Ebook Practical manual of medical microbiology: Part 1

Part 1 book “Practical manual of medical microbiology” has contents: Laboratory safety, first aid, hand washing, units, microscope, micrometry, sterilisation, hanging drop preparation, preparation and fixation of smears, methylene blue staining, indian ink staining, gram’s stain,… and other contents.

Practical Manual of

Medical Microbiology

(For Medical, Dental and Paramedical Students)

Practical Manual of

Medical Microbiology (For Medical, Dental and Paramedical Students)

CP Prince

MSc (Medical Microbiology), PhD, FAGE

LecturerDepartment of MicrobiologyMother Theresa Institute of Health Sciences

(A Government of Puducherry Institution)

e-mail: jaypee@jaypeebrothers.com, Website: www.jaypeebrothers.com

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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

Practical Manual of Medical Microbiology is aimed to help the teachersand students to conduct practical/demonstration classes and tosolve the difficulty of maintaining the practical record book

A sincere effort is made to provide brief knowledge on theprinciples and procedures of common laboratory experiments Thefigures and photographs especially the line diagrams illustrated inthis book will be useful to perform various experiments and writethe observations and reports The list of spotters and identificationpoints given in the last chapter will help the students to excel intheir practical examinations

The content of the book covers the syllabus requirements of manyUniversities and other regulatory bodies like Medical, Dental andNursing Councils The WHO recommended test procedures andquality assurance programmes which are mentioned in this book.These procedures and programmes may be helpful to standardiseand streamline the experiments in newly established medicalinstitutions all over the world

This book is tailored to the need of students of MBBS, BDS, BSc(MLT), BSc (Microbiology), DMLT and other paramedical coursesand those who work in the field of microbiology needing short andconcise information

I am extremely grateful to Dr V Balu, Dean, Mother TheresaInstitute of Health Sciences (MTIHS), Dr V Gopal, Principal, College

of Pharmacy, MTIHS and Dr Helen PS Mannuel, former Professor, Madras Medical College for their constant encouragementand support I owe special thanks and gratitude to my colleaguesand family for their support and help

Director-Readers’ suggestions and comments will help for furtherimprovement of the book in future editions

CP Prince

CODE OF PROFESSIONAL CONDUCT FOR

MEDICAL LABORATORY PERSONNEL

1 Place the well-being and service of the sick above your owninterests

2 Be loyal to your medical laboratory profession by maintaininghigh standards of work and striving to improve yourprofessional skills and knowledge

3 Work scientifically and with complete honesty

4 Do not misuse your professional skills or knowledge forpersonal gain

5 Never take anything from your place of work that does notbelong to you

6 Do not disclose to a patient or any unauthorised person theresults of your investigations

7 Treat with strict confidentiality any personal information thatyou may learn about a patient

8 Respect and work in harmony with the other members ofyour hospital staff or health centre team

9 Be, at all times, courteous, patient, and considerate to the sickand their relatives

10 Promote health care and the prevention and control of disease

11 Follow safety procedures and know how to apply First Aid

12 Do not drink alcohol during laboratory working hours orwhen 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 ofyour employment

1 Laboratory Safety 1

2 First Aid 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’s Stain (Acid Fast Stain) 53

14 Albert’s Stain 56

15 Leishman’s Stain 59

16 Preparation and Cleaning of Glassware 62

17 pH in Microbiology 67

18 Bacteriological Media 69

19 Inoculation of Culture Media 77

20 Anaerobic Cultivation 84

21 Important Bacterial Pathogens and the Diseases 88

22 Collection of Clinical Materials 95

for Microbiological Investigations 23 Biochemical Tests and Identification of Bacteria 106

24 O/F Test (Hugh and Leifson’s Test) 108

25 Catalase Test 110

26 Oxidase Test 112

27 Sugar Fermentation Test 115

28 Nitrate Reduction Test 118

29 Hydrogen Sulphide Production Test 120

30 Urease Test 122

31 Citrate Utilisation Test 124

32 Voges–Proskauer Test (VP Test) 126

33 Methyl Red Test (MR Test) 128

34 Indole Test 130

35 Bile Solubility Test 133

36 Coagulase Test 135

37 Antimicrobial Susceptibility Testing 138

38 Brucella Agglutination Test 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 Identification of Fungal Isolates 184

