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District

Laboratory

Practice in

Tropical

Countries

Part 2

Second Edition

Monica Cheesbrough

CAMBRIDGE UNIVERSITY PRESS

Cambridge, New York, Melbourne, Madrid, Cape Town, Singapore, São Paulo

Cambridge University Press

The Edinburgh Building, Cambridge CB2 8RU, UK

First published in print format

ISBN-13 978-0-521-67631-1

ISBN-13 978-0-511-34842-6

Every effort has been made in preparing this book to provide accurate and up-to-date

information which is in accord with accepted standards and practice at the time of

publication. Nevertheless, the authors, editors and publisher can make no warranties that

the information contained herein is totally free from error, not least because clinical

standards are constantly changing through research and regulation. The authors, editors

and publisher therefore disclaim all liability for direct or consequential damages resulting

from the use of material contained in this book. Readers are strongly advised to pay careful

attention to information provided by the manufacturer of any drugs or equipment that

they plan to use.

2006

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iii
Chapter 7 Microbiological tests
7.1 Microbiology  practice and quality assurance in district laboratories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pages 1–9
7.2 Features and  classiﬁcation of microorganisms of medical importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9–35
7.3 Microscopic al techniques used in microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35–45
7.4 Culturing  bacterial pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45–62
7.5 Biochemic al tests to identify bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62–70
7.6 Examination  of sputum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71–76
7.7 Examination  of throat and mouth specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76–79
7.8 Examination  of pus, ulcer material and skin specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80–85
7.9 Examination  of effusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85–90
7.10 Examination of urogenital specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90–97
7.11 Examination of faecal specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97–105
7.12 Examination of urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105–115
7.13 Examination of cerebrospinal ﬂuid (c.s.f.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116–124
7.14 Culturing blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124–130
7.15 Examination of semen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130–132
7.16 Antimicrobial susceptibility testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132–143
7.17 Water-related diseases and testing of water supplies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143–157
7.18 Summary of the clinical and laboratory features of microorganisms
Bacterial pathogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157–234
Fungal pathogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234–247
Viral pathogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248–266
COLOUR SECTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . between p. 266 and p. 267
Chapter 8 Haematological tests
8.1 Haematology  in district laboratories and quality assurance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268–271
8.2 Functions of  blood, haematopoiesis and blood disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271–295
8.3 Colle ction of blood. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295–299
8.4 Measurement  of haemoglobin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299–309
8.5 PCV  and red cell indices. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309–313
8.6 Counting  white cells and platelets. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313–319
8.7 Blood  ﬁlms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319–329
8.8 Erythrocyte  sedimentation rate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329–331
8.9 Reticulocyte  count. Methaemoglobin reduction test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331–334
8.10 Investigation of sickle cell disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334–340
8.11 Investigation of bleeding disorders. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340–347
Chapter 9 Blood transfusion tests
9.1 Blood  transfusion services at district level and quality assurance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348–351
9.2 Blood  donation and storage of blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352–361
9.3 Blood  grouping. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362–369
9.4 Compatibility  testing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369–378
Recommended Books. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
Details of Part 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
Appendix I
Preparation of reagents and culture media. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382–407
Appendix II
Useful addresses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410–416
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417–434
Part 2
Contents

Preface

Since the publication of the ﬁrst edition of Part 2 District Laboratory Practice in Tropical Countries in 2000,

the work of many district laboratories continues to be dominated by the on-going HIV/AIDS pandemic,

increases in the prevalence of tuberculosis and other HIV-related infections and more recently, the

requirement for laboratory monitoring of antiretroviral therapy.

This new edition includes an update on HIV disease/AIDS, recently developed HIV rapid tests to diagnose

HIV infection and screen donor blood, and current information on antiretroviral drugs and the laboratory

monitoring of antiretroviral therapy.

Information on the epidemiology and laboratory investigation of other pathogens has also been brought up

to date. Several new, rapid, simple to perform immunochromatographic tests to assist in the diagnosis of

infectious diseases are described, including those for brucellosis, cholera, dengue, leptospirosis, syphilis and

hepatitis. Recently developed IgM antibody tests to investigate typhoid fever are also described. The new

classiﬁcation of salmonellae has been introduced.

Details of manufacturers and suppliers now include website information and e-mail addresses. Websites are

also included that provide up to date information on water and sanitation initiatives, and diseases such as

tuberculosis, cholera, leptospirosis, mycetoma, HIV/AIDS and other sexually transmitted infections.

Where required the haematology and blood transfusion chapters have been updated, including a review of

haemoglobin measurement methods in consideration of the high prevalence of anaemia in developing

countries.

It is hoped that this new edition of Part 2 and recently published second edition of Part 1 District

Laboratory Practice in Tropical Countries will continue to help and motivate those working in district

laboratories and those responsible for the management of district laboratory services, training and

continuing education of district laboratory personnel.

Monica Cheesbrough, November 2005

Acknowledgements

The author wishes to thank all those who have corresponded and contributed their suggestions for this

second edition Part 2 District Laboratory Practice in Tropical Countries, particularly those working in district

laboratories and training laboratory personnel in tropical and developing countries.

Gratitude and thanks are also due to those who have helped to prepare the new edition:

Mr Steven Davies, Microbiology Specialist Advisor, Institute of Biomedical Sciences (IBMS) for reading

through and commenting on the microbiology chapter and contributing text on antimicrobials and the

Etest. Also acknowledged for their suggestions are Mr Stephen Mortlock, Member of the IBMS

Microbiology Advisory Panel and Mr Mark Tovey, Microbiology Department, Shefﬁeld.

Mr Simon Hardy, Senior Lecturer in Microbiology, University of Brighton, for also assisting in the revision

of the microbiology chapter.

Dr Eric Bridson, Microbiologist, for reading through the text and checking microbial nomenclature.

Dr Mohammed Toﬁq, NMK Clinic, for corresponding with the author and making suggestions for the

microbiology text.

In the preparation of the text covering laboratory monitoring of antiretroviral therapy, gratitude is

expressed to Dr Jane Carter, AMREF, Nairobi, Dr Steve Gerrish, Kara Clinic, Lusaka, Major Peter Disney,

Tshelanyemba Hospital, Zimbabwe and Mr Derryck Klarkowski, Laboratory Specialist, Médecins Sans

Frontières, for their helpful contributions.

Dr Henk Smits, Molecular Biologist, Biomedical Research Royal Tropical Institute, Amsterdam, for supplying

information on rapid tests for brucellosis and leptospirosis.

Professor Asma Ismail, Director Institute for Research in Molecular Medicine, University Sains Malaysia, for

providing text and artwork for the Typhirapidtest.

Ms M Marilyn Eales, Haematology Tutor, Paciﬁc Paramedical Training Centre, Wellington, New Zealand, for

reading through and commenting on the haematology and blood transfusion chapters.

The author also wishes to thank Fakenham Photosetting for their careful and professional preparation of the

new edition.

Acknowledgements for colour artwork: These can be found on page 267.

MICROBIOLOGICAL TESTS 1

7.1

7.1 Microbiology practice

and quality assurance in district

laboratories

In tropical and developing countries, there is an

urgent need to strengthen clinical microbiology and

public health laboratory services in response to:

 The high prevalence and increasing incidence of

infectious diseases.

HIV disease/AIDS, acute respiratory tract infections

(particularly pneumonia), typhoid, cholera, dysentery,

tuberculosis, meningitis, whooping cough, plague,

sexually transmitted diseases (including gonorrhoea and

syphilis), viral hepatitis, yellow fever, dengue, and viral

haemorrhagic fevers are major infectious diseases that

cause high mortality and serious ill health in tropical and

developing countries. Climatic changes, particularly

global warming and extreme rainfall, are increasing the

distribution of some infectious diseases, especially those

that are mosquito-borne and water-borne.

 The threat posed by the re-emergence and rapid

spread of diseases previously under control or in

decline such as tuberculosis, plague, diphtheria,

dengue, cholera and meningococcal meningitis.

 The emergence of opportunistic pathogens

associated with HIV, new strains of pathogens

such as Vibrio cholerae serotype 0139 and

viruses causing severe acute respiratory

syndrome (SARS) and avian inﬂuenza.

 The rapid rate at which bacterial pathogens are

becoming resistant to commonly available and

affordable antimicrobials.

Drug resistance is causing problems in the treatment and

control of infections caused by pathogens such as

Streptococcus pneumoniae, Haemophilus inﬂuenzae,

Staphylococcus aureus, Pseudomonas aeruginosa,

Neisseria gonorrhoeae, and enterococci. Some strains of

M.tuberculosis have developed multi-drug resistance.

 The need for reliable microbiological data to

develop and validate standard treatments and

control interventions, and ensure antimicrobial

drugs are purchased appropriately and used

correctly.

Infections are particularly prevalent where poverty,

malnutrition, and starvation are greatest, sanitation is

inadequate, personal hygiene poor, water supplies

are unsafe or insufﬁcient, health provision the least

developed, and disease control measures are lacking

or ineffective.

War and famine in developing countries have

greatly increased the number of people that have

become refugees, suffer illhealth and die prema-

turely from infectious diseases.

In rural areas, distances to health centres and

hospitals are often too great to be travelled by

patients or mothers with young children requiring

immunization.

In many countries, increasing urbanization has

resulted in an increase in the incidence of diseases

associated with inadequate and unsafe water, poor

sanitation, and overcrowded living conditions.

In areas of high HIV prevalence, major

pathogens such as M. tuberculosis and Streptococcus

pneumoniae and a range of opportunistic pathogens

associated with immunosuppression, are responsible

for infections, often life-threatening, in those infected

with HIV.

This subunit includes information on:

● Clinical microbiology and public health labora-

tory activities at district level.

● Quality assurance and standard operating

procedures (SOPs) in microbiology.

● Collection of microbiological specimens.

● Safe working practices.

CLINICAL MICROBIOLOGY AND PUBLIC HEALTH

LABORATORYACTIVITIE S AT DISTRICT LEVEL

A network of district microbiology and regional

public health laboratories is needed to provide to the

community, accessible microbiological services.

Microbiological tests

Important: District laboratories require the support of

the regional public health laboratory in the prep-

aration and implementation of microbiological

standard operating procedures (SOPs), safe working

practices, on-site training, quality assurance, and

provision of essential supplies (e.g. reagents, culture

media, controls, antisera).

Operating microbiological laboratory services

with minimal resources

The high cost of culture media and reagents, lack

of a rational approach to the selection and use of

microbiological investigations, and a shortage of

trained technical staff and clinical microbiologists are

important factors in preventing the establishment

and extension of essential microbiological services in

developing countries.

To ensure the optimal use of available resources,

it is important for health authorities to identify those

pathogens of greatest public health importance

2 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.1

which require microbiological investigations based

on a consideration of:

– local disease patterns,

– clinical relevance and frequency of isolation,

– severity of disease and outcome,

– possibility of effective intervention,

– need for surveillance to monitor drug resistance

and epidemic potential,

– cost beneﬁt ratio of isolation and, or, identiﬁ-

cation,

– laboratory capacity and resources available,

– availability of trained personnel to perform

microbiological investigations and ensure the

quality of work and reports.

Such an approach helps to target resources where

they are most needed, enables a list of essential

culture media and diagnostic reagents to be

identiﬁed, sourced and costed, and training in micro-

biology techniques and their application to be more

speciﬁc.

UALITY ASSURANCE AND SOPs IN MICROBIOLOGY

The principles of quality assurance (QA) and general

guidelines on how to prepare standard operating

procedures (SOPs) are described in subunit 2.4 in

Part 1 of the book.

Providing appropriate, reliable and

affordable microbiological services

Laboratory personnel, clinicians, community

health ofﬁcers and sanitary ofﬁcers must work

closely together in deciding the microbiological

services that are required and ensuring the

services provided are appropriate, reliable, and

affordable.

This involves identifying:

● The infectious diseases that require labora-

tory investigation (priority pathogens).

● Role of district laboratories in surveillance

work and the investigation of epidemics.

● Techniques (SOPs) to be used to collect

specimens, identify pathogens and perform

antimicrobial susceptibility tests.

● Most appropriate systems for reporting and

recording the results of microbiological

investigations, collating and presenting data

for surveillance purposes.

● Quality assurance.

● Training requirements, supervision, and on-

going professional support.

● Equipment and microbiological supplies

needed and systems for distribution of

supplies.

● Costs involved.

Need for quality assurance and SOPs in

microbiology

Microbiological investigations are important in

the diagnosis, treatment, and surveillance of

infectious diseases and policies regarding the

selection and use of antimicrobial drugs. It is

therefore essential that test reports:

– are reliable,

– standardized,

– provide the information that is required at

the time it is needed,

– in a form that can be understood.

Quality assurance is also required to minimize

waste and ensure investigations are relevant

and used appropriately.

WHO in its publication Basic laboratory procedures

in clinical bacteriology

states that quality assurance

in microbiology must be:

– comprehensive: to cover every step in the cycle

from collecting the specimen to sending the ﬁnal

report to the doctor as shown opposite;

– rational: to concentrate on the most critical steps

in the cycle;

– regular: to provide continuous monitoring of test

procedures;

– frequent: to detect and correct errors as they

occur.

The following apply to the QA of the pre-analytical,

analytical, and post-analytical stages of microbiologi-

cal procedures and should be incorporated in

microbiological SOPs.

Pre-analytical stage

SOPs need to describe:

● Selection and appropriate use of microbiological

investigations.

● Collection and transport of specimens.

● How to ﬁll in a request form correctly.

● Checks to be made when the specimen and

request form reach the laboratory.

Appropriate use of microbiological investigations

This aspect of QA requires collaboration between

laboratory personnel, clinicians, and public health

ofﬁcers as discussed at the beginning of this subunit.

The fewer the resources the more important it is to

establish priorities based on clinical and public

health needs. Clear guidelines should be provided

on the use and value of speciﬁc microbiological

investigations.

MICROBIOLOGICAL TESTS 3

7.1

Collection and transport of microbiological specimens

Specimens for microbiological investigation must be

collected correctly if pathogens are to be successfully

isolated and identiﬁed, reports are not to be mis-

leading, and resources are not to be wasted. Written

instructions for the collection of specimens must be

issued by the laboratory to all those responsible for

collecting microbiological specimens. The collection

of microbiological specimens is described at the end

of this subunit.

Request form

Each specimen must be accompanied by a request

form which details:

– the patient’s name, age (whether an infant, child,

adult), gender, outpatient or inpatient number,

ward or health centre, and home area/village.

– type and source of specimen, and the date and

time of its collection.

– investigation(s) required.

– clinical note summarizing the patient’s illness,

suspected diagnosis and information on any

antimicrobial treatment that may have been

started at home or in the hospital.

Note: The clinical note will help to report usefully the

results of laboratory investigations.

– name of the medical ofﬁcer requesting the

investigation.

Sampling

Storage

Macroscopic evaluation, odour

Microscopy, interpretation

Culture: choice of medium, temperature, atmosphere

Isolation of pure cultures, antibiogram

Identiﬁcation, interpretation

(contaminant, commensal, or pathogen)

Transport, labelling

PATIENT WITH INFECTION

Specimen, clinical

information

PRELIMINARY REPORT

TO PHYSICIAN

FINAL REPORT TO

PHYSICIAN

Steps in the laboratory investigation of an infected patient.

Reproduced from Basic Laboratory procedures in clinical bacteriology, 2nd edition, 2003, World Health Organization.

Checking a specimen and request form

SOPs should include the procedures to be followed

when specimens reach the laboratory, particularly

checks to ensure that the correct specimen has been

sent and the name on the specimen is the same as

that on the request form. Also included should be

how to handle and store specimens that require

immediate attention, e.g. c.s.f., blood cultures,

unpreserved urine, swabs not in transport media,

faecal specimens containing blood and mucus, and

wet slide preparations.

Examples of specimens which should not be

accepted for microbiological investigations include:

– dry faecal swabs,

– saliva instead of sputum,

– eye swabs that have not been freshly collected,

– any specimen not collected into a correct con-

tainer,

– a leaking specimen (sample may be contami-

nated).

Analytical stage

The following should be included in microbiological

SOPs, covering the analytical stage:

● Detailed procedures for examining different

specimens (described in subsequent subunits).

● Staining techniques and quality control (QC) of

stains (see following text).

● Aseptic techniques and safe handling of infec-

tious material as described in subunit 7.4.

● Preparation and QC of culture media and pres-

ervation of stock strains used in performance

testing (see subunit 7.4).

● Inoculation of broth and agar culture media and

plating out techniques (see subunit 7.4).

● Reading and interpretation of cultures (see

subunit 7.4).

● Techniques used to identify pathogens and the

QC of diagnostic reagents, strips, and discs as

described in subunit 7.5.

● Antimicrobial susceptibility testing and QC

of procedure and discs as described in

subunit 7.16.

● Cleaning and QC of equipment used in the

microbiology laboratory, e.g. microscope, incu-

bator, anaerobic jar, centrifuge, waterbath/heat

block, autoclave, hot-air oven, and refrigerator

(see following text).

● Immunological techniques and QC of antigen

and antibody reagents.

● Safe working practices (see end of this subunit).

4 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.1

● Disposal of specimens and cultures (see subunit

3.4 in Part 1 of the book).

● Cleaning of glassware, plasticware, etc (described

in subunits 3.4 and 3.6 in Part 1 of the book).

● Sterilization procedures and their control (see

subunits 3.4 and 4.8 in Part 1 of the book).

The sterilization of glassware by dry heat is

described in subunit 7.4.

Important: As part of QC, the performance of staff

must be monitored, all techniques must be demon-

strated to new members of staff, the results of QC

tests must be recorded and signed, and the work of

newly qualiﬁed staff supervized (see also subunit 2.4

in Part 1 of the book).

Control of stains and reagents

All stains and reagents must be clearly labelled,

dated, and stored correctly. The preparation, ﬁxation,

staining and reporting of smears as detailed in the

department’s SOPs must be followed exactly. Stains

and reagents should not be used beyond their

expiry date (where this applies) or when they show

signs of deterioration such as abnormal turbidity or

discoloration.

At regular intervals and whenever a new batch of

stain is prepared, e.g. basic fuchsin in the Ziehl-

Neelsen technique or crystal violet in the Gram

technique, control smears of appropriate organisms

should be stained to ensure correct staining reac-

tions. Control smears used in the Ziehl-Neelsen

technique should include smears with few to

moderate numbers of AFB. Smears for controlling

Gram staining can be prepared from a mixed broth

culture of staphylococci and Escherichia coli. All

control smears should be alcohol-ﬁxed and stored in

labelled, dated, airtight containers.

Use efﬁcient (non-leaking), preferably light-proof

stain dispensing containers to avoid stains being

wasted. Ensure containers can be closed when not

in use to avoid evaporation and contamination of

stains.

A common cause of poor staining is attempting

to stain a smear that is too thick, e.g. c.s.f. containing

many pus cells. When a smear is too thick, the decol-

orization process is often incomplete which can

result in Gram negative organisms being reported

as Gram positive. The QC of reagents used in bio-

chemical diagnostic tests is described in subunit 7.5.

Control of equipment

For each item of equipment there should be clear

operating and cleaning instructions, and service

sheets. Regular cleaning, servicing and maintenance

MICROBIOLOGICAL TESTS 5

7.1

care or the management of an epidemic must reach

the clinician or public health ofﬁcer/epidemiologist

as soon as possible. Those receiving the reports

should consult the laboratory when any part of the

report is unclear. Improvement in the quality and

usefulness of microbiological reports will only be

achieved by effective communication between those

requesting tests and laboratory staff.

A record of the results of all investigations must

be kept by the laboratory, e.g. as carbon copies,

work sheets, or in record books. Copies of work

sheets should be dated and ﬁled systematically each

day.

External quality assessment

Whenever possible the regional public health lab-

oratory should organize an external quality

assessment (EQA) scheme to help district microbiol-

ogy laboratories. An EQA scheme should include

testing for major pathogens. It should not be too

complicated, costly, or time-consuming for district

laboratories.

The main objective of an EQA scheme is to

conﬁrm that a laboratory’s SOPs and internal QC

procedures are working satisfactorily. EQA schemes

help to identify errors, improve the quality of work,

stimulate staff motivation, and assure users of the

service that the laboratory is performing to the

standard required to provide reliable results.

WHO advizes that an EQA scheme should

operate monthly or at least four times a year.

Instructions and a report form (to be returned with

results after 1 week) should be sent with the

specimens to each participating laboratory. Each

specimen should be examined in the same way as

routine clinical samples (not recognized as a QC

specimen). The District Laboratory Coordinator

should investigate and assist any poor performing

laboratory and where indicated, arrange for the

further training of staff. Refresher courses should be

held periodically to maintain competence and motiv-

ation and to introduce new tests.

Note:An excellent chapter on quality assurance in microbiol-

ogy can be found in the WHO publication Basic laboratory

procedures in clinical bacteriology.

COLLECTION OF MICROBIOLOGICAL SPECIMENS

The value and reliability of microbiological reports

are directly affected by the quality of the specimen

received by the laboratory and the length of time

between its collection and processing.

The collection of specimens must form part of

the department’s SOPs (see previous text), and the

laboratory should issue written instructions to all

are essential if equipment is to remain in good

working order and safe to use.

The operating temperature of a refrigerator,

incubator, heat block and water-bath should be

monitored and charted daily. Regular checks should

also be made of all glassware and reusable plastic

items to ensure that they are completely clean, not

damaged, and being sterilized correctly. Specimen

containers should be inspected regularly, especially

the caps of bottles and tubes for missing or worn

liners.

The use, care, maintenance, and performance

checks of microscopes are described in subunit 4.3

and of other items of equipment in subunits

4.4–4.12 in Part 1 of the book. Hazards associated

with the use of equipment and glassware are

covered in subunit 3.6 in Part 1. The use and control

of an autoclave are described in subunits 3.4 and

4.8, also in Part 1. The use and control of anaerobic

jars are covered in subunit 7.4.

Post-analytical stage

SOPs need to include:

● Reporting and verifying of microbiological test

results.

● Taking appropriate action(s) when a result has

serious patient or public health implications.

● Interpreting test reports correctly.

Reporting results

The terminology and format used in reporting

microscopical preparations, cultures, and antimicro-

bial susceptibility tests should be standardized and

agreed between laboratory personnel, clinicians, and

public health ofﬁcers. Any preliminary report of

microscopical ﬁndings or isolation of a pathogen

from a primary culture must be followed by a full

written report.

All reports must be concise and clearly pre-

sented. The use of rubber stamps can be helpful in

standardizing the report and making it easy to

understand, e.g. stamps that list the presence or

absence of recognized pathogens or that list the

antibiotics against which an isolate has been tested.

When using a stamp, care must be taken to position

it correctly and sufﬁcient ink must be used to repro-

duce clearly the entire stamp. The reporting of

cultures is discussed in subunit 7.4.

Verifying and interpreting reports

Before leaving the microbiology laboratory, all

reports must be checked for correctness and clarity

and signed by the person in charge of the depart-

ment. Reports which are urgently needed for patient

those responsible for the collection of specimens

from inpatients and outpatients. Such instructions

should include:

 The amount and type of specimen required, con-

tainer to use, and need for any preservative or

transport medium.

 Best time to collect a specimen.

 Aseptic and safe methods of collection to avoid

contamination and accidental infection.

 Labelling of the specimen container.

 Conditions in which specimens need to be kept

prior to and during their transport to the labora-

tory.

 Arrangements for processing specimens that are

urgent and those collected outside of normal

working hours, e.g. blood cultures collected by

medical staff.

Type of specimen

The correct type of specimen to collect will depend

on the pathogens to be isolated, e.g. a cervical not a

vaginal swab is required for the most successful

isolation of N. gonorrhoeae from a woman. Sputum

not saliva is essential for the isolation of respiratory

pathogens.

Time of collection

Specimens such as urine and sputum are best col-

lected soon after a patient wakes when organisms

have had the opportunity to multiply over several

hours. Blood for culture is usually best collected

when a patient’s temperature begins to rise. The

time of collection for most other specimens will

depend on the condition of the patient, and the

times agreed between the medical, nursing, and lab-

oratory staff for the delivery of specimens to the

laboratory.

Important: Every effort must be made to collect

specimens for microbiological investigation before

antimicrobial treatment is started and to process

specimens as soon after collection as possible.

Collection techniques

Detailed instructions on how to collect different

specimens can be found in the subsequent subunits

of this chapter.

The following apply to the collection of most

microbiological specimens:

● Use a collection technique that will ensure a

specimen contains only those organisms from

the site where it was collected. If contaminating

organisms are introduced into a specimen

6 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.1

during its collection or subsequent handling, this

may lead to difﬁculties in interpreting cultures

and delays in issuing reports.

A strictly sterile (aseptic) procedure is essential

when collecting from sites that are normally

sterile, e.g. the collection of blood, cerebrospinal

ﬂuid, or effusions. An aseptic technique is neces-

sary not only to prevent contamination of the

specimen but also to protect the patient.

● Avoid contaminating discharges or ulcer material

with skin commensals. The swabs used to

collect the specimens must be sterile and the

absorbent cotton-wool from which the swabs

are made must be free from antibacterial

substances.

● Collect specimens in sterile, easy to open, leak-

proof, dry containers, free from all traces of

disinfectant. Containers must be clean but need

not be sterile for the collection of faeces and

sputum.

To avoid breakages, whenever possible, con-

tainers made from autoclavable plastic should be

used providing these are leak-proof.*

*Autoclavable plastics used in the manufacture of bottles

include polypropylene, copolymer, polycarbonate, and

polymethylpentene.

The containers given to patients must be easy

for them to use. Patients should be instructed

in the aseptic collection of specimens and

asked to avoid contaminating the outside of

containers.

When contamination occurs, wipe the outside of

the container with a tissue or cloth soaked in dis-

infectant before sending the specimen to the

laboratory.

● Report any abnormal features, such as cloudiness

in a specimen which should appear clear,

abnormal coloration, or the presence of pus,

blood, mucus, or parasites.

The appearance of urine, pus, vaginal discharge,

faeces, effusions, and cerebrospinal ﬂuid should

be described routinely.

Labelling specimens

Each specimen must be clearly labelled with the date

and time of collection, and the patient’s name,

number, ward or health centre.

Slides with one end frosted (area of opaque glass

on which to write) should be used for making

smears so that a lead pencil can be used to label the

slides clearly.

