Guidelines On Standard Operating Procedures For Microbiology

SEA/HLM/324
Distribution: General
Guidelines on
Standard Operating Procedures
for Microbiology
Sudarshan Kumari
Regional Advisor, Blood Safety & Clinical Technology
WHO, SEARO
R.L. Ichhpujani
Consultant Microbiology and Head, Department of Zoonosis,
National Institute of Communicable Diseases, Delhi
World Health Organization
Regional Office for South-East Asia
New Delhi
May 2000
Standard Operating Procedures for Haematology
© World Health Organization 2000
This document is not a formal publication of the World Health Organization (WHO),
and all rights are reserved by the Organization. The document may, however, be freely
reviewed, abstracted, reproduced or translated, in part or in whole, but not for sale or
for use in conjunction with commercial purposes.
Page 3
The views expressed in documents by named authors are solely the responsibility of
those authors.
Page iii
Acknowledgements
WHO gratefully acknowledges the valuable contributions of Dr Rajesh Bhatia,
Consultant and Head, Department of Microbiology, National Institute of
Communicable Diseases, Delhi; Dr C.S. Bhaskaran (former Director, Institute
of Preventive Medicine, Hyderabad); Dr K.B. Sharma (former Regional Adviser,
Health Laboratory Services, WHO Regional Office for South-East Asia) and Mr
K.K. Khanna (former Joint Director, National Institute of Communicable
Diseases, Delhi) to this publication.
Page i
Preface
Laboratory services have become an integral and inseparable component of
modern medicine and public health. Laboratories play a decisive role in the
diagnosis, treatment, prognosis and monitoring of both communicable and
noncommunicable diseases. Intermediate and peripheral health facilities in
developing countries are crucial to primary health care. Therefore, reliable,
reproducible and rapid laboratory services, organized in a cost-effective
manner, will go a long way in providing quality health services at the district
hospitals and health centres.
Quality assurance in laboratory services, aimed at improving reliability,

efficiency and facilitating inter-laboratory comparability in testing, is the
backbone of quality health care delivery. The use of standard operating
procedures in laboratory testing is one of the most crucial factor in achieving
quality. This helps both in proper patient management and generates reliable
disease surveillance data. This publication provides guidelines on standard
operating procedures for diagnosing diseases of public health importance at
intermediate and peripheral levels. Guidelines on early warning signals about
epidemic-prone diseases based on laboratory data and on collecting and
effectively transporting the appropriate clinical specimens to the
referral/central laboratories for diseases for which diagnostic services are not
as yet developed at the intermediate laboratories are also provided.
The publication contains guidelines on the use of conventional

procedures which may be adapted as per local needs. The emphasis is on
providing feasible, practical, easy-to-reproduce, specific, simple and cost-
effective techniques for the diagnosis of communicable diseases. Necessary
biosafety guidelines have been provided and situations where referrals to
higher-level laboratories are indicated have also been identified.
It is hoped that this publication will be useful in achieving its objective of

improving the quality of laboratory services at intermediate and peripheral
levels.
Page iii
Guidelines on Standard Operating Procedures for Microbiology
Page iv
Contents
Page
Acknowledgements ........................................................................................................... i
Preface .............................................................................................................................. iii
SECTION A: GENERAL LABORATORY PRACTICES
1 Organization and Functions of Laboratories 1

Peripheral laboratory services ................................................................................... 1
Intermediate laboratory services............................................................................... 3
2 Collection and Transportation of Clinical

Clinical Specimens 5
Criteria for rejection of specimens............................................................................ 5
Collection of specimens............................................................................................ 6
Transportation of specimens .................................................................................... 9
3 Sterilization 11
Sterilization by heat .................................................................................................. 11
Disinfection .............................................................................................................. 13
Biohazard waste management .................................................................................. 15
4 Staining Techniques 17
Methylene blue staining............................................................................................ 17
Gram staining ........................................................................................................... 18
Albert’s staining ....................................................................................................... 19
India ink staining ...................................................................................................... 20
Ziehl Neelsen staining .............................................................................................. 20
Iodine staining for ova and cysts .............................................................................. 21
Quality control of stains ........................................................................................... 22
5 Bacteriological Media 23
Types of media ......................................................................................................... 23
Preparation of media and checking of pH ................................................................. 24
Nutrient broth........................................................................................................... 24
Nutrient agar ............................................................................................................ 25
Glucose broth ........................................................................................................... 25
Blood agar ................................................................................................................ 25
Chocolate agar ......................................................................................................... 25
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Page
XLD agar................................................................................................................... 25
Buffered glycerol saline ............................................................................................ 26
Loeffler serum medium ............................................................................................ 26
Blood tellurite agar ................................................................................................... 27
Salt broth (10%) ........................................................................................................ 27
Peptone water........................................................................................................... 28
Alkaline peptone water ............................................................................................. 28
MacConkey agar ....................................................................................................... 28
Bile salt agar ............................................................................................................. 28
Selenite F broth ........................................................................................................ 30
Media for carbohydrate fermentation ....................................................................... 30
Lowenstein Jensen medium ...................................................................................... 31
Cary and Blair transport medium .............................................................................. 32
Stuart transport medium .......................................................................................... 32
Venkataraman-Ramakrishnan (V.R.) holding medium............................................... 33
MacConkey broth for bacteriological examination of water ...................................... 33
Sabouraud Dextrose Agar (SDA) ............................................................................... 34
Performance of plated media .................................................................................... 36
6 Cultivation of Bacteria on Laboratory Media 39

Instrument for seeding media................................................................................... 39
Aseptic techniques ................................................................................................... 41
7 Antimicrobial Susceptibility Testing 43

Modified Kirby-Bauer method................................................................................... 44
Quality assurance in susceptibility test ..................................................................... 47
Biosafety................................................................................................................... 48
Referral..................................................................................................................... 48
When intermediate laboratories need not undertake suscpetibility tests? ................. 48
Salient features of quality assurance in antibiotic susceptibility testing.................... 48
8 Safety in Laboratories 53
Laboratory biosafety levels ....................................................................................... 53
Preventive measures against laboratory infections.................................................... 54
Pipetting ................................................................................................................... 54
Hypodermic syringes and needles ............................................................................ 54
Opening containers .................................................................................................. 55
Laboratory access ..................................................................................................... 56
Clothing.................................................................................................................... 56
Accidents in laboratory ............................................................................................. 56
Accidents and spills .................................................................................................. 57
Management of laboratory accidents ........................................................................ 57
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Page
General laboratory directions for safety .................................................................... 58
9 Quality Assurance 59
What is the objective of QA? ..................................................................................... 59
Factors affecting the quality ..................................................................................... 59
Maintenance of equipment ....................................................................................... 61
Performance tests on culture media ......................................................................... 62
Quality control for commonly-used tests ................................................................. 63
Quality control of immunological tests ..................................................................... 63
QA of antibiotic susceptibility testing ....................................................................... 63
In service training of staff ......................................................................................... 65
Participation in external quality assessment ............................................................. 65
SECTION B: STANDARD PROCEDURES FOR SPECIFIC

SPECIFIC DISEASES
10 Cholera 67
Causative organism .................................................................................................. 67
Specimens ................................................................................................................ 67
Collection, storage and transportation of specimens ................................................ 67
Materials................................................................................................................... 68
Procedure ................................................................................................................. 69
Quality assurance ..................................................................................................... 72
Antimicrobial susceptibility testing........................................................................... 72
Reporting of results .................................................................................................. 72
Referral..................................................................................................................... 72
Biosafety................................................................................................................... 72
11 Diphtheria 73
Collection and transportation of specimens.............................................................. 73
Storage ..................................................................................................................... 74
Processing of swabs ................................................................................................. 74
Staining of smears .................................................................................................... 74
Other significant organisms isolated from throat swabs ........................................... 76
Important points about diagnosis of diphtheria........................................................ 76
Susceptibility to antimicrobial agents ....................................................................... 77
Biosafety................................................................................................................... 77
Referral..................................................................................................................... 77
12 Enteric Fever 79
Collection of specimen ............................................................................................. 79
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Page
Isolation of the organism.......................................................................................... 79

Serodiagnosis ........................................................................................................... 81
Diagnosis of chronic typhoid carriers ....................................................................... 84
Biosafety................................................................................................................... 84
Referral..................................................................................................................... 84
13 Pyogenic Meningitis 87
Causative organisms................................................................................................. 87
Collection, storage and transportation of specimens ................................................ 88
Abnormalities associated with pyogenic meningitis.................................................. 89
Immunological tests ................................................................................................. 91
Biosafety................................................................................................................... 93
Referral..................................................................................................................... 93
14 Dysentery 95
Collection of faeces .................................................................................................. 95
Macroscopic examination ......................................................................................... 96
Microscopic examination .......................................................................................... 96
Isolation of organism................................................................................................ 96
Cautions ................................................................................................................... 97
Biosafety................................................................................................................... 97
Referral..................................................................................................................... 97
Antimicrobial sensitivity testing................................................................................ 97
15 Gonorrhoea 99
Causative organism .................................................................................................. 99
Specimen collection .................................................................................................. 99
Staining the smear and examination......................................................................... 10
0
Specimen transportation for culture ......................................................................... 10
1
Culture ..................................................................................................................... 10
1
Presumptive identification ........................................................................................ 10
2
Biosafety................................................................................................................... 10
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Page
3
3
3
3
Referral..................................................................................................................... 10
4
16 Syphilis 10
5
Direct demonstration................................................................................................ 10
5
7
Susceptibility testing ................................................................................................ 10
7
Serological evidence ................................................................................................. 10
7
Biological false positives in VDRL.............................................................................. 10
9
Quality assurance in VDRL testing ............................................................................ 11
0
Referral..................................................................................................................... 11
0
Biosafety................................................................................................................... 11
0
17 Tuberculosis 11
3
Morphology of M.tuberculosis .................................................................................. 11
3
Microscopy of sputum .............................................................................................. 11
3
Collection of sputum sample .................................................................................... 11
3
Storage and transportation of specimen ................................................................... 11
4
Preparation of smear and Ziehl Neelsen staining (AFB staining) ................................ 11
4
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Page
Culture for M. tuberculosis ....................................................................................... 11

5
Biosafety................................................................................................................... 11
6
7
Susceptibility to antituberculosis drugs .................................................................... 11
7
7
Referral..................................................................................................................... 11
7
18 Malaria 11
9
Preparation of blood smear ...................................................................................... 11
9
Recognition of the malaria parasite .......................................................................... 12
1
Common defects in making blood films ................................................................... 12
3
3
Referral..................................................................................................................... 12
3
3
Biosafety................................................................................................................... 12
4
19 Urinary
Urinary Tract Infection 12
7
Causative organisms................................................................................................. 12
7
Specimen .................................................................................................................. 12
7
Specimen transport................................................................................................... 12
9
Culture ..................................................................................................................... 12
9
Page x
Page
0
Susceptibility to antimicrobial agents ....................................................................... 13
0
0
Referral..................................................................................................................... 13
1
Biosafety................................................................................................................... 13
1
20 Parasitological
Parasitological Examination of Faeces 13
3
Collection of faecal sample ....................................................................................... 13
3
Transportation of samples........................................................................................ 13
4
Macroscopic examination ......................................................................................... 13
4
Microscopic examination (temporary wet mounts).................................................... 13
4
Concentration techniques ......................................................................................... 13
5
Formal ether sedimentation technique ..................................................................... 13
6
Biosafety................................................................................................................... 13
8
Disposal of morbid material ..................................................................................... 13
8
8
8
Referral..................................................................................................................... 13
8
21 Mycological Techniques 14
1
Collection and processing of samples....................................................................... 14
1
Presumptive identification based on direct microscopy ............................................ 14
3
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Page
Biosafety................................................................................................................... 14
5
5
Reporting.................................................................................................................. 14
6
Referral..................................................................................................................... 14
6
22 Bacteriological Examination of Water 14

7
Microbiological examination of water ....................................................................... 14
7
Frequency of examination ........................................................................................ 15
1
Standards ................................................................................................................. 15
1
Reporting.................................................................................................................. 15
2
Referral..................................................................................................................... 15
2
2
SECTION C: COLLECTION AND TRANSPORTATION

TRANSPORTATION OF CLINICAL
CLINICAL MATERIAL TO
REFERRAL
LABORATORIES
23 Bacterial Food Poisoning 15

3
Causative agents ...................................................................................................... 15
3
Collection of specimen ............................................................................................. 15
3
Transportation of specimens .................................................................................... 15
4
Interpretation ........................................................................................................... 15
5
Referral..................................................................................................................... 15
5
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Page
5
Biosafety................................................................................................................... 15
6
24 Acquired Immunodeficiency Syndrome 15