51 Germ Tube Test 195

52 Diagnosis of Virus Infections 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 Urinary Tract Infection 247

60 Spotters 253

Index 267

x Practical Manual of Medical Microbiology

Laboratory Safety 1

Laboratory safety is a vital component of functioning of anylaboratory Safety procedures and precautions to be followed in theMicrobiology laboratory should be designed to:

• Restrict microorganisms present in specimens or cultures to thevessels in which they are contained

• Prevent environmental microorganisms (normally present onhand, hair, clothing, laboratory benches or in the air) from enteringspecimens or cultures and interfering with the results of thestudies

Laboratory Biosafety Levels

Four Biosafety levels have been recommended based on the type ofmicrobes you are working with

Biosafety Level -1 (BSL-1): Adherence to standard microbiologicalpractices No special requirement as regards containment equipment.Biosafety Level-2 (BSL-2): In addition to the use of standardmicrobiological practice, laboratory coats, decontamination ofinfectious wastes, limited access, protective gloves and display ofbiohazard sign and partial containment equipment are therequirements for this level

Most peripheral and intermediate laboratories need BSL-1 orBSL-2 laboratory facilities

Laboratory Safety

1

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2 Practical Manual of Medical Microbiology

BSL-3: In addition to BSL-2, it has special laboratory clothing,controlled access to laboratory and partial containment equipment.BSL-4: BSL-3 plus entrance through change room where laboratoryclothing is put on, shower on exit, all wastes are decontaminatedbefore exit from the facility It requires maximum containmentequipment

Laboratory facilities in BSL-2 (Figs 1.1 and 1.2)

• Laboratory should be designed in such a way that it can be easilycleaned

• Laboratory contains a sink for washing

• Laboratory tops are impervious to water but resistant to acids,alkalis and organic solvents

• An autoclave to decontaminate infectious material is available

• Illumination is adequate for all laboratory activities

• Storage space is adequate

• Preventive measures against laboratory infections

These are aimed to protect workers, patients and cultures.Following steps are suggested:

• Perform adequate sterilization before washing or disposing waste

• Provide receptacle for contaminated glassware

• Provide safety hood

• Ensure that tissues are handled and disposed of properly

• Promote regular hand washing and cleaning of bench tops

• Ensure use of gloves

• Provide mechanical pipetting devices

• Protect patients from laboratory personnel with skin or upperrespiratory tract infections

• Provide special disposal containers for needles and lancets

Pipetting

Pipetting and suctioning have been identified as the significant andconsistent causes of occupational infections Various importantprecautions that must be taken while pipetting are:

• Develop pipetting techniques that reduce the potential for creatingaerosols

Laboratory Safety 3

Figure 1.1: Hazard warning symbols

Figure 1.2: Biological safety cabinets

4 Practical Manual of Medical Microbiology

• Plug pipettes with cotton

• Avoid rapid mixing of liquids by alternate suction and expulsion

• Do not forcibly expel material from a pipette

• Do not bubble air through liquids with a pipette

• Prefer pipettes that do not require expulsion of last drop of liquid

• Drop material having pathogenic organisms as close as possible

to the fluid or agar level

• Place contaminated pipettes in a container having suitabledisinfectant for complete immersion

A variety of pipettes are available Selection should depend uponthe ease of operation and the type of work to be performed

Hypodermic Syringes and Needles

Accidents involving the use of syringes and needles while drawingblood from patients or performing experiments on laboratory animalsare among the most common causes of occupational infections inlaboratories and health care facilities They account for almost 25%

of the laboratory-acquired infections that occur by accidents Thepractices which are recommended for hypodermic needle andsyringes are:

• Avoid quick and unnecessary movements of the hand holdingthe syringe

• Examine glass syringes for chips and cracks, and examine needlesfor barbs and plugs

• Use needle locking (Luer Lock type) syringes only and be surethat needle is locked securely

• Wear surgical or other gloves

• Fill syringes carefully to minimize air bubbles and frothing

• Expel excess air, liquid and bubbles vertically into a cotton pledgetmoistened with suitable disinfectant