Each specimen must be accompanied by a

correctly completed request form (see previous

text).

Specimens containing dangerous pathogens

Those delivering, receiving, and examining speci-

mens must be informed when a specimen is likely

to contain highly infectious organisms. Such a

specimen should be labelled HIGH RISK, and

whenever possible, carry a warning symbol such as

a red dot, star, or triangle which is immediately rec-

ognized as meaning that the specimen is dangerous

and must be handled with extra care.

Specimens which should be marked as HIGH RISK

include:

● Sputum likely to contain M. tuberculosis.

● Faecal specimen that may contain V. cholerae or

S. Typhi.

● Fluid from ulcers or pustules that may contain

anthrax bacilli or treponemes.

● Specimens from patients with suspected HIV

infection, viral hepatitis, viral haemorrhagic fever,

or plague.

Immediately after collection, a HIGH RISK specimen

should be sealed inside a plastic bag or in a con-

tainer with a tight-ﬁtting lid. The request form must

not be placed in the bag or container with the

specimen.

Note: Because any specimen may contain infectious

pathogens, it is important for laboratory staff to

handle all specimens with adequate safety precau-

tions and to wash their hands after handling

specimens (see also subunits 3.2–3.4 in Part 1 of the

book.

Preservatives and transport media for

microbiological specimens

In general, specimens for microbiological investi-

gationsshould be delivered to the laboratory without

delay and processed as soon as possible. This will

help to avoid the overgrowth of commensals.

When a delay in delivery is unavoidable, for

example when transporting a specimen from a

health centre to a hospital laboratory, a suitable

chemical preservative or transport culture medium

must be used. This will help to prevent organisms

from dying due to enzyme action, change of pH, or

lack of essential nutrients. A transport medium is

needed to preserve anaerobes.

Amies transport medium is widely used and

effective in ensuring the survival of pathogens in

specimens collected on swabs, especially delicate

organisms such as Neisseria gonorrhoeae. Amies

medium is a modiﬁcation of Stuart’s transport

MICROBIOLOGICAL TESTS 7

7.1

medium. Its preparation is described in No. 11

(Appendix I). An example of a preservative is boric

acid which may be added to urine.

Cary-Blair medium is used as a transport

medium for faeces that may contain Salmonella,

Shigella, Campylobacter or Vibrio species (see No.

22).

Note: Preservatives that contain formaldehyde solution, such

as merthiolate iodine formaldehyde (MIF) and formol saline,

must not be used for microbiological specimens because

formaldehyde kills living organisms.

Transport of microbiological specimens

collected in a hospital

As mentioned previously, specimens should reach

the laboratory as soon as possible or a suitable

preservative or transport medium must be used.

Refrigeration at 4–10°C can help to preserve

cells and reduce the multiplication of commensals in

unpreserved specimens. Specimens for the isolation

of Haemophilus, S. pneumoniae, or Neisseriaspe cies,

however, must never be refrigerated because cold

kills these pathogens.

Smears collected by ward staff or in outpatient

clinics for subsequent Gram staining, must be

placed in a safe place to dry, protected from dust,

ants, cockroaches, and ﬂies. The laboratory should

provide wards and outpatient clinics with petri dishes

(unsterile) or other containers in which to place and

transport slide preparations.

Dispatch of microbiological specimens

collected in health centres or district hospitals

without culture facilities

Specimens for dispatch must be packed well and

safely. When specimens are to be mailed, the regu-

lations regarding the sending of ‘Pathological

Specimens’ through the post should be obtained

from the Postal Service and followed exactly. When

dispatching microbiological specimens the following

apply:

● Keep a register of all specimens dispatched.

Record the name, number, and ward or health

centre of the patient, type of specimen, investi-

gation required, date of dispatch, and the

method of sending the specimen (e.g. mailing,

hand-delivery, etc). When the report is received

back from the microbiology laboratory, record

the date of receipt in the register.

● Check that the specimen container is free from

cracks, and the cap is leak-proof. Seal around the

container cap with adhesive tape to prevent loos-

ening and leakage during transit.

● Use sufﬁcient packaging material to protect a

specimen, especially when the container is a

glass tube or bottle (use a plastic container

whenever possible). Place the packaged con-

tainer in a strong protective tin or box, and seal

completely. When the specimen is ﬂuid, use suf-

ﬁcient absorbent material to absorb it should a

leakage or breakage occur.

● Mark all specimens that may contain highly

infectious organisms, ‘HIGH RISK’ (see previous

text). Do not mail such specimens.

● Dispatch slides in a plastic slide container or use

a strong slide carrying box or envelope.

● Label specimens dispatched by mail, ‘FRAGILE

WITH CARE – PATHOLOGICAL SPECIMEN’.

When a specimen is likely to deteriorate unless kept

cool, transport it in an insulated container, such as a

polystyrene box or thermos ﬂask containing ice

cubes. The specimen must be sealed inside a water-

proof bag or tin to prevent the label being washed

off when the ice cubes melt. Precautions must also

be taken to keep the request form dry.

Note: Details on international transport regulations

can be found on p. 66 in Part 1.

Collection of individual specimens

Subunit

Sputum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6

Throat specimens. . . . . . . . . . . . . . . . . . . . . . 7.7

Skin and ulcer specimens . . . . . . . . . . . . . . . 7.8

Skin and nasal smears for leprosy . . . . . . . . 7.18.30

Pus and effusions. . . . . . . . . . . . . . . . . . . . . . 7.9

Urogenital specimens . . . . . . . . . . . . . . . . . . 7.10

Faecal (stool) specimens . . . . . . . . . . . . . . . . 7.11

Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.12

Cerebrospinal ﬂuid (c.s.f.) . . . . . . . . . . . . . . . 7.13

Blood for culture . . . . . . . . . . . . . . . . . . . . . . 7.14

Seminal ﬂuid . . . . . . . . . . . . . . . . . . . . . . . . . 7.15

Note: Detailed and helpful guidelines on the

collection and dispatch of microbiological speci-

mens can be found in the WHO publication

Specimen collection and transport for microbiological

investigations.

Practice of virology in district laboratories

Viruses, particularly HIV, arboviruses, measles virus,

and viruses that cause respiratory and diarrhoeal

disease in young children, are major causes of death

and illness in tropical and developing countries. At

district level most virus diseases are presumptively

8 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.1

diagnosed clinically or remain undiagnosed. It is

usually only at central level that facilities exist for the

laboratory investigation of virus diseases based on

virus isolation, direct demonstration of virus or viral

components, and the serological diagnosis of virus

infections.

Inrecent years, however, rapid, simpleto perform

immunological assays have become available to

diagnose virus diseases such as dengue (see subunit

7.18.53),HIV infection (see subunit 7.18.55), andviral

hepatitis (see subunit 7.18.54). Where appropriate,

affordable, and available, these rapid techniques are

being increasingly used in district laboratories and

regional blood transfusion centres.

When needing to investigate a serious epidemic

caused by Ebola fever virus or other highly infec-

tious virus causing viral haemorrhagic fever, testing

must be performed in a virology laboratory or public

health laboratory having adequate containment

facilities with a specialist public health team (appro-

priately protected) collecting the samples.

Practice of mycology in district laboratories

The medically important fungi are listed in subunit

7.2 and the investigation of common fungal infec-

tions in district laboratories is described in subunits

7.18.38–7.18.52.

AFE WORKING PRACTICES

Health and safety in district laboratories, including

full coverage of microbial hazards, safe working

practices, and the decontamination of infectious

material and disposal of laboratory waste are

described in Chapter 3 in Part 1 of the book.

The following are some of the important points

which apply when working with infectious materials:

● Never mouth-pipette (see p. 63 in Part 1). Use

safe measuring and dispensing devices as

described in subunit 4.6 in Part 1.

● Do not eat, drink, smoke, store food, or apply

cosmetics in the working area of the laboratory.

● Use an aseptic technique when handling speci-

mens and culture (see subunit 7.4).

● Always wash the hands after handling infectious

material, when leaving the laboratory and before

attending patients. Cover any open wound with

a waterproof dressing.

● Wear appropriate protective clothing when

working in the laboratory. Ensure it is decon-

taminated and laundered correctly (see p. 59 in

Part 1).

● Wear protective gloves, and when indicated a

face mask, for all procedures involving direct

contact with infectious materials. When wearing

gloves, the hands should be washed with the

gloves on, particularly before using the tele-

phone or doing clerical work.

● Minimize the creation of aerosols. The common-

est ways infectious aerosols are formed are

detailed on pp. 61–62 in Part 1.

● Centrifuge safely to avoid creating aerosols.

Know what to do should a breakage occur when

centrifuging (see p. 63 in Part 1).

● Avoid practices which could result in needle-stick

injury.

● Do not use chipped or cracked glassware and

always deal with a breakage immediately and

safely (see p. 89 in Part 1).

● Avoid spillages by using racks to hold containers.

Work neatly and keep the bench surface free of

any unnecessary materials.

● Decontaminate working surfaces at the end of

each day’s work and following any spillage of

infectious ﬂuid. Know what to do when a spillage

occurs (see p. 63 in Part 1).

● Report immediately to the laboratory ofﬁcer in

charge, any spillage or other accident involving

exposure to infectious material.

● Know how to decontaminate specimens and

other infectious materials (see pp. 66–74 in

Part 1).

● Use and control an autoclave correctly (see p. 67

and subunit 4.8 in Part 1).

● Disposeof laboratory waste safely (see pp. 66–71

in Part 1).

● Do not overﬁll discard containers. Use appropri-

ate disinfectants (see pp. 67–70 in Part 1). Use

separate containers for ‘sharps’.

● Do not allow unauthorized persons to enter the

working area of the laboratory.

● Ensure technical and auxiliary staff working in

the laboratory receive appropriate immuniza-

tions. Those at increased risk of acquiring

infections, e.g. immunocompromised persons,

should not work in a laboratory handling infec-

tious material.

REFERENCES

1 Vandepitte J et al. Basic laboratory procedures in clinical

bacteriology. WHO, Geneva, 2nd edition, 2003.

Obtainable from WHO Publications, Geneva 1211, 27-

Switzerland.

MICROBIOLOGICAL TESTS 9

7.1–7.2

2 Engbaeck K et al. Specimen collection and transport

for microbiological investigation. WHO Regional

Publications, 1995. ISBN 92–9021–196– 2. Obtainable

from WHO Regional Ofﬁce for the Eastern

Mediterranean, PO Box 7608, Nasr City, Cairo, 11371,

Egypt.

7.2 Features and classiﬁcation

of microorganisms of medical

importance

Most microorganisms are free-living and perform

useful activities that beneﬁt animal and plant life.

Microorganisms that have the ability to cause

disease are called pathogens. They include:

 Bacteria (singular, bacterium)

 Viruses (singular, virus)

 Fungi (singular, fungus)

 Protozoa (singular, protozoon)*

*The protozoa of medical importance are described in

Chapter 5 in Part 1 of the book.

Infection occurs when a pathogen is able to establish

itself in a person. Not all infections, however, result

in clinical infection, i.e. a person falling ill. Frequently

a person displays no symptoms of disease (asymp-

tomatic). Such an infection is referred to as

subclinical.

Virulence is the term used to describe the

degree of pathogenicity of an organism. It is mainly

dependent on the invasiveness and, or, the ability of

the organism to produce toxins (poisonous sub-

stances). The infectiousness or communicability of

an organism refers to its capacity to spread.

Epidemiology is the study of the spread, distribution,

prevalence, and control of disease in a community.

Endemic, epidemic and pandemic disease

Endemic: This refers to the constant presence of a disease or

agent of disease in a community or region. A sporadic disease

is one which breaks out only occasionally.

Epidemic: This usually means an acute outbreak of disease in

a community or region, in excess of normal expectancy, and

derived from a common or propagated source. Many endemic

diseases can rapidly become epidemic if environmental or

host inﬂuences change in a way which favour transmission.

Pandemic: This refers to a disease which spreads to several

countries and affects a large number of people. HIV disease,

inﬂuenza and cholera are examples of diseases that have

caused or currently are the cause of pandemics.

The control and prevention of outbreaks of infec-

tious disease depend on knowing the reservoirs,

sources, routes of transmission, and effective control

measures to use.

Factors that contribute to the spread of

communicable diseases in developing

countries

Most microbial diseases are transmitted by:

– ingesting pathogens in contaminated food or

water as in cholera, typhoid and paratyphoid

fever, bacillary dysentery, hepatitis A, or ingest-

ing pathogens in unpasteurized milk and dairy

products as in brucellosis, or Campylobacter

infections.

– inhaling pathogens in air-borne droplets as in

tuberculosis, whooping cough, measles,

inﬂuenza, pneumonia, meningitis, SARS.

– pathogens being transferred by direct contact

from one person to another as in HIV infection,

syphilis, gonorrhoea, ringworm infection.

– pathogens entering the blood and tissues

through the bite of an arthropod vector as in

bubonic plague, rickettsial infections, dengue, rift

valley fever.

– pathogens entering wounds, cuts, or burns by

way of contaminated hands or unsterile instru-

ments as in infections of the skin such as boils

and abscesses and tetanus (via contaminated soil

or dust).

– transfer of pathogens in contaminated blood or

blood products as in HIV infection, viral hepatitis

(HBV, HCV).

– pathogens transmitted from mother to child

during pregnancy or childbirth as in HIV

infection, congenital syphilis, Chlamydia infec-

tion, herpes infection, congenital rubella,

gonococcal conjunctivitis, cytomegalovirus

neonatal infection.

In persons with inadequate immune responses,

infections can also be caused by the body’s normal

microbial ﬂora (organisms that naturally colonize

certain areas of the body, see later text).

Important factors which inﬂuence the transmission

and spread of communicable diseases in tropical

and developing countries include:

● Inadequate surveillance, preventive and control

measures, and lack of health care facilities in rural

areas to detect and treat patients with communi-

cable diseases.

● Socioeconomic factors including increasing

urbanization, poverty, unemployment, poorly

10 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.2

constructed houses, overcrowding, malnutrition

(particularly protein and vitamin deﬁciencies),

and starvation brought about by drought, crop

failure, ﬂooding, war, and mass migration.

● Inadequate and contaminated water supplies,

inadequate sewage disposal and unhygienic

practices.

● Climatic factors including extreme rainfall and

ﬂooding leading to pollution of water supplies

and greater numbers of insect vectors. During

the dry season, an increase in dust-borne par-

ticles can lead to increased transmission, e.g.

meningococci.

● Ineffective control of mosquitoes and other insect

vectors.

● Geographical factors including the difﬁculties of

vaccination teams and health workers in reaching

remote villages.

● Unavailability of drugs and non-compliance by

patients.

● Ineffective health education or lack of access to

health education.

● Particularly involving young children: disruption

to vaccine programmes, malnutrition, and co-

existing infection, e.g. malaria.

Human carriers: A carrier is a person who is colo-

nized by a pathogen but experiences no disease or

only minor symptoms from it. Such a person can

excrete the pathogen he or she is carrying over a

long period and be a source of infection to others

without realizing it. The carrier state is particularly

important in the transmission of typhoid fever and

also occurs in a proportion (about 10%) of those with

hepatitis B infection.

Body’s defence mechanisms

Although the human body continually comes into

contact with potentially pathogenic microorganisms,

infection and disease are usually prevented or

minimized because a healthy body has a range

of defence mechanisms to protect it. These consist

of:

● Non-speciﬁc defences

● Speciﬁc immune responses

Non-speciﬁc defences: Although referred to as

natural or innate immunity, these defences are non-

immunological. They include the body’s natural

barriers to infection (skin, mucous membranes,

antimicrobial secretions), phagocytosis of pathogens

involving polymorphonuclear neutrophils (poly-

morphs) and macrophages, complement, the

inﬂammatory process, and the actions of natural

killer (NK) cells.

Phagocytosis: Phagocytic cells ingest and kill invading

pathogens. Polymorphs circulate in the blood and respond

rapidly to infection (form ‘pus cells’). Many microorganisms

produce chemical substances that attract phagocytes.

Macrophages circulate in the blood as monocytes and are

present in tissues as ﬁxed or free macrophages. The engulﬁng

of pathogens by phagocytes is facilitated by antibody and, or,

complement (opsonization effect). Cytokines released from T

lymphocytes (in immune responses) increase the phagocytic

action of macrophages.

Inﬂammatory response: (local accumulation of ﬂuid and

cells, redness, swelling, pain): This is a protective response

to the presence of pathogens or other foreign bodies in

tissue. Phagocytes are attracted to the infection site. They

engulf and kill the pathogens and a ﬁbrin clot forms to

prevent the infection from spreading. Polymorphs predomi-

nate in acute pyogenic infections and macrophages and

helper T lymphocytes in chronic or granulomatous

infections. Cytokines and other substances assist in the

inﬂammatory process.

Complement: Consists of a set of proteins which participate in

both non-speciﬁc and speciﬁc immune defences. In non-

speciﬁc defences, complement can be activated by bacterial

peptidoglycan and lipopolysaccharide (alternative pathway).

Some Gram negative bacteria are lyzed by complement

binding to their surface. In the laboratory, complement in

serum can be inactivated at 56C for 30 minutes (antibody is

not inactivated at this temperature).

Natural killer cells: These are lymphocytes which can kill virus

infected cells (and tumour cells) without antigenic stimulation

although antibody enhances their activity. They destroy cells

by secreting cytotoxins. They have no immunological

memory.

Speciﬁc immune responses: Occur following

contact with a ‘foreign’ antigen, e.g. invading

pathogen or its products. Speciﬁc immunity involves

antibody production by B lymphocytes, cell

mediated immune responses by T lymphocytes, and

the production of memory cells that enable the body

to respond rapidly should infection by the same

pathogen recur. Also involved are phagocytic cells,

complement, and cytokines (helper factors) which

include interleukins, interferons, and tumour

necrosis factor.

Antibody mediated immunity (humoral immunity)

Antibodies are produced following the antigenic

stimulation of B lymphocytes. The antigen binding

receptor on a B lymphocyte is an immunoglobulin

(Ig). When ﬁrst stimulated the B cell proliferates and

differentiates into:

– plasma cells which produce speciﬁc antibody.

MICROBIOLOGICAL TESTS 11

7.2

– memory B cells which enable the immune

system to react rapidly should the same antigen

be encountered in the future (secondary

response).

Antibody mediated immunity is the body’s main

defence against extracellular pyogenic (pus-forming)

bacteria such as staphylococci and streptococci,

against capsulated bacteria such as pneumococci,

Haemophilus and Neisseria species, and against

toxin-producing bacteria such as Clostridium tetani,

Vibrio cholerae, and Corynebacterium diphtheriae.

Antibody immunity is also important in some virus

infections, e.g. hepatitis B virus infection.

Antibodies: The ﬁrst antibodies produced in infection

(primary response) are immunoglobulins (Ig) of the IgM

class, becoming detectable about 1 week after infection and

persisting for about 6 weeks. IgM antibody is a large molecule

with up to ten antigen binding sites. It is a good complement

ﬁxing antibody and therefore aids lysis of microbial cells. It

also acts as an opsonin. It forms the main antibody response

in many Gram negative bacterial infections.

About 2 weeks after infection, IgG antibody is produced

and is long lasting. IgG antibody is the main antibody formed

in a secondary response. It has two antigen binding sites. It

also ﬁxes complement, and acts as an opsonin. It passes easily

from the blood into tissue spaces and is the only class of Ig

that can cross the placenta from mother to fetus.

Other classes of antibody involved in protecting the

body are IgA, IgD and IgE. None of these classes of

immunoglobulin ﬁx complement or opsonize. IgA is the

main immunoglobulin in secretions. It prevents bacteria and

virusesattaching to mucous membranes. IgD is found on the

surface of many B lymphocytes and in serum but little is

knownof its functions. IgE is concentrated in the submucosa

andbinds to mast cells and basophils. It is the main antibody

involved in immediate type hypersensitivity anaphylactic

reactions. High levels of serum IgE are found in patients

with asthma and also in helminth infections such as

schistosomiasis, ascariasis, hookworm disease, toxocariasis,

and ﬁlariasis. IgE causes the release of enzymes from

eosinophils.

Immunization: Active antibody mediated immunity can also

be induced in a person by immunization using vaccine

consistingof live bacteria or organisms that have been treated

so that they are harmless while remaining antigenic, or dead

organisms or their products (e.g. toxins) that have been

chemically or physically altered so that they cannot cause

harm but can stimulate the body to produce antibodies.

Examples of microbial diseases that can be prevented by

artiﬁcial immunization include diphtheria, whooping cough,

mumps, cholera, anthrax, poliomyelitis, rubella, tetanus,

tuberculosis, typhoid, and hepatitis B. Immunization against

the ﬁrst three diseases provides life-long immunity. For the

other diseases, revaccination may be required every few

monthsor years.

Passive immunity: This is when antibodies that have been

formed in another human or animal are introduced into the

body,e.g. diphtheria antitoxin to neutralize circulating toxin.

Passive immunity occurs naturally when antibodies (IgG)

from a mother are transferred across the placenta or

maternal antibody is transferred in breast milk after birth.

These antibodies protect an infant during the ﬁrst few

months of its life until it begins to make its own protective

antibodies.

Cell mediated immunity

The cells involved are macrophages, helper T

lymphocytes and cytotoxic T lymphocytes. Cell-

mediated immunity is mainly directed at virus

infected cells, intracellular fungi, and intracellular

bacteria such as Mycobacterium tuberculosis,

Mycobacterium leprae, and Brucella species.

Helper T cells (CD4 positive)

CD4



helper T cells carry CD4 glycoprotein markers on their

surface. They are important cells in cellular immunity. They

release cytokines, help to activate B lymphocytes, and

modulate cellular immune responses. Helper T cells recognize

antigen bound to MHC (major histocompatibility complex)

class II protein.

Cytotoxic T cells (CD8 positive)

CD8



cytotoxic T cells carry CD8 glycoprotein markers

on their surface. They recognize antigen bound to MHC I

class protein, mainly on virus-infected cells. They produce

cytotoxins which destroy cells infected with viruses and other

intracellular organisms. Cytotoxins are also important in elim-

inating tumour cells.

Effective and correctly regulated immune responses

are dependent on there being the correct ratio of

helper CD4



T cells to cytoxic CD8



T cells

(normally CD4: CD8 cells  1.5). When there are

insufﬁcient helper T cells, e.g. in HIV disease in

which CD4



T cells are destroyed, immune

responses become impaired. This leads to increased

susceptibility to infection with pathogens such as

Mycobacterium tuberculosis, and the development of

opportunistic infections and certain tumours (see

also subunit 7.18.55).

How microorganisms overcome the body’s

defences and cause disease

The following are some of the ways developed by

pathogens to overcome the body’s defence mech-

anisms, become established in tissues, multiply, and

cause disease:

● Adherence ﬁmbriae (pili)

● Production of enzymes that facilitate the spread

of pathogens

● Mechanisms that interfere with phagocytosis

● Production of beta-lactamases

● Mechanisms that destroy or neutralize antibodies

● Production of exotoxin

12 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.2

● Release of endotoxin

Adherence ﬁmbriae (pili): These are small hairs that enable

some pathogens to attach and adhere easily to cell surfaces,

particularly mucous membranes. Bacteria possessing pili

include Neisseria gonorrhoeaeand some strains of Escherichia

coli, Salmonellaand Shigella species.

Enzymes that help pathogens to spread: For example,

hyaluronidase produced by Clostridium perfringensand some

streptococci and staphylococci, helps organisms to spread

through the body by breaking down the hyaluronic acid of

connective tissue.

Mechanisms that interfere with phagocytosis:Bacteria such as

Streptococcus pneumoniae, Klebsiella pneumoniae,

Haemophilus inﬂuenzae, and Neisseria meningitidis secrete a

capsule around their cell wall which helps to prevent

opsonization and phagocytotis. The M protein in the cell wall

of Streptococcus pyogenes is anti-phagocytic and the

haemolysins and leucocidins of streptococci and staphylococci

interfere with the functioning of phagocytes and destroy poly-

morphs and macrophages.

Production of beta-lactamases: These penicillin-destroying

enzymes are produced by many bacteria including some

strains of Staphylococcus aureusand Neisseria gonorrhoeae.

Mechanisms that destroy or neutralize antibodies: For

example, the destructive IgA protease of Pseudomonas aerug-

inosa. Other pathogens produce soluble antigen which

neutralizes antibody before it is able to bind to the surface of

bacteria.

Production of exotoxin: Several Gram positive and a few

Gram negative bacteria secrete powerful poisons called

exotoxins that are capable of destroying or injuring host cells.

They tend to be speciﬁc in their action, e.g. the exotoxin of

Clostridium tetaniis a neurotoxin. Other important exotoxin-

producing pathogens include Clostridium botulinum,

Clostridium perfringens, Corynebacterium diphtheriae,

enterotoxigenic Escherichia coli(ETEC), Shigella dysenteriae,

and Vibrio cholerae. Toxin produced by enteric pathogens is

known as enterotoxin.

Exotoxins are highly antigenic. By special chemical prep-

aration, exotoxins can be made into non-toxic toxoids which

can be used to immunize and protect individuals against

speciﬁc diseases.

Release of endotoxin:The cell walls of Gram negative organ-

isms contain endotoxin (O antigen). Unlike exotoxin,

endotoxin is not usually secreted by an organism but is

released only when the organism is destroyed. When endo-

toxin is released into the blood circulation, the resulting

toxaemia may cause rigor, chills, and shock. Endotoxin from

some pathogens may also have clotting properties and lead to

disseminated intravascular coagulation (DIC). Endotoxin

release may also lead to a marked leucocytosis. In contrast to

exotoxin, endotoxin is only weakly antigenic. It is also more

heat stable than exotoxin.

Other factors which determine whether a pathogen

will cause disease include:

– Transmission route

– Number of bacteria that invade

– State of health of the person infected

For a pathogen to c ause disease it must enter the

body by a route which will enable it to reach a site

where it can establish itself and multiply, e.g. the

Clostridium organism which causes gas gangrene

must reach deep tissues to ﬁnd the anaerobic

conditions necessary for its growth. Other

organisms such as Staphylococcus aureus can cause

several different diseases depending on whether

the organism is ingested (e.g. food-poisoning),

infects the skin (e.g. boils), or reaches the lung (e.g.

pneumonia).

