to inoculate Koser’s citrate broth to prevent any carry overofmedium. If using a wire loop to subculture colonies, hold the bottle or tube at an angle and rub the loop against the side of the container below the level of the fluid. Labelling of Inoculated Media Using a grease pencil or marker pen, label inoculated media with the date and the patient’s number. Always label the base of a culture plate. A slope should be labelled on the underside of the media so that the wording does not obscure the culture. A stab culture should be labelled above the level of the agar. If a plate is to be incubated anaerobically it should be marked ‘An’ or if in a carbon dioxide atmosphere it should be marked ‘CO2’. Incubation of Cultures Inoculated media should be incubated as soon as possible. A delay in incubation can effect the viability of pathogens especially anae- robes, pneumococci, meningococci, gonococci, and Haemophilus influenzae. It can also increase the risk of plates becoming contaminated from small insects and dust especially in the dry season. Uninoculated and inoculated media must be protected from sunlight. Microorganisms require incubation at the temperature and in the humidity and gaseous atmosphere most suited to their
  90. 90. Inoculation of Culture Media 83 metabolism. The length of time of incubation depends on how long an organism takes to develop the cultural characteristics by which it is recognized (Fig. 19.5). Temperature of Incubation The temperature at which a microorganism grows best is referred to as its optimum temperature. The temperature below which growth stops (not necessarily resulting in death) is called the minimum temperature, and that above which growth stops and death occurs is called the maximum temperature. Note: The thermal death point of microorganism is the lowest temperature above the optimum at which death will occur in a given time. It is influenced among other factors by the culture medium, age of culture, and moisture. The temperature selected for routine culturing is 35-37o C with most microbiologists recommending 35o C in preference to 36o C or 37o C. In general, the growth of microorganisms is more affected by slight rises above their optimum temperature than by reductions- below it.
  91. 91. Depending on oxygen requirement, miroorganisms can be classified into4groups: 1. OBLIGATE AEROBES Thosebacteriawhichrequirefreeoxygenfortheirgrowth. Ex.: a. Alkaligenesfaecalis. b. Moraxella. c. Brucella 2. OBLIGATE ANAEROBES Those that can live and grow only in the absence of oxygen, i.e. under high reducing intensity and low redox potentials Ex.: a. Treponema denticola b. Clostridiumtetani c. Clostridium botulinum. 3. FACULTATIVE ANAEROBES Thesebacteriawhichcanliveandgrowinthepresenceaswellasin the absence of oxygen. Most of the bacteria come under this group. Anaerobic Cultivation 20 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
  92. 92. Anaerobic Cultivation 85 Ex.: a. Staphylococci. b. Esch.coli. 4. MICROAEROPHILIC ORGANISMS Thosethatgrowjustinthepresenceoftraceofoxygen. Ex.: a. Vibrio sputorum b. Campylobacter. SOURCE OF ANAEROBES Anaerobes occur in a variety of human body sites. They are usually present as normal commensals without causing any harmful effects. But under predisposing factors, i.e. whenever there is a low-redox potential, they can act as pathogens, thus producing endogenous infections.Duringinfection,theyareisolatedfromvariousspecimens like,Faeces,Blood,Pus,Exudates,etc. Methods of Creating Anaerobiosis I. Byusingcertaingases,like, a. 100% H2 b. 90% H2 + 10% CO2 c. 80% H2 + 10% N2 + 10% CO2 II. Additionofreducingagentstothemedia,like, a. Thioglycollate(Fig.20.1) b. 10%. Ascorbic acid. c. Meat particles as in Robertson’s cooked medium. d. Palladium Chloride. e. Cysteine,etc. III. Growing anaerobes deep in agar butts. IV. Simultaneous growth of aerobe and an anaerobe. V. Biological method by employing germinating seeds. VI. A Simple method, devised by Buchner employs a filter paper havingthesamediameterasapetridish.Thisfilterpaperis placedonthetopofonehalfofthepetridishandamixtureof pyrogallic acid, and Sodium hydroxide in dry powder form isspreadonit.Thentheotherhalfofthepetridishcontaining
  93. 93. 86 Practical Manual of Medical Microbiology themediawithinoculatedspecimenisinvertedoverthefilter paper and the edge is sealed tight with molten wax. The alkaline-pyrogallate mixture will absorb all the O2 present withintheclosedsystem,thuscreatingcompleteanaerobiosis. VII. UtilizationofAnaerobicJars(Fig.20.2). Ex.: 1. McIntosh and Filde’s Jar 2. Gaspak jar Figure 20.2: Candle jar (left) anaerobic jar (right) Figure 20.1: Thyoglycollate medium (For colour version, see Plate 5)
  94. 94. Anaerobic Cultivation 87 McIntosh and Filde's Anaerobic Jar This jar is used to provide complete anaerobic environment for the cultivationofanaerobicbacterialikeclostridia,etc.Itisastout metallicorglassjarwithametallid.Inoculatedcultureplatesare placedinsidethejarandthelidisclampedtight.Thelidhas2tubes: (1) Outlet tube which is connected to a vacuum pump to evacuate theairinside,(2)Gasinlet:whichisthenconnectedtoahydrogen supply. The lid also has 2 electric terminals, leading from which andsuspendedontheundersideofthelidisasmallporcilianspool, around which is wrapped a layer of palladinised asbestos. After fillingthejarwithH2,theelectricterminalsareconnectedtoacurrent supplytoheatthepalladinishedasbestos.Thisactsasacatalystfor the combination of H2 with residual O2 present in the jar to form water. An indicator, i.e. reduced Methylene blue is employed for verifying anaerobic condition in the jar, which remains colourless anaerobically, but turns blue on exposure to O2. The jar with the complete anaerobiosis is incubated at 37o C for 48 hours and growth of anaerobic organisms is noted.