7
Collection of sample of blood ................................................................................... 15
8
9
Referral..................................................................................................................... 15
9
Biosafety................................................................................................................... 16
0
0
25 Viral Hepatitis 16
3
Collection and transportation of specimens.............................................................. 16
3
5
5
Biosafety................................................................................................................... 16
6
Referral..................................................................................................................... 16
6
26 Poliomyelitis 16
7
Collection of stool specimen from a case with acute flaccid paralysis (poliomyelitis) 16
7
8
Referral..................................................................................................................... 16
9
9
27 Dengue Fever and Dengue Haemorrhagic Fever 17
Page xiii
Page
3
Laboratory diagnosis ................................................................................................ 17
3
Recognizing cases of DF/DHF/DSS ........................................................................... 17
5
6
6
Biosafety................................................................................................................... 17
6
Referral..................................................................................................................... 17
6
Index ................................................................................................................................. 17
7
Page xiv
Page xv
Section A
General Laboratory Practices
1. Organization and Functions of
Laboratories
The organization of laboratories in any country is usually a three or four tier
system with various possible functional linkages between them. One possible
way of networking of laboratories is shown in Fig 1.
Figure 1: Networking of
Cen tral o r N atio n al Referen ce Lab o rato ry
Regio n al Regio n al Regio n al

Referral Lab Referral Lab Referral Lab
D istrict Lab D istrict Lab D istrict Lab
Perip h eral Lab . Perip h eral Lab . Perip h eral Lab .
Peripheral laboratory services

Peripheral laboratories are located at the point of first contact of patients with
the health care services. In most developing countries these are available
only at primary health centre or community health centre (upgraded primary
health centre) level. These laboratories provide technical support for
preventive, curative and promotive services for the individual as well as the
community.
Staff
The staff in peripheral laboratories should include one technician and one
laboratory assistant/attendant.
Space
The space available in peripheral laboratories should include at least one
laboratory-cum-office/record room (approx. 5 meters x 3 meters) and one
store-room which can be used for other services also (approx. 5 meters x 3
meters).
Page
Pa ge 1
Other facilities
Other necessary facilities include
➢ supply of safe water

➢ reliable source of energy (battery, electricity, solar or kerosene)
➢ sterilization/disinfection facilities
➢ waste disposal facilities
There must also be transport and communication facilities between the
peripheral and intermediate laboratories for referral of samples and patients,
procurement of supplies and personal discussion.
Equipment and supplies

Necessary equipment and supplies include good microscopes, centrifuges,
autoclaves, refrigerators, balances, pH meters, incubators, water bath,
transport media, glassware, sterile swabs, reagents for staining (eg. Gram,
Albert, Ziehl Neelsen, Romanowsky), reagents for chemical examination of
urine, kits and reagents for rapid diagnostic tests, sterilized syringes and
needles, micropipettes and tips as well as sterile collection bottles for
blood/serum and water analysis.
Tests to be performed
Peripheral laboratories are expected to undertake tests of public health as
well as clinical relevance. Among the tests of public health relevance,
diseases of greater epidemiological importance should be accorded priority.
Testing of environment samples (especially water) also falls into the priorities
of public health relevance. Certain rapid serological tests may be of use in
studying epidemiological patterns of important diseases and the same can
also be performed at peripheral laboratories.
The tests to be performed by peripheral laboratories are subject to the

availability of resources, manpower, technology and prevalence of various
diseases in the area catered to by the laboratory. A suggested list is provided
in Table 1.
Table 1: Suggested tests to be performed at peripheral laboratories

Procedure/Specimen For detection/diagnosis of
Urine examination Pus cells, RBCs
Albumin
Sugar
Stool examination Ova and cysts
Stained smears
smears
Throat specimen Diphtheria
Sputum Tuberculosis
CSF (pyogenic and tubercular) Meningitis
Peripheral blood smear Malaria, filariasis
Page 2
Rapid diagnostic tests HIV

Hepatitis B surface Ag
Syphilis
Meningococcal disease
Intermediate laboratory
laboratory services
In most developing countries, intermediate laboratories are located at district
or the regional headquarters and may act as clinical as well as public health
laboratories. The following functions are expected to be performed by these
laboratories:
1. Laboratory support to clinical diagnosis/public health

Quality assurance
Logistic and technical support
Training of staff for peripheral laboratories
2. Supervision and monitoring of peripheral laboratories
Intermediate laboratories help in the diagnosis and treatment of the

individual patient and are also used as public health laboratories for
epidemiological surveillance and control of diseases in the community. These
laboratories also serve as links between peripheral laboratories and the
state/central laboratory for the following:
➢ Collection, storage and analysis of data.

➢ Distribution of reagents, media, laboratory manuals.
➢ Purchase of equipment.
➢ Supervision of peripheral laboratories.
➢ To conduct external quality assessment scheme (EQAS) for peripheral
laboratories.
➢ To take part in EQAS organized by the state/central laboratories.
➢ To send samples to higher/reference laboratories for
characterization of isolate/confirmation of diagnosis.
Staff
Qualified pathologist/ microbiologist
(Doctor of Medicine/diploma in clinical pathology) 1
Technicians –
DMLT (diploma in medical laboratory technology) with experience 2
Laboratory Assistants (DMLT) 1
Laboratory attendants 2
Cleaner 1
Clerk-cum-storekeeper 1
Since it may not be possible to have a full-time epidemiologist, at least

part time help of an epidemiologist should be available.
Page 3
Space
Microbiology/Serology laboratory (approx.8 metersx5 meters) 1
Sterilization, disinfection and media preparation laboratory
(approx. 6 metersx4 meters) 1
Store-room (approx.3 metersx5 meters) 1
Office (approx. 3 metersx5 meters) 1
Equipment
Binocular microscope 2 Colorimeter 1
Dark-field microscope 1 Refrigerator 1
Inoculating chamber 2 Balances 2
Centrifuge 2 pH meter 1
Autoclave 2 Inspissator 1
Incubator 2 Distil water apparatus 1
Hot air oven 1 Micropipettes as per
workload
Water bath 2 Tips for pipettes as per
workload
VDRL shaker 1
This manual describes most of the tests that have been suggested to be
performed at intermediate-level laboratories.
Further
Further reading
1. Kumari S, Bhatia Rajesh, Heuck CC. Quality Assurance in Bacteriology and
Immunology. WHO Regional Publication, South East Asia Series No 28, 1998.
2. Kumari S, Sharma KB et al. Health Laboratory Services in support of Primary Health

Care in South-East Asia, WHO Regional Publication, South East Asia Series No 24,
2nd Ed, 1999, New Delhi.
Page 4
2. Collection and Transportation of
Clinical Specimens
The laboratory diagnosis of an infectious disease begins with the collection of
a clinical specimen for examination or processing in the laboratory (the right
one, collected at the right time, transported in the right way to the right
laboratory). Proper collection of an appropriate clinical specimen is the first
step in obtaining an accurate laboratory diagnosis of an infectious disease.
Guidelines for the collection and transportation of specimens should be made
available to clinicians in a lucidly written format. The guidelines must
emphasize two important aspects:
➢ Collection of the specimen before the administration of antimicrobial

agents.
➢ Prevention of contamination of the specimen with externally present

organisms or normal flora of the body.
General rules for collection and transportation of specimens are

summarized in Table 1.
Table1: Collection and transportation of specimens
• Apply strict aseptic techniques throughout the procedure.

• Wash hands before and after the collection.
• Collect the specimen at the appropriate phase of disease.
• Make certain that the specimen is representative of the infectious
process (e.g. sputum is the specimen for pneumonia and not saliva) and
is adequate in quantity for the desired tests to be performed.
• Collect or place the specimen aseptically in a sterile and/or appropriate
container.
• Ensure that the outside of the specimen container is clean and
uncontaminated.
• Close the container tightly so that its contents do not leak during
transportation.
• Label and date the container appropriately and complete the requisition
form.
• Arrange for immediate transportation of the specimen to the
laboratory.
Page 5
Criteria for rejection of specimens

Criteria should be developed by a laboratory on the basis of which the
processing of a specimen may not be done by the laboratory. The following
are some examples:
➢ Missing or inadequate identification.

➢ Insufficient quantity.
➢ Specimen collected in an inappropriate container.
➢ Contamination suspected.
➢ Inappropriate transport or storage.
➢ Unknown time delay.
➢ Haemolysed blood sample.
Collection of specimens
The clinical state of the patient will not necessarily be reflected by the result
of laboratory investigation despite correct laboratory performance unless the
specimen is in optimal condition required for the analysis. Some of the
important specimens and their proper collection and transportation methods
are described here so as to ensure quality.
Blood
Whole blood is required for bacteriological examination. Serum separated
from blood is used for serological techniques. Skin antisepsis is extremely
important at the time of collection of the sample. Tincture of iodine (1-2%),
povidone iodine (10%) and chlorhexidine (0.5% in 70% alcohol) are ideal
agents. However, some individuals may be hypersensitive to iodine present in
some of these. While collecting blood for culture,the following points must be
remembered:
➢ Collect blood during the early stages of disease since the number of
bacteria in blood is higher in the acute and early stages of disease.
➢ Collect blood during paroxysm of fever since the number of bacteria

is higher at high temperatures in patients with fever.
➢ In the absence of antibiotic administration, 99% culture positivity can

be seen with three blood cultures.
➢ Small children usually have higher number of bacteria in their blood

as compared to adults and hence less quantity of blood needs to be
collected from them (Table 2).
Table 2:: Volume of blood to be collected at different ages

Age Volume
Volume in 2 bottles
< 2 years 2 ml
Page 6
2-5 years 8 ml
6-10 years 12 ml
>10 years 20 ml
Cerebrospinal fluid (CSF)

Examination of CSF is an essential step in the diagnosis of any patient with
evidence of meningeal irritation or affected cerebrum. Almost 3-10 ml of CSF
is collected and part of it is used for biochemical, immunological and
microscopic examination and remaining for bacteriological or fungal
examination. The following important precautions need to be taken for CSF
collection and transportation:
➢ Collect CSF before antimicrobial therapy is started.

➢ Collect CSF in a screw – capped sterile container and not in an
injection vial with cotton plug.
➢ Do not delay transport and laboratory investigations.

➢ Transport in a transport medium if delay in processing is
unavoidable.
➢ CSF is a precious specimen, handle it carefully and economically. It

may not be possible to get a repeat specimen.
➢ Perform physical inspection immediately after collection and indicate

findings on laboratory requisition form.
➢ Store at 37oC, if delay in processing is inevitable.
The characteristics of the appearance of CSF are outlined in Table 3.
Table 3: Appearance and interpretations of CSF

Clear and colourless Normal
Clear with Tyndall effect High protein content
(sparkling appearance against incident
light)
Clear yellowish Old haemolysis
Clear red Fresh haemolysis
Turbid blood-stained Haemorrhage
Turbid white High cell or protein content
Turbid clot (after overnight storage) Fibrin clots
Sputum
Sputum is processed in the laboratory for aetiological investigation of
bacterial and fungal infections of the lower respiratory tract. It is of utmost
importance in the diagnosis of pulmonary tuberculosis.
Page 7
➢ Select a good wide-mouthed sputum container, which is preferably

disposable, made of clear thin plastic, unbreakable and leak proof
material.
➢ Give the patient a sputum container with the laboratory serial

number written on it. Show the patient how to open and close the
container and explain the importance of not rubbing off the number
written on the side of the container.
➢ Instruct the patient to inhale deeply 2-3 times, cough up deeply

from the chest and spit in the sputum container by bringing it closer
to the mouth.
➢ Make sure the sputum sample is of good quality. A good sputum

sample is thick, purulent and sufficient in amount (2-3 ml).
Give the patient an additional container with laboratory serial number

written on it for an early morning specimen. Explain to the patient to rinse
his/her mouth with plain water before bringing up the sputum.
Urine
Under normal circumstances urine is sterile. The lower part of the urethra and
the genitalia are normally colonised by bacteria, many of which may also
cause urinary tract infection. Since urine is a good growth medium for all
sorts of bacteria, proper and aseptic collection assumes greater importance
for this specimen.
For microbiological examination urine must be collected as a “clean

catch-mid-stream” specimen.
Urine specimens should be transported to the laboratory within one hour

for bacteriological examination, because of the continuous growth of bacteria
in vitro thus altering the actual concentration of organisms.
Stool
Faecal specimens for the aetiological diagnosis of acute infectious diarrhoeas
should be collected in the early stage of illness and prior to treatment with
antimicrobials. A stool specimen rather than a rectal swab is preferred.
➢ The faeces specimen should not be contaminated with urine.