Certain organisms may require a vector for their

development and transmission, e.g. rickettsiae

develop in an arthropod such as a tick, mite, ﬂea or

louse, and are transmitted to man when the arthro-

pod bites and the organisms are injected into the

blood.

For some bacteria, the entry of large numbers of

organisms may be necessary before a healthy

person’s defence mechanisms are overcome,

whereas only a few organisms may be required to

produce disease in a person already in poor health,

a malnourished person (especially a child) or a

person with immunosuppression caused for

example by HIV disease. Particularly virulent

bacteria, however, need only be present in very

small numbers to cause disease, e.g. Shigella dysen-

teriae.

LABORATORYINVESTIGATIONOF MICROBIAL

INFECTIONS

The laboratory investigation of microbial diseases

involves:

 Examining specimens to detect, isolate, and

identify pathogens or their products using:

– Microscopy

– Culture techniques

– Biochemical methods

– Immunological (antigen) tests

 Testing serum for antibodies produced in

response to infection, i.e. serological response.

Examination of specimens for microorganisms

MICROSCOPY

To assist in the diagnosis of microbial infections,

microorganisms can be examined microscopically

for their motility, morphology, and staining reac-

tions.

MICROBIOLOGICAL TESTS 13

7.2

Examples

● Motile Vibrio cholerae in a rice water faecal specimen

from a person with cholera.

● Treponema pallidum in chancre ﬂuid (using dark-ﬁeld

microscopy), establishing a diagnosis of primary syphilis.

● Fungal hyphae and arthrospores in a sodium hydroxide

preparation of skin from a person with ringworm.

● Gram negative reaction and characteristic morphology of

Neisseriae gonorrhoeae (intracellular diplococci) in a

urethral discharge from a man with gonorrhoea.

● Gram positive reaction and morphology of pneumococci

in cerebrospinal ﬂuid from a patient with pneumococcal

meningitis.

● Gram positive reaction and morphology of yeast cells in a

vaginal discharge from a woman with vaginal candidiasis.

● Acid fast reaction of Mycobacterium tuberculosis in

Ziehl-Neelsen stained sputum from a person with pul-

monary tuberculosis.

Note: Microscopical techniques are described in

subunit 7.3 and in subsequent subunits covering the

examination of different specimens.

CULTURE TECHNIQUES

The culture of pathogens enables colonies of pure

growth to be isolated for identiﬁcation and, when

required, antimicrobial susceptibility testing.

Note: The cultural requirements of pathogens,

preparation, inoculation and quality control of

culture media are described in subunit 7.4, and the

use and reporting of cultures in the subunits describ-

ing the examination of different specimens.

Antimicrobial susceptibility testing is described in

subunit 7.16.

BIOCHEMICAL METHODS

Following culture, biochemical tests are often

required to identify pathogens including the use of

substrates and sugars to identify pathogens by their

enzymatic and fermentation reactions.

Examples

● Catalase test to differentiate staphylococci which produce

the enzyme catalase from streptococci which are non-

catalase producing.

● Oxidase test to help identify Vibrio, Neisseria, Pasteurella

and Pseudomonas species, all of which produce oxidase

enzymes.

● Coagulase test to help identify Staphylococcus aureus

which produces the enzyme coagulase (coagulates

plasma).

● Fermentation tests to differentiate enterobacteria, e.g.

use of glucose and lactose in Kligler iron agar medium to

assist in the identiﬁcation of Shigella and Salmonella

organisms.

● Indole test to detect those organisms that are able to

break down tryptophan with the release of indole. It is

mainly used to differentiate Escherichia coli from other

enterobacteria.

● Urease test to assist in the identiﬁcation of organisms

such as Proteus species which produce the enzyme

urease.

Note: Biochemical test methods are described in

subunit 7.5.

While kits for the identiﬁcation of bacteria and fungi

(visual, chart, and computer-based) are available, e.g.

API system, they are expensive and not always

needed. In this publication, conventional biochemi-

cal testing methods are described, using reagents

that can be prepared in the laboratory or are easily

and economically available as ready-made reagents

or as strip, disc, or tablet reagents.

IMMUNOLOGICAL (ANTIGEN) TESTS

Antigen tests often enable an early diagnosis or pre-

sumptive diagnosis of an infectious disease to be

made. They involve the use of speciﬁc antibody

(antisera or labelled antibody):

– To identify a pathogen that has been isolated by

culture, e.g. identiﬁcation of Salmonella serovars,

Shigella species, and Vibrio cholerae by direct

slide agglutination.

– To identify pathogens in specimens using direct

immunoﬂuorescence, e.g. identiﬁcation of respi-

ratory viruses, rabies virus, cytomegalovirus,

Pneumocystis jiroveci, and Chlamydia. Fluoresc-

ence techniques are more difﬁcult to perform in

district laboratories.

– To identify antigens of microbial origin that can

be found in serum or plasma, cerebrospinal ﬂuid,

urine, specimen extracts and washings, or ﬂuid

cultures. Highly speciﬁc monoclonal antibody

reagents are often used. Techniques to identify

soluble microbial antigens include agglutination

techniques (direct, latex, coagglutination),

enzyme immunoassays (EIA), or more recently

developed immunochromatographic (IC) tests

and dipstick dot immunoassays. Examples are

listed in chart 7.2. Because many of these antigen

tests are rapid, simple to perform, and have good

stability, they are becoming increasingly used

at district level. Adequate controls must be

used.

PRINCIPLES OF ANTIGEN TESTS

Direct slide agglutination

This is used to identify bacteria following culture on a carbo-

hydrate-free medium. A bacterial colony of pure growth is

emulsiﬁed in physiological saline on a slide and antiserum

containing speciﬁc antibody is added. The antibody binds to

the bacterial antigen, resulting in the agglutination of the bac-

terial cells.

Antiserum  Bacteria → Bacteria AGGLUTINATED

14 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.2

Latex agglutination

Latex particles are coated with speciﬁc antibody. The

specimen containing microbial antigen is mixed with the latex

reagent, resulting in agglutination of the latex particles.

Antibody latex  Antigen in → Latex particles

reagent specimen AGGLUTINATED

Coagglutination (COAG)

Speciﬁc antibody is bound to the surface protein A of staphy-

lococci (Cowan type 1 strain of Staphylococcus aureus).

Soluble microbial antigen in the specimen is mixed with the

COAG reagent, resulting in the agglutination of the staphylo-

coccal cells.

Antibody COAG  Antigen in → Staphylococcal cells

reagent specimen AGGLUTINATED

Direct immunoﬂuorescence

Speciﬁc antibody is conjugated (joined) to a ﬂuorochrome

such as ﬂuorescein isothiocyanate and applied to the

specimen containing the pathogen on a slide. The ﬂuoro-

chrome antibody conjugate binds to the pathogen (antigen).

When examined by ﬂuorescence microscopy the pathogen is

seen to ﬂuoresce (e.g. yellow-green or orange) against a dark

background. Because of the specialized equipment and exper-

tise required to prepare and read ﬂuorescence preparations,

immunoﬂuorescence techniques are not often performed in

district laboratories.

Fluorochrome  Antigen → Pathogen

antibody reagent (pathogen) FLUORESCES

Enzyme immunoassays (EIA)* to detect antigen

*Enzyme assays are also referred to an enzyme linked

immunosorbent assays (ELISA).

Antibody against the antigen to be detected is ﬁxed to the well

of a microtitration plate or membrane of an individual test

device such as a plastic block. Soluble microbial antigen in the

specimen binds to the antibody. After washing, antibody con-

jugated to an enzyme (e.g. horseradish peroxidase) is added.

This binds to the captured antigen. After another wash, a

chromogenic (colour-producing) substrate such as hydrogen

peroxide joined to an indicator is added. The enzyme

hydrolyzes the substrate, producing a colour reaction. The

colour can be read visually (membrane EIA) or spectropho-

tometrically (microtitration plate EIA).

1 Fixed antibody  Antigen in → Antigen binds

specimen to antibody

2 Enzyme conjugated → Binds to antigen–antibody

antibody added complex

3 Chromogenic substrate added → COLOUR produced

Note: Most ﬂow-through membrane EIAs are rapid, usually

have built-in controls, require no additional equipment, and

enable specimens to be tested individually.

Immunochromatographic (IC) techniques to detect antigen

Most IC tests are produced in strip or cassette form with the

immunological reagents ﬁxed on the strip or cassette

membrane. When using an IC strip, the lower end is immersed

in the specimen or if using an IC cassette, the specimen is

applied to an absorbent pad. Antigen in the specimen ﬁrst

meets speciﬁc antibody conjugated to colloidal gold (pink-

mauve) particles and the antigen binds to the antibody. The

antigen–antibody colloidal gold complex migrates up the strip

(or along the membrane) where it becomes bound (captured)

by a line of speciﬁc antibody, producing a pink line in the test

result area. A further pink line. i.e. inbuilt positive control, is

produced above the test line, showing that the test has per-

formed satisfactorily.

1 Antigen in the  Antibody colloidal → Antigen binds

specimen gold conjugate to antibody

2 Antigen–antibody .... Meets .... Antibody line → Complex

colloidal gold on strip captured

PINK LINE

produced

Dipstick comb immunoassays to detect antigen

These assays involve dipping a plastic comb in the specimen

and reagent solutions. Each comb is designed for testing up to

6 specimens and controls although the comb can be cut when

there are fewer specimens. When used for antigen detection,

speciﬁc antibody is ﬁxed to the ends of the comb teeth. The

comb is dipped in the specimen and antigen in the specimen is

captured by the antibody. After washing, the comb is dipped

in a colloidal gold antibody conjugate. This binds to the

antibody–antigen complex. After washing, a pink dot is

produced, indicating a positive test. Although easy to

perform, dipstick assays are not as rapid as most IC strip or

cassette immunoassays.

1 Antibody on teeth of comb  Antigen in specimen →

Antigen binds to antibody

2 Colloidal gold conjugate applied → Binds to antigen–

antibody complex

PINK DOT produced

Immunodiagnostics developed by PATH: Some of

the most affordable, available, stable, and rapid

antigen (and antibody) tests are the IC tests and

comb dipstick tests developed by PATH (Program

for Appropriate Technology for Health). They are

produced and distributed by several manufacturers

under licence from PATH.Some of these manufac-

turers are listed in chart 7.2.PATH is continuing to

develop new products to diagnose major bacterial,

viral, and parasitological diseases and details of

these can be obtained from PATH (see Appendix

11).

Testing serum for antibodies (serological tests)

In district laboratories, serological testing in which

antigen is used to detect and measure antibody in a

person’s serum is used mainly:

– To help diagnose a microbial disease when the

pathogen or microbial antigen is not present in

routine specimens or if present is not easily

isolated and identiﬁed by other available tech-

niques, e.g. dengue, brucellosis, rickettsial

infections, syphilis, leptospirosis.

– To test individuals and screen donor blood for

antibody to HIV-1 and HIV-2.

– To measure antibody levels to determine the

MICROBIOLOGICAL TESTS 15

7.2

prevalence of infectious disease in a community

and immune status of individuals.

– To screen for rises in anti-streptolysin O, e.g. in

the investigation of rheumatic fever, acute

glomerulonephritis, and other complications of

Group A streptococcal infection.

– To screen pregnant women for infections such as

syphilis and HIV infection.

Demonstrating active infection in clinical diagnosis

For many common infections, antibodies (IgG)

against the pathogens involved will often be present

in a person’s serum from a previous infection or fol-

lowing natural or acquired immunization. Levels of

such antibodies are usually low.

To diagnose active infection it is necessary to

detect a particularly high level of antibody or prefer-

ably, when possible, to demonstrate a four-fold

increase in IgG antibody in paired sera (acute i.e.

soon after the onset of symptoms, and convalescent

samples), taken 10–14 days apart. Alternatively,

active infection can be shown by demonstrating IgM

antibodies in the serum which are produced early in

an infection and do not persist for more than a few

weeks.

When previous exposure is unlikely, e.g. rare

infections, the ﬁndingof antibody in serum collected

during the infection is signiﬁcant and testing a

second serum is often not required. In diagnosing

neonatal infections, it is ne cessary to test for IgM

antibody bec ause this will show that the antibody

has been produced bythe infant and is not maternal

IgG antibody (IgM antibody does not cross the

placenta).

Antibody titre: The level of antibody in a serum can

be determined by testing dilutions of the serum

using a double dilution technique, e.g. 1 in 2, 1 in

4, 1 in 8, 1 in 16 etc. The last dilution to show

antibody activity gives the titre (strength) of

antibody, e.g. if the end-point dilution is 1 in 8, the

antibody titre is 8. A four-fold rise in titre to e.g. 32

in a convalescent serum, would be an indication of

active infection. Sometimes, however, the titre is

slow to rise or may show no rise depending on

when the sera are collected.

Prozone effect: When testing a serum (agglutination tech-

nique) that has a high antibody level, e.g. from a patient with

acute brucellosis, it is possible for only the higher dilutions,

e.g. over 1 in 40 or 1 in 80 to show agglutination. This is

referred to as a prozone reaction and is probably caused by

excess protein coating the antigen particles.

Collection of blood for antibody testing

Sufﬁcient serum for most antibody tests can be

16 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.2

Chart 7.2 Examples of antigen and antibody tests

PATHOGEN/MICROBIAL ANTIGEN DETECTED TEST MANUFACTURER

● Salmonella, Shigella, Vibrio cholerae identiﬁcation Direct 1, 2, 4, 9, 19, 22, 25, 26, 31

from culture agglutination

● Vibrio cholerae from faeces IC Dipstick 19

● Neisseria meningitidis, Streptococcus pneumoniae, Latex 4, 8, 25

Haemophilus inﬂuenzae, Escherichia coli antigens COAG 9

in c.s.f.

● Cryptococcus neoformans in c.s.f. and serum Latex 5, 25

● Hepatitis B surface antigen (HbsAg) in serum or Latex 1, 10, 24

plasma IC strip 15, 22, 24, 29, 32

IC cassette 6, 12, 21*, 22, 23, 32

● Beta-haemolytic streptococci cell wall antigens Latex 1, 2, 19, 10, 25, 31

(Lanceﬁeld grouping) COAG 9

EIA membrane 4

● Streptococcus pyogenes from throat swab IC cassette 15, 16, 28, 29, 30

COAG 9

● Neisseria gonorrhoeae identiﬁcation from culture COAG 4, 9

IC membrane 14

● Staphylococcus aureus identiﬁcation from culture Latex 1, 2, 8, 9, 10, 25, 31

● Pregnancy direct test to detect HCG in urine Latex 1, 10, 24

IC 6, 10, 13, 15, 20, 22, 29, 32

● Chlamydia from urogenital specimens IC 11, 20, 29, 30, 32

Note: Other antigen tests are also available from several of the manufacturers listed in this chart. This type of technology is

developing rapidly but the cost of most antigen tests is high.

Antigen Tests

DISEASE BEING INVESTIGATED TEST MANUFACTURER

● Dengue IC cassette 14, 17, 22

IC Strip 32

● S. Typhi IgM antibodies I C membrane 14

IC Dipstick 32, 33

● Brucellosis Direct 1, 4, 10, 22, 24, 25

agglutination

● Syphilis: – Cardiolipin antibody (e.g. RPR) Flocculation 1, 4, 7, 10, 24, 26, 28

– Speciﬁc treponemal antibody IC strip 13*, 22, 32

TPHA 1, 2, 7, 10, 24, 31

TPPA 3

Antibody Tests

obtained from 3–5 ml of venous blood. For some

micro-techniques, a smaller volume of blood may be

adequate and it may be possible to use capillary

blood collected on to ﬁlter paper (the testing labora-

tory should always provide written instructions to

those collecting blood).

Collect the blood in a dry leak-proof glass tube

or bottle (avoid plastic because blood does not clot

well in a plastic container). A sterile container should

be used when the blood is sent to a referral labora-

tory. Store the blood at 4–6C. If unable to test the

specimen within 48 hours, separate the serum from

the cells. To do this, allow the blood to clot and after

the clot has retracted and sedimented (or centrifuge

the blood), use a plastic or glass Pasteur pipette to

transfer the serum (cell-free) to a leak-proof plastic or

glass container. Label with the patient’s name,

identity number, and date of collection. Haemolyzed

and lipaemic serum samples are unsuitable for

antibody testing. Precautions to avoid haemolysis are

described in subunit 8.3.

Serological techniques used in district

laboratories

Serological techniques most frequently used in

district laboratories are those that can be performed

simply and economically, use stable reagents, do not

require specialist equipment and enable specimens

to be tested individually or in small numbers. Such

techniques include agglutination tests, ﬂocculation

tests, enzyme immunoassays (membrane based

EIA), immunochromatographic strip, cassette and

card tests, and dipstick comb immunoassays.

Examples are listed in Chart 7.2.

MICROBIOLOGICAL TESTS 17

7.2

PRINCIPLES OF ANTIBODY TESTS

Agglutination techniques

To detect antibody, the following agglutination techniques are

used:

– Direct slide and tube agglutination tests in which a bac-

terial antigen reagent is used to agglutinate antibody in

serum.

Bacterial  Antibody in → AGGLUTINATION

suspension patient’s serum

– Latex agglutination tests in which latex particles are

coated with antigen to detect antibody in the patient’s

serum

Latex antigen  Antibody in → Latex particles

reagent patient’s serum AGGLUTINATED

– Indirect (passive) haemagglutination (IHA) test in which

antigen is coated on treated red cells (often bird cells

because these are nucleated and sediment rapidly).

Antigen-coatedred cells are referred toas sensitized cells.

Most IHA tests are performed in microtitration plates.

Thesensitized cells are added to dilutions of the patient’s

serum. Antibody in the serum agglutinates the cells and

theysettle forming an even covering in the bottom of the

well.When there is no antibody, the cells are not aggluti-

natedand they forma red button in thebottom of the well.

Sensitized  Antibody in → Red cells

red cells patient’s AGGLUTINATED

serum Smooth covering in well

Flocculation tests

A soluble antigen reagent is used to react with antibodies in

the patient’s serum to form ﬂoccules (clumps of precipitate).

Antigen  Antibody in → FLOCCULATION

reagent patient’s serum

Enzyme immunoassays (EIA) to detect antibody

In EIA (ELISA) techniques to detect antibody, antigen is

● Leptospirosis IC membrane 14

Agglutination 27

● Rickettsial infections Agglutination 1, 10, 22

IC cassette 32

● HIV infection IC cassette 21, 32

Agglutination 3, 21

Comb dipstick 6*, 18*, 19, 22*, 26*

Flow through membrane 7, 12, 15, 21, 22, 24

● Detection of anti-streptolysin O (ASO) Latex 1, 3, 10, 24, 26

● Detection of rheumatoid factor (RA) Latex 1, 10, 24

*PATH collaboration in development of test (see previous text).

Manufacturers (addresses in Appendix 11): 1 HD Supplies, 2 Oxoid, 3 Fujirebio, 4 BP Diagnostics, 5 Immuno-Mycologics

6 Laboratorium Hepatika, 7 Abbott Diagnostika, 8 bioMérieux, 9 Boule Diagnostics, 10 Plasmatec, 11 Diagnostics for the Real

World, 12 Mitra J, 13 Quest Diagnostics, 14 Zephyr Biomedicals (Tulip Group), 15 Acorn Laboratories, 16 Biomerica, 17 PanBio,

18 Concept Foundation, 19 Span Diagnostics, 20 Teco Diagnostics, 21 Trinity Biotech, 22 Paciﬁc Biotech 23 Orchid Biomedical,

24 Tulip Group, 25 Bio-Rad Laboratories, 26 Wiener Laboratories, 27 KIT Biomedical, 28 Binax, 29 Savyon Diagnostics,

30 Unipath, 31 Mast Group, 32 Standard Diagnostics Inc, 33 Malaysian Bio-Diagnostic Research Sdn.

bound to the cell well of a microtitration plate or ﬁlter

membrane (EIA membrane test) and the patient’s serum

added. Antibody binds to the antigen. After washing, anti-

human immunoglobulin (AHG) conjugated with an enzyme is

added which binds to the antibody–antigen complex. After a

further wash, a chromogenic enzyme substrate is added, pro-

ducing a colour reaction which is read spectrophotometrically

(microtitration plate EIA) or visually (membrane EIA).

Controls are run with the tests. Microtitration plate EIAs

require specialist equipment and training and to be performed

economically, specimens need to be tested in batches.

1 Antigen in well  Antibody in → Antibody

or on membrane patient’s serum captured

2 Enzyme conjugated → Binds to antibody antigen

to AHG added complex

3 Chromogenic substrate added → COLOUR produced

Note: Most ﬂow-through membrane immunoassays to detect

antibody are not enzyme based. Like IC assays (explained in

the following text), most membrane immunoassays use a col-

loidal gold conjugate to visualize the antigen–antibody

reaction.

Immunochromatographic (IC) strips, cassettes and cards to

detect antibody

Rapid easy to use IC strips, cassettes and cards to detect

antibody are becoming increasingly available for the diagnosis

of microbial infections. They are similar in format to those

described previously for antigen detection. To detect

antibody, the lower end of an IC strip is immersed in the

patient’s serum or the sample is added to a samples well.

Antibody in the specimen reacts with speciﬁc antigen bound

to colloidal gold particles and the antibody binds to the

antigen. The antibody–antigen colloidal gold complex

migrates along the membrane where it becomes captured by a

line of speciﬁc antigen, producing a pink line in the test area.

A further pink line, i.e. positive control is produced above

this, showing that the test has performed satisfactorily.

1 Antibody in  Antigen bound → Antibody binds

serum to colloidal gold to antigen

2 Antibody–antigen . . Meets . . Antigen line → Complex

colloidal gold captured

complex PINK LINE

produced

Dipstick comb immunoassays to detect antibody

The format of dipstick combs to detect antibody is similar to

that previously described for antigen detection except speciﬁc

antigen not antibody is ﬁxed to the end of each tooth. The

antigen captures the antibody in the patient’s serum. After

washing the comb is dipped in a protein A colloidal gold con-

jugate which binds to the antibody–antigen complex,

producing a pink dot.

1 Antigen on teeth  Antibody in → Antibody binds

of comb serum to antigen

2 Protein A colloidal gold → Binds to antibody–antigen

applied complex

PINK DOT produced

Sensitivity and speciﬁcity of immunoassays

When comparing the performances of different

immunoassays and selecting which test to use, it is

important to know how sensitive and speciﬁc a par-

ticular assay is so that the most appropriate test is

18 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.2

selected. The sensitivity of an assay refers to its ability

to identify all those that are infected. The speciﬁcity

of an assay is its ability to identify correctly all those

not infected. For example, a highly sensitive test

should be used to screen donor blood for antibod-

ies to HIV to ensure a positive test result is obtained

from all sera that contain anti-HIV1 and anti-HIV2

antibodies. Deﬁnitive tests should be speciﬁc to

minimize false positive test results. Most manufac-

turers supply details of the sensitivity and speciﬁcity

of their assays and also information on a test’s limi-

tations and possible cross-reactions.

Even when a test is highly sensitive and speciﬁc

and correctly performed with appropriate controls,

there is still a possibilityof false positive results when

theprevalence ofa diseaseis low.Conﬁrmatory testing

becomesimport antin thesesituations. The higherthe

predictive value of a test,the higher the possibility in

anypopulation that a positive testmeans disease.

Note: Further information on predictive values and

how to calculate the speciﬁcity and sensitivity of tests

(expressed as percentages) can be found in subunit

2.2 in Part 1 of the book.

Nucleic acid tests to diagnose microbial infections

Recent advances in nucleic acid probe technologies and gene

ampliﬁcation techniques (e.g. polymerase chain reaction,

PCR), have resulted in the development of a new generation

of rapid, highly sensitive and speciﬁc tests to identify

pathogens in clinical specimens and cultures, often at an

earlier stage than by other tests. As yet only a few manufac-

turers are producing these new technologies and because of

their very high cost, more demanding technique, and special-

ist training required, only a few research and specialist

laboratories are using them.

FEATURESAND CLASSIFICATIONOF BACTERIA

Bacteria form a large group of unicellular parasitic,

saprophytic and free-living microorganisms, varying

in size from 0.1–10 m long. They have a simple cell

structure, contain both deoxyribonucleic acid (DNA)

and ribonucleic acid (RNA), and multiply by binary

ﬁssion. They are classiﬁed by their morphology,

staining reactions, cultural characteristics, biochemi-

cal reactions, antigenic structure, and increasingly by

their genetic composition using specialized molecu-

lar biology techniques.

Bacterial structure

Bacteria have a simple cell structure consisting of:

● Cytoplasm containing the bacterial chromosome

(genome), ribosomes, stored energy inclusions,

and often plasmids (extra-chromosomal frag-

ments).

● Cytoplasmic membrane and mesosomes.

● Cell wall (except bacteria with deﬁcient cell walls).

● External structures, including (depending on

species) a capsule, ﬁmbriae (pili), and ﬂagella.

Spores are produced by Bacillus and Clostridium

species of bacteria.

Genome:Bacteria are prokaryotes, i.e. their genetic material

is not organized into chromosomes inside a nuclear

membrane but consists of a single usually circular chromo-

some of double-stranded DNA which lies coiled in the

cytoplasm, attached to a septal mesosome.

Plasmids: These are small, self-replicating, double-stranded

circular DNA molecules which enable genetic material to be

exchanged within and between bacterial species through

specialized sex pili (see later text). Depending on the genes

containedinthe plasmid,one bacteriummay conferon another,

propertiessuch asantimicrobial resistance ortoxin production.

Differentplasmids can befound in the samebacterium.

Ribosomes:These are sites of protein production distributed

in the cytoplasm. They are composed of RNA and proteins.

Inclusion granules: Composed of volutin, lipid and polysac-

charide. These cytoplasmic inclusions are sources of stored

energy.

Cytoplasmic membrane and mesosomes: The cytoplasmic

membrane acts as a semi-permeable membrane controlling

the movement of water, nutrients, and excretory substances in

and out of the cell. It also secretes extracellular enzymes and

toxins. Mesosomes appear as convoluted indentations in the

cytoplasmic membrane (see Fig 7.1). They are sites of respi-

ratory enzyme activity and assist with cell reproduction.