• Do not use syringe to forcefully expel infectious fluid into anopen vial for mixing Mixing with a syringe is appropriate only ifthe tip of the needle is held below the surface of the fluid in thetube

• Do not bend, shear, recap or remove the needle from syringe byhand

Laboratory Safety 5

• Place used needle-syringe units directly into a puncture-resistantcontainer and decontaminate before disassembly, reuse ordisposal

Opening Containers

The opening of vials, flasks, petri dishes, culture tubes, embryonatedeggs, and other containers of potentially infectious materials posespotential but subtle risks of creating droplets, aerosols orcontamination of the skin or the immediate work area The mostcommon opening activity in most health care laboratories is theremoval of stoppers from containers of clinical materials It isimperative that specimens should be received and opened only bypersonnel who are knowledgeable about occupational infection risks.Various precautions that can be taken in this regard are:

• Open containers with clinical specimens in well-lighted anddesignated areas only

• Wear a laboratory coat and suitable gloves

• If possible, use a plastic-backed absorbent paper towel to:– Facilitate clean-up

– Reduce generation of aerosols

• Specimens which are leaking or broken may be opened only insafety cabinets

Tubes containing bacterial cultures should be handled with care.Vigorous shaking of liquid cultures creates a heavy aerosol When asealed ampoule containing a lyophilized or liquid culture is opened,

an aerosol may be created Ampoules should be opened in a safetycabinet (Fig 1.3)

• The universal biohazard symbol shall be displayed at specificlaboratories in which manipulations of organisms with moderate

6 Practical Manual of Medical Microbiology

and heavy risk are being carried out Only authorized visitorsshall enter the laboratory showing universal biohazard sign(Fig 1.4) Doors displaying biohazard symbol shall not be proppedopen, but shall remain closed when in use

Figure 1.4: Universal biohazard sign Figure 1.3: Biological safety cabinet

Laboratory Safety 7 Clothing

• All employees and visitors in microbiological laboratories shallwear laboratory clothing and laboratory shoes or shoe covers

• Disposable gloves shall be worn wherever radiological, chemical,carcinogenic materials or virus preparations of moderate to highrisk are handled

• Laboratory clothing including shoes shall not be worn outsidethe work area

Accidents in Laboratory

In the microbiological laboratory, infections pose the most frequentrisk The important pathogens are:

Hepatitis B virus, Shigella spp

Leptospires Yersinia pestis

Mycobacteria spp

Histoplasma

Accidents and Spills

The order of priorities is as follows:

• Protection of personnel

• Confinement of contamination

• Decontamination of personnel

• Decontamination of area involved

Decontamination of skin: The area is washed thoroughly with soapand water Detergents or abrasive materials must not be used andcare must be taken not to damage the skin

Decontamination of cuts\eyes: These are irrigated with water takingcare to prevent the spread of contamination from one area to another.Decontamination of clothing: Contaminated garments should beremoved immediately and placed in a container They should not beremoved from the spill location until contamination has beenmonitored

8 Practical Manual of Medical Microbiology

Decontamination of work surfaces

• Flood the total spillage area including the broken container withdisinfectant

• Leave undisturbed for 10 minutes

• Mop with cotton wool or absorbent paper

• Wear disposable gloves, apron and goggles

• If a dustpan and brush or forceps have been used these too requiredisinfection

• For blood or viruses, hypochlorites (10 gm/L) are used

• Do not use hypochorite solution in centrifuges

• Use activated gluteraldehyde (20 gm/L) on surfaces for viraldecontamination

• Place all potentially contaminated materials in a separatecontainer and retain until monitored

• Restrict the entry to such an area until contamination monitoringhas been carried out

Management of Laboratory Accidents

An adequately equipped first-aid box should be kept in thelaboratory in a place that is known and accessible to all members ofstaff The box must be clearly marked and preferably be made ofmetal or plastic to prevent from damage by pests A medical officershould be consulted regarding the contents of the box A first-aidchart giving the immediate treatment of cuts, burns, poisoning, shockand collapse, should be prepared and displayed in the laboratory

General Laboratory Directions for Safety

The salient general laboratory directions which must be obeyed byall are:

• Long hair should be bound back neatly away from shoulders

• Do not wear any jewellery to laboratory sessions

• Keep fingers, pencils, bacteriological loops, etc out of your mouth

• Do not smoke in the laboratory

• Do not lick labels with tongue (use tap water)

• Do not drink from laboratory glassware

• Do not place contaminated pipettes on the bench top.