➢ Do not collect the specimen from bed pan.
➢ Collect the specimen during the early phase of the disease and as far
as possible before the administration of antimicrobial agents.
➢ 1 to 2 gm quantity is sufficient.
➢ If possible, submit more than one specimen on different days.
➢ The fresh stool specimen must be received within 1-2 hours of
passage.
Page 8
➢ Store at 2-8oC.
➢ Modified Cary and Blair medium (see chapter 5) is recommended as a
good transport medium. It is a very stable medium and can be stored
for use in screw – capped containers. It is a semi-solid transport
medium. At least two swabs should be inoculated. Most pathogens
will survive for up to 48 hours at room temperature. Specimens are
unacceptable if the medium is held for more than one week or if
there is detectable drying of the specimen.
Alternative transport media are Venkataraman-Ramakrishnan
medium (V-R fluid) or alkaline peptone water. VR fluid should be
prepared in 30 ml (1 oz) screw capped bottles (MacCartney bottles).
It preserves vibrios for more than six weeks and has also proved to
be a very convenient medium for transportation as it can be kept at
room temperature after collection of the specimen.
Throat swab
➢ Depress the tongue with a tongue blade.
➢ Swab the inflammed area of the throat, pharynx or tonsils with a
sterile swab taking care to collect the pus or piece of membrane.
➢ Transport in sterile transport tube.
Bone marrow
Bone marrow is collected by a doctor who is well trained in this procedure
➢ Decontaminate the skin overlying the site from where specimen is to

be collected with 70% alcohol followed by 2% tincture of iodine.
➢ Aspirate 1 ml or more of bone marrow by sterile percutaneous

aspiration.
➢ Collect in a sterile screw-cap tube.

➢ Send to laboratory immediately.
Rectal swab
➢ Insert swab at least 2.5 cm beyond the anal sphincter so that it
enters the rectum.
➢ Rotate it once before withdrawing.

➢ Transport in Cary and Blair or other transport medium.
Transportation of specimens
Specimens to be sent to other laboratories require special attention for safe
packing of the material. Guidelines are usually issued by national authorities
and the same should be strictly followed. For hand-carried transportation
Page 9
over a short distance, the specimen should be placed upright in appropriate

racks. For long distance transportation, it should be placed in three
containers, i.e:
➢ A primary container which has the specimen and is leakproof with a

screw-cap.
➢ A secondary container which is durable, waterproof and made of

metal or plastic with a screw-cap. It should have enough absorptive
material to absorb the contents of the primary container should the
latter break or leak. On its outside, the details of the specimen
should be pasted.
➢ A tertiary container is usually made of wood or cardbox. It should be

capable of withstanding the shocks and trauma of transportation.
Dry ice can be kept between this and the secondary container along
with sufficient absorbents and provision for the escape of
carbondioxide to prevent a pressure build-up inside (Fig 1).
In general, most specimens should be processed in the laboratory within

1 to 2 hours after collection. In practice, a 2-to 4-hour time limit is probably
more practical during a normal working day. The laboratory must be
organized to permit processing of the specimens as soon as they arrive, and
the collection of most specimens should be limited to the working hours of
the laboratory. However, some arrangements must be made to allow for the
initial handling of the few specimens that have to be collected outside of the
laboratory’s working hours.
Page 10
Figure 1: Transportation container
A continuous effort must be made in order to ensure proper collection

and transportation of clinical specimens. Full cooperation of nursing staff and
others concerned with specimen collection is required and can be achieved
once they are made aware of the principles involved and the significance of
what they are being asked to do.
Further reading
1. Lennette HE, Balows A, Hauser WJ et al. Collection, Handling and Processing of
Specimen. In Manual of Clinical Microbiology, 4th Ed, ASM, Washington, DC, 73-
98, 1985.
2. El-Nageh, Heuck CC, Appel W, Vandepitte et al. Basics of quality assurance in

peripheral laboratories. WHO EMRO Series No 2, Alexandria, 1992.
Page 11
3. Sterilization
Sterilization is defined as the destruction or removal (by filtration) of all
microorganisms and their spores, whereas 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.
Sterilization 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.
Sterilization by heat
This can be achieved by autoclaving, by exposing articles to dry heat in hot
air ovens or boiling.
Autoclaving
Autoclaving
Autoclaves can sterilize anything that can withstand a temperature of 121oC
for 30 minutes. A pressure cooker used in homes for cooking purposes can
also be used as a makeshift autoclave.
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 sterilizers are rarely
satisfactory for laboratory work. There are two varieties of laboratory
autoclaves:
➢ Pressure cooker type.

➢ Gravity displacement models with automatic air and condensate
discharge.
Pressure-
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
Page 11
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.
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 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 pressure
gauge has reached zero.
➢ 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.
➢ 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.
➢ 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
80oC is reached.
Page 12
➢ 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 a fan to
ensure uniform temperature inside. The required temperature for sterilization
is generally 160oC for one hour.
➢ 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 160oC.
➢ 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 detergent
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-
Page 13
used chemical disinfectants include chlorine releasing compounds; ethyl and

isopropyl alcohol, quaternary ammonium compounds and gluteraldehyde.
The synopsis of a few commonly-used disinfectants is given in Table 1.
Preferred methods of sterilization for common articles are given in Table

2.
Decontamination of some of the commonly reusable equipment has been

briefly presented in Table 3.
Page 14
Table 1: Disinfectants and their mode of application*

Strength to
use Time of
Target Disinfectant Application
(disinfectant/ exposure
material V/V)
Skin Ethanol 70% Direct 2 minutes

Iodine 1% Direct 2 minutes
Povidone iodine 1% Direct 2 minutes
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

instrumen Isopropanol 70% Direct 4 hours
ts
* Based upon: Basics of quality assurance: WHO/EMRO, 1992, page 162
Table 2: Preferred methods of sterilization for common

common--use articles
Autoclaving Hot air oven

Animal cages Glass ware
Sugar tubes Beakers
Lab. coats Flasks
Cotton Petridish
Filters Pipette
Instruments Slides
Culture media Glass syringes
Test tubes
Powders
Rubber Wood
Gloves, stopper, tubing Tongue depressor, applicator
Page 15
Glass
Slides, syringes, test tubes
Enamel metal trays
Wire baskets
Table 3: Disinfection of specific equipment

Method of choice for Alternative method of
Container/material
decontamination decontamination
Reusable stool container Autoclaving 121oC for 30 Fill the jar having stool
minutes with 5% solution of
phenol and keep for
24hours
Empty into lavatory* Empty into lavatory*
Reusable containers of Autoclaving Boiling in detergent

CSF, pus, sputum
Urine bottles (after Autoclaving Fill with 2% phenol or 1%

emptying in lavatory*) 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 slides** Autoclaving Soak overnight in 5%

phenol
* 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.
Biohazard
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.
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, it may be necessary to double-bag
the waste to prevent leakage. 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.
Page 16
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, decontaminate
with chemical disinfectants or by boiling for 20 minutes before
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 121oC 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 160-170oC 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 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 decontaminating process per se. However, it does prevent the
infectious material from becoming a reservoir of infection 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.
Further reading
El-Nageh MM et al. Basics of Quality Assurance for Intermediate and Peripheral
Laboratories. WHO Regional Publication, Eastern Mediterranean Series No 2, 156-166,
1992.
Page 17
4. Staining Techniques
Staining of the clinical material or the bacteria from colonies on laboratory
media provide a direct visualization of the morphology of the organisms as
well as their reactions to the chemicals present in stains. This is an invaluable
and easy-to-use tool for establishing the identity of various microorganisms.
Some of the commonly-used staining techniques are:
➢ Methylene blue staining

➢ Gram staining
➢ Albert staining
➢ Ziehl Neelsen staining (Acid fast staining)
➢ India ink staining
➢ Iodine staining for ova and cysts in faeces
Methylene blue staining

Ingredients and preparation
Methylene blue 0.3 gm
Distilled water 100 ml
Dissolve the dye in water. Filter through a filter paper.
Staining procedure
➢ 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.
Page 17
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.
Gram staining
This is the most extensively used differential stain that divides bacteria into
two major groups. Those which retain crystal violet dye after treatment with
iodine and alcohol appear purple or bluish purple and are designated as Gram
positive. Those bacteria which lose the crystal violet show the colour of the
counter stain employed. The commonly-used counter stain is saffranin which
gives a pink/red colour to bacteria and these organisms are labelled as Gram
negative.

Crystal violet
Solution A
Crystal violet 2.0 gm

Ethanol, 95% 20 ml
Solution B
Ammonium oxalate 0.8 gm

Mix solutions A and B. Store for 24 hours before use.
Gram iodine
Iodine crystals 1.0 gm

Potassium iodide 2.0 gm
Grind the dry iodine and potassium iodide in a mortar. Add water, a few
ml at a time, and grind thoroughly after each addition until the iodine and
iodide dissolve. Rinse the solution into an amber glass bottle with the
remainder of the distilled water.
Saffranin solution
Stock solution
Saffranin O 2.5 gm
Ethanol, 95% 100 ml
Page 18
Working solution
Stock solution 10 ml
Staining procedure
➢ Make a thin smear on a clean glass slide, dry it in air and fix by
passing through flame of a burner.
➢ Cover the smear with crystal violet, keep for one minute.
➢ Wash the slide with water, then cover with Gram iodine and let it
stand for one minute.
➢ Wash the slide with water.

➢ Decolour with acetone/alcohol, rocking the slide gently for 10-15
seconds till the violet colour comes off the slide.
➢ Wash with water immediately.

➢ Counterstain with saffranin. Let the counterstain stand for 30
seconds.
➢ Wash with water, blot dry and examine under the oil immersion lens
of a microscope.
Uses
Widely used in diagnostic bacteriology mainly to differentiate organisms on
the basis of morphology and Gram reaction.
Albert staining
Ingredients and preparations
preparations
Albert stain I
Toluidine blue 0.15 gm

Malachite green 0.20 gm
Glacial acetic acid 1.0 ml
Alcohol(95%) 2.0 ml
Grind and dissolve the dyes in alcohol, add water and then add acetic
acid. Let the mixture stand for 24 hours and then filter.
Albert stain II
Page 19
Iodine 2.0 gm
Potassium iodide 3.0 gm
Dissolve iodine and potassium iodide in water by grinding in a mortar

with a pestle. Filter through a filter paper.
Staining procedure
➢ Cover the heat-fixed smear with Albert stain I. Let it stand for two
minutes.
➢ Wash with water.

➢ Cover the smear with Albert stain II. Let it stand for two minutes.
➢ Wash with water, blot dry and examine.
Uses
To demonstrate metachromatic granules in C.diphtheriae. These granules
appear bluish black whereas the body of bacilli appear green or bluish green.
India ink staining

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.
Use
To demonstrate the capsule which is seen as an unstained halo around the
organisms distributed in a black background. This is employed for fungal
diagnostics especially for Cryptococcus neoformans.
Page 20
Ziehl Neelsen staining

Ingredients and preparations
Carbol fuchsin 1%
Sulphuric acid 25%
Methylene blue 0.1%
➢ Select a new, unscratched slide and label the slide with a Laboratory
Serial number.
➢ Make a smear from yellow purulent portion of the sputum using a

bamboo stick. A good smear is spread evenly, 2 cms x 3 cms in size
and is neither too thick nor too thin. The optimum thickness of the
smear can be assessed by placing the smear on a printed matter, the
print should be readable through the smear.
➢ Let the smear air-dry for 15-30 minutes.

➢ Fix the smear by passing the slide over the flame 3-5 times for 3-4
seconds each time.
➢ Place the fixed slide on the staining rack with the smeared side
facing upwards.
➢ Pour filtered 1% carbol fuchsin over the slide so as to cover the entire
slide.
➢ Heat the slide underneath until vapours start rising. Do not let carbol
fuchsin to boil or the slide to dry. Continue the process up to five
minutes.
➢ Allow the slide to cool for 5-7 minutes.

➢ Gently rinse the slide with tap water to remove the excess carbol
fuchsin stain. At this point, the smear on the slide looks red in
colour.
➢ Decolor the stained slide by pouring 25% sulphuric acid on the slide
and leaving the acid for 2-4 minutes.
➢ Lightly wash away the free stain. Tip the slide to drain off the water.
➢ If the slide is still red, reapply sulphuric acid for 1-3 minutes and
rinse gently with tap water.
➢ Counter stain the slide by pouring 0.1% methylene blue solution onto
the slide and let it stand for one minute.
➢ Gently rinse the slide with tap water and tip the slide to drain off the
water.
➢ Place the slide in the slide tray and allow it to dry.
Page 21
➢ Examine the slide under a microscope using 40 x lens to select the

suitable area of the slide and examine under 100 x lens using a drop
of immersion oil.
Uses
Distinguishes acid fast bacilli such as Mycobacterium tuberculosis and
M.leprae from other non-acid fast bacilli.
Iodine staining for ova and cysts

➢ On a clean glass slide place one drop of normal saline and one drop
of 2% iodine solution at two different sites.
➢ Mix a portion of stool first with normal saline and then with iodine
solution with the help of a wire loop or applicator.
➢ Place coverslips on both the emulsions.