Cell wall: This provides the bacterial cell with rigidity and

protectsagainst osmotic damage. The cell wallis strengthened

bya mucopeptide polymer calledpeptidoglycan. Based on dif-

ferencesinthe composition ofbacterial cellwalls, most bacteria

when stained by the Gram staining technique (described in

subunit7.3) can be divided into those that are Gram positive,

i.e. retain the stain crystal violet, and those that are Gram

negative,i.e. are decolorized andtake up the red counterstain.

The cell wall of Gram negative bacteria contains a smaller

amount of peptidoglycan and there is an outer membrane

which contains toxic lipopolysaccharides (endotoxins).

Note: Spirochaetes (Treponema, Borrelia, Leptospiraspecies)

have a ﬂexible thin cell wall.

External structures (ﬂagella, ﬁmbriae, capsule): Motile

bacteria possess one or more thread-like ﬂagella. Some

bacteria such as Salmonella species are identiﬁed by the

speciﬁc antibodies formed against ﬂagellar proteins.

Many Gram negative bacteria and some Gram positive

bacteria have hair-like structures called ﬁmbriae (pili). They

enable organisms to adhere to host cells and to one another.

Specialized sex ﬁmbriae enable genetic material to be trans-

ferred from one bacterium to another, a process called

conjugation.

Manybacteria secretearound themselves apolysaccharide

substance (or sometimes protein) which may become sufﬁ-

MICROBIOLOGICAL TESTS 19

7.2

ciently thick to form a deﬁnitecapsule. Possessinga capsule

usually increases the virulence of an organism. Special tech-

niques are required to demonstrate bacterial capsules, e.g.

Indiaink preparation or dark-ﬁeldmicroscopy. Capsular poly-

saccharide antigens (K antigens)enable pneumococci to be

identiﬁedserologically.

Spores: When conditions for vegetative growth are not

favourable, especially when carbon and nitrogen become

unavailable, bacteria of the genera Bacillus and Clostridium

are able to survive by forming resistant endospores. Spore for-

mation involves a change in enzyme activity and morphology.

The spore may be positioned at the end (terminal) of the

bacterium or centrally (median). It may be round, oval, or

elongate. Endospores being dense and thick-walled, are able

to withstand dehydration, heat, cold, and the action of disin-

fectants. A spore is unable to multiply but when conditions for

vegetative growth return, it is able to produce a bacterial cell

which is capable of reproducing.

Morphology of bacteria

Morphologically bacteria can resemble:

● Cocci (Singular: coccus)

● Bacilli (rods) (Singular: rod, bacillus)

● Vibrios (Singular: vibrio)

● Spirilla (Singular: spirillum)

● Spirochaetes (Singular: spirochaete)

Note: Several species of bacteria are able to change

their form, especially after being grown on artiﬁcial

media. Organisms which show variation in form are

described as pleomorphic.

Cocci: These are round or oval bacteria measuring

about 0.5–1.0 m in diameter. When multiplying,

cocci may form pairs, chains, or irregular groups:

– cocci in pairs are called diplococci, e.g. meningo-

cocci and gonococci.

– cocci in chains are called streptococci, e.g.

Streptococcus pyogenes.

– cocci in irregular groups are called staphylococci,

e.g. Staphylococcus aureus.

Gram reaction: Staphylococci and streptococci are Gram

positive, whereas diplococci can be Gram positive or Gram

negative.

Rods (bacilli): These are stick-like bacteria with

rounded, tapered(fusiform), square, or swollen ends.

They measure 1–10 m in length by 0.3–1.0 m

in width. The short rods with rounded ends are

often called coccobacilli. When multiplying, bacterial

rods do not usually remain attached to one another,

but separate. Occasionally, however, they may:

– form chains, e.g. Streptobacillus species.

– form branching chains, e.g. lactobacilli.

– mass together, e.g. Mycobacterium leprae.

– remain attached at various angles resembling

Chinese letters, e.g. Corynebacterium diphthe-

riae.

As explained previously, the rods of the genera

Bacillus and Clostridium are able to form resistant

spores when conditions for vegetative growth are

unfavourable. Many rods are motile having a single

20 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.2

ﬂagellum, or several ﬂagella, at one or both ends or

surrounding the entire organism.

Gram reaction: Many rods are Gram negative such as the

large group of enterobacteria. Gram positive rods include

Clostridium species, Corynebacterium species, Bacillus

anthracis, and Listeria monocytogenes.

Note: Some coccobacilli, such as Yersinia species, show

bipolar staining when stained with methylene blue or Giemsa.

Cocci in groups Streptococci Various diplococci

Vibrios Bacilli Bi-polar staining Bacilli in chains

Actinomycetes Terminal

Spores

Central Subterminal

Treponemes Leptospires Borreliae

10μm

Fig 7.1 Morphological features of bacteria.

Vibrios: These are small slightly curved rods mea-

suring 3–4 m in length by 0.5m in width. Most

vibrios are motile with a single ﬂagellum at one end.

They show a rapid darting motility, e.g. Vibrio

cholerae.

Gram reaction: Vibrios are Gram negative.

Spirilla: These are small, regularly coiled, rigid

organisms measuring about 3–4 m in length. Each

coil measures about 1 m. Spirilla are motile with

groups of ﬂagella at both ends. An example of a

spirillum is Spirillum minus.

Gram reaction: Spirilla are Gram negative.

Spirochaetes: These are ﬂexible, coiled, motile

organisms. They progress by rapid body move-

ments. Most are not easily stained by the Gram

method. Spirochaetes are divided into three main

groups:

– treponemes, which are thin delicate spirochaetes

withre gulartight coils, measuring from 6–15m

by 0.2 m in width. Examples include

Treponemapallidum and Treponema pertenue.

– borreliae, which are large spirochaetes with irreg-

ular open coils, measuring 10–20 m in length

by about 0.5 m in width. Examples include

Borrelia duttoni and Borrelia vincenti.

– leptospires, which are thin spirochaetes with

many tightly packed coils that are difﬁcult to dis-

tinguish. They measure 6–20 m in length by

0.1m in width and have hooked ends. The lep-

tospire of medical importance is Leptospira

interrogans (contains many serovars).

Note: Diseases carried by medically important cocci, rods,

vibrios and spirochaetes are summarized in Chart 7.4.

Rickettsiae

Although classiﬁed as bacteria, rickettsiae resemble

viruses in that they replicate only in living cells and

are unable to survive as free-living organisms. They

can just be seen with the light microscope (red par-

ticles in Giemsa preparations). Unlike viruses,

rickettsiae contain both RNA and DNA, multiply by

binary ﬁssion and have cell walls composed of pep-

tidoglycan. They show sensitivity to antiseptics and

some antibiotics.

Note: The important diseases caused by Rickettsiaspecies and

rickettsia-like microorganisms are described in subunit

7.18.35.

Chlamydiae

Chlamydiaeare small (250–500 nm) Gram negative

bacteria but resemble viruses in being unable to

replicate outside of living cells. They contain both

MICROBIOLOGICAL TESTS 21

7.2

DNA andR NAand have their own enzyme systems.

The energy required for metabolic activities is

supplied by the host cell. Chlamydiae develop and

reproduce in a special way. The infectious form is

called an elementary body. Following infection of

a host cell, the elementary body develops into a

reticulate body. This reproduces by binary

ﬁssion, producing microcolonies within a large cyto-

plasmic inclusion (chlamydial inclusion). Elementary

particles are produced and released to infect new

cells when the host cell ruptures (48–72 h after

infection).

Note: The diseases caused by Chlamydiaspecies are described

in subunit 7.19.37.

Prion particles

Neither bacteria nor viruses, prions are thought to

be infectious self-replicating protein particles that

cause a range of rare fatal degenerative neurological

diseases (transmissible degenerative spongiform

encephalopathies) which have long incubation

periods. They include:

– Scrapie in sheep (disease known for many years).

– Bovine spongiform encephalopathy (BSE) in

cattle.

– Kuru in humans (found only in Papua New

Guinea, associated with ritual cannibalism, now a

rare occurrence).

– Creutzfeld-Jakob disease (CJD) affecting mainly

elderly people, and new variant CJD affecting

younger people. Infection of brain tissue causes

vacuolation of neurones with a sponge-like

appearance of the tissue. Inﬂammatory cells are

absent and there is no antibody response.

Prions are particularly resistant to heat and some

chemical agents. They are inactivated by hypochlo-

rite and by autoclaving at 134C for 18 minutes.

Bacteria lacking cell walls

Four types of bacteria with deﬁcient cell walls are

recognized:

– Mycoplasma species

– L-forms

– Spheroplasts

– Protoplasts

Mycoplasmas: These are naturally occurring stable

bacteria that lack a rigid cell wall. They are among

the smallest living microorganisms capable of inde-

pendent existence, ranging in size from 0.1–2 m.

Species of medical importance include Mycoplasma

pneumoniae and Ureaplasma urealyticum.

L-forms:These are mutant bacteria without a cell wall, usually

produced in the laboratory but sometimes formed in the body

of patients being treated with penicillin. They can reproduce

on ordinary culture media.

Protoplasts:These unstable cell deﬁcient forms originate arti-

ﬁcially. The cell wall is lost due to the action of lysozyme

enzymes which destroy peptidoglycan. Protoplasts are meta-

bolicallyactive but unable toreproduce. They are easily lyzed.

Spheroplasts: Derived from Gram negative bacteria, sphero-

plasts are bacteria with a damaged cell wall. The damage is

caused by a toxic chemical or antibiotic such as penicillin.

They are able to change back to their normal form when the

toxic agent is removed.

Reproduction of bacteria

Bacteria multiply by simple cell division known as

binary ﬁssion (splitting into two). The single piece of

double-stranded DNA reproduces itself exactly. The

information required to make the cell’s protein is

encoded in the bacterial genome. Messenger (m)

RNA is transcribed from the DNA chromosome and

the proteins translated from the mRNA are assem-

bledby the ribosomes. Several enzymesare involved

in DNA replication and protein production. Bacterial

mutations (chemical alteration in DNA) or transmis-

sible bacterial variations involving gene transfer may

occur in response to environmental changes.

Gene transfer

Where fragments of chromosomal DNA from one bacterium

are transferred into another bacterial cell by phage (virus that

infects a bacterium) this is referred to as transduction. It can

only occur between closely related bacterial strains. The main

way genetic material can be exchanged between bacterial cells

is by conjugation involving plasmids (see previous text). Less

commonly, some bacteria are able to take up soluble DNA

molecules from other closely related species directly across

their cell wall (transformation).

When a bacterial species produces several forms

each with its own characteristics, these variations are

called strains.

Cultural characteristics

Most medically important bacteria can be grown

artiﬁcially in the laboratory provided the atmospheric

conditions and temperature are correct and the

culture medium used contains the required nutri-

ents (described in detail in subunit 7.4).

Differences in the effect of oxygen on bacterial

growth provide a further way of classifying bacteria:

● aerobes, which require free oxygen to grow,

● anaerobes, which are unable to grow in free

oxygen,

● facultative anaerobes, which can grow in con-

ditions in which oxygen is present or absent,

● microaerophiles which grow best in conditions of

reduced oxygen concentration.

Note: Chart 7.3 is a basic classiﬁcation of the med-

ically important bacteria based on their Gram

22 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.2

reaction, morphology, whether they are sporing or

non-sporing (Gram positive bacteria) and whether

they are aerobes, facultative anaerobes, anaerobes,

or microaerophiles. The diseases caused by these

pathogens are summarized in Chart 7.4.

Chart 7.3 Basic classiﬁcation of medically important bacteria

GRAM POSITIVE BACTERIA

Aerobe or Staphylococcus

facultative Streptococcus

Cocci

anaerobe Enterococcus

Anaerobe Peptostreptococcus

Corynebacterium

Listeria

Aerobe or

Lactobacillus

facultative

Nocardia

Rods

anaerobe

Mycobacterium

Bacillus

Actinomyces

Anaerobe

Clostridium

GRAM NEGATIVE BACTERIA

Cocci Aerobe Neisseria

Aerobe Pseudomonas

Salmonella

Shigella

Klebsiella

Proteus

Rods

Facultative

Escherichia

anaerobe

Yersinia

Bordetella

Haemophilus

Brucella

Pasteurella

Vibrio

Bacteroides

Anaerobe Fusobacterium

Prevotella

Microaerophile Campylobacter

Aerobe Leptospira

Spirochaetes

Borrelia

Anaerobe

Treponema

Normal microbial ﬂora

The normal microbial ﬂora are those organisms that

make their home on or in some part of the body. In

a healthy person such organisms rarely cause

disease. Microorganisms of the normal ﬂora consist

of symbionts, commensals, and opportunists.

Symbionts: These are organisms that usually beneﬁt the

person infected, e.g. the enteric bacteria that form part of the

normal ﬂora of the intestine, assist in the synthesis of vitamin

K and some of the vitamins of the B complex.

Commensals: These organisms form the largest group of the

normal microbial ﬂora of the body. They live on skin and the

mucous membranes of the upper respiratory tract, intestines,

and vagina. They are mostly neither beneﬁcial nor harmful to

their host, and can protect by competing with potential

pathogens.

Opportunists: These are the organisms that can, if a suitable

opportunity arises, become pathogenic and cause disease.

Such an opportunity may arise following:

– The transfer of a commensal from its usual habitat to

another part of the body where it can establish itself and

cause disease, e.g. Escherichia coliis a normal inhabitant

of the intestinal tract but if it enters the urinary tract it can

cause urinary infection.

– The weakening of a person’s natural immunity due to

poor health, malnutrition, previous surgery, infection with

HIV, or drug therapy, e.g. Staphylococcus aureus is a

normal commensal in the nose but it may become a

pathogen and cause pneumonia in a child with measles or

inﬂuenza.

Opportunistic organisms are often the cause of what are

called nosocomial infections, i.e. infections accidentally

acquired by patients during a hospital stay due to their

defence mechanisms being weakened.

Factors which inﬂuence the sites in the body selected

by the organisms of the normal ﬂora include tem-

perature, pH, and available nutrients. When

optimum conditions for the balanced growth of the

body’s normal ﬂora become disturbed, for example,

due to intensive broad spectrum antibiotic treat-

ment, this can lead to those organisms not affected

by the antibiotic increasing in numbers and causing

ill health. The range of organisms that make up a

person’s normal ﬂora is dependent on a number of

factors including age, gender, hormonal activity,

race, environment, diet and nutrition.

In the laboratory investigation of microbial infec-

tions, it is important to be aware of those sites in the

body that are normally colonized by micro-

organisms because specimens originating from or

collected via these sites are likely to contain com-

mensals which can make it difﬁcult to interpret

cultures. One of the ways of preventing the growth

of unwanted commensals is to use a selective culture

medium (or selective and enrichment medium)

which will inhibit the growth of commensals while

supporting the growth of the pathogen(s) suspected

of causing the infection.

Sites of the body having a normal microbial ﬂora

include the skin, axilla and groin, conjunctiva,

external ear, mouth, nose and nasopharynx, large

intestine, anterior urethra and vagina. The following

text lists those specimens in which commensals are

likely to be found and those specimens which in

MICROBIOLOGICAL TESTS 23

7.2

health do not contain microorganisms and should

not contain contaminants providing an aseptic col-

lection technique and sterile container are used.

Chart 7.4 Bacterial pathogens and diseases they cause

Disease Bacterial pathogen

RESPIRATORY INFECTIONS AND MENINGITIS

Tuberculosis Mycobacterium tuberculosis

Lobar pneumonia Streptococcus pneumoniae

Bronchopneumonia Streptococcus pneumoniae

Haemophilus inﬂuenzae

Occasionally Staphylococcus

aureusand coliforms (E. coli,

Proteus, Klebsiella, Pseudomonas)

Atypical pneumonia Mycoplasma pneumoniae

Coxiella burnetii

Pneumocystis Pneumocystis jiroveci

pneumonia

Empyema Streptococcus pneumoniae

Staphylococcus aureus

Whooping cough Bordetella pertussis

(Pertussis) Occasionally B. parapertussis

Chronic Haemophilus inﬂuenzae

bronchitis Streptococcus pneumoniae

Occasionally Moraxellacatarrhalis

Acute epiglottitis Haemophilus inﬂuenzae

(Croup syndrome)

Sore throat Streptococcus pyogenes

(Pharyngitis) Post-streptococcal immunological

complications include rheumatic

fever and acute glomerulo-

nephritis

Peri-tonsillar Streptococcus pyogenes

abscess

SPECIMENS CONTAINING COMMENSALS

● Sputum

● Throat and mouth specimens

● Nasopharyngeal and nasal specimens

● Eye discharges

● Skin and ulcer specimens

● Urogenital specimens

● Faeces and rectal swabs

● Urine (small numbers of commensals)

Note: The commensals which may be present in each

specimen are listed in those subunits in which the exam-

ination of each specimen is described.

SPECIMENS NOT CONTAINING COMMENSALS*

● Pus (wounds, abscesses, burns, sinuses)

● Cerebrospinal ﬂuid

● Blood

● Serous ﬂuids (synovial, pericardial, ascitic, hydro-

cele)

*Note:Special care must be taken not to introduce con-

taminants into these specimens.

Ulcerative tonsillitis Borrelia vincenti and Gram

(Vincent’s angina) negative anaerobes

Diphtheria Corynebacterium diphtheriae

Otitis media Haemophilus inﬂuenzae

(middle ear Streptococcus pyogenes

infection) Streptococcus pneumoniae

Staphylococcus aureus

Pseudomonasspecies

Proteusspecies

Bacteroides fragilis

Meningitis Neisseria meningitidis

– pyogenic Streptococcus pneumoniae

Haemophilus inﬂuenzae

Note: Neonatal meningitis may

also be caused by Escherichia coli,

Klebsiellaspecies, Proteus species,

Listeria monocytogenes, and

Streptococcus agalactiae(Group B).

– tuberculous Mycobacterium tuberculosis

EYE INFECTIONS

Stye, blepharitis Staphylococcus aureus

Conjunctivitis Haemophilus inﬂuenzae(pink eye)

Staphylococcus aureus

Streptococcus pneumoniae

Neonatal eye Neisseria gonorrhoeae

infections Chlamydia trachomatis (D–K)

Staphylococcus aureus

Trachoma Chlamydia trachomatis(A–C)

SEPTICAEMIA and BACTERAEMIA

Associated with Salmonella Typhi, other Salmonella

generalized infection: serovars

Neisseria meningitidis

(meningococcal septicaemia)

Mycobacterium tuberculosis(HIV

coinfection)

Yersinia pestis(septicaemic plague)

Brucellaspecies

Listeria monocytogenes

(immunocompromised)

Pathogens entering Haemophilus inﬂuenzae

from localized Staphylococcus aureus

infections: Streptococcusspecies

Escherichia coli

Salmonellaserovars (common with

HIV coinfection)

Shigella dysenteriae

Enterococcusspecies

Pseudomonasspecies

Proteusspecies

Klebsiellaspecies

Bacteroides fragilis

Clostridium perfringens

Bartonellosis Bartonella bacilliformis

ARTHRITIS AND BONE INFECTIONS

Arthritis Staphylococcus aureus

Neisseria gonorrhoeae

Neisseria meningitidis

24 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.2

Streptococcus pneumoniae

Osteomyelitis Staphylococcus aureus

Occasionally Haemophilus

inﬂuenzae, Streptococcus pyogenes,

coliforms, Mycobacterium

tuberculosis, Brucellaspecies

DIARRHOEAL DISEASES

Bacillary dysentery Shigella species

Campylobacter Campylobacter jejuni

enterocolitis Campylobacter coli

Escherichia coli Enteroinvasive E. coli (EIEC)

dysentery Enterohaemorrhagic E. coli

(EHEC)

Enteroaggregative E.coli (EAEC)

Salmonella Salmonella serovars

enterocolitis

Yersinia Yersinia enterocolitica

enterocolitis (rare infection)

Cholera Vibrio cholerae O1, 0139

Vibriogastroenteritis Vibrio parahaemolyticus

Infantile E.coli Enteropathogenic E. coli (EPEC)

diarrhoea Enterotoxigenic E. coli (ETEC)

(also cause of traveller’s

diarrhoea)

Salmonellafood- Salmonellaserovars

poisoning

Clostridial food- Clostridium perfringens

poisoning

Staphylococcal food- Staphylococcus aureus

poisoning (enterotoxin-producing strains)

Campylobacter Campylobacter jejuni

enteritis Campylobacter coli

Bacillusfood- Bacillus cereus and other

poisoning species

Antibiotic-associated Clostridium difﬁcile(may also

diarrhoea (rare) cause pseudomembranous colitis)

Botulism Clostridium botulinum

Disease caused by exotoxin in

contaminated food

Gastric and Helicobacter pylori

duodenal ulceration

URINARY TRACT INFECTIONS (UTI)

Common cause Escherichia coli

Less common cause Proteus mirabilis

Enterococcus faecalis

Klebsiellaspecies

UTI in sexually Staphylococcus saprophyticus

active women

UTI associated with Staphylococcus aureus

catheterization or Pseudomonas aeruginosa

instrumentation Proteus Klebsiella

Renal tuberculosis Mycobacterium tuberculosis

SEXUALLY TRANSMITTED INFECTIONS

Venereal syphilis Treponema pallidum

Gonorrhoea Neisseria gonorrhoeae

Soft chancre Haemophilus ducrei

Granuloma inguinale Klebsiella granulomatis

(donovanosis)

Lymphogranuloma Chlamydia trachomatis (L1–L3)

inguinale

Vaginosis Gardnerella vaginalis and

Bacteroides

Non-speciﬁc Chlamydia trachomatis(D–K)

urethritis Ureaplasma urealyticum

Pelvic inﬂammatory Neisseria gonorrhoeae

disease Chlamydia trachomatis (D–K)

SKIN AND WOUND INFECTIONS

Boils, abscesses, stye Staphylococcus aureus

pustules, carbuncles

Impetigo Staphylococcus aureus

Streptococcus pyogenes

Occasionally Corynebacterium

diphtheriae

Erysipelas Streptococcus pyogenes

Cellulitis Streptococcus pyogenes

Wound infections

– Surgical Staphylococcus aureus

Escherichia coli

Proteusspecies

Klebsiellaspecies

Enterococcusspecies

Pseudomonas aeruginosa

Clostridium perfringens

Bacteroides fragilis

Anaerobic cocci

– Puerperal sepsis and Streptococcus pyogenes

septic abortion Streptococcus agalactiae

Clostridium perfringens

– Burns Streptococcus pyogenes

Pseudomonas aeruginosa

– Gas gangrene Clostridium perfringens

Occasionally Clostridium novyi,

Clostridium septicum

Peritonitis Coliforms, Enterococcusspecies,

Bacteroides fragilis. Occasionally

Clostridium perfringens

Tetanus Clostridium tetani

Buruli ulcer Mycobacterium ulcerans

Tropical ulcer Borrelia vincentiwith fusiform

bacilli

Streptococcus pyogenes

Leprosy Mycobacterium leprae

Actinomycosis Actinomyces israeli

Nocardiosis Nocardia asteroides

Mycetoma Nocardia brasiliensis

(Madura foot) Nocardia caviae

Yaws (framboesia) Treponema p.pertenue

MICROBIOLOGICAL TESTS 25

7.2

Pinta Treponema carateum

ZOONOSES

Brucellosis Brucella species

Plague Yersinia pestis

Anthrax Bacillus anthracis

Leptospirosis Leptospira interrogans

Rat bite fever Spirillum minus

Streptobacillus moniliformis

Listeriosis Listeria monocytogenes

Food-poisoning Salmonella serovars

Campylobacterspecies

Escherichia coli(EHEC:0157)

Mesenteric adenitis, Yersinia pseudotuberculosis

enteritis Yersinia enterocolitica

Lyme disease Borrelia burgdorferi

Tularaemia Francisella tularensis

Q fever Coxiella burnetii

Enteritis necroticans Clostridium perfringens(C)

(Pigbel)

ARTHROPOD-BORNE INFECTIONS

Louse-borne Borrelia recurrentis

relapsing fever

Tick-borne Borrelia duttoniand other

relapsing fever Borrelia species

Epidemic typhus Rickettsia prowazekii

Vector: Louse

Endemic typhus Rickettsia typhi

Vector: Flea

Scrub typhus Orientia tsutsugamushi

Vector: Mite

Tick-borne typhus Rickettsia conorii

Vector: Tick

Rocky Mountain Rickettsia rickettsii

spotted fever Vector: Tick

Trench fever Bartonella quintana

Vector: Louse, Reservoir: Possibly

rodent

Note: The laboratory features of the bacteria listed in this

chart are described in subunits 7.18.1–7.18.37 and in the

subsequent subunits describing the examination of specimens.

FEATURESAND CLASSIFICATIONOF VIRUSES

Features which make viruses different from other

microorganisms are their small size, non-cellular

structure, genome containing either deoxyribonu-

cleic acid (DNA) or ribonucleic acid (RNA) but not

both, and their inability to replicate outside of living

cells. Because viruses replicate inside cells, fewer

drugs are available to treat virus infections compared

with bacterial infections, although vaccines are avail-

able against virus diseases such as inﬂuenza, yellow

fever, poliomyelitis, measles, mumps, rubella, hepa-

titis A and B, and rabies.

Structure of viruses

Virus particles, or virions (infectious particles), are too

small to be seen by the light microscope, measuring

only 20–300 nm (0.02–03 m). They can however

be seen by the electron microscope (specialist

virology laboratories).

All viruses consist of a mass (core) of nucleic acid

(DNA or RNA) surrounded by a protective protein

coat called a capsid. For RNA viruses, the genome

can be single stranded, double stranded or frag-

mented. The genome of most DNA viruses is

double stranded. The nucleic acid together with the

capsid form the nucleocapsid. The capsid is anti-

genic and also contains the receptors which enable

a virus to attach to the surface of its speciﬁc host cell.

The capsid consists of a number of identical units

called capsomeres. The symmetry, or pattern, of

capsides is one of the features used to classify

viruses.

Capsid symmetry

Capsid symmetry is described as being:

● Icosahedral in which the capsomeres are arranged to form

a symmetrical icosahedran surrounding the nucleic acid.

Small size (below 50 nm) icosahedral viruses appear

spherical.

● Helical in which the capsomers are arranged around a

spiral of nucleic acid. Helical viruses can appear spherical,

elongated, or ﬁlamentous.

● Complex, meaning the capsid symmetry is neither icosa-

hedral nor helical.