• Do not discard contaminated cultures, glassware, pipettes, tubes

or slides in wastepaper basket or garbage can

• Avoid dispersal of infectious materials

• Operate centrifuges, homogenizer and shakers safely

• Immunize the laboratory workers against vaccine-preventablediseases such as hepatitis B, meningococcal meningitis, rabies, etc

Knowledge of first aid can help to reduce suffering and consequences

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 ofaccidents which may occur in the laboratory

First Aid Box

An adequately equipped first aid box should be kept in thelaboratory The box should be clearly marked with a red cross Thecommon items kept in a First Aid Box are given below The laboratoryincharge should regularly check the items; replace the missing items

or medicines that may have expired

1 First aid manual

8 Antibiotic cream (triple antibiotic ointment)

9 Antiseptic solution (like hydrogen peroxide)

First Aid

2

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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)

“Soap and common sense can prevent 80% of Nosocomial ofinfections” This fact points to the importance of soap and handwashing in the control of infectious diseases Hand hygiene hasbeen described as the single most effective means of preventingspread of infections

The aim of hand washing is to remove transient microorganisms(not commensal organisms) as soon as possible following acquisitionfrom contact with possible sources, or immediately before performinginvasive procedures and touching susceptible patients or susceptiblesites

Method

Proper hand washing can easily achieve by thorough hand washingtechnique with soap and water using mechanical friction for 10-15seconds followed by drying

Surgical hand wash involves washing both hands and fore armsusing a defined technique for at least 2 minutes (Fig 3.1)

In order to disinfect clean hands, alcohol rub can be appliedfollowing washing Alternatively, alcohol based product can be usedwhich are shown to be very effective when running water, soap and

Hand Washing

3

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Commonly used disinfectant solutions are chlorhexidine,povidone iodine and commercially available antiseptic solutionslike Dettol or Savlon.

Figure 3.1: Hand washing procedure

The international system of units has been developed and agreedinternationally in the interest of world health It overcomes languagebarriers, enabling an exchange of health information with in acountry 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 theseseven base units Some SI derived units have been given specialnames

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

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

6 Volt V Electric potential difference

7 Degree oC Celsius temperature

SI Unit Prefixes

To enable the measurement of larger or smaller units, SI system alsoincludes a set of prefixes The use of a prefix makes a unit larger orsmaller

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)

16 Practical Manual of Medical Microbiology

Prefix Symbol Function Multiplied by

Microscope was invented by Antony Van Leuwenhoek (1632-1723)

He was a Dutch lens maker and was the first person to observeBacteria

Microscope is an essential optical instrument of Microbiologylaboratory It consists of combination of lenses which will give a

magnified image of minute objects or micro organisms like Bacteria,

Fungi, and Protozoa.

6 Dark ground microscope

7 Phase contrast microscope

18 Practical Manual of Medical Microbiology

Microscope that is suitable for the study of microorganisms isthe light compound Microscope This consists of two converginglenses fixed at the ends of a brass tube The lens which is nearer tothe object is called “Objective” and the lens which is close to the eye

is called Eyepiece or “Ocular” The final image can be observedthrough the ocular

The objective magnifies the specimen to a definite amount andproduces a real, inverted intermediate image of the object, which lieswithin the principle focus of the eyepiece The eyepiece furthermagnifies 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 andeyepiece are engraved on each part The final image seen is thusinverted, magnified and virtual

Parts of Compound Microscope

Based on the manufacturer the parts and adjustments may slightlyvary from each other Microscopes with fixed stage and mechanicalstage are available Microscope with mechanical stage is moreconvenient for microbiological studies (Fig 5.1)

I Mechanical parts Base (Foot)