➢ Examine the preparations under 10x and 40x of the microscope for
various ova and cysts.
Quality control of stains

Test all stains at appropriate intervals for their ability to distinguish positive
and negative organisms and document the results. The performance
standards for Ziehl-Neelsen and Gram staining are as given below:
Control organism/
Stain ATCC No* Expected result
material
Ziehl-Neelsen Mycobacterium 25177 Pink red bacilli
spp. 25922 Blue bacilli
E. coli
Gram E. coli 25922 Gram -ve bacilli
S. aureus 25923 Gram +ve cocci
Iodine solution Formalin treated Visible cyst nuclei
stool specimen
with cysts
* If no standard strains are available, known laboratory strains should be used as controls.
The quality control procedure for stains needs to be performed on a

weekly basis and also as and when a new lot of reagents for staining is
procured/prepared.
Further reading
1. Manual of basic techniques for a health laboratory, WHO, 1980.
Page 22
2. Bailey & Scott’s Diagnostic Microbiology by Baron, Peterson and Finegold, 9th Ed,
Mosby, 1994.
Page 23
5. Bacteriological Media
The role of suitable quality culture media for cultivation of microorganisms
cannot be over emphasised. On it depends the very success of isolation of
aetiological agents. 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 require
growth- promoting ingredients such as blood, glucose, serum, egg, etc. The
media containing ingredients which enhance their 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)
(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
Page 23
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.
The pH determination can be conveniently done with the use of Lovibond

comparator with phenol red indicator disc.
➢ Take two clean test tubes and add 5 ml of the medium to each of the
tubes. One serves as a blank while phenol red indicator is added to
the other tube.
➢ Compare the colour of the medium with the phenol red indicator at
the appropriate pH marking.
➢ Add N/10 NaOH or N/10 HCl, drop by drop till the colour of the
medium matches the colour of the disc at the required pH reading.
➢ Calculate the volume of the NaOH or HCL of 1/10 strength for 5 ml of

the medium to get the required pH.
➢ Based on the calculation, the volume of 1N NaOH or IN HCl required

for the total volume of medium can be calculated and added.
➢ 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. Hence the method of preparation is
given likewise:
Page 24
Nutrient broth
Meat extract 10.0 gm
Peptone 10.0 gm
Sodium chloride 5.0 gm
Mix the ingredients and dissolve them by heating in a steamer. When

cool, adjust the pH to 7.5-7.6.
Nutrient
Nutrient agar
To the ingredients as in nutrient broth, add 15 gm agar per litre. Dissolve the
agar in nutrient broth and sterilize by autoclaving at 121oC 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 l00oC for one hour.
Blood agar
Nutrient agar 100 ml
Sheep blood (defibrinated) 10 ml
➢ Melt the sterile nutrient agar by steaming, cool to 45oC.

➢ 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.
Page 25
➢ Melt the sterile nutrient agar by steaming and cool to about 75oC.
➢ Add blood to the molten nutrient agar and allow to remain at 75oC
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
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 56oC
and pour in plates.
Buffered glycerol saline

Glycerol 300 ml
Disodium hydrogen phosphate 10.0 gm
Na2 H PO4 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 115oC for 15 minutes.
Loeffler serum medium

Nutrient broth 100 ml
Serum (sheep or horse or ox) 300 ml
Glucose 1.0 gm
Page 26
➢ Dissolve glucose in nutrient broth and sterilize at 121oC 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
82oC 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
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 121oC for 15 minutes.
Glycerolated blood tellurite mixture

Sterile defibrinated sheep blood 14 ml
Sterile glycerol 6 ml
Sterile potassium tellurite solution
(1% in water) 4 ml
Sterilize the glycerol in hot air oven at 160oC for 60 minutes and the
tellurite solution by autoclaving at 115oC for 20 minutes. Mix the ingredients
in a sterile flask, incubate for 1-2 hrs. at 37oC, then refrigerate. Haemolysis is
complete after 24 hrs. The mixture keeps 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 45oC, add blood and tellurite and pour in sterile
petri dishes.
Page 27
Salt broth (10%)

Peptone 10.0 gm
Prepare as for nutrient broth, distribute and sterilize at 121oC for 15

minutes.
Peptone water
Peptone 10.0 gm
Dissolve peptone and sodium chloride in distilled water by heating.

Adjust pH to 7.2. Distribute in test tubes and sterilize by autoclaving at 121oC
for 15 minutes.
Alkaline peptone water

Prepare peptone water as described above. Adjust pH of peptone water (7.2 to
9.2). Distribute in test tubes and sterilize at 121oC for 15 minutes.
MacConkey agar
Sodium taurocholate 5.0 gm
Peptone 20.0 gm
Lactose 10.0 gm
Agar 15.0 gm
Neutral red (2% solution in 50% ethanol) 3.5 ml
➢ Mix 5 gm sodium taurocholate or bile salts, 20 gm of peptone, 5 gm

sodium chloride and 15 gm agar with 1000 ml water.
➢ Steam until the solids are dissolved.

➢ Cool to about 50oC, and at this temperature adjust reaction to pH 7.5
to 7.8. Autoclave at 121oC for 15 minutes and filter while hot through
a good grade of filter paper, or a plug of cotton wrapped in gauze
placed in the funnel.
➢ Adjust reaction of the filtrate to pH 7.3 at 50oC or pH 7.5 at room

temperature. Add 10 gm lactose and 3.5 ml of 2% solution of neutral
red in 50% ethanol.
Page 28
➢ Mix thoroughly, distribute in flasks and sterilize in the autoclave at

121oC for 15 minutes.
➢ For use, melt in the steamer, pour into sterile petri dishes and allow
to set.
Bile salt agar

Peptone 10.0 gm
Meat extract 5.0 gm
Sodium taurocholate 5.0 gm
Agar 15.0 gm
➢ Dissolve by steaming in 1000 ml of water, 10 gm peptone, 5 gm meat

extract, 5 gm sodium chloride, 15 gm agar and 5 gm sodium
taurocholate.
➢ Adjust pH to 8.5 with sodium hydroxide solution.

➢ Cool and filter through a good grade filter paper or absorbent cotton
wool previously wetted with water.
➢ Distribute into sterile flasks in convenient amounts and sterilize by

autoclaving at 121oC for 15 minutes.
➢ Plates are made by melting the stock medium and pouring into sterile
petri dishes.
Agar base
Peptone (proteose) 20.0 gm
Agar 90.0 gm
Lactose 40.0 gm
Neutral red (2% solution in 50% ethanol) 5.0 gm
Distilled water 4.0 litre
Solution A
Sodium citrate, 2H2O 17.0 gm
Sodium thiosulphate 17.0 gm
Distilled water 100.0 ml
Solution B
Page 29
Agar base
➢ Dissolve by heating 20 gm meat extract in 200 ml water and make
the solution just alkaline with 50 per cent sodium hydroxide solution.
➢ Boil and filter through filter paper.

➢ Adjust to pH 7.3, make upto 200 ml and add 20 gm proteose
peptone.
➢ In another vessel, dissolve 90 gm agar in 3700 ml water by steaming.

➢ Filter the agar solution, add the meat extract peptone solution to it
and mix.
➢ Add 5 ml neutral red solution in 50% ethanol and 40 gm lactose.

➢ Make up to 4 litres with water.
➢ Mix, bottle accurately in lots of 100 ml, and sterilize in an autoclave
by free steaming for one hour and then at 121oC for 10 minutes.
Solution A
Dissolve 17 gm sodium citrate, 17 gm sodium thiosulphate and 2 gm ferric
ammonium citrate (green scales) in 100 ml of sterile distilled water with
heating.
Solution B
Dissolve 10 gm sodium desoxycholate in 100 ml of sterile distilled water.
➢ Sterilize solution A and B at 60oC in water bath for one hour.

➢ For preparing desoxycholate citrate agar medium, melt 100 ml of the
agar base in a water bath and add 5 ml each of the solutions A and B
in the order given, using separate pipettes.
➢ Mix well after each addition.

➢ Cool the tube at 50 to 55oC.
➢ Pour into sterilized petri dishes and allow to set.
➢ Dry the surface of the medium in the incubator before use.
Selenite F broth
Sodium hydrogen selenite 4.0 gm
Peptone 5.0 gm
Lactose 4.0 gm
Disodium hydrogen phosphate 9.5 gm
(Na2HPO4,12H2O)
Sodium dihydrogen phosphate 0.5 gm
Page 30
➢ Dissolve 4 gm sodium hydrogen selenite, 5 gm peptone, 4 gm

lactose, 9.5 gm disodium hydrogen phosphate and 0.5 gm sodium
dihydrogen phosphate in 1000 ml of sterile water (water autoclaved
at 121oC for 30 minutes) with sterile precautions and distribute the
yellowish solution in 10 ml amounts in sterile screw-capped bottles.
➢ Steam at 100oC for 30 minutes. The medium should not be

autoclaved.
➢ There may be a slight red precipitate in the medium, but this does not
interfere with the action of the medium.
➢ The pH of the medium as prepared should be 7.1 and the quantity of

phosphate added may be varied slightly to achieve this.
Media for carbohydrate fermentation

(a) Peptone 10.0 gm
(b) Carbohydrate base 10.0 gm
(c) Andrade’s indicator 10 ml
➢ Dissolve 10 gm peptone and 5 gm sodium chloride in 900 ml water.

➢ Adjust pH to 7.0 to 7.3 so that after addition of 10 ml of Andrade’s
indicator the pH should be 7.5.
➢ Sterilize at 121oC for 15 minutes.

➢ Dissolve 10 gm of the requisite sugar in 90 ml water and steam for
30 minutes or sterilize by filtration.
➢ With sterile precautions, add 90 ml of this sugar solution and 10 ml

of Andrade’s indicator solution to 900 ml of the sterile peptone water
solution. Distribute into sterile test tubes containing inverted Durham
fermentation tubes. Steam for 30 minutes.
➢ Prepare Andrade’s indicator solution by adding 1 N sodium hydroxide

solution to 0.5% aqueous solution of acid fuchsin until the colour of
the indicator solution is just yellow.
Lowenstein-
Lowenstein-Jensen medium
Salt solution
Potassium dihydrogen phosphate (KH2PO4) 2.40 gm
Magnesium sulphate (MgSO4.7H2O) 0.24 gm
Magnesium citrate 0.60 gm
Asparagine L(Pure) 3.60 gm
Glycerol 12.0 ml
Page 31

Whole egg 1000 ml
Malachite green 2% 20 ml
Dissolve the salts and glycerol in water over a water bath. Autoclave for
15 minutes at 121oC, cool to room temperature.
Beaten egg
➢ Use only fresh eggs preferably not more than three days old.
➢ Using a soft brush and soap and with soda solution clean the outside
of the egg’s wall. Leave the eggs in 5% soap and soda solution for 30
minutes.
➢ Place in running tap water till the water is perfectly clear. Drain and
dry the eggs.
➢ Just before breaking, clean the outside of the eggs with a piece of
sterile gauze dipped in alcohol.
➢ Break the eggs into a sterile flask aseptically.

➢ Close with a rubber stopper and shake vigorously until well
homogenized. Filter through two or three layers of sterile gauze
stretched over a sterile funnel and collect the filtrate in a sterile
container.
Malachite green solution

➢ Dissolve l gm malachite green in 50 ml distilled water in a screw
capped bottle and sterilise at 121oC for 15 minutes.
➢ Add the eggs to the salt solution aseptically and mix well.
➢ Add malachite green and mix.
➢ Pour into a sterile aspirator jar and let it stand for 30 minutes for the
air bubbles to escape.
➢ Fill into tubes or bottles.

➢ Inspissate for 40 minutes at 82oC to 85oC.
➢ While inspissating, the bottle should be kept flat but the tubes have
to be sloped.
Cary and Blair transport medium

Sodium thioglycollate 1.5 gm
Disodium phosphate 1.1 gm
Agar 5.0 gm
Page 32
Calcium chloride, (1% solution) 9.0 ml

➢ Dissolve by heating 1.5 gm sodium thioglycollate, 1.1 gm disodium

phosphate, 5 gm sodium chloride and 5 gm agar in 990 ml distilled
water.
➢ Cool to 50oC and add 9 ml of one per cent aqueous solution of

calcium chloride. Adjust the pH to 8.4.
➢ Dispense in 7 to 10 ml amounts in wide mouth screw capped bottles.

➢ Steam for 15 minutes, cool and tighten the caps.
Stuart transport medium

Thioglycolic acid 2 ml
Sodium hydroxide, (IN NaOH) 12-15 ml
Sodium glycerophosphate, 100 ml
20% aqueous
Calcium chloride, CaCl2, 1% aqueous 20 ml
Mix the ingredients, adjust pH to 7.2 with IN sodium hydroxide solution.
Agar solution
Agar 6.0 gm
Dissolve by steaming
Preparation of complete medium

Anaerobic salt solution 900 ml
Agar solution 11 ml
Methylene blue, 0.1% aqueous 4 ml
➢ Melt the agar and add the salt solution.