Virus envelope

Many helical viruses and a few icosahedral viruses

are surrounded by an envelope. This is a lipoprotein

membrane composed of lipid and virus-speciﬁc gly-

coproteins (antigens) that project as spikes from the

surface of the envelope. Compared with non-

enveloped (naked) viruses, enveloped viruses are

more sensitive to heat, detergents, and lipid solvents.

Infection of cells by viruses

Because viruses possess neither a cellular structure

nor organelles, they are dependent on their host

cells for energy and replication. Outside of living

cells, viruses are metabolically inert.

The information required for a virus to replicate

is contained in its nucleic acid (genome). Following

infection, a virus ‘takes over’ the synthesizing activi-

ties of its host cell, directing the cell to transcribe

and, or, translate its genetic information to produce

the protein and nucleic acid components required to

26 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.2

make new virions. Most DNA viruses replicate and

are assembled in the nucleus of the host cell

whereas most RNA viruses replicate and are assem-

bled in the cytoplasm.

Mature virions are released from host cells either

by rupture of the cell membrane as occurs with most

unenveloped viruses, or by a process called budding

in which enveloped virions are extruded from the

cell membrane.

Viruses that infect bacteria are called bacterio-

phages, or phages. They infect only a narrow range

of bacteria. They are important because they are

able to transfer bacterial DNA from one bacterial cell

to another and also incorporate viral DNA into the

bacterial chromosome. This can result in bacteria

producing new proteins, e.g. toxins. Bacteriophages

that lyze bacteria are referred to as virulent phages.

Phage typing using lytic phages can help to identify

bacterial strains, e.g. salmonellae (in specialist

bacteriology centres).

Effects of viruses on cells

When a virus infects a cell it usually replicates and causes the

death of its host cell. The observable changes which lead to

the death of a host cell are called cytopathic effects (CPE).

These may include the formation of inclusion bodies (sites of

virus replication) or syncytia (virus-infected cells fused

together). In viruses cultured in the laboratory, CPE is

observed by a shrinking or enlargement of infected cells, and

the detachment of dead cells from the glass surface on which

they are growing.

Occasionally, viruses infect cells and replicate without

causing the immediate death of their host cells. New viruses

are extruded through the cell membrane of the infected cells.

Examples of such viruses include rubella virus, parainﬂuenza

viruses, and hepatitis B virus.

Some viruses after infecting cells do not replicate, or they

become active for a time and then become inactive (latent).

Viruses that can cause latent infections include herpes viruses

where viral nucleic acid remains in the cytoplasm of the host

cell, and HIV where DNA copy of viral nucleic acid becomes

part of the host cell genome. In response to certain stimuli,

latent viruses can be reactivated and start to replicate.

Some viruses are able to change, or transform, their host

cells from normal cells into tumour producing cells. Such

viruses are said to have neoplastic, or oncogenic properties.

RNA neoplastic viruses usually replicate in their transformed

cells (without causing cytolysis) whereas DNA neoplastic

viruses usually do not replicate in the cells they have trans-

formed.

Transmission of viruses

Human virus diseases are caused by:

 Viruses for which man is the natural or most

important maintenance host.

Examples: rotaviruses, polioviruses, hepatitis

viruses, HIV, rubella virus, rhinoviruses, measles

virus, papillomaviruses, and several herpes

viruses.

Transmission routes for human viruses

– By direct contact, e.g. sexually transmitted viruses such as

HIV, herpes simplex virus 2, and hepatitis B virus.

– By ingesting viruses in food or water contaminated with

faeces, e.g. enteroviruses, rotaviruses, and hepatitis A

virus.

– By inhaling viruses in airborne droplets, e.g. inﬂuenza

viruses, measles virus, adenoviruses, respiratory syncytial

virus, and rhinoviruses. Overcrowding greatly assists in

the spread of droplet infections.

– By contact with an article, such as a ﬂoor mat contami-

nated with papillomavirus (wart-producing virus) or a

towel contaminated with a virus that causes eye infection.

– By a mother infecting her child during pregnancy, e.g.

cytomegalovirus or rubella virus. Such infections may

cause abortion, stillbirth, congenital abnormalities, or ill-

health of the newborn. Hepatitis B virus and HIV can be

transmitted from mother to baby during birth.

– By transfusion of virus infected blood, e.g. HIV 1 and 2,

hepatitis B virus, and hepatitis C virus.

Note: Human viruses can also be carried from one person

to another on the bodies of houseﬂies or bedbugs.

 Viruses for which arthropods (mosquitoes, sand-

ﬂies, ticks) and vertebrate animals, especially

rodents, birds, monkeys, are the natural or main

reservoir hosts and humans only accidental or

secondary hosts.

Examples: rabies virus, viruses that cause viral

haemorrhagic fever, and the large number of

arthropod-borne viruses which cause diseases

such as yellow fever, dengue, and Rift Valley

fever.

Transmission routes for arthropod and animal viruses

– By the bite of an infected, blood-sucking mosquito,

sandﬂy, tick or midge. Arthropod-borne viruses are

referred to as arboviruses although they belong to several

different virus groups. They are major causes of fever,

encephalitis and viral haemorrhagic fever (VHF) in

tropical and developing countries (see later text).

– By the bite of an animal host, e.g. rabies virus is transmit-

ted to man through the bite of an infected dog or other

rabid animal.

– By man coming into contact with vegetation, food, or

articles that have been contaminated with the excretions

of infected animals, especially rodents, e.g. Lassa fever is

transmitted via rodent urine. Infection can occur if the

virus enters damaged skin, is inhaled in aerosols, or is

ingested.

– By the direct transfer of viruses from one person to

another, especially highly infectious viruses such as Ebola

and Marburg viruses. The viruses are present in the saliva,

urine and blood of infected persons.

Seasonal changes in climate can also inﬂuence the

rate of transmission and spread of virus diseases, e.g.

increases in mosquito numbers during the rainy

season and times of ﬂooding, increase the incidence

of mosquito-borne infections such as dengue,

MICROBIOLOGICAL TESTS 27

7.2

O’nyong, and Rift Valley fever. Lack of effective

vector control, late response to epidemics, the

creation of habitats that favour vector breeding or

bring people into closer contact with vectors (e.g.

deforestation or poorly planned irrigation schemes)

are also important factors that increase the incidence

and spread of arthropod-borne virus infections.

Opportunistic virus infections

Several viruses cause opportunistic infections in

those with defective or inadequate immune

responses, e.g. AIDS patients or those receiving

treatment with immunosuppressive drugs. Such

viruses include herpes simplex viruses (HHV-1,

HHV-2), cytomegalovirus, varicella zoster virus,

papovavirus, and HHV-8 which has been linked to

Kaposi’s sarcoma.

Laboratory transmission of virus infections

Laboratory transmission of viruses can occur by acci-

dentally inhaling viruses in aerosols, ingesting

viruses from contaminated ﬁngers, or by viruses

entering damaged skin (e.g. through cuts, scratches,

insect bite wounds, eczematous areas) or acciden-

tally through needle stick injuries or occasionally by

contamination of the eye or membranes of the nose

and mouth. Viruses can also be transmitted by way

of contaminated laboratory coats.

To avoid laboratory infection with highly virulent

and, or, infectious viruses such as Lassa, Marburg

and Ebola viruses, Crimean-Congo virus, Kyasanur

forest disease virus, and hepatitis B virus, every

possible safety precaution must be taken when col-

lecting, handling and testing specimens, especially

blood, urine, body ﬂuids and exudates. Specimens

from patients with suspected viral haemorrhagic

fever must be tested in a specialist virology labora-

tory with adequate containment facilities. Laboratory

staff should know the effects of heat and chemical

agents on viruses and which disinfectants are the

most effective inactivating agents.

Effects of physical and chemical agents on viruses

● Heat: Most viruses are inactivated at 56C for 30 minutes

or at 100C for a few minutes.

Cold: Viruses are stable at low temperatures. Most can be

satisfactorily stored frozen although some viruses are par-

tially inactivated by freezing and thawing.

● Ultraviolet (UV) irradiation: Inactivates viruses.

● Chloroform, ether and other organic solvents: Viruses sur-

rounded by an envelope are inactivated. Unenveloped

viruses are resistant (see Chart 7.5).

● Oxidizing and reducing agents: Chlorine, iodine,

hydrogen peroxide, and formaldehyde, all inactivate

viruses.

● Phenols: Most viruses are relatively resistant.

● Virus disinfectants: Include hypochlorite solutions and

glutaraldehyde. Tissues, however, may protect viruses by

quenching disinfectant.

28 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.2

Classiﬁcation of medically important viruses

Virusesare classiﬁed bytheir genome (RNA orDNA),

morphology (capsid symmetry), size, presence or

absence of anenvelope and method of replication.

Chart 7.5 RNA and DNA viruses of medical importance

RNA VIRUSES

Family Form/Size Envelope Viruses Disease Transmission to humans

Togavirus Icosahedral  Rubivirus Rubella, congenital Respiratory droplets,

60–70 nm malformations transplacental

Alphaviruses

● Chikungunya virus Fever, arthritis, rash

● Western and Fever,

Venezuelan equine encephalitis

encephalitis viruses

● Sindbis virus Fever, rash Mosquitoes

● Ross River virus Fever, arthritis, rash

● Mucambo virus Fever

● Mayaro virus Fever, rash

● O’nyong-nyong virus Fever, arthralgia, rash

Bunyavirus Helical  ● Rift Valley fever virus Fever, VHF Mosquitoes

90–120 nm

● Hantaan virus VHF, renal disease, Rodent saliva

pulmonary syndrome and urine

● Crimean-Congo Fever, VHF, rash Ticks

haemorrhagic fever virus

● Hazara virus VHF Ticks

● Bunyamwera group

● Bwamba group

Fever Mosquitoes

● C group

● Guama group

● Oropuche virus Fever, encephalitis Mosquitoes

● Sandﬂy fever virus Fever Sandﬂies

Flavivirus Complex  ● Hepatitis C virus Hepatitis C (HCV), Blood, sexual, mother to

40–50 nm liver carcinoma child

● Hepatitis G virus Hepatitis G (HGV) Blood

● Dengue (1– 4) viruses Dengue, DHF, DSS

● Yellow fever virus Fever, jaundice, VHF

● Japanese encephalitis Fever, encephalitis

virus Mosquitoes

● West Nile fever virus Fever, encephalitis,

● Murray River virus rash

● Rocio virus Fever, encephalitis

● Kyasanur Forest Fever, VHF Ticks

disease virus

Filovirus Helical  ● Marburg virus Person to person

80 1000 nm ● Ebola virus VHF (Rodents probably

natural hosts)

Arenavirus Complex  ● Lymphocytic Meningitis Rodent urine

50–300 nm choriomeningitis virus

● Lassa virus

● Machupo virus

VHF

Rodent urine

● Junin virus

● Suanarito virus

Rhabdovirus Helical  ● Rabies virus Rabies Bite of rabid animal

75–180 nm (dog, wolf or bat)

MICROBIOLOGICAL TESTS 29

7.2

Paramyxovirus Helical  ● Morbillivirus (measles) Measles

120–300 nm virus

● Parinﬂuenza virus Bronchiolitis, croup

(infants), colds

● Mumps virus Mumps

Respiratory droplets

● Pneumovirus (respiratory Bronchiolitis and

syncytial virus) pneumonia (infants)

Orthomyxovirus Helical  ● Inﬂuenza A (including, Inﬂuenza Respiratory droplets

80–120 nm new avian strain), B, C

viruses

Coronavirus Helical  ● Coronaviruses Respiratory infection, Respiratory droplets

60–220 nm common cold. SARS

(severe acute respiratory

syndrome)

Retrovirus Icosahedral  ● HIV-1, HIV-2 viruses AIDS/HIV disease Sexual, blood, mother to

About 100 nm

● HTLV-1, II viruses T cell leukaemia,

child

lymphoma, paresis As above

Picornavirus Icosahedral  ● Hepatitis A virus Hepatitis A (HAV) Faecal–oral

22–30 nm

● Rhinoviruses Colds Respiratory droplets

Enteroviruses

● Poliovirus, 1, 2, 3 Poliomyelitis Faecal–oral

encephalitis

● Coxsackie A (1– 24), Respiratory infection Faecal–oral

B (1–6) viruses rash, meningo- Respiratory droplets

● Echoviruses 1– 34 encephalitis,

myocarditis

● Enteroviruses 68– 71 Respiratory disease, Respiratory droplets

haemorrhagic

conjunctivitis

Reovirus Icosahedral  ● Rotavirus Gastroenteritis Faecal–oral

70–80 nm

Calicivirus Icosahedral  ● Hepatitis E virus Hepatitis E (HEV) Faecal–oral

27–38 nm

● Norwalk virus Gastroenteritis Faecal–oral

DNA VIRUSES

Family Form/Size Envelope Viruses Disease Transmission

Poxvirus Complex  Orthopox viruses

250–400 nm ● Variola virus Smallpox (now

extinct)

● Monkeypox virus Mild smallpox-like Contact with infected

disease monkeys/squirrels

● Cowpox virus Ulcerating lesions Milking infected cows

Parapox viruses

● Orf virus Orf (pustular Contact with infected

dermatitis) sheep or goats

● Molluscum contagiosum Skin nodules Skin contact. Also

virus opportunistic

Herpesvirus Icosahedral  Human herpes viruses (HHV)

120–150 nm ● HHV-1 (herpes simplex Cold sores, oral and Saliva skin contact

virus 1) eye infections,

encephalitis

Virus diseases in tropical countries

The following are among the virus diseases that can

cause epidemics and, or, are important causes of ill-

health and mortality in tropical and developing

countries:

● HIV disease and AIDS

● Virus hepatitis (HAV, HBV, HCV)

● Dengue and dengue haemorrhagic fever

● Japanese encephalitis

30 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.2

● Viral haemorrhagic fever caused by Ebola virus,

Marburg virus, Lassa virus and other viruses as

listed in Chart 7.5

● Yellow fever

● Rift Valley fever

● Infections caused by herpes viruses

● Virus infections causing gastroenteritis, particu-

larly in young children

● Measles

● HHV-2 (herpes simplex Genital herpes, Sexual, mother to child

virus 2) neonatal infection

● HHV-3 (varicella zoster Chickenpox, shingles Respiratory droplets,

virus) reactivation

● HHV-4 (Epstein-Barr Glandular fever Oral, saliva

virus)

Associated with Co-factors involved

Burkitt’s lymphoma, in tumour development

nasopharyngeal

carcinoma

● HHV-5 (Cytomegalovirus) Glandular fever, Body ﬂuids,

congenital infection, transplacental

disseminated infection

in AIDS and immuno-

compromised,

pneumonitis and

hepatitis

● HHV-6 Roseola infantum, Primary and reactivation

mononucleosis

● HHV-8 virus Associated with Unknown

Kaposi’s sarcoma

Hepadnavirus Icosahedral  ● Hepatitis B virus Hepatitis B (HBV) Blood, sexual, mother to

About 42 nm acute and chronic, child, close contact

liver carcinoma

Deltavirus Helical  ● Hepatitis D virus* Hepatitis D (HDV) As for HBV

About 37 nm

* Found only in those

infected with hepatitis B virus

Adenovirus Icosahedral  ● Adenoviruses 1–49 Sore throat, Respiratory droplets

70–90 nm pneumonia,

conjunctivitis

Papovavirus Icosahedral  ● Papillomavirus (70) Warts, carcinoma Skin contact,

45–55 nm of cervix, rectum, genital

penis

● JC virus Rare neurological Opportunistic

disease in

immunocompromised

Parvovirus Icosahedral  ● B19 virus Childhood fever, Respiratory route,

About 22 nm rash (cheeks), transplacental

aplastic crisis,

hydrops fetalis

Abbreviation:VHF  Viral haemorrhagic feversymptoms, DHF  Dengue haemorrhagic fever, DSS  Dengueshock syndrome

Further information: For detailed information on the pathogenesis of virus infections and host responses, readers are referred to

textbooks of clinical microbiology such as those listed under Recommended Reading at the end of this chapter. Further infor-

mation on virus infections in tropical countries can be found in Mansons Tropical Diseases, 21st edition, 2003.

● Rabies ● Poliomyelitis

● Burkitt’s lymphoma

SARS (severe acute respiratory syndrome) is caused by a

newly identiﬁed coronavirus, SARS-CoV. Between 2002

(when SARS pneumonia was ﬁrst reported from China) and

2004, more than 8400 people have been reported as having

SARS with an estimated 910 persons having died from it. The

virus is transmitted by infectious respiratory droplets. The

incubation period is 2–12 days. Most infections have been in

China, Taiwan, Singapore, Vietnam and Canada with inter-

national travel contributing to the rapid spread of the virus.

An ELISA and western blot assay have been developed to

detect antibodies to the virus.

Note: Based on the tests that can be performed in

district laboratories, investigation of the following

virus infections are included in this publication:

– HIV infection, described in subunit 7.18.55

– Hepatitis, described in subunit 7.18.54

– Dengue, described in subunit 7.18.53

– Examination of imprint smears for Burkitt’s

lymphoma cells, described in subunit 8.2.

Pathogenic arboviruses and viruses that cause viral

haemorrhagic fever (some are arboviruses) are of

particular importance in tropical countries.

Arboviruses

Arboviruses require an arthropod, e.g. mosquito,

tick, or sandﬂy for their transmission. Their natural

hosts include rodents, birds and monkeys.

Arboviruses are classiﬁed into three main groups:

alphavirus (formerly group A arboviruses) belonging

to the family togavirus, ﬂavivirus (formerly group B

arboviruses) and bunyavirus. Those of importance in

tropical countries are as follows:

ALPHAVIRUSES

Mosquito-borne Distribution

● Western equine

encephalitis virus South America

● Venezuelan equine South America

encephalitis virus

● Mucambo virus Brazil

● Mayaro virus South America

● Chikungunya virus Africa, India,

Southeast Asia

● Sindbis virus Africa, India,

Southeast Asia

● Ross River virus South Paciﬁc

● O’nyong-nyong virus Central Africa, East Africa

FLAVIVIRUSES

Mosquito-borne:

● Dengue virus Latitude 30 N to 40S

(4 types, see 7.18.53)

● Yellow fever virus Africa, Central and

South America

● Japanese encephalitis Far East, Japan

virus Bangladesh, India

● West Nile fever virus Africa, India

MICROBIOLOGICAL TESTS 31

7.2

● Murray River virus New Guinea

● Rocio virus Brazil

Tick borne:

● Kyasanur India

Forest disease virus

BUNYAVIRUSES

Mosquito-borne:

● Rift Valley fever virus Africa

● Bunyamwera group* Africa, South and

Central America

● Bwamba group* Africa

C group* South America

Central America

● Oropouche virus Brazil

● Guama group* South America

Tick-borne:

● Crimean-Congo Africa, Central Asia

haemorrhagic fever virus Pakistan, Middle East

● Hazara virus Pakistan

Sandﬂy-borne:

● Sandﬂy fever virus Africa, Asia, Middle East

*Details of the viruses in these groups can be found in

Mansons Tropical Diseases(see Recommended Reading).

Viruses causing viral haemorrhagic fever

Viruses that cause viral haemorrhagic fever

(VHF) include some arboviruses, arenaviruses and

ﬁloviruses as follows:

ARBOVIRUSEScausing VHF

● Dengue virus (see also subunit 7.18.53)

● Rift Valley fever virus

● Yellow fever virus

● Kyasanur Forest virus

● Crimean-Congo haemorrhagic fever virus

Note: See above text for the distribution of these viruses.

ARENAVIRUSEScausing VHF

Transmittedbyrodent excretionsand contactwith infectedperson:

● Lassa virus West and Central Africa

● Machupo virus

(Bolivian VHF)

● Junin virus

(Argentinian VHF) South America

● Guanarito virus

(Venezuelan VHF)

● Sabia virus

FILOVIRUSEScausing VHF

Transmitted from person to person (highly infectious)

● Marburg virus* East and South Africa

● Ebola virus* Zaire, Sudan, Gabon,

Ivory Coast

*These viruses are known to infect monkeys but these are not

thought to be the natural hosts (probably rodents).

Clinical features associated with infections caused by

arbovirusesand the listed arenavirusesand ﬁloviruses

include (depending on virus), fever, rash, haemor-

rhagic symptoms (viruses that cause VHF), liver

damage, encephalitis,and renal damage. Diseases to

exclude when investigating serious arboviral

and VHF infections include malaria, typhoid,

brucellosis, plague, leptospirosis, septicaemia,

bacterial meningitis, and rickettsialinfe ctions.

Laboratory ﬁndings in encephalitis: In the early

stages of infection there is usually a low white cell

count, followed by a mild leucocytosis with lympho-

cytosis. The cerebrospinal ﬂuid is under pressure, the

total protein is usually raised and up to 1000  10

cells/litre (1000/mm

) may be found. The glucose

concentration is not altered.

Laboratoryﬁndings in VHF: Haematological, bio-

chemical and urineabnormalities are summarized in

the following text:

Haematological tests

Haemoglobin. . . . . . . . . . . . . . . . . . . . . Reduced

Platelet count. . . . . . . . . . . . . . . . . . . . . Reduced

White cell count. . . . . . . . . . . . . . . . . . . Low

Blood ﬁlm . . . . . . . . . . . . . . . . . . . . . . . Reactive lymphocytes

Coagulation tests

Bleeding time . . . . . . . . . . . . . . . . . . . . Prolonged

Clotting time . . . . . . . . . . . . . . . . . . . . . Prolonged

Prothrombin time . . . . . . . . . . . . . . . . . Prolonged

Partial thromboplastin time test . . . . . Prolonged

Fibrinogen . . . . . . . . . . . . . . . . . . . . . . . Low

FDP’s . . . . . . . . . . . . . . . . . . . . . . . . . . . Raised

Biochemical tests

Aspartate aminotransferase (AST) . . Raised

Serum bilirubin . . . . . . . . . . . . . . . . . . . Raised

Blood urea . . . . . . . . . . . . . . . . . . . . . . . Raised

Serum creatinine. . . . . . . . . . . . . . . . . . Raised

Urine tests

Protein . . . . . . . . . . . . . . . . . . . . . . . . . . Present

Haemoglobin or red cells. . . . . . . . . . . Detected

Microscopy. . . . . . . . . . . . . . . . . . . . . . . Granular casts and

usually red cells

Notes: 1 In the later stages, there may be an increase in white

cells. 2 Fibrin ﬁbrinogen degradation products (FDPs)

become raised when there is disseminated intravascular coag-

ulation (DIC). 3 Other enzymes are also raised.

Caution: Strict safety precautions must be taken

when handling, transporting and testing blood,

secretions, urine and other specimens which may

contain VHF viruses, particularly highly virulent

viruses such as Ebola, Lassa, and Marburg.

Protective gloves and a gown must be worn and

where possible specimens should be handled in the

laboratory in a safety cabinet. Needles, syringes,

pipettes, and all glassware and other equipment

used in collecting and testing the specimens must

be safely decontaminated (see subunit 3.4 in Part 1

32 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.2

of the book). Any barrier (isolation) nursing pro-

cedures that are in place must be followed carefully

by laboratory personnel when visiting the wards.

Important: District laboratory staff working in areas

where VHF infections occur, should consult their

regional or central public health laboratory regard-

ing the safety procedures to follow when collecting,

transporting, testing, and disposing of specimens,

appropriate tests to perform, and the reporting of

test results.

BASIC FEATURES AND CLASSIFICATION OF FUNGI

Fungi are saprophytic, parasitic or commensal

organisms. Most live in the soil on decaying matter

helping to recycle organic matter. Unlike bacteria,

fungi have a eukaryotic cell structure, i.e. their

genetic material is differentiated into chromosomes

which are enclosed by a nuclear membrane and the

cell contains ribosomes and mitochondria. The cell

wall consists of polysaccharides, polypeptides and

chitin, and the cell membrane contains sterols which

prevent many antibacterial antibiotics being effective

against fungi. The majority of fungi are obligate

aerobes and can be grown in the laboratory on

simple culture media.

The study of fungi is called mycology. Fungi can be

divided into:

 Yeasts

 Filamentous fungi, also referred to as moulds

 Dimorphic fungi (having yeast and ﬁlamentous

forms)

Yeasts

These are round, oval or elongate unicellular fungi,

measuring 3–15 m. They reproduce by asexual

budding. A daughter cell called a blastoconidia

(often called a blastospore) is formed on the surface

of the parent cell. Elongated budding cells often link

together forming branching chains which are

referred to as pseudohyphae. A few pathogenic

yeasts form a capsule, e.g. Cryptococcus neoformans.

Yeasts of medical importance

Candida albicans Malassezia furfur*

Cryptococcus neoformans Trichosporon beigelii

*Can also co-exist in ﬁlamentous form.

Moulds

These are multicellular fungi that form branching ﬁl-

aments called hyphae. A mass of interwoven hyphae

is called a mycelium. The hyphae of many patho-

genic moulds are septate, i.e. the hyphae are divided

into cells by cross-walls called septa. Hyphae without

septa are referred to as aseptate (feature of

zygomycetes). Moulds reproduce and survive

adverse conditions by forming conidia and spores.

These are of value in identifying species of fungi,

especially following culture.

Conidia

Various types of conidia are produced:

– angular or parcel-shaped arthroconidia (often referred to

as arthrospores), formed directly from the hyphae by sep-

tation followed by hyphal fragmentation.

– thick walled, spherical, resistantchlamydoconidia (usually

referredto as chlamydospores) formed from pre-existing

cellsin the hyphae, along the side, or at the tip of hyphae.

– small spherical unicellular microconidia (often seen in

clusters) formed directly on the side of hyphae or at the

end of a specialized hyphal strand called a conidiophore*.

– large club or spindle-shaped macroconidia arising from

the hyphal tip or wall and borne on a short or long conid-

iophore*.

* With some fungi, the conidiophore may branch into sec-

ondary segments called phialides. With Aspergillus species,

the conidiophore ends in a swollen vesicle from which

phialides bearing clusters of conidia develop. In Penicillium

species the conidiophore branches into a structure called a

penicillus with conidia being produced from phialides formed

from secondary branches.

Spores

Spores are produced either sexually after the fusion of nuclei

followed by meiosis or asexually by mitosis. Many pathogenic

fungi are ‘imperfect’, i.e. they are known only to produce veg-

etative non-sexual spores and conidia. Asexual spores called

sporangiospores are produced by species of Mucor, Absidia,

Rhizopusand other fungi of the class Zygomycota. The spo-

rangiospores are produced within a sac-like structure called

a sporangium which is borne on a sporangiophore.