Limb (Arm)StageAdjustment knobsRevolving nose piece

II Magnifying parts Objective

OcularIII Illuminating parts Sub stage condenser

Iris diaphragmFilter holderMirrorElectric 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

Microscope 19

b Limb: It forms the arm which bears the illuminating parts, stage

and the observation tube In some microscopes the limb is attached

to the foot by a hinged joint so that the microscope can be set at acomfortable 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 thespecimen to be moved smoothly backwards, forwards orsideways Sometimes a scale is fitted to two sides of the stage

to show the extent of the movement This is called the VernierScale and it is useful to trace a part of the blood film or sputumsmear that you need to re-examine or show to your supervisor

d Adjustment knobs: They are used to focus the object two types of

knobs will be present (1) Coarse adjustment for initial adjustmentand (2) Fine adjustment for getting a clear image

Figure 5.1: Compound microscope

Eye piece

Limb

Coarse adjustment Fine adjustment

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

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 theworking distance The working distance is the distance between thefront lens of the objective and the specimen on the stage (when thespecimen is in focus) The higher the magnifying power of theobjective the shorter is the working distance Working distances forthe standard objectives are likely to be indicated as follows

of condenser diminishes illumination whereas raising the condenserincreases the illumination While using oil immersion objective, thecondenser is completely raised as it requires more light When theother objectives are used, it is to be lowered suitably.

Iris Diaphragm

Immediately below the condenser and incorporated in the samemount is the sub stage iris diaphragm operated by a small leverwhich protrudes to one side Opening or closing of this irisdiaphragm controls the amount of light reaching the object Irisdiaphragm is opened widely when the oil-immersion objective isused as it requires maximum light and closed partially when theother 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 theexcessive 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 substage condenser The flat surface (Plane mirror) is used wheneverthe oil immersion objective is employed The concave mirror isemployed with low and high power objectives

22 Practical Manual of Medical Microbiology

Electric Bulb

Instead of mirror in newer models there will be an electric bulb whichwill 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 objectachieved by the microscope The total magnification achieved byvarious 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

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

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 underoil immersion lens Rack oil immersion objective down till itstip dips in the oil Using the fine adjustment focus the object

5 After this, remove the oil from the objective and the objectwith 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 preventsrefraction 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, whichbinds the lenses

• Direct sunlight on to the mirror should be avoided

• For prolonged work, artificial light of particular wavelengths areadvisable

• When not used, protect it from dust and damages

The measurement of objects using a calibrated eye piece scale(micrometer) is micrometry This is used to measure the size of objectsobserved under microscope Measurement of size is useful for theidentification of microorganism especially cyst and Ova of Parasites

Method

1 Caliberation of eye piece micrometer

2 Measuring an object with calibrated eye piece micrometer

Micrometry

6

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Micrometry 25

Calibration of Eye Piece Micrometer

The eyepiece micrometer will require calibration for each objective

of the microscope at which measurements will be required

For parasitology, the scale should be calibrated for the 40 ×objective and for Bacteriology it is useful to calibrate for the 100 ×objective A table can be prepared for each objective givingmeasurement 1 to 50 divisions of the eye piece Scale

1 Remove the normal eyepiece and insert the eye piece micrometer

in the tube of the microscope

2 Place the stage micrometer slide on the stage of the microscope

3 Focus the stage micrometer scale using the required objective

4 Adjust the field until the 0 line of the eye piece Scale aligns exactlywith the ‘0’ line of the stage

5 Look along the Scales and note where a division of the eye piecescale aligns exactly with a division of stage scale

Figure 6.1: Micrometry: Stage micrometer scale (upper) and ocular

micrometer (lower)

26 Practical Manual of Medical Microbiology

6 Measure the distance between ‘0 point and where the alignmentoccurs The measurements Calibration Scale are 0.1mm to 2.0 mmeach small division measures 0.01mm

7 Count the number of divisions of the eye piece Scale coveredbetween the ‘0’ point and where the alignment occurs

8 Calculate the measurement of 1 of the divisions of the eye pieceScale in 1 μm

Measuring the Object

• Remove the stage micrometer

• Place the object slide

• With eye piece micrometer measure divisions covered for the object(e.g Cyst or ova)

• Refer the prepared table for the objective being used to obtain themeasurement of size

Sterilisation is defined as the destruction or removal of allmicroorganisms and their spores.