➢ Adjust the pH to 7.3-7.4.
➢ Add the methylene blue and distribute in small bottles filling nearly to
capacity. Autoclave at 121oC for 15 min. and immediately tighten
caps.
➢ When cool, the medium should be colourless.
Page 33
Venkataraman-
Venkataraman-Ramakrishnan (VR) holding
medium
➢ Dissolve 12.4 gm. boric acid and 14.9 gm potassium chloride in 800
ml hot distilled water, cool and make up to 1000 ml with distilled
water.
➢ To 250 ml of this stock solution, add 133.5 ml N/5 sodium hydroxide

solution. Make up to 1000 ml with distilled water and add 20 gm
common salt. Shake and dissolve.
➢ Filter through filter paper and dispense in 10 to 15 ml amounts in

wide- mouthed screw capped bottles.
A simple modification is as follows:
➢ Dissolve 20 gm of crude sea salt and 5 gm of peptone in distilled

water to make one litre.
➢ Adjust the pH to 8.6 to 8.8.

➢ Dispense 10-15 ml amounts in wide-mouthed bottles with screw-
caps.
➢ About 1-3 ml of stool specimen should be inoculated into the

medium.
MacConkey broth for bacteriological

examination of water
This medium is used for detecting the presence of coliform organisms in
water.
Single strength
Sodium taurocholate (commercial) 5 gm
Peptone (any good make) 20 gm
Sodium chloride (NaCl) 5 gm
Lactose 10 gm
Bromocresol purple, 1%, 5 ml
solution in ethanol
or Neutral red, 1% aqueous solution 5 ml
Water 1000 ml
➢ Dissolve the bile salt, peptone and sodium chloride.

➢ Steam for two hours, cool and transfer to the refrigerator overnight.
➢ Add lactose and when dissolved, filter cold through filter paper.
➢ Adjust the reaction to pH 7.4.
➢ Add indicator.
Page 34
➢ Distribute in 5 ml amounts in 1-oz. bottles or 15 cm x 1.5 cm test

tubes with Durham tubes.
➢ Autoclave at 121oC for 15 minutes.
Double strength
➢ Prepare as above, but with half the amount of water.
➢ Distribute in 50 ml amounts in 5-oz bottles using 3x3/8 inch test
tubes with Durham tubes, and in 10 ml amounts in 1-oz bottles
using 2X0.25 inch Durham tubes.
Sabouraud Dextrose Agar (SDA)

Ingredients
Original SDA Emmon's modification
Dextrose Peptone 40 gm 20 gm
Neopeptone 10 gm 10 gm
Agar 20 gm 20 gm
Distilled water 1000 ml 1000 ml
Adjust final pH 5.6 6.8 to 7.0
Preparation
Mix the ingredients in water by heating. Adjust the pH. Sterilize in the
autoclave at 121oC for 10 minutes.
10% Potassium Hydroxide

Potassium hydroxide 10 gm
Dissolve 10 grams of potassium hydroxide in 100 ml of distilled water.
Lactophenol cotton blue mounting medium

Phenol crystals 20 gm
Lactic acid 20 gm
Glycerol 40 gm
Cotton blue (poirrier's blue) 0.05 gm
Melt the phenol crystals in water bath. Mix water and phenol crystals. Add
0.05 gram of cotton blue into a mortar and grind it with a pestle. Add water and
phenol little by little and make it a nice paste. Add the remaining amount of
water and phenol, mix well. Then add lactic acid and glycerol and mix well.
Filter through a filter paper.
Page 35
Desoxycholate Citrate Agar (DCA)

Ingredients
Proteose peptone, No.3 10.0 gm
Lactose 10.0 gm
Sodium citrate 20.0 gm
Bacto agar 15.0 gm
Neutral red (1% solution) 2.3 ml
Beef infusion broth 1000 ml
pH 7.4
Preparation
Add Proteose peptone, sodium citrate, ferric ammonium citrate and bacto
agar to the beef Infusion broth, pH of which has already been adjusted.
Recheck pH. Keep it in a water bath for 30 minutes for dissolving. Then add
lactose, sodium desoxycholate and neutral red solution and mix. Then pour in
plates.
Bismuth Sulfite Agar (BSA) (Wilson & Blair medium)

Ingredients
Polypeptone peptone 10.0 gm
Beef extract 5.0 gm
Dextrose 5.0 gm
Disodium phosphate 4.0 gm
Ferrous sulfate 0.3 gm
Bismuth sulfite indicator 8.0 gm
Brilliant green 0.025 gm
Agar 20.0 gm
pH 7.5 + 0.2
Preparation
Mix the ingredients in distilled water. Allow to stand for five minutes and mix
thoroughly. When a uniform suspension has been obtained, heat with
frequent agitation and boil for one minute. Cool to about 50oC. Mix well and
pour into sterile plates.
Mueller-
Mueller-Hinton Agar (MHA)
Ingredients
Page 36
Beef extract 2.0 gm

Acidicase Peptone 17.5 gm
Starch 1.5 gm
Agar 17.0 gm
Final pH 7.4 + 0.2
Preparation
Dissolve the ingredients in one liter of distilled water. Mix thoroughly. Heat
with frequent agitation and boil for one minute. Dispense and sterilize by
autoclaving at 121oC for 15 minutes. Do not overheat. When remelting the
sterile medium, heat as briefly as possible.
Saponin lysed blood agar plus VCNT (A) inhibitors

A selective medium for the growth of N.gonorrhoeae using GC agar base
GC agar base (Difco) 36 gm

Saponin lysed horse/sheep blood 90 ml (9% final concentration)
VCN inhibitor (10 ml vial) 1 vial
Trimethoprim (100 mg/L stock) 2 ml
(Amphotericin (1000 mg/L stock) 1 ml (optional))
Method
The GC agar base medium is prepared at single strength which is half the
strength recommended by the manufacturer, since the addition of equal
volume of 2% haemoglobin is replaced by adding lysed blood. Suspend 36 gm
GC agar base in one litre of distilled water. Mix well, then steam or boil gently
until it dissolves completely. Sterilize by autoclaving at 15 psi (121oC) for 15
minutes. Cool to 52-54oC in a waterbath. When the molten GC agar medium
has cooled to this temperature, aseptically add the saponin lysed horse or
sheep blood. VCN inhibitor solution, trimethoprim (and amphotericin if used).
Mix well and pour 25 ml volumes in 90 mm diameter petri dishes. Allow the
agar to set, then store the plates in an inverted position in the laboratory
refrigerator (2-8oC) until required.
Performance of plated media

Samples of plates from each batch are selected for performance-testing and
are inoculated with the appropriate stock cultures. For each type of medium,
at least two or three microorganisms having growth characteristics with
‘positive’ and ‘negative’ results for the medium should be used. The size of
inoculum and method of inoculating the test plates must be standardized as
closely as possible. In general, control organisms should be selected from an
actively growing broth culture and a standard loopful of culture seeded
Page 37
directly onto the test medium, which is then streaked so as to obtain isolated
colonies. After appropriate incubation, the results of the performance test are
recorded. The medium is released for use in the clinical laboratory only if the
results indicate satisfactory performance. In initiating a quality control
programme, one must establish some priorities, such as beginning by testing
those media that are most likely to demonstrate deficiencies. Top priority
should be given to blood agar, chocolate agar and Thayer Martin agar media.
Secondary priority should be accorded to selective enteric media such as
MacConkey agar, XLD and bile salt agars.
A quantitative approach may be more useful for testing of performance

of selective or inhibitory media such as Thayer Martin agar. N gonorrhoeae
and N.meningitidis usually grow on Thayer Martin agar when the inoculum is
heavy, but when a fairly light inoculum is used, the pathogens might be
inhibited. Consequently, a somewhat quantitative performance test could
detect deficiencies that would be overlooked if one simply inoculated test
plates with undiluted stock cultures.
Further reading
Balows A, Hausler WJ, Herrman KL et al. Manual of Clinical Microbiology 5th Ed,
American Soceity of Microbiologists, Washington, 1991.
Page 38
6. Cultivation of Bacteria on
Laboratory Media
Inoculation of Culture Media
For microbiological investigations it is essential to learn the skills of
inoculating specimens onto culture media and subculturing from one medium
to another.
Instrument for seeding

seeding media
This is selected according to the nature of the medium and inoculum.
Platinum or nichrome wires of different gauges are used. Nichrome has
oxidizing properties and hence in some of the tests where this property of
bacterium is to be tested (e.g. oxidase test), platinum wire, instead of
nichrome should be used. This wire is sterilized by holding it vertically in the
flame of the burner so that the whole length of wire becomes red hot. It is
allowed to cool down before it touches any material suspected to be having
bacteria to avoid the heat killing the organisms. Presterilized disposable loops
are now available commercially. The wire can be used as a:
➢ Straight wire to stab the culture, picking of single colonies as well as

for inoculating the liquid media,
➢ Thick wire which is useful for lifting the viscid material such as
sputum, and
➢ Wire loop which is usually of 2 mm diameter is most useful of all

inoculating wires. These are preferred to seed a plate of medium as
the straight wire usually cuts the agar.
Seeding a culture plate

There are three commonly employed techniques for seeding culture plates.
The most common is shown in Fig. 1.
The inoculum from the clinical material or another plate is first spread
out in the form of a primary inoculum (as at A in Fig 1) which is also called as
‘well-inoculum’ or only ‘well’. The successive series of strokes B, C, D and E
are made with the loop sterilized between each sequence. At each step the
inoculum is derived from the most distal part of the immediately preceding
Page 39
strokes so as to gradually reduce the number of bacteria. This helps in

obtaining isolated colonies.
In an alternative plating procedure one edge of a large loop is used to

make a secondary well (see B in Fig 1). The other edge is then used to make
succession of strokes across the remaining unseeded area.
When the inoculum is small or the medium is selective it can be more

heavily inoculated (Fig 2). Several loop-fulls of the specimen are used to
spread the primary inoculum (see A in Fig 2).
After sterilizing the loop, it is recharged by rubbing it over area A and the
plate is seeded in parallel strokes.
Figure1: Seeding a culture plate Figure 2: Seeding with heavy

inoculum
Seeding a liquid medium

If the tubes have got cotton plugs, the mouth of the tubes should be heated
in flame before and after any handling of tube to prevent contamination from
the rims of tubes getting into the medium. It is not required when metal caps
and screw-capped tubes are handled. Incline the tube containing the liquid
medium to 45o and deposit the inoculum on its wall above the surface of the
liquid at its lower end. Return the tube to a vertical position. Now the
inoculum shall be below the surface of the liquid.
Subculture from a solid medium to solid medium

Using a sterile wire or loop, a representative colony is touched and
subcultured onto appropriate solid medium by touching the wire or loop onto
the surface of the medium.
Important points about inoculation of culture media

➢ Aseptic technique is important to avoid contamination.
Page 40
➢ When more than one medium is inoculated, follow a particular order.

Inoculate media without inhibitors, followed by indicator and then
selective media.
➢ While processing fluid specimen inoculate liquid media first to reduce

the chances of carry over from contaminated solid media.
➢ Prepare smears for staining after all media have been inoculated.
➢ Properly label the media to be inoculated to avoid any mix-up of the
specimens.
➢ Inoculate the media with clinical specimens as soon as possible.

➢ Minimize the aerosol production by opening the caps of liquid media
slowly, avoiding vigorous shaking of the specimen and avoiding the
expulsion of the last drop from the pipette.
Inoculation of carbohydrate fermentation media

These are inoculated as liquid media and incubated at 37oC for 18-24 hours.
When the particular sugar is fermented, acid is produced which changes the
pH of the medium thus turning phenol red into yellow. In case the
fermentation is with the production of gas, a bubble of air is visible in
Durham tube.
Seeding solid media in test tubes

Slopes of solid media are inoculated by streaking the surface of the agar with
loop in a zig zag manner. Stab cultures are inoculated by plunging the wire
into the centre of the medium.
Aerobic Incubation of cultures

For bacteria of medical importance, incubation is uniformally done at 37oC.
Depending upon the workload a laboratory may have a tabletop incubator
(suitable for peripheral laboratories) or a walk-in incubator. For prolonged
incubations, as are required for the growth of Mycobacterium tuberculosis,
screw-capped bottles should be used instead of petri dishes or tubes to
prevent the drying of medium.
Incubation in an atmosphere with added carbon dioxide

Extra carbon dioxide is needed for optimal growth of organisms such as
Brucella abortus, pneumococci and gonococci. The concentration of
additional carbon dioxide needed is 5-10 per cent. The simplest method for
having this environment is to put the plates in a container and generate CO2
inside by lighting a candle in it just before putting on the lid. Pure CO2 can
also be introduced in a container. Carbon dioxide-generating kits are now
available and so are incubators which can provide a predetermined and
Page 41
regulated amount of this gas. Special CO2 incubators (also called capnoeic
incubators) are available commercially.
Aseptic techniques
Aseptic techniques are important to protect the worker from infection from
the clinical specimen and also to prevent contamination of the material under
process. Aseptic conditions can be achieved by following steps:
➢ Open the caps and lids of the containers containing the specimen for
the briefest period required.
➢ Do not keep the lids on the workbench.