Zygomycetes also reproduce sexually forming zygospores.

Fungi that have a sexual phase of reproduction in their life

cycle are referred to as ‘perfect fungi’.

Moulds of medical importance

Dermatophytes Aspergillus species

Fungi causing mycetoma Penicillium marneffei

Fungi causing Zygomycetes: Mucor,

chromomycosis Absidia, Rhizopus,

Basidiobolusspe cies

Dimorphic fungi

These are fungi that are able to grow as yeasts or

moulds depending on environmental conditions and

temperature. The yeast form is found in infected

tissue and when the fungus is cultured at 35–37C

and the ﬁlamentous form is found in the soil and

when the fungus is cultured at ambient tempera-

tures (20–30C).

Dimorphic fungi of medical importance

Blastomyces dermatitidis Paracoccidioides brasiliensis

Histoplasma species Coccidioides immitis

Sporothrix schenckii

MICROBIOLOGICAL TESTS 33

7.2

Fungi of medical importance in tropical

countries

Fungal infections are called mycoses. While fungal

pathogens do not cause widespread or dangerous

epidemics like bacteria and viruses, they are a major

cause of individual distress, disability, and disﬁgure-

ment, and the cause of severe life-threatening

conditions in those with immunosuppression caused

for example by AIDS or treatment with immuno-

suppressive drugs. The widest range and most

serious of fungal infections and diseases caused by

fungal toxins (mycotoxicoses) are found in tropical

and developing countries. Heat, humidity, inade-

quate water supplies, poor living conditions,

malnutrition, co-infection with HIV, and lack of diag-

nostic and care facilities for those with mycoses, all

contribute to the high prevalence and severity of

fungal infections in these countries.

Based on the sites of the body affected, the principal

mycoses can be described as:

 Superﬁcial mycoses, affecting the skin, hair or

nails, e.g. dermatophytosis (ringworm) and pityri-

asis versicolor. They are conﬁned to the body

surfaces and do not directly involve living tissues.

 Subcutaneous mycoses, which are referred to as

mycoses of implantation, e.g. mycetoma, chro-

momycosis, subcutaneous phycomycosis,

rhinosporidiosis, and sporotrichosis. They are

acquired when the pathogen is inoculated

through the skin by minor cuts or scratches or by

thorn or splinter wounds.

 Systemic mycoses which are often referred to as

deep mycoses, e.g. histoplasmosis, blastomyco-

sis, paracoccidioidomycosis, aspergillosis, and

coccidioidomycosis. They are acquired by inhala-

tion and may spread from the lung and involve

any part of the body. Widespread infections can

be fatal. Skin lesions are often present.

Opportunistic mycoses

An increasing number of fungal species are being

reported as causing infections, often severe and life-

threatening in those with HIV disease and other

conditions causing immunosuppression. While a few

species form part of the normal microbial ﬂora (e.g.

Candida species), most opportunistic mycoses are

caused by saprophytic fungi. The most widespread

and, or, serious opportunistic mycoses are listed in

Chart 7.6 and described in subunits 7.18.47–7.18.52.

Mycotoxicoses

Mycotoxicoses are caused by ingesting mycotoxins

in mouldy food, such as grain which has been stored

under damp humid conditions. The toxins are

released by certain moulds as they grow, e.g. aﬂa-

toxins are produced by Aspergillus ﬂavus when

growing on peanuts and grains. Aﬂatoxin poisoning

can cause hepatitis and hepatic carcinoma.

Fungal allergies

Inhalation of fungal spores, particularly those of

Aspergillus, can cause strong hypersensitivity reac-

tions in susceptible persons previously sensitized,

including asthma (type 1 immediate reaction) eosin-

ophilia, and type 3 hypersensitivity skin reaction.

Spores that germinate can lead to hyphae colonizing

the bronchial tree (living tissue is not invaded),

causing pneumonitis.

Chart 7.6 Fungi of importance in tropical countries

FUNGI CAUSING SUPERFICIAL

MYCOSES Disease

● Microsporum species Dermatophytosis

● Trichophyton species (ringworm, tinea)

● Epidermophyton ﬂoccosum

● Malassezia furfur Pityriasis versicolor

(tinea versicolor)

FUNGI CAUSING SUBCUTANEOUS MYCOSES

● Phialophora, Fonsecaea and Chromoblastomycosis

Cladosporiumspecies (Chromomycosis)

Mycetoma:

● Madurella grisea – Black granule

● Madurella mycetomatis – Dark red granule

● Exophiala jeanselmei – Black granule

● Leptosphaeria senegalensis – Black granule

● Pseudallescheria boydii – White granule

● Aspergillus nidulans – White-yellow granule

● Basidiobolus species Subcutaneous

zygomycosis

● Rhinosporidium seeberi Rhinosporidiosis

● Sporothrix schenckii Sporotrichosis

FUNGI CAUSING SYSTEMIC MYCOSES

● Histoplasma capsulatum Histoplasmosis (classical)

● Histoplasma C. duboisii African histoplasmosis

● Blastomyces dermatitidis Blastomycosis

● Paracoccidioides brasiliensis Paracoccidioidomycosis

● Coccidioides immitis Coccidioidomycosis

FUNGI CAUSING OPPORTUNISTIC MYCOSES

● Candida albicans and related Candidiasis

yeasts

● Cryptococcus neoformans Cryptococcal meningitis

● Aspergillus species Aspergillosis

● Pneumocystis jiroveci Pneumocystis pneumonia

● Mucor, Absidia, Rhizopus, Mucormycosis

Rhizomucorspecies (zygomycosis)

● Penicillium marneffei Systemic penicilliosis

● Histoplasma species Histoplasmosis

● Sporothrix schenckii Sporotrichosis

Note: Features of these fungi and their laboratory identiﬁ-

cation can be found in subunits 7.18.38 to 7.18.52.

34 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.2

LABORATORYDIAGNOSIS OF FUNGAL SPECIM ENS

Many of the fungi that cause superﬁcial and subcu-

taneous mycoses and some that cause systemic

mycoses can be detected and occasionally identiﬁed

microscopically:

– in wet specimen preparations, e.g. Aspergillus

hyphaein sputum or Cryptococcus neoformans in

cerebrospinal ﬂuid (mixed with India ink or

examined by dark-ﬁeld microscopy).

– in potassium hydroxide (KOH) cleared speci-

mens, e.g. dermatophytes (ringworm fungi) in

skin scrapings, nails or hair.

– in stained preparations, e.g. Candida albicans in

Gram stained smears of vaginal discharge or

Pneumocystis jiroveci in Giemsa or other stained

preparations of broncho-alveolar lavage or

induced sputum.

Use of calcoﬂuor white and ﬂuorescence microscopy to

demonstrate fungi

Calcoﬂuor white (colourless) is a ﬂuorochrome which can be

used to detect rapidly, yeast cells, pseudohyphae, and hyphae

in specimens when examined by ﬂuorescence microscopy. The

ﬂuorochrome binds to the cellulose and chitin present in

fungal cell walls. Depending on the wavelength of the exciter

light, the fungi appear bright apple green or blue-white. When

used to look for ringworm fungi, the calcoﬂuor white can be

mixed with potassium hydroxide. Calcoﬂuor white (also

known as Fluorescent Brightner 28, Code F 3543 from Sigma),

is prepared by dissolving 1 g of the ﬂuorochrome in 100 ml

distilled water. From this a working solution is made by

diluting 10 ml in 90 ml of 0.05% Evans blue stain. For use, 1

drop is mixed with 1 drop of 20% KOH (without dimethyl

sulphoxide).

Culture: This is indicated when it is not possible to

diagnose a serious fungal infection microscopically

or a species identiﬁcation needs to be established or

conﬁrmed. The appearances of fungal cultures, how

quickly a fungus grows and at what temperature,

and particularly the morphology of the conidia and

spores produced, can help to identify fungal

pathogens. Culturing of fungi is best carried out in a

public health laboratory or specialist mycology

centre where facilities exist for the safe handling of

cultures and staff have training in mycological tech-

niques and the recognition of fungal pathogens.

District laboratory staff should request instructions

from their mycology referral laboratory regarding

the collection and sending of specimens for fungal

culture. It may take several weeks before a culture

report is received because some fungal pathogens

are slow-growing.

Biopsies (preserved in 10%

formol saline,

Reagent No. 38): Require processing and examining

in a histopathological laboratory, e.g. for the diag-

nosis of histoplasmosis, rhinosporidiosis and

sporotrichosis.

Serology Antibody tests are not often used to

diagnose fungal infections due to cross-reactions, the

inability of tests to demonstrate active infection, and

inadequate antibody responses in severe immuno-

suppression. Several rapid and simple to perform

immunological tests, however, have been developed

to detect fungal antigen in specimens, e.g. antigen

test to detect Cryptococcus neoformans antigen in

c.s.f. and serum. (see subunit 7.18.48).

Note: In district laboratories, the main method of

investigating fungal infections is the microscopical

examination of specimens directly and in KOH and

stained preparations.

MICROBIOLOGICAL TESTS 35

7.2–7.3.1

 Polychrome Loefﬂer methylene blue staining of

anthrax bacilli is described in subunit 7.18.6.

7.3.1 Examining pathogens

in wet preparations

In district laboratories the examination of wet prepa-

rations is mainly used:

– to examine specimens and cultures for motile

bacteria.

– to examine c.s.f. for capsulated yeast cells.

– to examine specimens for fungi.

Detecting motile bacteria

Knowingwhether anorganism is motileor non-motile

can oftenassist in its identiﬁcation, e.g. most serovars

of Salmonellaare motile whereas Shigella species are

non-motile.Vibrio and Campylobacterspecies show a

distinctive motility. The movement of spirochaetes is

alsocharacteristic.

Technique using transmitted light microscopy

The simplest way of examining a bacterial suspen-

sion for motile bacteria is as follows:

1 Place a small drop of suspension on a slide and

cover with a cover glass. Avoid making the prep-

aration too thick. It is advisable to seal the

preparation with nail varnish or molten petro-

leum jelly to prevent it drying out.

Hanging drop preparation: Placing a drop of suspension

on a cover glass and inverting this over a cavity slide or

over a normal slide supported on a ring of plasticine is not

recommended. Vibration of the ﬂuid makes the prep-

aration difﬁcult to examine.

2 Examine the preparation microscopically for

motile organisms, using the 10 and 40

objectives. Make sure the iris diaphragm of

the condenser is sufﬁciently closed to give

good contrast otherwise the organisms will

not be seen. Bring the preparation into focus

by focusing ﬁrst on the edge of the cover

glass.

Note: The movement of small motile bacteria must

be distinguished from the on-the-spot vibratory

movement (Brownian movement) which is shown

by all microorganisms and particles when sus-

pended in a ﬂuid. True bacterial motility is the ability

of an organism to move itself in different directions

or a single direction.

7.3 Microscopical techniques

used in microbiology

Information provided by microscopical techniques

can often provide a rapid presumptive diagnosis of

an infection, e.g. pulmonary tuberculosis using the

Ziehl-Neelsen staining technique. Other techniques

can help to identify a pathogen, e.g. the Gram

staining technique can indicate whether an

organism is Gram positive or Gram negative, a

coccus or bacillus. Such information is particularly

helpful when investigating diseases such as menin-

gitis and gonorrhoea. Useful information can also be

provided from the microscopical examination of wet

preparations, e.g. when looking for motile vibrios in

a faecal specimen or capsulated C. neoformans in

cerebrospinal ﬂuid (c.s.f.).

This subunit describes:

 Examination of pathogens in wet preparations

 How to prepare and ﬁx smears prior to staining

 Precautions to take when staining smears

 Gram technique

 Ziehl-Neelsen technique to detect AFB

 Auramine-phenol technique to detect AFB

 Methylene blue technique

 Wayson’s bipolar staining of bacteria

 Albert staining of volutin granules

 Giemsa technique

 Acridine orange ﬂuorochrome staining.

Other staining techniques

 Toludine blue-0 staining of P. jiroveci cysts is

described in subunit 7.18.52.

Dark-ﬁeld microscopy

Because the refractive index of unstained organisms

in a ﬂuid medium is not very different from the ﬂuid

medium which surrounds them, other forms of

microscopy such as phase contrast and dark-ﬁeld are

recommended in preference to transmitted light

microscopy when examining organisms in

unstained wet preparations. Dark-ﬁeld microscopy is

required to detect Treponema pallidum spirochaetes

in specimens.

The equipment for phase contrast microscopy is

expensive and not usually found in district labora-

tories. Although the equipment needed to examine

preparations by dark-ﬁeld microscopy using a 100

objective is also expensive, a simple system which

uses a dark-ﬁeld stop is suitable for examining most

specimens for motile bacteria, including spirochaetes

(when using 10and 40 objectives). The use of a

dark-ﬁeld stop and technique for examining wet

preparations by dark-ﬁeld microscopy are described

on pp. 122–123 in Part 1 of the book.

Note: The identiﬁcation of Treponema pallidum is

described in subunit 7.18.32 and the examination of

specimens and cultures for Vibrio cholerae is

described in subunit 7.18.19.

Examining wet preparations for capsulated

organisms

When cryptococcal meningitis is suspected, the

examination of c.s.f. for capsulated yeast cells is an

important investigation. Capsulated C. neoformans

yeast cells are best detected in thin preparations of

sediment from centrifuged c.s.f., using India Ink

(Pelikan drawing ink) or dark-ﬁeld microscopy (see

subunit 7.18.48).

7.3.2 How to prepare and

ﬁx smears

If smears are to provide reliable information they

must be prepared, labelled, and ﬁxed correctly prior

to being stained.

Labelling slides

Every slide must be labelled clearly with the date

and the patient’s name and number. Whenever

possible, smears should be spread on slides which

have one end frosted for labelling. With the

increased use of such slides in recent years, their

price is now little more than slides without a frosted

36 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.3.1–7.3.2

end. A lead pencil should be used for writing on the

frosted area because pencil marks, unlike biro and

grease pencil marks, will not be washed off during

the staining process.

Caution: Slides from positive AFB smears should

always be discarded and never reused. Scratched,

chipped, and discolored slides should also be dis-

carded.

How to make smears

Smears should be spread evenly covering an area of

about 15–20 mm diameter on a slide. The precau-

tions which should be taken when handling

infectious material are described on pp. 61–64 in

Part 1 of the book. The techniques used to make

smears from different specimens are as follows:

● Purulent specimen: Using a sterile wire loop,

make a thin preparation. Do not centrifuge a

purulent ﬂuid, e.g. c.s.f. containing pus cells.

● Non-purulent ﬂuid specimen: Centrifuge the ﬂuid

and make a smear from a drop of the well-mixed

sediment.

● Culture: Emulsify a colony in sterile distilled water

and make a thin preparation on a slide. When a

broth culture, transfer a loopful to a slide and

make a thin preparation.

● Sputum: Use a piece of clean stick to transfer and

spread purulent and caseous material on a slide.

Soak the stick in a phenol or hypochlorite disin-

fectant before discarding it.

● Swabs: Roll the swab on a slide. This is particu-

larly important when looking for intracellular

bacteria such as N. gonorrhoeae (urethral,

cervical, or eye swab). Rolling the swab avoids

damaging the pus cells.

● Faeces: Use a piece of clean stick to transfer pus

and mucus to a slide. Decontaminate the stick

before discarding it. Spread to make a thin prep-

aration.

● Skin smears: Making skin smears for M. leprae is

described in subunit 7.18.30.

Drying and ﬁxing smears

After making a smear, leave the slide in a safe place

for the smear to air-dry, protected from dust, ﬂies,

cockroaches,ants, and direct sunlight. When asmear

requires urgent staining, it can be dried quickly

using gentleheat. Smears taken from in-patients and

duringout-patient clinics must always be transported

to the laboratory in a covered container.

The purpose of ﬁxation is to preserve micro-

organisms and to prevent smears being washed

from slides during staining. Smears are ﬁxed by

heat, alcohol, or occasionally by other chemicals.

Microorganisms are not always killed by heat

ﬁxation, e.g. M. tuberculosis.

Heat ﬁxation

This is widely used but can damage organisms

and alter their staining reactions especially when

excessive heat is used. Heat ﬁxation also damages

leucocytes and is therefore unsuitable for ﬁxing

smears which may contain intracellular organisms

such as N. gonorrhoeae and N. meningitidis.

When used, heat ﬁxation must be carried out

with care. The following technique is recommended:

1 Allow the smear to air-dry completely.

2 Rapidly pass the slide, smear uppermost, three

times through the ﬂame of a spirit lamp or pilot

ﬂame of a Bunsen burner.

Note: After passing the slide through the ﬂame

three times, it should be possible to lay the slide

on the back of the hand without the hand feeling

uncomfortably hot. When this cannot be done,

too much heat has been used.

3 Allow the smear to cool before staining it.

Alcohol ﬁxation

This form of ﬁxation is far less damaging to micro-

organisms than heat. Cells, especially pus cells, are

also well preserved. Alcohol ﬁxation is therefore rec-

ommended for ﬁxing smears when looking for

Gram negative intracellular diplococci. Alcohol

ﬁxation is more bactericidal than heat (e.g. M. tuber-

culosis is rapidly killed in sputum smears after

applying 70% v/v alcohol).

A method of alcohol ﬁxing smears is as follows:

1 Allow the smear to air-dry completely.

2 Depending on the type of smear, alcohol-ﬁx as

follows:

– For the detection of intracellular Gram

negative diplococci (N. gonorrhoeae or

N. meningitidis), ﬁx with one or two drops of

absolute methanol or ethanol.

– For the detection of other organisms includ-

ing M. tuberculosis, ﬁx with one or two drops

of 70% v/v methanol or ethanol (absolute

methanol can also be used but a 70% v/v

solution is adequate).

3 Leave the alcohol on the smear for a minimum

of 2 minutes or until the alcohol evaporates.

Other chemical ﬁxatives

Other chemicals are sometimes necessary to ﬁx

MICROBIOLOGICAL TESTS 37

7.3.2–7.3.3

smears which contain particularly dangerous organ-

isms to ensure all the organisms are killed, e.g.

40 g/l potassium permanganate is recommended

for ﬁxing smears which may contain anthrax bacilli.

Formaldehyde vapour is sometimes rec-

ommended for ﬁxing smears which may contain

Mycobacterium species. Formaldehyde ﬁxed smears,

however, tend to stain poorly and the chemical itself

is toxic with an injurious vapour.

7.3.3 Precautions to take

when staining smears

● Use a staining rack. Do not immerse slides in

containers of stain because this can lead to con-

tamination of stains and transfer of organisms

from one smear to another.

Staining rack: This can be made by joining two pieces of

glass or metal rod at each end with rubber or plastic

tubing. The length of the rods will depend on the width of

the sink or staining container.

Caution: When using a staining container/tray, empty it

regularly to reduce the risk of ﬁre from ﬂammable chem-

icals.

● Do not attempt to stain a smear that is too thick.

This is one of the commonest causes of poor

staining and incorrect reporting of smears.

● To dispense stains, alcoholic and acetone

reagents, use dropper bottles (e.g. TK type, see

p. 167 in Part 1 of the book) or other spouted

containers that can be closed between use. This

will avoid evaporation, deterioration of stains and

reagents and any build-up of toxic and ﬂam-

mable fumes in the laboratory.

● Label clearly stains and reagents. Indicate when

a stain or reagent is Toxic, Flammable,or

Corrosive. Write this on the dispensing container

or use the appropriate biohazard symbol (see

p.75 in Part 1of the book). Makesure ﬂammable

stains and reagents are kept well away from an

open ﬂame, e.g. from a lighted swab or ﬂame of

a spiritlamp or Bunsen burner. Use atray to hold

the dispensing bottles as this will help to contain

any spillage of a reagent.

● Follow exactly a staining technique, particularly

staining and decolorizing times, to ensure correct

and reproducible staining reactions.

● When washing smears of c.s.f. sediment and

other specimens which can be easily washed

from a slide, direct the water from a wash bottle

on the back of the slide, not directly on the

smear.

● After staining, place the slides at an angle in a

draining rack for the smears to air-dry. Do not

blot smears dry with ﬁlter or blotting paper

(which is expensive and inappropriate to use).

When a report is required urgently, dry a smear

carefully over the pilot ﬂame of a Bunsen burner

or ﬂame of a spirit lamp.

● To check staining results, use quality control

smears of organisms, particularly when a new

batch of stain is used (see subunit 7.1: Control of

stains and reagents).

7.3.4 Gram technique

The Gram staining reaction is used to help identify

pathogens in specimens and cultures by their Gram

reaction (Gram positive or Gram negative) and mor-

phology. Pus cells can also be identiﬁed in Gram

smears.

Gram positive bacteria: Stain dark purple with

crystal violet (or methyl violet) and are not decol-

orized by acetone or ethanol. Examples include

species of:

Staphylococcus Actinomyces

Streptococcus

Clostridium

Corynebacterium

Gram negative bacteria: Stain red because after

being stained with crystal violet (or methyl violet)

they are decolorized by acetone or ethanol and take

up the red counterstain (e.g. neutral red, safranin, or

dilute carbol fuchsin). Examples include species of:

Neisseria Klebsiella

Haemophilus Brucella

Salmonella Yersinia

Shigella Coliforms

Vibrio

Gram reaction

Differences in Gram reaction between bacteria is thought to

be due to differences in the permeability of the cell wall of

Gram positive and Gram negative organisms during the

staining process. Following staining with a triphenyl methane

basic dye such as crystal violet and treatment with iodine, the

dye–iodine complex is easily removed from the more perme-

able cell wall of Gram negative bacteria but not from the less

permeable cell wall of Gram positive bacteria. Retention of

crystal violet by Gram positive organisms may also be due in

38 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.3.3–7.3.4

part to the more acidic protoplasm of these organisms binding

to the basic dye (helped by the iodine).

Gram staining technique

Required

– Crystal violet stain

Reagent No. 28

– Lugol’s iodine Reagent No. 53

– Acetone–alcohol decolorizer

Reagent No. 1

– Neutral red, 1 g/l (0.1% w/v)

Reagent No. 60

Notes

Gentian violet or methyl violet can also be used.

Some workers prefer to use acetone by itself, ethanol 95%

v/v, or ethanol–iodine as the decolorizing solution. A mixture

of acetone and alcohol is recommended because it decolorizes

more rapidly than ethanol 95% v/v, and is less likely to over-

decolorize smears than acetone without alcohol added.

Neutral red is selected as the counterstain because it stains

well gonococci and meningococci. Safranin can also be used.

The use of dilute carbol fuchsin (1 in 10) is recommended for

staining Vincents’ organisms, Yersinia, Haemophilus, Campy-

lobacter, and Vibriospecies.

Method

1 Fixthe dried smearas explained in subunit7.3.2.

Note: When the smear is for the detection of

gonococci or meningococci, it should be ﬁxed

with methanol for 2 minutes (avoids damaging

pus cells).

2 Cover the ﬁxed smear with crystal violet stain

for 30–60 seconds.

3 Rapidly wash off the stain with clean water.

Note: When the tap water is not clean, use

ﬁltered water or clean boiled rainwater.

4 Tip off all the water, and cover the smear with

Lugol’s iodine for 30–60 seconds.

5 Wash off the iodine with clean water.

6 Decolorize rapidly (few seconds) with

acetone–alcohol. Wash immediately with clean

water.

Caution: Acetone–alcohol is highly ﬂammable,

therefore use it well away from an open ﬂame.

7 Cover the smear with neutral red stain for 2

minutes.

8 Wash off the stain with clean water.

9 Wipe the back of the slide clean, and place it in

a draining rack for the smear to air-dry.

10 Examine the smear microscopic ally, ﬁrst with

the 40 objective to check the staining and to

see the distribution of material, and then with

the oil immersion objective to report the

bacteria and cells.

Results

Gram positive bacteria . . . . . . . . . . . . . Dark purple

Yeast cells . . . . . . . . . . . . . . . . . . . . . . . Dark purple

Gram negative bacteria. . . . . . . . . . Pale to dark red

Nuclei of pus cells . . . . . . . . . . . . . . . . . . . . . . . Red

Epithelial cells . . . . . . . . . . . . . . . . . . . . . . . . Pale red

Reporting Gram smears

The report should include the following information:

● Numbers of bacteria present, whether many,

moderate, few, or scanty

● Gram reaction of the bacteria, whether Gram

positive or Gram negative

● Morphology of the bacteria, whether cocci, diplo-

cocci, streptococci, rods, or coccobacilli. Also,

whether the organisms are intracellular.

● Presence and number of pus cells

● Presence of yeast cells and epithelial cells.

Example

A urethral smear report might read:

‘Moderate numbers Gram negative intracellular

diplococci and many pus cells.’

Note: Colour plates 7, 15, 24, 25, 28, 38, 43, 45, 48

show bacteria in Gram stained preparations.

Variations in Gram reactions

 Gram positive organisms may lose their ability to

retain crystal violet and stain Gram negatively for the

following reasons:

– Cell wall damage due to antibiotic therapy or

excessive heat-ﬁxation of the smear.

– Over-decolorization of the smear.

– Use of an iodine solution which is too old, i.e.

yellow instead of brown in colour (always

store in a brown glass or other light opaque

container).

– Smear has been prepared from an old

culture.

 Gram negative organisms may not be fully

decolorized and appear as Gram positive when a

smear is too thick.

Control: Always check new batches of stain and

reagents for correct staining reactions using a smear

containing known Gram positive and Gram negative

organisms.

MICROBIOLOGICAL TESTS 39

7.3.4–7.3.5

7.3.5 Ziehl-Neelsen

technique for M. tuberculosis

and M. ulcerans

Ziehl-Neelsen staining for M. leprae is described in subunit

7.18.30

The Ziehl-Neelsen (Zn) technique is used to stain

Mycobacterium species including M. tuberculosis,

M. ulcerans, and M. leprae. Mycobacteria, unlike

most other bacteria, do not stain well by the Gram

technique. They can however be stained with carbol

fuchsin combined with phenol. The stain binds to

the mycolic acid in the mycobacterial cell wall. After

staining, an acid decolorizing solution is applied.