Disinfection is the destruction of many microorganisms but notusually the bacterial spores Sterilization is usually achieved withthe help of heat whereas chemical agents are employed to effectdisinfection

Sterilisation and disinfection are part of the daily routine ofmicrobiological laboratories and constitute a vital activity whichensures that cultures, containers, media and equipment are treated

in such a way that only the inoculated organisms will grow whileall others will be eliminated

Sterilisation by Heat

This can be achieved by autoclaving, by exposing articles to dryheat in hot air ovens or boiling

Autoclave

Autoclaves can sterilise anything that can withstand a temperature

of 121oC for 30 minutes A pressure cooker used in homes for cookingpurposes can also be used as a makeshift autoclave (Fig 7.1)

Sterilisation

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28 Practical Manual of Medical Microbiology

The containers having clinical material are subjected to heattreatment in the autoclave after which these are emptied and washedand put back into service

Only autoclaves designed for laboratory work and capable ofdealing with a mixed load should be used Porous load and bottlefluid sterilisers are rarely satisfactory for laboratory work There aretwo varieties of laboratory autoclaves

1 Pressure cooker type

2 Gravity displacement models with automatic air and condensatedischarge

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 befastened down and sealed with a rubber gasket An air and steamdischarge tap, pressure gauge and a safety valve are fitted in the lid.Water in the bottom of the autoclave is heated by external gas burners,

an electric immersion heater or a steam coil (Fig 7.2)

Figure 7.1: Steam circulation in a double jacketed autoclave

Sterilisation 29

Operating Instructions

• Ensure that there is sufficient water inside the chamber

• Load the autoclave and fasten the lid keeping the discharge tapopen

• Adjust the safety valve to the required temperature and turn theheat on

• Allow the mixture of air and steam to pass out freely till all air hasbeen discharged

• Close the air discharge tap and let the steam pressure rise withinthe chamber till it attains a temperature of 121oC (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 pressuregauge has reached zero

Figure 7.2: Vertical autoclave

30 Practical Manual of Medical Microbiology

• Allow the material to cool before these are handled (usually agarbottles 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 and7.3)

Figure 7.3: Horizontal autoclave

Figure 7.4: Candle filter

Sterilisation 31 Operating Instructions

• Bring the jacket of the autoclave to operating temperature

• Load the chamber, close the door and open the steam valve sothat steam can freely enter the top of the chamber Air andcondensate shall automatically flow out through the drain at thebottom (Fig 7.4)

• When the drain thermometer reaches the required temperature,allow further period for the load to reach that temperature (thishas 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 80oC is reached

• Gradually and softly open the autoclave enabling the steam toescape and allow the load to cool further

Hot Air Oven

A hot air oven is electrically operated and should be equipped with

a fan to ensure uniform temperature inside The required temperaturefor sterilization is generally 160oC for one hour (Fig 7.5)

Figure 7.5: Hot air oven

32 Practical Manual of Medical Microbiology

Operating Instructions

• Arrange the material to be sterilized loosely and evenly on theracks of the oven allowing free circulation of air and thereby evenheating 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 theoven 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 160oC

• Do not overheat since it would char the cotton plugs and paperwrappings

Autoclaves and hot air ovens can be used for disinfection ofinfectious waste before it is discarded In addition, waste can bedisposed of by boiling in detergent or by burial

Boiling

In the absence of an autoclave, most specimen containers can beboiled in water having detergents to decontaminate This processkills the vegetative bacteria but fails to destroy the spores and certainviruses 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 bigcontainer and boil specimen containers in it for a minimum of 30minutes

Disinfection

Disinfection can be undertaken either chemically or by boiling.Boiling is an effective method to disinfect equipment, e.g needlesand syringes, if autoclaving facilities are not available Equipmentwhich has already been cleaned should be boiled for 20 minutes.Chemical disinfection is used for heat-sensitive equipment that isdamaged at high temperatures Commonly-used chemical disinfec-tants include chlorine releasing compounds; ethyl and isopropylalcohol, quaternary ammonium compounds and gluteraldehyde