➢ Inoculating loops should be put through the flame properly prior to
introducing them into the specimen container.
➢ While working on the infectious material, keep the specimen away

from the face.
➢ Loops should not contain fluid or large particles of matter that may
splatter when placed in the flame.
➢ Avoid vigorous shaking of the specimen prior to opening; open the

caps slowly to minimize aerosol production.
➢ Perform homogenization and grinding procedures involving tissue or

biopsy specimen in safety cabinet.
➢ Keep all specimens, tubes and bottles of media in racks to reduce the
risk of accidental spillage.
➢ Mop up the workbench clean with any disinfectant at the start and
close of work.
➢ Wash hands with soap and water before and after handling infectious
specimens.
Further reading
1. Isenberg HD(Ed) Clinical Microbiology Procedures handbook. American Society for
Microbiology, Washington, DC, Vol 1, Section 1.4, 1992.
2. Collins CH, Lyne PM and Grange JM. Microbiological Methods. Butterworths,
London, 94-96, 1995.
3. PHLS Standard Operating Procedures – Inoculation of culture media No B.SOP 54
Version:1, 1998.
Page 42
7. Antimicrobial Susceptibility Testing
Antibiotic susceptibility testing has become a very essential step for the
proper treatment of infectious diseases. It is used
➢ To guide the clinician in selecting the best antimicrobial agent.

➢ To accumulate epidemiological information on the resistance of
microorganisms of public health importance.
The choice of drugs used in a routine antibiogram is governed by various

considerations since only a few antimicrobial agents can be tested. Table 1
suggests the drugs to be tested in various situations. The drugs in Table 1 are
divided into two sets. Set 1 includes drugs that are available in most hospitals
and for which routine testing should be carried out for every strain. Tests for
drugs in set 2 are to be performed only at the special request of the physician,
or when the causative organism is resistant to the first-choice drugs, or when
other reasons (allergy to a drug, or its unavailability) make further testing
justified.
Table 1: Basic sets of drugs for routine susceptibility

susceptibility tests
Set 1 Set 2
Staphylococcus Benzyl penicillin Gentamicin

Oxacillin Amikacin
Erythromycin Co-trimoxazole
Tetracycline Clindamycin
Chloramphenicol
Intestinal Ampicillin Norfloxacin
Chloramphenicol
Co-trimoxazole
Nalidixic acid
Tetracycline
Enterobacteriaceae Sulfonamide Norfloxacin
Trimethoprim Chloramphenicol
Urinary
Co-trimoxazole Gentamicin
Ampicillin
Nitrofurantoin
Nalidixic acid
Tetracycline
Blood and tissues Ampicillin Cefuroxime
Chloramphenicol Ceftriaxone
Cotrimoxazole Ciprofloxacin
Tetracycline Piperacillin
Gentamicin Amikacin
Pseudomonas Piperacillin Amikacin
aeruginosa Gentamicin
Page 43
Tobramycin
Antimicrobial susceptibility tests measure the ability of an antibiotic or

other antimicrobial agent to inhibit bacterial growth in vitro. This ability may
be estimated by either the dilution method or the diffusion method. The
recommended method for intermediate and peripheral laboratories is the
modified Kirby-Bauer method, the methodology of which is given below:
Modified Kirby-
Kirby-Bauer method
method
Reagents
Mueller-
Mueller-Hinton agar
➢ Mueller-Hinton agar should be prepared from a dehydrated base
according to the manufacturer’s recommendations. The medium
should be such that with standard strains, zone sizes within the
acceptable limits are produced. It is important not to overheat the
medium.
➢ Cool the medium to 45-50oC and pour into plates. Allow to set on a
level surface, to a depth of approximately 4 mm. A 9 cm diameter
plate requires approximately 25 ml of the medium.
➢ When the agar has solidified, dry the plates for immediate use for 10-
30 minutes at 36oC by placing them in an upright position in the
incubator with the lids tilted.
➢ Any unused plates may be stored in a plastic bag, which should be

sealed and placed in a refrigerator. Plates stored in this way can be
kept for two weeks.
➢ In order to ensure that the zone diameters are sufficiently reliable for
testing susceptibility to sulfonamides and co-trimoxazole, the
Mueller-Hinton agar must have low concentrations of the inhibitors
thymidine and thymine. Each new lot of Mueller-Hinton agar should
therefore be tested with a control strain of Enterococcus faecalis
(ATCC 29212 or 33186) and a disc of co-trimoxazole. A satisfactory
lot of medium will give a distinct inhibition zone of 20 mm or more
that is essentially free of hazy growth or fine colonies.
➢ For testing the susceptibility of fastidious organisms, 5% blood

should be added to the Mueller-Hinton agar base.
Antibiotic discs
Any commercially available discs with the proper diameter and potency can be
used. Stocks of antibiotic discs should preferably be kept at -20oC, or the
freezer compartment of a home refrigerator is convenient. A small working
supply of discs can be kept in the refrigerator for up to one month. On
removal from the refrigerator, the containers should be left at room
Page 44
temperature for about one hour to allow the temperature to equilibrate. This
procedure reduces the amount of condensation that occurs when warm air
reaches the cold container.
Turbidity standard
Prepare the turbidity standard by pouring 0.6 ml of a 1% (10 gm/L) solution of
barium chloride dihydrate into a 100-ml graduated cylinder, and filling to 100
ml with 1% (10 ml/L) sulphuric acid. The turbidity standard solution should
be placed in a tube identical to the one used for the broth sample. It can be
stored in the dark at room temperature for six months, provided it is sealed
to prevent evaporation.
Swabs
A supply of cotton wool swabs on wooden applicator sticks should be
prepared. These can be sterilized in tins, culture tubes, or on paper, either in
the autoclave or by dry heat.
Procedure
➢ To prepare the inoculum from the primary culture plate, touch with a
loop the tops of each of 3-5 colonies, of similar appearance, of the
organism to be tested.
➢ When the inoculum has to be made from a pure culture, a loopful of

confluent growth is similarly suspended in saline. Inoculum from
colonies of streptococci cannot be made by emulsification. Hence,
with streptococci, after inoculation the culture tubes are incubated
for 4-6 hours to get uniform turbidity which should be matched with
the turbidity standards.
➢ Compare the tube with the turbidity standard and adjust the density
of the test suspension to that of the standard by adding more
bacteria or more sterile saline. Proper adjustment to the turbidity of
the inoculum is essential to ensure that the resulting lawn of growth
is confluent or almost confluent.
➢ Inoculate the plates by dipping a sterile swab into the inoculum.

Remove excess inoculum by pressing and rotating the swabs firmly
against the side of the tube above the level of the liquid.
➢ Streak the swab all over the surface of the medium three times,
rotating the plate through an angle of 60o after each application.
Finally, pass the swab round the edge of the agar surface. Leave the
inoculum to dry for a few minutes at room temperature with the lid
closed. The antibiotic discs may be placed on the inoculated plates
using a pair of sterile forceps.
Page 45
➢ A sterile needle tip may also be used to place the antibiotic discs on
the plate. Alternatively, an antibiotic disc dispenser can be used to
apply the discs to the inoculated plate.
➢ A maximum of seven discs can be placed on a 9-10 cm diameter

plate. Six discs may be spaced evenly, approximately 15 mm from the
edge of the plate, and one disc placed in the centre of the plate. Each
disc should be pressed down gently to ensure even contact with the
medium.
➢ The plates should be placed in an incubator at 35oC within 30

minutes of preparation. Temperatures above 35oC invalidate the
results for oxacillin/ methicillin.
➢ Do not incubate in an atmosphere of carbon dioxide.

➢ After overnight incubation, the diameter of each zone(including the
diameter of the disc) should be measured and recorded in mm. The
results should then be interpreted according to the critical diameters
by comparing with standard tables (Table 2).
➢ The measurements can be made with a ruler on the under surface of

the plate without opening the lid.
➢ The end-point of inhibition is judged by the naked eye at the edge

where the growth starts, but there are three exceptions:
– With sulfonamides and co-trimoxazole, slight growth occurs
within the inhibition zone; such growth should be ignored.
– When β-lactamase producing staphylococci are tested against
penicillin, zones of inhibition are produced with a heaped-up,
clearly defined edge; these are readily recognizable when
compared with the sensitive control, and regardless of the size of
the zone of inhibition, they should be reported as resistant.
– Certain Proteus spp. may swarm into the area of inhibition around
some antibiotics, but the zone of inhibition is usually clearly
outlined and the thin layer of swarming growth should be ignored.
Table 2*: Zone sizes with different antimicrobial agents

Zone diameter nearest whole mm
When testing Disc
Antimicrobial agent Intermediat
against content Resistant Susceptible
e
ß-LACTAMS
Ampicillin Enterobacteriaceae 10 µg <13 14-16 >17
Staphylococci 10 µg <28 >29
Enterococci 10 µg <16 >17
Carbenicillin Pseudomonas 100 µg <13 14-16 >16
Gram negatives 100 µg <19 20-22 >23
Methicillin Staphylococci 5 µg <9 10-13 >14
Page 46

When testing Disc
e
Mezlocillin Pseudomonas 75 µg <15 >16

Other Gram negatives 75 µg <17 18-20 >21
Nafcillin Staphylococci 1 µg <10 11-12 >13
Oxacillin Staphylococci 1 µg <10 11-12 >13
Penicillin Staphylococci 10 units <28 >29
Enterococci 10 units <14 >15
Piperacillin Pseudomonas 100 µg <17 >18
Ticarcillin Pseudomonas 75 µg <14 >15
ß-LACTAM/ß-
LACTAM/ß-
LACTAMASE INHIBITOR
COMBINATIONS
Amoxycillin/clavulan Staphylococci 20/10 µg <19 >20
ic acid
Other organisms 20/10 µg <13 14-17 >18
Ampicillin/sulbacta Staph & Gram -ve 10/10 µg <11 12-14 >15
m
Piperacillin/tazobact Pseudomonas 100/10 µg <17 >18
am
Other Gram negatives 100/10 µg <17 18-20 >21
Staphylococci 100/10 µg <17 >18
Ticarcillin/clavulanic Pseudomonas 75/10 µg <14 >15
acid
Other Gram negatives 75/10 µg <14 15-19 >20
Staphylococci 75/10 µg <22 >23
CEPHEMS
Cefamandole 30 µg <14 15-17 >18
Cefazolin 30 µg <14 15-17 >18
Cefotaxime 30 µg <14 15-22 >23
Cefoxitin 30 µg <14 15-17 >18
Ceftazidime 30 µg <14 15-17 >18
Ceftizoxime 30 µg <14 15-19 >20
Ceftriaxone 30 µg <13 14-20 >21
Cefuroxime oral 30 µg <14 15-22 >23
Cefuroxime 30 µg <14 15-17 >18
parentral
Cephalothin 30 µg <14 15-17 >18
CARBAPENEMS
Page 47

When testing Disc
e
Imipenem 10 µg <13 14-15 >16

MONOBACTAMS
Aztreonam 30 µg <15 16-21 >22
GLYCOPEPTIDES
Telcoplanin 30 µg <10 11-13 >14
Vancomycin Enterococci 30 µg <14 15-16 >17
Other Gram positives 30 µg <9 10-11 >12
AMINOGLYCOSIDES
Amikacin 30 µg <14 15-16 >17
Gentamicin except high resistant 10 µg <12 13-14 >15
enterococci
Kanamycin 30 µg <13 14-17 >18
Netilmycin 30 µg <12 13-14 >15
Streptomycin 10 µg <11 12-14 >15
Tobramycin 10 µg <12 13-14 >15
MACROLIDES
Azithromycin 15 µg <13 14-17 >18
Clarithromycin 15 µg <13 14-17 >18
Erythromycin 15 µg <13 14-22 >23
TETRACYCLINES
Doxycycline 30 µg <12 13-15 >16
Minocycline 30 µg <14 15-18 >19
Tetracycline 30 µg <14 15-18 >19
QUINOLONES
Ciprofloxacin 5 µg <15 16-20 >21
Enoxacin 10 µg <14 15-17 >18
Lomefloxacin 10 µg <18 19-21 >22
Nalidixic acid 30 µg <13 14-18 >19
Norfloxacin 10 µg <12 13-15 >16
Ofloxacin 5 µg <12 13-15 >16
OTHERS
Chloramphenicol 30 µg <12 13-17 >18
Clindamycin 2 µg <14 15-20 >21
Nitrofurantoin 300 µg <14 15-16 >17
Rifampin 5 µg <16 17-19 >20
Sulfonamides 250/300 <12 13-16 >17
µg
Page 48