This removes the red dye from the background cells,

tissue ﬁbres, and any organisms in the smear except

mycobacteria which retain (hold fast to) the dye and

are therefore referred to as acid fast bacilli, or simply

AFB. Following decolorization, the smear is counter-

stained with malachite green or methylene blue

which stains the background material, providing a

contrast colour against which the red AFB can be

seen.

Note: Some actinomycetes, corynebacteria, and bacterial

endospores are also acid fast.

Differences between the acid fastness of

Mycobacterium species

– M. tuberculosis and M. ulcerans are strongly acid

fast. When staining specimens for these species,

a 3% v/v acid solution is used to decolorize

the smears (as describe d in the following

technique).

– M. leprae is only weakly acid fast. A 1% v/v acid

decolorizing solution is therefore used for

M. leprae smears and also different staining and

decolorizing times as described in subunit

7.18.30.

‘Acid and alcohol fast bacilli’:The acid decolorizing reagents

used in the Zn staining technique also contain alcohol

(ethanol). It is not true, however, that mycobacteria can be

differentiated by whether they are acid fast or acid and

alcohol fast. As Collins et alremark, ‘There is no basis for the

old story that tubercle bacilli are acid and alcohol fast while

other bacteria are only acid fast. Acid-fastness varies with the

physiological state of the organisms. The alcohol in the decol-

orizing solution merely gives a cleaner stained smear’.

Hot and cold Zn techniques

In the ‘hot’ Zn technique (as described in this publi-

cation), the phenolic-carbol fuchsin stain is heated to

enable the dye to penetrate the waxy mycobacterial

cell wall. Techniques which do not heat the stain are

referred to as ‘cold’ techniques. In these, penetration

of the stain is usually achieved by increasing the con-

centrations of basic fuchsin and phenol and

incorporating a ‘wetting agent’ chemical. Com-

parisons between the ‘hot’ and ‘cold’ methods have

shown that both M. leprae and M. tuberculosis stain

less well by the ‘cold’ method and stained smears

fade rapidly.

Note: In the paper of Ridley, MJ and Ridley, DS.

the

authorsreport that after staining smears for leprosy bacilli at

room temperature, examination was always difﬁcult because

of pallor. Where bacilli were few, some were missed

altogether.

Ziehl-Neelsen technique for M. tuberculosis

and M. ulcerans

The preparation of sputum smears for the detection

of M. tuberculosis is described in subunit 7.6, and

cerebrospinal ﬂuid preparation in subunit 7.13. In

HIV-infected patients, AFB may be detected in buffy

coat smears prepared from EDTA anticoagulated

blood. The collection of ulcer material to detect

M. ulcerans is described in subunit 7.18.29.

Required

– Carbol fuchsin stain (ﬁltered) Reagent No. 21

– Acid alcohol, 3% v/v Reagent No. 4

– Malachite green, 5 g/l Reagent No. 55

(0.5% w/v)*

*If preferred, methylene blue, 5 g/l may be used instead of

malachite green.

Method

1 Heat-ﬁx the dried smear as described in subunit

7.3.2.

Alcohol-ﬁxation: This is recommended when the smear

has not been prepared from sodium hypochlorite

(bleach) treated sputum and will not be stained immedi-

ately. M. tuberculosis is killed by bleach and during the

staining process. Heat-ﬁxation of untreated sputum

will not kill M. tuberculosis whereas alcohol-ﬁxation is

bactericidal.

2 Cover the smear with carbol fuchsin stain.

3 Heat the stain until vapour just begins to rise

(i.e. about 60C ). Do not overheat. Allow the

heated stain to remain on the slide for 5

minutes.

Heating the stain: Great care must be taken when

heating the carbol fuchsin especially if staining is carried

out over a tray or other container in which highly ﬂam-

mable chemicals have collected from previous staining.

Only a small ﬂame should be applied under the slides

using an ignited swab previously dampened with a few

drops of acid alcohol or 70% v/v ethanol or methanol.

40 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.3.5

Do not use a large ethanol soaked swab because this is a

ﬁre risk.

4 Wash off the stain with clean water.

Note: When the tap water is not clean, wash the

smear with ﬁltered water or clean boiled rain-

water.

5 Cover the smear with 3% v/v acid alcohol for 5

minutes or until the smear is sufﬁciently decol-

orized, i.e. pale pink.

Caution: Acid alcohol is ﬂammable, therefore

use it with care well away from an open ﬂame.

6 Wash well with clean water.

7 Cover the smear with malachite green stain for

1–2 minutes, using the longer time when the

smear is thin.

8 Wash off the stain with clean water.

9 Wipe the back of the slide clean, and place it in

a draining rack for the smear to air-dry (do not

blot dry).

10 Examine the smear microscopic ally, using the

100  oil immersion objective. When available,

use 7 eyepieces because these will give a

brighter image. Scan the smear systematically

as shown in Fig. 7.2.

Note: Do not touch the smear with the end of

the oil dispenser because this could transfer

AFB from one preparation to another. After

examining a positive smear, the oil must be

wiped from the objective.

Fig 7.2 Method of examining a Ziehl-Neelsen stained

sputum smear for AFB.

Results

AFB. . . . . . . . . . . . . . . . . . . . Re d, straight or slightly

curved rods, occurring singly

or in small groups,

may appear beaded.

Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Green

Background material . . . . . . . . . . . . . . . . . . . . Green

Note: The appearance of M. tuberculosis in a Ziehl-

Neelsen stained smear is shown in colour Plates 56

and 57.

Reporting of sputum smears

When any deﬁnite red bacilli are seen, report the

smear as ‘AFB positive’, and give an indication of the

number of bacteria present as follows:

More than 10 AFB/ﬁeld. . . . . . . . . . . report 

1–10 AFB/ﬁeld . . . . . . . . report 

10–100 AFB/100 ﬁelds . . report 

1–9 AFB/100 ﬁelds . . . . . report the

exact number

When very few AFB are seen: e.g. when only one or

two AFB are seen, request a further specimen to

examine. Tap water and deionized water (using ‘old’

resin) sometimes contain AFB that resemble tubercle

bacilli, and occasionally stained scratches on a slide

can be mistaken for AFB although these tend to

be in a different focal plane from the smear.

Occasionally AFB can be transferred from one

smear to another when the same piece of blotting

paper is used to dry several smears.

When no AFB are seen after examining 100 ﬁelds:

Report the smear as ‘No AFB seen’. Do not report

‘Negative’ because organisms may be present but

not seen in those ﬁelds examined. Up to three speci-

mens (one collected as an early morning specimen)

may need to be examined to detect M. tuberculosis

in sputum.

Quality control

At regular intervals, and always when a new batch of

stain is started, two sputum smears of known high

and low AFB positivity should be stained with the

routine smears to check that the carbol fuchsin,

staining method, and the microscopical examination

of smears are satisfactory.

REFERENCES

1 Collins CH, Grange JM, Yates MD. Tuberculosis bacteri-

ology, 2nd edition, 1997. Butterworth Heinemann, ISBN

0 7506 2458 2.

2 International Union Against Tuberculosis and Lung

Disease (IUATLD). Technical Guide – Sputum examin-

ation for tuberculosis by direct microscopy in low income

countries, 5th edition, 2000.

3 Ridley MJ, Ridley DS. Stain techniques and morphology

of Mycobacterium leprae. Leprosy Review, 42, pp. 88–95,

1971.

7.3.6 Auramine-phenol

technique

The auramine-phenol ﬂuorochrome staining tech-

nique can be used to detect M. tuberculosis in

MICROBIOLOGICAL TESTS 41

7.3.5–7.3.6

sputum, cerebrospinal ﬂuid, and other specimens

when facilities for ﬂuorescence microscopy are avail-

able. Compared with the Ziehl-Neelsen technique,

the auramine-phenol ﬂuorochrome technique

enables a more rapid examination of smears

because the 40 objective can be used. When

tubercle bacilli are few they are more likely to be

successfully detected in auramine-phenol stained

smears.

Auramine-phenol to demonstrate AFB

Auramine ﬂuoresces when illuminated (excited) by blue-

violet or ultra-violet (UV) light. It can be used to demonstrate

AFB because it binds to the mycolic acid in the mycobacterial

cell wall. No heating of the stain is required. After being

stained with auramine, the smear is decolorized with acid

alcohol which removes the dye from the background. The

smear is then washed with a weak solution of potassium per-

manganate to darken the background. Tubercle bacilli

ﬂuoresce white-yellow against a dark background.

Note: The principle of transmitted and incident

ﬂuorescencemicroscopy is describedon pp. 123–125

in Part1 of the book.

Auramine-phenol ﬂuorochrome staining

technique

Required

– Auramine-phenol stain Reagent No. 14

(ﬁltered)

– 1% acid alcohol Reagent No. 3

– Potassium permanganate, Reagent No. 70

1g/l (0.1% w/v)

Method

Whenever possible use a sodium hypochlorite tech-

nique to concentrate the bacilli prior to staining (see

subunit 7.6).

1 Heat-ﬁx the dried smear as described in subunit

7.3.2.

2 Cover the ﬁxed smear with the auramine-phenol

stain for 10 minutes. Always include a positive

control smear.

3 Wash off the stain with clean water.

Note: When the tap water is not clean, wash the

smear with ﬁltered or clean boiled rainwater.

4 Decolorize the smear by covering it with 1% v/v

acid alcohol for 5 minutes.

Caution: Acid alcohol is ﬂammable, therefore use

it with care well away from an open ﬂame.

5 Wash off the acid alcohol with clean water.

6 Cover the smear with the potassium perman-

ganate solution for about 10 seconds, followed

by several rinses with clean water.

7 Wipe the back of the slide clean and place it in a

draining rack for the smear to dry. Do not blot-

dry. To prevent fading of the ﬂuorescence,

protect the stained smear from sunlight and

bright light.

8 Systematically examine the smear for AFB by

ﬂuorescencemicroscopy using the40 objective.

Results

Acid fast bacilli (AFB) . . . . . . . . . White-yellow rods

glowing against a

dark background

Reporting sputum smears

When ﬂuorescent AFB (conﬁrmed by Zn staining)

are seen, report the smear as ‘AFB positive’, and give

an indication of the number of bacilli present in plus

signs ( to ).

42 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.3.6

When no ﬂuorescent rods are seen, report the smear

as ‘No AFB seen’.

Note: Up to three specimens may need to be

examined to detect the organisms.

Quality control

Whenever a new batch of stain is started, two

sputum smears of known high and low positivity

should be stained with the routine smears to check

that the auramine-phenol stain and staining tech-

nique are satisfactory. At least one positive control

smear should be included each time smears are

stained by the auramine-phenol technique.

Availability of a low cost ﬂuorescence microscopy

system

The system shown in Plate 7.1 has been designed by

Portable Medical Laboratories Inc. as a low cost easy

Viewing aid

FluoreslenS (40x) objective with

built in dichroic beam-splitting

mirror, exciter filter and barrier filter

Light guide

Halogen 180W lamp

100x objective

5x ocular

Plate 7.1 Low cost easy to use ﬂuorescence system suitable for a wide range of ﬂuorescence antibody techniques, including

examining sputum smears for AFB. Courtesy of WR Sanborn,Portable Medical Laboratories Inc.

to operate ﬂuorescence system. The specially con-

structed FluoreslenS objective attaches to a standard

microscope. This houses the ﬂuorescence dichroic

mirror, exciter ﬁlter, and barrier ﬁlter. When

used with the ﬁbre optic light source (180 W

halogen lamp), incident ﬂuorescence microscopy

can be performed to demonstrate AFB in sputum

(auramine 0 ﬂuorochrome) and a wide range of

other ﬂuorescence techniques.

Availability

The complete system is shown in Plate 7.1. Items are also

available separately, e.g. FluoreslenS objectives (40 and

100), 180 W quartz halogen lamp unit with ﬁbre optic guide

and light guide adaptors. Full details of the FluoreslenS ﬂuo-

rescence microscopy equipment can be obtained from

Portable Medical Laboratories Inc. (see Appendix II).

7.3.7 Methylene blue

technique

The methylene blue technique is a rapid method

which can be used to show the basic morphology of

bacteria and the bipolar staining of organisms. It is

also useful for staining leucocytes in faecal prepara-

tions.

Important: Polychromed methylene blue (see

text below) is required to stain the capsules of

Bacillusanthracis (McFadyean’s reaction), see subunit

7.18.6.

Required

Loefﬂer’s methylene blue Reagent No. 51

Polychrome Loefﬂer methylene blue.

Methylene blue stains

Loefﬂer’s methylene blue is an alkaline stain that can be easily

prepared in the laboratory using methylene blue powder

(Reagent No. 51). It can also be purchased ready-made from

Merck/BDH or other manufacturers of stains.

When polychromed, Loefﬂer’s methylene blue is suitable for

staining the capsules of B.anthracis (McFadyean reaction). In

its polychrome form, however, Loefﬂer’s methylene blue is

only available as a ready-made stain, not in powder form. It is,

however, stable for several years if kept in a dark bottle out of

direct light. It should be ordered as ‘Methylene blue,

McFadyean stain’.

What is referred to as ‘Polychrome methylene blue’ is avail-

able as a powder, but this is not suitable for staining

B. anthracis. It is used mainly in the preparation of

Romanowsky stains.

Method

1 Fix the dried smear as described in subunit 7.3.2.

MICROBIOLOGICAL TESTS 43

7.3.7–7.3.8

When anthrax is suspected, ﬁx the smear with

potassium permanganate (Reagent No. 71) for

10 minutes.

2 Cover the smear with the stain for 1 minute.

Note: When staining anthrax bacilli, use

Loefﬂer’s polychrome methylene blue (see above

text).

3 Wash off with clean water. When the tap water is

not clean, use ﬁltered water or clean boiled rain-

water.

4 Wipe the back of the slide clean, and place it in

a draining rack for the smear to air-dry.

5 Examine the smear microscopically, ﬁrst with the

40 to see the distribution of material, and then

with the oil immersion objective to look for

bacteria.

Results

Bacterial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . Blue

Nuclei of leucocytes . . . . . . . . . . . . . . . . . . . . . Blue

Capsular material . . . . . . . . . . . . . . . . Mauve-purple

(If polychrome Loefﬂer’s stain has been used)

Note: B. anthracis is shown in colour Plate 55.

Stained faecal leucocytes are shown in colour Plate

7.3.8 Wayson’s bipolar

staining

Wayson’s staining technique is a rapid method

which shows clearly the bipolar staining morphology

of bacteria such as Yersinia pestis.

Required

Wayson’s stain Reagent No. 86

Method

1 Fix the dried smear as described in subunit 7.3.2.

2 Cover the smear with Wayson’s stain for 10–20

seconds.

3 Wash off the stain with clean water. When the

tap water is not clean, use ﬁltered water or clean

boiled rainwater.

4 Wipe the back of the slide clean, and place it in

a draining rack for the smear to air-dry.

5 Examine the smear microscopically, ﬁrst with the

40 objective to see the distribution of material

and then with the oil immersion objective to look

for bipolar stained bacteria.

Results

Bacteria . . . . . . . . . . . . . . . . . . . Blue with pink ends

Note: The bipolar staining of Y. pestis is shown in

colour Plate 54 (Giemsa preparation).

7.3.9 Albert staining

of volutin granules

The Albert technique is used to stain the volutin, or

metachromatic, granules of C. diphtheriae. The

granules are most numerous after the organism has

been cultured on a protein-rich medium such as

Dorset egg or Loefﬂer serum (see subunit 7.18.7).

Note: Metachromatic granules can also be found in

other Corynebacterium species and occasionally in

some Bacillus species. The presence of granules is of

no signiﬁcance regarding virulence.

Required

– Toluidine blue-malachite green Reagent No. 83

– Albert’s iodine Reagent No. 7

Method

1 Fix the dried smear using alcohol (see subunit

7.3.2)

2 Cover the smear with the toluidine blue-

malachite green stain for 3–5 minutes.

3 Wash off the stain with clean water. When the

tap water is not clean, use ﬁltered water or clean

boiled rainwater.

4 Tip off all the water.

5 Cover the smear with Albert’s iodine for

1 minute. Wash off with water.

6 Wipe the back of the slide clean, and place it in

a draining rack for the smear to air-dry.

7 Examine the smear microscopically, ﬁrst with the

40 objective to check the staining and to see

the distribution of material and then with the oil

immersion lens to look for bacteria containing

metachromatic granules.

Results

Bacteria cells . . . . . . . . . . . . . . . . . . . . . . Pale green

Metachromatic granules . . . . . . . . . . . . Green-black

44 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.3.9–7.3.10

Note: An Albert stained smear of C. diphtheriae

showing metachromatic granules is shown in colour

in Plate 32.

7.3.10 Giemsa technique

Giemsa is a Romanowsky stain that is widely used in

parasitology to stain malaria and other blood para-

sites. In microbiology, the Giemsa technique can be

used to stain Chlamydia trachomatis inclusion bodies

(see subunit 7.18.37), Borrelia species (see subunit

7.18.34), and when Wayson’s stain is not available, to

stain Yersinia pestis (see subunit 7.18.22). It is also

used to stain Histoplasma species (se e subunit

7.16.43), the internal bodies of Pneumocystis jiroveci

cysts (see subunit 7.18.52), Klebsiella granulomatis

(see subunit 7.10), Penicillium marneffei (see subunit

7.18.50), and occasionally bacterial capsules.

Note: For staining chlamydiae, a weaker solution of

Giemsa and a longer staining time are used (see fol-

lowing text).

Required

– Giemsa stain Reagent No. 39

– Buffered water, pH 7.0–7.2 Reagent No. 20

Method

1 Fix the dried smear by covering it with methanol

(methyl alcohol) for 2–3 minutes. Allow the

smear to air-dry.

2 Dilute the Giemsa stain in the buffered water as

follows:

C. trachomatis, dilute the stain 1 in 40:

– Fill a small cylinder to the 19.5 ml mark with

the buffered water.

– Add 0.5 ml of Giemsa stain, i.e. to the 20 ml

mark.

Other organisms, dilute the stain 1 in 20:

– Fill a small cylinder to the 19 ml mark with

the buffered water.

– Add 1 ml of Giemsa stain, i.e. to the 20 ml

mark.

3 Place the slide, smear downwards, in a petri dish

or other small container, supported on each side

by a thin piece of stick.

4 Pour the diluted stain into the dish and cover

with a lid.

Note: This inverted method of staining avoids

stain being deposited on the smear.

5 Leave the smear to stain as follows:

C. trachomatis, stain 1 –2 hours.

Other organisms, stain 25–30 minutes.

6 Wash the stain from the dish and rinse the smear

with buffered water.

7 Wipe the back of the slide clean, and place it in

a draining rack for the smear to air-dry.

8 Examine the smear microscopically, ﬁrst with the

40 objective to see the distribution of material

and to select a suitable part of the smear to

examine with the oil immersion lens.

Results

C. trachomatis

Inclusion . . . . . . . . . . . . Blue-mauve to dark purple,

bodies depending on stage

of development

Nuclei of host cells . . . . . . . . . . . . . . . . Dark purple

Cytoplasm of host cells . . . . . . . . . . . . . . . Pale blue

Eosinophil granules . . . . . . . . . . . . . . . . . . . . . . Re d

Melanin granules . . . . . . . . . . . . . . . . . Black-green

Bacteria . . . . . . . . . . . . . . . . . . . . . Pale or dark blue

Borrelia species

Borrelia spirochaetes . . . . . . . . . . . . . . . . Mauve-blue

Red cells . . . . . . . . . . . . . . . . . . . . . . . . . Mauve-blue

Nuclei of white cells . . . . . . . . . . . . . . . . Dark purple

Cytoplasm of white cells . . . . Pale blue or grey-blue

Y. pestis

Coccobacilli. . . . . . . . . . . . . . Blue with dark stained

ends (bipolar staining)

Note: The appearance of Y. pestis is shown in colour

Plate 54, Borrelia species in colour Plate 64, C.

trachomatis in colour Plate 67, K. granulomatis in

colour Plate 46, P. marneffei in colour Plate 71,

and P. Jiroveci in colour Plate 77.

7.3.11 Acridine orange

technique

Acridine orange is a ﬂuorochrome that causes

deoxyribonucleic acid (DNA) to ﬂuoresce green and

ribonucleic acid (RNA) to ﬂuoresce orange-red. It

has been recommended for the rapid identiﬁcation

of Trichomonas vaginalis, yeast cells, and clue cells in

vaginal smears. It can also be used to detect intra-

cellular gonococci, meningococci, and other bacteria

particularly in blood cultures.



MICROBIOLOGICAL TESTS 45

7.3.11–7.4

Required

– Acridine orange acid stain Reagent No. 6

– Alcohol saline solution Reagent No. 9

– Sodium chloride, 8.5 g/l Reagent No. 68

(physiological saline)

Method

1 Cover the unﬁxed dried smear with the acridine

orange acid stain for 5–10 seconds.

Note: The acid ﬁxative is contained in the stain.

2 Wash off the stain, and decolorize the smear with

alcohol saline solution for 5–10 seconds.

3 Rinse the smear with physiological saline, and

place the slide in a draining rack.

4 Add a drop of saline or distilled water to the

smear, and cover with a cover glass.

5 Examine the smear by ﬂuorescence microscopy

using a BG 12 exciter ﬁlter and No. 44 and No.

53 barrier ﬁlters.

Examine ﬁrst with the 10obje ctive to see the

distribution of ﬂuorescing material, and then with

the 40 objective to identify T. vaginalis and to

detect yeast cells, and bacteria.

Results

T. vaginalistrichomonads . . . . . . . . Orange-red with

yellow-green nucleus

Yeast cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . Orange

Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Orange

Leucocytes (pus cells) . . . . . . . . . . . . . . Yellow-green

Epithelial cells*. . . . . . . . . . . . . . . . . . . . Yellow-green

* In bacterial vaginosis, the orange staining bacteria adhering

to the green epithelial cells (clue cells) can be clearly seen.

Note: The appearance of T. vaginalis in an acridine

stained smear is shown in colour Plate 44.

Further information: The value of acridine orange in micro-

biology work and information on low cost ﬂuorescence

microscope systems can be obtained from Dr Warren

Sanborn, Portable Medical Laboratories (see Appendix 11).

7.4 Culturing bacterial

pathogens

The purpose ofusing cultural techniques in microbi-

ology is to demonstrate the presence of organisms

which may be causing disease, and when indicate d,

totest the susceptibility of pathogens to antimicrobial

agents.

This subunit describes:

 Different types of culture media

 How to prepare, sterilize, and test culture media

 Sterilizing glassware used in culture work

 How to dispense culture media

 Inoculating plates, tubes, and bottles of culture

media

 Incubation of inoculated media

 Reporting cultures

DIFFERENT TYPES OF CULTURE MEDIA

For a culture medium to be successful in growing

the pathogen sought it must provide all essential

nutrients, ions, and moisture, maintain the correct

pH and osmotic pressure, and neutralize any toxic

materials produced. It is also essential to incubate

the inoculated medium in the correct atmosphere, at

the optimum temperature and for an adequate

period.

The main types of culture media are:

● Basic

● Enriched

● Selective

● Indicator

● Transport

● Identiﬁcation

Basic media: These are simple media such as nutrient agar

and nutrient broth that will support the growth of micro-

organisms that do not have special nutritional requirements.

They are often used in the preparation of enriched media, to

maintain stock cultures of control strains of bacteria, and for

subculturing pathogens from differential or selective media

prior to performing biochemical and serological identiﬁcation

tests.

Enriched media: Enriched media are required for the growth

of organisms with exacting growth requirements such as

H. inﬂuenzae, Neisseria species, and some Streptococcus

species. Basic media may be enriched with whole or lyzed

blood, serum, peptones, yeast extract, vitamins and other

growth factors. An enriched medium increases the numbers of

a pathogen by containing all the necessary ingredients to

promote its growth. Such a medium is often used for speci-

mens collected from sites which are normally sterile to ensure

the rapid multiplication of a pathogen which may be present

only in small numbers.

Enrichment media: This term is usually applied to ﬂuid selec-

tive media which contain substances that inhibit the growth of

unwanted organisms, e.g. Rappaport-Vassiliadis broth which

is often used as an enrichment medium for Salmonella

serovars in faeces.

Selective media: These are solid media which contain sub-

stances (e.g. bile salts or other chemicals, dyes, antibiotics)

which inhibit the growth of one organism to allow the growth

of another to be more clearly demonstrated. A selective

medium is used when culturing a specimen from a site having

46 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.4

a normal microbial ﬂora to prevent unwanted contaminants

overgrowing a pathogen. Media made selective by incorporat-

ing antibiotics are usually expensive.

Other ways to select organisms

Incubation conditions may be used to select organisms, e.g.

P.aeruginosa is inhibited by anaerobic conditions. Also the

pH of a medium may make it selective for a particular

organism, e.g. V. cholerae can be isolated on an alkaline

medium such as TCBS agar. Temperature may also help to

select an organism e.g. Listeria monocytogenes can grow at

4C whereas other organisms are inhibited. Growth, however,

is slow.

Indicator (differential) media:These are media to which dyes

or other substances are added to differentiate micro-

organisms. Many differential media distinguish between

bacteria by incorporating an indicator which changes colour

when acid is produced following fermentation of a speciﬁc

carbohydrate e.g. MacConkey agar.

Note: Many media used to isolate pathogens are both selective

and enrichment or both selective and differential.

Transport media: These are mostly semisolid media that

contain ingredients to prevent the overgrowth of commensals

and ensure the survival of aerobic and anaerobic pathogens

when specimens cannot be cultured immediately after collec-

tion. Their use is particularly important when transporting

microbiological specimens from health centres to the district

microbiology laboratory or specimens to the Regional Public

Health Laboratory. Examples of transport media include

Cary-Blair medium for preserving enteric pathogens and

Amies transport medium for ensuring the viability of gono-

cocci.

Identiﬁcation media:These include media to which substrates

or chemicals are added to help identify bacteria isolated on

primary cultures. Examples include peptone water sugars,

urea broth, and Kligler iron agar. Organisms are mainly ident-

iﬁed by a change in the colour of the medium and or the

production of gas. Organisms used to inoculate identiﬁcation

media must be ﬁrst isolated in pure culture.

Choice of culture media

The choice of culture media to use in microbiology

laboratories will depend on:

– The major pathogens to be isolated, their growth

requirements, and the features by which they are

recognized.