  95. 95. BACTERIAL PATHOGENS AND RELATED DISEASES Organism Morphology Important diseases Gram Positive Cocci Staphylococcus Cocci in groups Boils, abscesses, secondary aureus infections bacteraemia, pneumonia, meningitis, conjunctivitis in newborns, food-poisoning Staphylococcus Cocci in groups Urinary tract infections saprophyticus Streptococcus Cocci in chains Sore throat, scarlet fever, pyogenes bacteraemia, otitis media, (Group A meningitis,cellulitis,puerperal Streptococcus) sepsis. Post-Streptococcal glomerulonephritis, and rheumatic fever leading to heart disease Contd... Important Bacterial Pathogens and the Diseases 21 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○
  96. 96. Important Bacterial Pathogens and the Diseases 89 Contd... Viridans Streptococci Cocci in chains Bacterial endocarditis, bacteraemia, tooth decay, abscesses. Streptococcus Capsulated Meningitis, lobar pneumonia, pneumoniae diplococci otitis media, pleurisity (Diplococcus pneumoniae) Streptococcus Cocci in chains Urinarytractinfections,wound faecalis and pairs and ulcer infections, (Group D septicaemia Streptococci) Streptococcus Cocci in chains Septicaemia, Pneumonia, agalactiae and pairs neonatal meningitis (Group B Streptococcus) Gram Positive Bacilli Bacillus anthracis Large, spore Anthrax forming, capsulated, bacilli which tend to form chains Corynebacterium Pleomorphic, Diphtheria of the throat and diphtheriae non-motile rods, skin often seen joined at angles Listeria Small rods with Meningitis, still-birth, monocytogenes tumbling motility bacteraemia at low temperatures Clostridium Non-motile Severe food-poisoning botulinum pleomorphic rods, with oval subterminal spores Contd...
  97. 97. 90 Practical Manual of Medical Microbiology Contd... Clostridium tetani Non-motile, long Tetanus (Drum-stick bacillus) thin rods with oval subterminal spores Clostridium Non-motile, thick, perfringens brick-shaped rods, (Clostridium welchii) Spores are rarely Gas gangrene, septicaemia, seen food poisoning Clostridium difficile Non-motile, thick, Antibiotic-associated brick-shaped rods, diarrhoea and colitis Spores are rarely seen Gram Negative Rods Haemophilus Small, non-motile Acute respiratory infections, influenzae coccobacilli or meningitis, cellulitis, rods, pleomorphic, ear infections Usually capsulated Haemophilus Small, non-motile Infectious conjunctivitis aegyptius rods or cocco (pink-eye), respiratory bacilli infections. Haemophilus Small, non-motile Soft chancre ducreyi rods often in pairs (Chancroid) or chains Bordetella pertussis Small, non-motile Whooping cough coccobacilli, may be capsulated Brucella species Small, non-motile Brucellosis coccobacilli may (undulant fever) show bipolar staining Yersinia pestis Non-motile, Bubonic, septicaemic capsulated and pneumonic cocco-bacilli, plague showing bipolar staining Contd...
  98. 98. Important Bacterial Pathogens and the Diseases 91 Contd... Yersinia Motile Acute mesenteric pseudotuberculosis pleomorphic lymphadenitis, septicaemia rods or cocco- bacilli showing bipolar staining Yersinia Motile Gastroenteritis, peritonitis, enterocolitica pleomorphic bacteraemia, abscesses rods or cocco bacilli showing bipolar staining Francisella tularensis Small, non-motile, Skin infections, capsulated, lymphadenitis, eye pleomorphic rods infections, lymphoid-like or coccobacilli, illness, and respiratory showing bipolar infections. staining Bacteroides species Non-motile small Bedsores, abscesses, pleomorphic rods abdominal and pelvic infections, bacteraemia Escherichia coli Motile rods Urinary infections, wound infections, bacteraemia, gastroenteritis Klebsiella Non-motile Chest infections, urinary pneumoniae capsulated rods infections, wound infections bacteraemia, meningitis, endocarditis Klebsiella rhino- Non-motile Rhinoscleroma of the upper scleromatis capsulated rods, respiratory tract with foam cells Proteus species Motile rods Urinary infection, respiratory infections, ear and wound infectinos, burns infections (often hospital-acquired), septicaemia Pseudomonas Motile rods Melioidosis pseudomallei showing bipolar (pneumoenteritis) staining Contd...
  99. 99. 92 Practical Manual of Medical Microbiology Contd... Shigella species Non-motile rods Bacillary dysentery Salmonella species Motile rods Enteric fever (typhoid and paratyphoid), food-poisoning, septicaemia, meningtis, bone infections, abscesses Vibrio cholerae Motile, slightly Cholera curved rods Vibrio parahae- Motile, slightly Gastroenteritis molyticus curved rods Campylobacter Motile, curved Gastroenteritis jejuni/coli spiral rods Legionella Non-motile rods Severe pneumonia pneumophila Gram Negative Cocci Neisseria Intracellular Meningitis and meningitidis diplococci septicaemia Neisseria Intracellular Gonorrhoea, eye infection in gonorrhoeae diplococci newborns Acid Fast Bacilli Mycobacterium Non-motile Tuberculosis tuberculosis bacilli Mycobacterium Non-motile Skin ulcers (Buruli ulcer) ulcerans bacilli Mycobacterium Non-moitle bacilli Leprosy leprae often in groups (globi) Spirochaetes and Other Organisms Treponema Motile delicate Syphilis pallidum1 treponemes Treponema Motile delicate Yaws pertenue1 treponemes Contd...

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