When testing Disc
e
Trimethoprim 5 µg <10 11-15 >16

Trimethoprim/ 1.25/ <10 11-15 >16
sulfamethoxazole 23.75 µg
* For Vibrio cholerae, the results of disc diffusion tests for ampicillin, tetracycline and
trimethoprim/sulfamethoxazole correlate with results obtained by broth microdilution methods.
Results
The result of the susceptibility test, as reported to the clinician, is the
classification of the microorganism in one of two or more categories of
susceptibility. The simplest system comprises only two categories,
susceptible and resistant. This classification, although offering many
advantages for statistical and epidemiological purposes, is too inflexible for
the clinician to use. Therefore, a three-category classification is often
adopted. The Kirby-Bauer method and its modifications recognize three
categories of susceptibility and it is important that both the clinicians and the
laboratory workers understand the exact definitions and the clinical
significance of these categories.
➢ Susceptible: An organism is called “susceptible” to a drug when the

infection caused by it is likely to respond to treatment with this drug,
at the recommended dosage.
➢ Intermediate susceptibility covers two situations. It is applicable to

strains that are “moderately susceptible” to an antibiotic that can be
used for treatment at a higher dosage because of its low toxicity or
because the antibiotic is concentrated at the focus of infection (e.g.
urine). The term also applies to those strains that are susceptible to a
more toxic antibiotic that cannot be used at a higher dosage. In this
situation the intermediate category serves as a buffer zone between
susceptible and resistant.
➢ Resistant: This term implies that the organism is expected not to

respond to a given drug, irrespective of the dosage and the location
of the infection.
For testing the response of staphylococci to benzylpenicillin, only the

categories ‘susceptible’ and ‘resistant’ (corresponding to the production of β-
lactamase) are recognized. Staphylococci that are resistant to methicillin or
oxacillin are also resistant to other penicillins or cephalosporins even though
they show a zone of inhibition against these drugs.
Zone diameters, to the nearest whole mm for various antimicrobial

agents with disc content specified for each one for interpretation as
susceptible, intermediate and resistant are given in Table 2.
Page 49
Quality assurance in susceptibility test

The precision and accuracy of the test are controlled by the parallel use of a
set of control strains, with known susceptibility to antimicrobial agents.
These quality control strains are tested using exactly the same procedure as
for the test organisms. The zone sizes shown by the control organisms
should fall within the range of diameters given in Table 3. When the results
regularly fall outside this range, they should be regarded as evidence that a
technical error has been introduced into the test, or that the reagents are at
fault. Each reagent and each step in the test should then be investigated until
the cause of the error has been found and eliminated.
The quality assurance programme should use standard reference strains

of bacteria that are tested in parallel with the clinical culture. They should
preferably be run every week, or with every fifth batch of tests, and in
addition, every time that a new batch of Mueller-Hinton agar or a new batch
of discs is used. The standard strains are:
Staphylococcus aureus (ATCC 25923)

Escherichia coli (ATCC 25922)
Pseudomonas aeruginosa (ATCC 27853)
Culture for day-to-day use should be grown on slants of nutrient agar

(tryptic soya agar is convenient) and stored in a refrigerator. These should be
subcultured onto fresh slants every two weeks.
Table 3: Quality Control – Susceptibility of Control Strains

Zone diameter of inhibition (mm)
Disc potency S.aureus E.coli P.aeruginosa
Antibiotic
(IU or µg) (ATCC 25923) (ATCC25922) (ATCC 27853)
Amikacin 30 20-26 19-26 18-26

Ampicillin 10 27-35 16-22 -
Ceftriaxone 30 22-28 29-35 17-23
Cephalothin 30 29-37 15-21 -
Chloramphenicol 30 19-26 21-27 -
Ciprofloxacin 5 22-30 30-40 25-33
Clindamycin 2 24-30 - -
Erythromicin 15 22-30 - -
Gentamicin 10 19-27 19-26 16-21
Nalidixic acid 30 - 22-28 -
Nitrofurantoin 300 18-22 20-25 -
Norfloxacin 10 17-28 28-35 -
Oxacillin 1 18-24 - -
Penicillin G 10 26-37 - -
Page 50
Disc potency S.aureus E.coli P.aeruginosa

Antibiotic
(IU or µg) (ATCC 25923) (ATCC25922) (ATCC 27853)
Piperacillin 100 - 24-30 25-33

Tetracycline 30 19-28 18-25 -
Tobramycin 10 19-29 18-26 19-25
Trimethoprim 5 19-26 21-28 -
Trimethoprim- 25 24-32 24-32 -
sulfamethoxazol
e
Biosafety
➢ Observe good laboratory practices as outlined in Chapter 8.
➢ Antimicrobial susceptibility of organisms such as Yersinia pestis and
Bacillus anthracis should be performed under BSL-3 environment.
➢ The culture plates with organisms such as Bacillus anthracis be

appropriately autoclaved after chemical treatment with hypochlorite
solution before disposal.
Referral
➢ In the event of disease outbreak the susceptibility results must be
verified by the reference laboratory.
➢ When unusual susceptibility patterns are encountered.

➢ As a part of quality assurance programme.
When intermediate laboratories need not

undertake suscpetibility tests?
➢ In organisms with predictable susceptibility patters e.g. Streptococcus
haemolyticus, Corynebacterium diphtheriae and Treponema pallidum
infections.
➢ In cases where susceptibility tests are technically more demanding

such as Mycobacterium tuberculosis and Neisseria spp.
Salient features of quality assurance in

antibiotic susceptibility testing
➢ Use antibiotic discs of 6 mm diameter.
➢ Use correct content of antimicrobial agent per disc.
Page 51
➢ Stock the supply of antimicrobial discs at -200C.

➢ Use Mueller-Hinton medium for antibiotic sensitivity determination.
➢ Use appropriate control cultures.
➢ Use standard methodology for the test.
➢ Use coded strains from time to time for internal quality control.
➢ Keep the antibiotic discs at room temperature for one hour before
use.
➢ Incubate the sensitivity plates for 16-18 hours before reporting.

➢ Incubate the sensitivity plates at 350C.
➢ Space the antibiotic discs properly to avoid overlapping of inhibition
zone.
➢ Use inoculum size that produces near confluent growth.

➢ Ensure an even contact of the antibiotic disc with the inoculated
medium.
➢ Measure the zone sizes precisely.

➢ Interpret the zone sizes by referring to standard charts.
Further reading
NCCLS: Performance Standards for Antimicrobial Susceptibility Testing: Eighth
Informational Supplement.M100-S8, Vol 18 No 1 January 1998, NCCLS, Pennysylvania,
USA.
Page 52
8. Safety in Laboratories
Laboratory safety is a vital component of functioning of any laboratory. Safety
procedures and precautions to be followed in the microbiology laboratory
should be designed to:
➢ Restrict microorganisms present in specimens or cultures to the

vessels in which they are contained.
➢ Prevent environmental microorganisms (normally present on hand,

hair, clothing, laboratory benches or in the air) from entering
specimens or cultures and interfering with the results of the studies.
Laboratory biosafety levels

Four biosafety levels have been recommended based on the infectiousness of
the agent/s.
Biosafety Level -1 (BSL-1): Adherance to standard microbiological practices.

No special requirement as regards containment equipment.
Biosafety Level-
Level-2 (BSL-2): In addition to the use of standard microbiological
practice, laboratory coats, decontamination of infectious wastes, limited
access, protective gloves and display of biohazard sign and partial
containment equipment are the requirements for this level.
Most peripheral and intermediate laboratories need BSL-1 or BSL-2

laboratory facilities.
BSL-
BSL-3: In addition to BSL-2, it has special laboratory clothing, controlled
access to laboratory and partial containment equipment.
BSL-
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-

BSL-2
➢ Laboratory should be designed in such a way that it can be easily
cleaned.
➢ Laboratory contains a sink for washing.
➢ Laboratory tops are impervious to water but resistant to acids,
alkalies and organic solvents.
Page 53
➢ 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 handwashing and cleaning of bench tops.
➢ Ensure use of gloves.
➢ Provide mechanical pipetting devices.
➢ Protect patients from laboratory personnel with skin or upper
respiratory tract infections.
➢ Provide special disposal containers for needles and lancets.
Pipetting
Pipetting and suctioning have been identified as the significant and consistent
causes of occupational infections. Various important precautions that must be
taken while pipetting are:
➢ Develop pipetting techniques that reduce the potential for creating

aerosols.
➢ 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 suitable

disinfectant for complete immersion.
Page 54
A variety of pipettes are available. Selection should depend upon 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 blood from
patients or performing experiments on laboratory animals are among the
most common causes of occupational infections in laboratories and health
care facilities. They account for almost 25% of the laboratory-acquired
infections that occur by accidents. The practices which are recommended for
hypodermic needle and syringes are:
➢ Avoid quick and unnecessary movements of the hand holding the

syringe.
➢ Examine glass syringes for chips and cracks, and examine needles for
barbs and plugs.
➢ Use needle locking (Luer Lock type) syringes only and be sure that
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 pledget
moistened with suitable disinfectant.
➢ Do not use syringe to forcefully expel infectious fluid into an open

vial for mixing. Mixing with a syringe is appropriate only if the tip of
the needle is held below the surface of the fluid in the tube.
➢ Do not bend, shear, recap or remove the needle from syringe by

hand.
➢ Place used needle-syringe units directly into a puncture-resistant

container and decontaminate before disassembly, reuse or disposal.
Opening containers
The opening of vials, flasks, petri dishes, culture tubes, embryonated eggs,
and other containers of potentially infectious materials poses 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
personnel who are knowledgable about occupational infection risks. Various
precautions that can be taken in this regard are:
➢ Open containers with clinical specimens in well-lighted and

designated areas only.
Page 55
➢ 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 in safety

cabinets.
Tubes containing bacterial cultures should be handled with care.

Vigorous shaking of liquid cultures creates a heavy aerosol. When a sealed
ampoule containing a lyophilized or liquid culture is opened, an aerosol may
be created. Ampoules should be opened in a safety cabinet.
Laboratory access
➢ As far as possible children and pregnant women visitors should not
enter the microbiological laboratories.
➢ Appropriate signs should be located at points of access to laboratory

areas directing all visitors to a receptionist or receiving office for
access procedures.
➢ The universal biohazard symbol (Fig 1) shall be displayed at specific

laboratories in which manipulations of organisms with moderate and
heavy risk are being carried out. Only authorized visitors shall enter
the laboratory showing universal biohazard sign. Doors displaying
biohazard symbol shall not be propped open, but shall remain closed
when in use.
Figure 1: Universal biohazard sign
Clothing
➢ All employees and visitors in microbiological laboratories shall wear
laboratory clothing and laboratory shoes or shoe covers.
Page 56
➢ Disposable gloves shall be worn wherever radiological, chemical,

carcinogenic materials or virus preparations of moderate to high risk
are handled.
➢ Laboratory clothings including shoes shall not be worn outside the

work area.
Accidents in laboratory
In the microbiological laboratory, bacterial infections pose the most frequent
risk. The important diseases/organisms are:
Hepatitis B virus Shigella spp.

HIV Salmonella spp. including S typhi
Brucella spp. Bacillus anthracis
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.
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.
Decontamination of cuts\
cuts\eyes.These
eyes. are irrigated with water taking care to
prevent the spread of contamination from one area to another.
Decontamination of clothing.Contaminated
clothing. garments should be removed
immediately and placed in a container. They should not be removed from the
spill location until contamination has been monitored.
Decontamination of work surfaces

➢ Flood the total spillage area including the broken container with
disinfectant.
➢ Leave undisturbed for 10 minutes.

➢ Mop with cotton wool or absorbent paper.
Page 57
➢ Wear disposable gloves, apron and goggles.

➢ If a dustpan and brush or forceps have been used these too require
disinfection.
➢ 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 viral
decontamination.
➢ Place all potentially contaminated materials in a separate container

and retain until monitored.
➢ Restrict the entry to such an area until contamination monitoring has

been carried out.
Management of laboratory accidents

An adequately equipped first-aid box should be kept in the laboratory in a
place that is known and accessible to all members of 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 chart giving the immediate treatment of cuts,
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 all 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 glasswares.
➢ Do not wander about the laboratory; uncontrolled activities cause:
– accidents
– distract others
– promote contamination
➢ Do not place contaminated pipettes on the bench top.

➢ Do not discard contaminated cultures, glasswares, pipettes, tubes or
slides in wastepaper basket or garbage can.
Page 58
➢ Avoid dispersal of infectious materials.

➢ Operate centrifuges, homogenizer and shakers safely.
➢ Immunize the laboratory workers against vaccine-preventable
diseases such as hepatitis B, meningococcal meningitis, rabies, etc.
Further reading
1. Guidelines for Preventing HIV, HBV and other Infections in the Health Care Setting,
SEARO WHO, Delhi 1996.
2. World Health Organization: Laboratory biosafety manual, 2nd Edition, WHO, 1993.
3. Miller B.M. (et al). Laboratory safety: principles and practices, Washington DC,
American Society for Microbiology, 322, 1986.
Page 59
9. Quality Assurance
Quality Assurance (QA) is a wide ranging concept covering all matters that
individually or collectively influence the quality of a product. It denotes a
system for continuously improving reliability, efficiency and utilization of
products and services. In the context of quality assurance two important
definitions need to be clearly understood:
(i) Internal Quality Control (IQC): which denotes a set of procedures

undertaken by the staff of health facility (medical, paramedical
workers as well as laboratorians) for continuously and concurrently
assessing laboratory work so that quality results are produced by the
laboratory for supporting quality health care at patient and
community levels.
(ii) External Quality Assessment (EQA): is a system of objectively

assessing the laboratory performance by an outside agency. This
assessment is retrospective and periodic but is aimed at improving
the IQC.
IQC and EQA are complementary in ensuring the reliability of the

procedures, results and quality of the product.
What is the objective of QA?

QA programmes are required for the following reasons:
➢ To improve the quality of health care.

➢ To generate reliable, reproducible results.
➢ To establish inter-laboratory comparability in laboratory testing.
➢ To establish the credibility of the laboratory among doctors and the
public at large.
➢ Motivating the staff for further improvement.

➢ Prevention of legal complications which may follow poor quality
results.
Page 59
Factors affecting the quality

It is commonly believed that the quality of laboratory results solely depends
upon the laboratory undertaking this analysis. However, there are many pre-
analytical and post-analytical factors which influence the quality of the end
results to a very significant extent. The principle of “GIGO” – “Garbage in
Gabage Out” very well applies to the laboratory tests also. Some of the
important factors influencing quality are listed here:
(i) Specimen: This is the single most important factor. Selection of the
right sample, collection in a right manner, adequate quantity, proper
transportation to the laboratory, and processing of the sample before
testing, are crucial factors.
(ii) Personnel: The quality of the laboratory results generated is directly

proportional to the training, commitment and motivation of the
technical staff.
(iii) Environmental factors: Inadequate lighting, workspace or

ventilation or unsafeworking conditions may influence the laboratory
results.
(iv) Analytical factors: The quality of reagents, chemicals,

glassware, stains, culture media,use of standard procedures and
reliable equipment all influence laboratory results. Failure to examine
a sufficient number of microscope fields can lead to false negative
results.
(v) Post analytical factors: Transcription errors, incomplete reports, and

improper interpretation can adversely influence the laboratory results.
IQC – the mainstay of QA

The backbone of a good quality assurance programme is a good IQC.
Intermediate and peripheral laboratories must put in place various IQC
procedures and may participate in any EQAS that is in operation.
Requirements of IQC
➢ Comprehensive: Cover all steps from collection of sample to
reporting.
➢ Regular continuous monitoring.

➢ Rational: Focus more on critical factors.
➢ Practical: Should not atttempt to evaluate everything.
➢ Economical: Should be cost-effective and within the provided budget.
Page 60
Each laboratory should have Standard Operating Procedure Manuals

(SOPMs) which should include the following information about the
infrastructure of a laboratory
➢ Biosafety precautions
➢ Disposal of infectious waste
➢ Collection, transport and storage of specimens
➢ Criteria of rejection of samples
➢ Processing of specimens
➢ Maintenance of equipment
➢ Recording of results
➢ Reporting of results
➢ Procedure of quality control
➢ Referral
SOPMs should be periodically reviewed and revised and religiously
followed in the laboratories.
Most of the above-mentioned factors have been described in various

chapters of this document. The remaining have been presented briefly here.
Maintenance of equ
equipment
ipment
Good quality equipment is absolutely essential to generate quality results.
Care of the equipment purchased is also crucial. The quality control steps for
some of the commonly-used equipment at the intermediate/peripheral
laboratory level is depicted in Table 1.
Table 1: Suggested maintenance of commonly

commonly--used equipment
Equipment Maintenance Instructions
Autoclave Clean and change water monthly
Adjust water level before each run
Record time, temperature and pressure for each run
Inspect gasket in the lid weekly
Technical inspection every six months
Incubator Clean inside walls once in a month

Record temperature at the start of each working day
Technical maintenance every six months
Hot air oven Clean the inside at least once a month

Record time and temperature with every run
Technical inspection every six months
Page 61
Equipment Maintenance Instructions

Microscope Wipe lenses with lens paper at the end of each day’s
work
Protect the microscope from dust, vibrations and
moisture
Place a shallow plate containing dry blue silica gel in
a box to absorb moisture
Check alignment of the condenser once a month
Technical inspection once in a year
Balance Keep the balance and weights clean and dry

Always use a container or weighing paper, do not
put material directly on the pan
Prevent the balance from drafts of air
Refrigerator Place at least 10 inches away from the wall

Clean and defrost at least every two months
Record temperature daily
Technical service at least once a year
Water Bath Check water level daily

Check temperature before and during use
Clean monthly
Technical inspection once in six months
Inspissator Check temperature daily

Clean after each batch of culture media prepared
Centrifuge Wipe inner walls with antiseptic solution weekly

Check brushes and bearings every six months
Glassware Discard chipped glassware

Ensure these are free of detergents
Do not store sterile glassware for more than three
weeks before it is used.
Performance tests on culture media

Culture media may be prepared from the individual ingredients or may be
prepared from dehydrated powders available commercially. The important
points in QC of media are listed here:
➢ Do not over-stock the media. Store the required quantities only which
can be used in 6-12 months.
Page 62
➢ Store the media away from moisture by securing the caps of all the
containers tightly.
➢ Store in a dark, cool and well-ventilated place.

➢ Keep a record of the receipt, and opening of the media container.
➢ Discard all dehydrated media that are either darkened or caked.
Rotate the stock of media, following the principle of “first in, first
out”.
➢ For preparation of media adhere strictly to the manufacturer’s

instructions.
➢ Prepared media should be protected from sunlight and heat.

➢ Sterility testing, performance testing and pH test of the prepared
media should be done as listed in Table-2.
Table 2: Performance tests on commonly

commonly--used media
Medium Incubation Control Organism Expected Result
Blood Agar 24h, CO2 S. aureus Growth and beta-
haemolysis
S.pneumoniae Growth and alpha-
haemolysis
Chocolate agar 24h, CO2 H.influenzae Growth
MacConkey agar 24h E.coli Red colonies
With crystal violet
P.mirabilis Colourless colonies
(no swarming)
E.faecalis No growth
Methyl 48h E.coli Positive/negative
red/Voges-
Proskauer
K.pneumoniae Negative/positive
Mueller-Hinton 24h E.coli ATCC 25922 Acceptable zone

sizes
P.aeruginosa ATCC Acceptable zone
27853 sizes
Peptone water 24h E.coli Positive
(indole)
K.pneumoniae Negative
Simmons citrate 48h E.coli No growth
(incubate with
loose screwcap)
K.pneumoniae Growth, blue colour
Thiosulfate 24h Vibrio spp. (non Yellow colonies

citrate bile salt agglutinable
(TCBS) agar
Thayer Martin 24h, CO2 N.meningitidis Growth
Agar
N.gonorrhoeae Growth
Page 63
Staphylococci No growth
E.coli No growth
Quality control for commonly-

commonly-used tests
The QC for commonly-used tests at the intermediate/peripheral level is listed
in Table 3.
Quality control of immunological tests

Quality control procedures used for the detection of antigen or antibodies by
various test methods are listed in Table 4.
QA of antibiotic susceptibility testing

For details refer to chapter 7.
Table 3:: QC for commonly

commonly--used tests
Procedure/result Test Control organism Expected reaction

Catalase S. aureus + Bubbling reaction
Streptococcus spp. – No bubbling
Coagulase S. aureus + Clot formation in 4 hours
Indole E. coli + Red ring at surface
E. aerogenes – Yellow ring at surface
Methyl red E. coli + Instant red colour
E.. aerogenes – No colour change
Oxidase P.aeruginosa + Purple colour in 20 seconds
E. coli – No colour in 20 seconds
Voges E. aerogenes + Red colour
Proskauer E. coli – No colour change
Bacitracin disc Streptococcus + Zone of inhibition

group A
E. faecalis – Zone of inhibition
Optochin disc S. pneumoniae + Zone of inhibition
S. viridans – No zone of inhibition
Oxidase disc P.aeruginosa + Purple colour in 30 seconds
E. coli – No change in colour

The testing should be done each time a new batch of working solution is prepared.
Page 64
Table 4: QC procedures for immunological tests
Test Control procedures Expected results

required
1. Flocculation test (RPR) Nonreactive serum control No clumping
Weakly reactive serum Clumping of graded

control activity
Reactive serum control Clumping of graded

activity
2. Latex agglutination test Negative control serum No clumping

(ASO)
Positive control serum Clumping
3. Direct agglutination Antigen control No clumping

(Widal test)
Negative control serum No clumping
Positive control serum Clumping
4. Capsular Quellung Pneumococci Capsular swelling

reaction (Omni serum,
Haemolytic streptococci No reaction
H.influenzae type b)
H.influenzae type b Capsular swelling
Acinetobacter anitratum No reaction
In service training of staff

Periodic updating of the skills and knowledge of the laboratory workers is
essential for maintaining quality. Course-curriculum of such trainings should
focus on the issues highlighted above.
Participation in external quality assessment

Participation in EQAS reassures about the correctness of the results generated
by the laboratory and finds out whether IQC is in place or not. The control or
referral laboratories should organise EQAS in some commonly used tests.
Further reading
1. Sudarshan Kumari, Rajesh Bhatia, CC Heuck: Quality Assurance in Bacteriology &
Immunology WHO Regional Publication, South-East Asia Series No. 28, 1998.
2. Bhatia Rajesh & lchhpujani R.L.: Quality Assurance in Microbiology CBS Publishers
and Distributors, New Delhi, 1995.
3. Howaritz PJ, Kowanitz JH: Laboratory quality assurance (McGraw Hill Book
Companry, New York (1987).
Page 65

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SEA/HLM/324

© World Health Organization 2000

Quality assurance in laboratory services, aimed at improving reliability,

The publication contains guidelines on the use of conventional

It is hoped that this publication will be useful in achieving its objective of

SECTION A: GENERAL LABORATORY PRACTICES

1 Organization and Functions of Laboratories 1

2 Collection and Transportation of Clinical

6 Cultivation of Bacteria on Laboratory Media 39

7 Antimicrobial Susceptibility Testing 43

General laboratory directions for safety .................................................................... 58

SECTION B: STANDARD PROCEDURES FOR SPECIFIC

Isolation of the organism.......................................................................................... 79

Culture for M. tuberculosis ....................................................................................... 11

22 Bacteriological Examination of Water 14

SECTION C: COLLECTION AND TRANSPORTATION

23 Bacterial Food Poisoning 15

24 Acquired Immunodeficiency Syndrome 15

27 Dengue Fever and Dengue Haemorrhagic Fever 17

Cen tral o r N atio n al Referen ce Lab o rato ry

Regio n al Regio n al Regio n al

D istrict Lab D istrict Lab D istrict Lab

Perip h eral Lab . Perip h eral Lab . Perip h eral Lab .

Peripheral laboratory services

➢ supply of safe water

Equipment and supplies

The tests to be performed by peripheral laboratories are subject to the

Table 1: Suggested tests to be performed at peripheral laboratories

Rapid diagnostic tests HIV

1. Laboratory support to clinical diagnosis/public health

2. Supervision and monitoring of peripheral laboratories

Intermediate laboratories help in the diagnosis and treatment of the

➢ Collection, storage and analysis of data.

Since it may not be possible to have a full-time epidemiologist, at least

2. Kumari S, Sharma KB et al. Health Laboratory Services in support of Primary Health

➢ Collection of the specimen before the administration of antimicrobial

➢ Prevention of contamination of the specimen with externally present

General rules for collection and transportation of specimens are

Table1: Collection and transportation of specimens

• Apply strict aseptic techniques throughout the procedure.

Criteria for rejection of specimens

➢ Missing or inadequate identification.

➢ Collect blood during paroxysm of fever since the number of bacteria

➢ In the absence of antibiotic administration, 99% culture positivity can

➢ Small children usually have higher number of bacteria in their blood

Table 2:: Volume of blood to be collected at different ages

Cerebrospinal fluid (CSF)

➢ Collect CSF before antimicrobial therapy is started.

➢ Do not delay transport and laboratory investigations.

➢ CSF is a precious specimen, handle it carefully and economically. It

➢ Perform physical inspection immediately after collection and indicate

➢ Store at 37oC, if delay in processing is inevitable.

The characteristics of the appearance of CSF are outlined in Table 3.

Table 3: Appearance and interpretations of CSF

➢ Select a good wide-mouthed sputum container, which is preferably

➢ Give the patient a sputum container with the laboratory serial

➢ Instruct the patient to inhale deeply 2-3 times, cough up deeply

➢ Make sure the sputum sample is of good quality. A good sputum

Give the patient an additional container with laboratory serial number