– Whether the specimens being cultured are from

sterile sites or from sites having a normal micro-

bial ﬂora. Although a selective medium is usually

more expensive than a non-selective one, it often

avoids subculturing, isolates a pathogen more

quickly, and makes it easier to differentiate and

interpret bacterial growth.

– Cost, availability, and stability of different media

in tropical countries.

– Training and experience of laboratory staff in

preparing, using, and quality controlling culture

media.

Solid, semi-solid and ﬂuid culture media

Culture media can be classiﬁed by consistency as:

● Solid

● Semi-solid

● Fluid

Solid culture media

Media are solidiﬁed by incorporating a gelling agent

such as agar or gelatin.

Agar

Agar (polysaccharide extract obtained from seaweed) is

commonly used to solidify culture media because of its high

gelling strength, its setting temperature of 32–39C and

melting temperature of 90–95C. Most agars used in bac-

teriological work produce a ﬁrm gel at an agar concentration

of 1.5% w/v. The low gelling temperature allows heat-sensi-

tive nutrients such as whole blood to be added safely at

45–50C. At a concentration of 0.4–0.5% w/v, agar is added

to transport media such as Amies medium to give a semisolid

gel.

Solid media are used mainly in petri dishes as plate

cultures. Also in bottles or tubes as stab (deeps) or

slope cultures. The inoculation of plates, slopes and

deeps is described later in this subunit. The purpose

of culturing on solid medium is principally to isolate

discrete colonies of each organism present in the

specimen. This will enable pure cultures to be

produced for identiﬁcation and sensitivity testing.

The colonial appearances and changes in the media

made by colonies may provide valuable identiﬁ-

cation information.

Appearances of bacterial colonies on solid media

Bacterial colonies should be examined in a good light. Use

oblique lighting when examining for iridescent colonies. A

low power magnifying lens can help to see morphological

detail.

When viewed from above: Colonies may appear round, irreg-

ular, crenated, or branching. They may be transparent or

opaque and their surface may be smooth or rough, dull or

shiny. The colonies of capsulated species appear mucoid.

Mature colonies of pneumococci have a ringed appearance.

When viewed from the side: Colonies may appear ﬂat or raised

in varying degrees sometimes with bevelled edges or with a

central elevation or depression.

When touched with a wire loop: Some colonies are soft and

easily emulsiﬁed such as Staphylococcus aureus, whereas

others are difﬁcult to break up such as Streptococcus

pyogenes.

The colour of colonies: This also helps to identify bacteria,

especially when using differential media containing indi-

cators.

Changes which may occur in the medium when bacteria are

cultured on solid agar

These include haemolytic reactions, pigment production,

colour changes surrounding carbohydrate fermenting

colonies, and blackening due to hydrogen sulphide produc-

tion.

MICROBIOLOGICAL TESTS 47

7.4

An example of a pigment forming organism is Pseudomonas

aeruginosa which produces a yellow-green colour in media

such as blood agar and MacConkey agar.

An example of an organism that produces a colour change is

Vibrio choleraewhich is sucrose-fermenting, giving a yellow

colour in TCBS agar. Blackening due to hydrogen sulphide

production is seen with many salmonellae cultured in Kligler

iron agar.

Haemolytic reactions in blood agar are seen with beta-

haemolytic streptococci and alpha-haemolytic pneumococci.

Morphological appearances of colonies can vary depending

on the species of blood used, e.g. horse, sheep, or goat blood.

Semi-solid culture media

This form of culture medium is prepared by adding

a small amount of agar (0.4–0.5% w/v) to a ﬂuid

medium. Semi-solid media are used mainly as

transport media, and for motility and biochemical

tests.

Fluid culture media

Fluid media are most commonly used as enrichment

where organisms are likely to be few e.g. blood

culture. Some organisms produce a surface growth

on the medium in which they are growing e.g.

Vibrio cholerae when growing in alkaline peptone

water. Fluid media may also be used for biochemical

testing e.g. peptone water sugars or the use of

media containing tryptophan to detect indole pro-

duction by some enterobacteria. A good inoculation

technique is important as the introduction of a single

contaminating organism may produce an incorrect

result. The inoculation of a ﬂuid culture medium is

described later in this subunit.

OW TO PREPARE, STERILIZE, ANDTESTCULTURE

MEDIA

If pathogens are to be isolated successfully, standard

operating procedures (SOPs) are needed which

detail for each culture medium used in the labora-

tory, its purpose, from where it can be obtained, its

preparation, and how it is sterilized, dispensed,

labelled, stored and performance tested.

SOPs: General guidelines on how to prepare SOPs can be

found in subunit 2.4 in Part 1 of the book.

Use of dehydrated culture media

In the preparation of complex culture media, it is

advisable for district laboratories to use ready-made

standardized dehydrated media to ensure good per-

formance and reproducibility. In most instances it will

also be less expensive than buying the individual

chemical constituents. Some chemicals may also be

difﬁcult to obtain, not available in small amounts,

and may have a short shelf-life.

Dehydrated media is hygroscopic, i.e. it absorbs

water.When exposed to moisture, itrapidly becomes

unﬁt for use. A hard massis forme dwhich alters the

chemical and microbiological properties of the

medium. This can be a serious problem for tropical

countrieswith humid climates. Adequate precautions

must be taken to prevent dehydrated culture media

deteriorating and having to be discarded before it is

ﬁnished. Such precautions include:

● Weighing the medium rapidly, and tightly

capping the bottle as soon as possible after

removing the approximate amount. Do not

return small amounts of medium to the stock

bottle (it is best to close the bottle quickly).

Whenever possible, use containers or tubes that

have been pre-marked to hold the amount

required. This will reduce the time the culture

medium container needs to be open.

Formedia available only in large quantitiesand

supplied in wide-necked containers, it may be

advisable to transfer the media into several

containers (clean sterile with air-tight c aps, and

clearly labelled) to avoid the stock container

being opened repeatedly and absorbing

moisture.

● Sealing the cap of the container with adhesive

tape. When the cap cannot be easily taped,

seal the container in an airtight plastic bag

(squeeze out most of the air before sealing the

bag).

● Storing media in the coolest driest place available

and always out of sunlight, e.g. do not store

dehydrated media in the same room as used for

steam sterilizing, boiling materials, or cleaning

glassware etc.

Important: Whenever possible, the Central or

Regional Public Health Laboratory should supply

district laboratories with ready-made or easy to make

culture media. This will promote standardization and

enable media to be purchased economically in

500 g amounts.

To minimize costs, the different types of media

should be kept to a minimum, e.g. the same

medium can be used to prepare blood agar, the

base medium used to make a selective medium to

isolate N. gonorrhoeae, and to make the agar slope

in blood culture media. Columbia agar is

recommended.

When culture media are purchased by the

Central or Regional Laboratory, it is more efﬁcient

and cost-effective for the purchasing laboratory to

48 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.4

perform the required quality control of each culture

mediumprior to itsdistribution to district laboratories.

Preparation of culture media

Full details of the preparation and quality assurance

of all culture media used in the laboratory must be

included in SOPs and a record kept of stock items,

sources of materials, and the dates when different

media are prepared.

The following are important when preparing

culture media:

● As discussed previously, prepare media made

from dehydrated products in as damp-free an

environment as possible. To prevent the risk of

inhaling ﬁne particles of dehydrated media, wear

a dust mask while handling dehydrated media.

powder or use granulated media (granulated

culture media are available from Merck, see

Appendix 11).

● Wash the hands immediately after preparing

media.

● Once the ingredients are weighed, do not delay

in making up the medium. Follow exactly the

manufacturer’s instructions.

● Use completely clean glassware, plastic or stain-

less steel equipment that has been rinsed in pure

water. The container in which the medium is

prepared should have a capacity of at least twice

the volume of the medium being prepared.

● Use distilled water from a glass still. Deionized

water can also be used providing the exchange

resins do not contain substances inhibitory to

bacteria (preparation of deionized and distilled

water in district laboratories is described in

subunit 4.4 in Part 1 of the book).

Water containing chlorine, lead, copper, or deter-

gents must not be used. Besides containing

substances harmful to bacteria, impure water can

alter the pH of a medium or cause a precipitate

to form.

● Add the powdered or granular ingredients to the

water and stir to dissolve. Do not shake a

medium but mix by stirring or by rotating the

container.

● When heating is required to dissolve the

medium, stir while heating and control the heat

to prevent boiling and foaming which can be

dangerous and damage the medium, e.g. DCA

orTCBS agar. Overheatinga medium can alter its

nutritional andgelling properties, and also its pH.

● Autoclave a medium only when the ingredients

are completely dissolved. Always autoclave at the

correct temperature and for the time speciﬁed

(see later text).

● Dispense medium in bottles or tubes in amounts

convenient for use. Know the length of time

prepared media can be stored without deterio-

rating (take into account storage temperature).

Checking the pH of a culture medium

The pH of most culture media is near neutral. An

exception is alkaline peptone water. The simplest

way of testing the pH of a culture medium is to

use narrow range pH papers or a pH meter (see

pp. 173–174 in Part 1 of the book).

A ﬂuid medium can be tested by dipping a

narrow range pH paper into a sample of the

medium when it is at room temperature and com-

paring the colour of the paper against the pH colour

chart provided. An agar medium can be tested by

pouring a sample of the molten medium into a small

beaker or petri dish and when it has solidiﬁed, laying

a narrow range pH paper on its surface. The colour

of the paper is then compared against the pH colour

chart.

The pH of a dehydrated medium should not

require adjustment providing it has been prepared

correctly using pure water and clean equipment,

and it has not been over-autoclaved. The manufac-

turer’s instructions must be followed exactly.

The pH of other media should be adjusted as

directed in the method of preparation. Minor adjust-

ments should be carried out using 0.1 mol/l (N/10)

sodium hydroxide when the medium is too acid,

and 0.1 mol/l (N/10) hydrochloric acid when too

alkaline. Use 1 mol/l (1N) sodium hydroxide

(Reagent No. 75) to adjust the pH of alkaline

peptone water.

When adjusting the pH of a large volume of

medium it is best to measure the amount of acid

that needs to be added to adjust 10 ml of the

medium, and then calculate the amount required to

adjust the remaining volume.

Sterilizing culture media

The following methods are used to sterilize culture

media:

– Autoclaving

– Steaming at 100C

– Filtration

Autoclaving

The majority of culture media are sterilized by being

autoclaved. This ensures the destruction of bacterial

endospores as well as vegetative cells.

It is important to sterilize a medium at the correct

temperature and for the correct length of time as

MICROBIOLOGICAL TESTS 49

7.4

instructed in the method of preparation. Under-

autoclaving can result in an unsterile medium which

will need to be discarded. Over-autoclaving can

cause precipitation, alteration of pH, and the

destruction of essential components in a medium.

Note: The principles of autoclaving, the speciﬁ-

cations of autoclaves and pressure cookers

appropriate for use in district laboratories, and how

to autoclave culture media safely and correctly are

described in subunit 4.8 in Part 1 of the book.

Steaming at 100



This is used to sterilize media containing ingredients

that would be broken down or inactivated at tem-

peratures over 100C, e.g. Cary-Blair transport

medium. Steaming is also used to re-melt previously

bottled sterile agar media.

Steaming can be performed in an autoclave with

the lid left loose, or in any form of steam sterilizer

such as an Arnold or Koch steamer. The bottles of

media with loosened caps are placed on perforated

trays above the boiling water. After sterilization and

the medium has cooled, the bottle tops are tight-

ened. Steaming times vary according to the type of

medium, e.g. 15 minutes for Cary-Blair medium.

Filtration

This provides a means of removing bacteria from

ﬂuids. It is used mainly to sterilize additives that are

heat-sensitive and cannot be autoclaved, or less

stable substances that need to be added to a sterile

medium immediately before it is used. Examples

include serum and solutions containing urea and

certain carbohydrates.

Several different types of ﬁlters can be used

including those made from sintered glass or inert

cellulose esters. Cellulose ﬁlters are referred to as

membrane ﬁlters. They are preferred to other types

of ﬁlters because they ﬁlter more rapidly, do not

affect the ﬁltrate in any way, and absorb very little of

the substance being ﬁltered.

Membrane ﬁlters are particularly suitable for ﬁl-

tering small volumes of ﬂuid because they can be

placed in a Swinnex type ﬁlter holder which can be

attached to a syringe as shown in Plate 7.2. Swinnex

ﬁlter holders are available to take membrane ﬁlters

of diameter sizes 13 mm, 25 mm, and 47 mm.

Swinnex holders made from polypropylene and

polycarbonate can be autoclaved and used many

times. Membrane ﬁlters made from cellulose nitrate

are also autoclavable. They are available in a variety

of pore sizes, with 0.22 m being required for

sterile ﬁltration. The ﬂuid being ﬁltered should be

relatively clear to pass through a 0.22 m porosity

ﬁlter. Cloudy ﬂuids should ﬁrst be passed through a

less ﬁne ﬁlter.

Availability: Autoclavable polypropylene ﬁlter holders (25

mm diameter) and cellulose nitrate membranes (25 mm

diameter 0.22 m porosity) can be obtained from Millipore

Corporation (see Appendix 11). Membranes are available in

pore sizes from 0.025 m to 8 m. A range of autoclavable

ﬁlter holders suitable for sterilizing larger volumes of ﬂuid

by membrane ﬁltration are also available from Millipore

Corporation.

50 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.4

with standardized tested media. When this is not

possible, each district laboratory should set up its

own qualitycontrol of the media it prepares. A set of

control organisms (stable stock strains) will need to

be obtained from the Regional or Central Public

Health Laboratory (or from a commercial source)*

and these organisms maintained with regular sub-

culturing as indicated in Chart 7.7.

*Sources of well-characterized stable strains of control

bacteria

● National Collection of Type Cultures (NCTC), Central

Public Health Laboratory Service, Colindale Avenue,

London NW9 5HT, UK.

E-mail: nctc@phls.nhs.uk

Website: www.phls.co.uk

● American Type Culture Collection (ATCC), 12301

Parklawn Drive, Rockville, MD 20852, USA

E-mail: help@atcc.org

Website: www.atcc.org

● Centers for Disease Control (CDC) Bacterial Diseases

Division, Atlanta GA 30333, USA.

● Mast Diagnostics (see Appendix 11) supply QC sticks

consisting of lyophilized gelatin pellets of microorganisms

derived from ATCC or NCTC strains. They are sold, 3

QC sticks per pack. They have a 2 year shelf-life.

● Oxoid (see Appendix 11) supply quality control organ-

isms (ATCC strains) on loops, i.e. Culti-Loopsin packs of

5 loops (Remel) or packs of 100 loops (Oxoid).

Control of nutrient agar, blood agar,

chocolate agar

Use appropriate control species as listed in Chart 7.7.

Inoculate slopes or quarter plates of the medium to

be tested with a 5 hour broth culture of each control

organism. Use a straight wire to inoculate the

medium and a wire loop to spread the inoculum.

Depending on the species, incubate aerobically or in

a carbon dioxide enriched atmosphere at 35–37C.

After overnight incubation, examine the cultures

for the degree of growth, size of colonies, and other

characteristics such as alpha-orbeta-haemolysis.

Recordthe resultsof each controlspecies andcompare

withthe results of previous performancetests.

Control of a differential medium

Use control species that show the differential

features of the medium as listed in Chart 7.7.

Inoculate quarter plates of the medium to be tested

with a 5 hour broth culture of each control species.

Use a straight wire to inoculate the medium and a

wire loop to spread the inoculum. Incubate aerobi-

cally at 35–37C.

After overnight incubation, examine the cultures

for the differential characteristics of the medium.

Record the results of each control species and

compare with the results of previous tests.

Plate 7.2 Autoclavable Swinnex ﬁlter holders attached to

syringes. Courtesy of Millipore Corporation.

Sterility testing

Sterility test routinely media to which blood or other

substances have been added after autoclaving. For

‘sterile’ media in screw-cap tubes or bottles, the

simplest way to test for contamination is to incubate

5% of the batch at 35–37C overnight.

Contamination by microorganisms capable of

overnight growth, will be shown by a turbidity in a

ﬂuid medium and growth on or in a solid medium.

Media in petri dishes are best examined for con-

tamination immediately before use.

Important: All media, even those that have been

sterility tested at the time of preparation, should

always be checked visually immediately before

being inoculated for any change in appearance that

could indicate contamination or deterioration. This is

particularly important during the hot season and

when the humidity is high.

Performance testing

Whenever possible,the Central or Re gionalmicrobi-

ology laboratory should supply district laboratories

Control of a selective medium

Use the control species that show the selectivity and

inhibitory properties of the medium as listed in

Chart 7.7.

Technique of testing a selective medium

1 Prepare a 5 h broth culture of the organism to be selected

and a 5 h mixed broth culture of the organism(s) to be

inhibited.

2 Take three sterile tubes and label 1 to 3. Using a sterile

Pasteur pipette, place in each tube, 5 drops of the broth

culture containing the organism to be selected.

3 Using a second sterile Pasteur pipette, add to each tube

the following drops of the mixed broth culture containing

the organisms to be inhibited:

Tube 1: 5 drops

Tube 2: 10 drops

Tube 3: 15 drops

4 Divide a plate of the medium to be tested into three

segments and label 1, 2, and 3. Using a small sterile loop,

inoculate the appropriate segments of the plate with a

loopful of the organism suspension from the tubes (i.e. 1

loopful from tube 1 in segment 1, etc).

5 Inoculate a second plate of the medium with a loopful of

the pure 5 h broth culture of the organism to be selected.

6 After overnight incubation at 35–37 C, record the degree

of selectivity of the medium (from the segmented plate)

and the size and appearance of the colonies of the

selected organism (from the pure culture plate). Compare

with the results of previous performance tests.

Control of a biochemical testing medium

Most biochemical testing media are controlled at the

time they are used. The medium is inoculated with

bacterial species of known positive and negative

reactions as explained in subunit 7.3.

Control of a transport medium

Immerse in the medium a swab of the specimen

containing the pathogen(s) to be preserved (e.g.

urogenital swab containing N. gonorrhoeae in

Amies medium, or a faecal swab containing Shigella

or Salmonella in Cary-Blair medium).

Leave the inoculated transport medium at room

temperature (protected from direct light) for the

length of time the medium is intended to preserve

the viability of the pathogen(s) it contains. After this

time, inoculate the swab on an appropriate medium

to check for viability of the pathogen.

Control species

Chart 7.7 lists some of the microorganisms that are

suitable for testing the performance of different

culture media (see previous text for sources of

control organisms). Also listed are media rec-

ommended for the maintenance of control cultures

and the recommended frequency of subculturing.

MICROBIOLOGICAL TESTS 51

7.4

Most species of bacteria required to control the

culture media used in district laboratories, can be

maintained in nutrient agar deeps covered with

sterile mineral oil, in semisolid nutrient agar, on

slopes of Dorset egg medium, or in cooked meat

medium or Amies transport medium. Control

species of anaerobes are best preserved in cooked

meat medium.

Long-term preservation of control strains

This is possible by storing in 16% v/v glycerol broth at 20C.

Glycerol nutrient broth is prepared by mixing 16 ml of

glycerol with 84 ml of nutrient broth, dispensing in 5 ml

amounts in bottles, and autoclaving at 115C for 20 minutes.

Prepare the control organisms on blood agar. Using a sterile

swab, subculture the entire growth of an overnight pure

culture in 5 ml of sterile 16% v/v glycerol broth, and freeze

immediately.

After 24 hours, check the viability of the organism by

thawing the suspension at 35–37C, and inoculating it on a

plate of blood agar. If satisfactory growth occurs, re-freeze the

suspension and store at 20C or below. Some bacterial

species can be maintained for several years by this method.

Record the colonial, biochemical, and other characteristics of

each control strain both before and after storage.

Note: In reference laboratories, control strains of bacteria are

usually stored in lyophilized form. This ensures stability and

viability of bacterial strains for many years.

To reduce the risk of contamination and changes in

the growth characteristics of control strains, stock

cultures should not be subcultured more than is

necessary. Several subcultures should be prepared

at one time. Guidelines for the frequency of subcul-

turing of the different control species is given in

Chart 7.7.

Labelling and storage of culture media and

additives

As previously discussed, dehydrated culture media

and dry ingredients such as peptone, tryptone, and

carbohydrates (solid form) should be stored at an

even temperature in a cool dry place away from

direct light. Container tops must be tight-ﬁtting and

in humid climates, tape-sealed.

Additives such as blood, serum, antimicrobials in

solid form, urea and carbohydrate solutions, require

storage at 2–8C. All additives should be allowed to

warm to room temperature before being used.

Antimicrobial solutions should be stored frozen at

20C in the amounts required.

Plates of culture media should be stored at

2–8C, preferably in sealed plastic bags. Most

media in screw-cap tubes or bottles can be stored at

room temperature (20–28C). Prepared media

should be stored in the dark. When in use, the

media must be protected from direct light, especially

sunlight.

52 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.4

Chart 7.7 Quality control of commonly used culture media

Culture medium Recommended Control species Maintenance medium Subculturing interval

Alkaline peptone water Enriched: Nutrient agar 6 months

Vibrio species semisolid or deep

(Use small inoculum)

Inhibited controls: As above As above

Escherichia coli

Proteusspecies

Blood agar Streptococcus pyogenes Cooked meat medium 3 months

Streptococcus pneumoniae

Haemophilus inﬂuenzae Chocolate agar slope 1 month

(With S.aureus streak) (35–37 C)

Chocolate agar Haemophilus inﬂuenzae Chocolate agar slope 1 month

(35–37C)

Cooked meat medium Clostridium sporogenes Cooked meat medium 12 months

Cystine lactose Staphylococcus aureus Nutrient agar 6 months

electrolyte deﬁcient Proteus mirabilis semisolid or deep

agar (CLED)

Kligler iron agar Citrobacter freundii Nutrient agar 6 months

(KIA) or Triple Proteus vulgaris

sugar iron agar Alcaligenes faecalis

MacConkey agar Escherichia coli Nutrient agar 6 months

Proteus mirabilis semisolid or deep

Modiﬁed New York Selected: Amies medium 2 weeks

City (MNYC) medium Neisseria gonorrhoeae (Use heavy inoculum

or other selective and store at 2–8C)

medium to isolate

Inhibited control: Nutrient agar 6 months

N.gonorrhoeae

Proteus vulgaris semisolid or deep

Thioglycollate broth Clostridium species or Cooked meat medium 12 months

Bacteroidesspecies

Thiosulphate citrate bile Selected: Nutrient agar 6 months

salt sucrose (TCBS) agar Vibrio cholerae semisolid or deep

(Nonpathogenic strain NCTC 11218)

Inhibited control: As above As above

Escherichia coli

Columbia agar diphasic Streptococcus pyogenes Cooked meat medium 3 months

medium Staphylococcus aureus

Haemophilus inﬂuenzae Chocolate agar slope 1 month

(35–37C)

Xylose lysine deoxycholate Salmonella Typhimurium Dorset egg medium 12 months

(XLD) agar

Escherichia coli Nutrient agar 6 months

semisolid or deep

Notes

● Use well-characterized stable strains of control organisms (for sources, see previous text).

● When subculturing from solid control cultures, take growth from several colonies. Prepare several subcultures (up to 6 for entero-

bacteria).

● Do not attempt to use a control culture that has become contaminated.

● Label clearly control cultures with the name of the species (and strain if known) and date of inoculation.

● Store control cultures in a secure place away from light. Most controls can be stored at room temperature (20–28 C).

● Make sure the bottle tops of control cultures are screwed tightly.

● The preparation of nutrient agar deeps, semisolid nutrient agar, Dorset egg medium, and cooked meat medium are described in

Appendix 1.

All culture media and additives must be clearly

labelled. When colour codes are used, an identiﬁ-

cation chart should be prepared and displayed. Each

batch of prepared medium should be given a

number and the date of its preparation recorded.

Sterilizing glass petri dishes, tubes and other

glassware in a hot air oven

To sterilize glassware by dry heat, a temperature of

160 C held for 45–60 minutes is required, timed

from when the items in the oven have reached this

temperature. A heating up time (of up to 1 hour)

must therefore be allowed. A cooling time is also

necessary to enable the items in the oven to cool

slowly. The oven door must notbe opene d until the

temperature inside the oven has fallen to above

50C. This will avoid cracking the glassware and air,

which may contain contaminating organisms, being

drawn into the oven.

For the needs of most district microbiological lab-

oratories, a small capacity hot air oven of the

convection type is adequate. To enable maintenance

and any repairs to be carried out locally, an oven

ﬁtted with a simple hydraulic thermostat and

analogue (dial) thermometer is recommended in

preference to a microprocessor controlled oven. The

oven must be ﬁtted with a protective over-heat cut

out device. The more expensive microprocessor

controlled ovens are usually ﬁtted with a fan, tem-

perature chart recorder, port for thermocouples, and

a door interlock.

Availability: An example of a convection type hot air oven

ﬁtted with an hydraulic thermostat, external thermometer and

safety cut-out, is the model E28, manufactured by Binder (see

Appendix 11). It is shown in Plate 7.3. Internally the oven

measures 400 mm wide  250 mm deep  280 mm high

(external dimensions: 580  425  402 mm high). Its power

consumption is about 800 W and it weighs 18 Kg. It has a

temperature range of 60–230C, with an accuracy of 1.5 C

and variation of 3C. A pilot light shows when the oven is

switched on.

Items suitable for sterilizing in a hot air oven (at

160 C) include:

● glass or aluminium petri dishes (not plastic

dishes).

● glass tubes (rimless) ﬁtted with aluminium caps

or with non-absorbent cotton wool plugs.

● bottles with aluminium caps lined with silicone

rubber (not red or black rubber). Autoclaving is

also suitable for bottles.

● glass ﬂasks and cylinders (cover the open end

with aluminium foil or paper, tied on with

string).

● glass pipettes (graduated and Pasteur) with ends

MICROBIOLOGICAL TESTS 53

7.4

plugged to a depth of about 20 mm with non-

absorbent cotton wool.

● nylon or glass syringes (not polypropylene or

other plastic).

● metal needles, lancets, and forceps (not plastic).

● dry swabs in tubes, plugged with non-absorbent

cotton wool.

Items for dry heat sterilizing must be dry. They can

be wrapped individually in brown (Kraft) paper (X-

ray ﬁlm wrapping paper can also be used and

reused) or placed in aluminium or copper canisters,

e.g. petri dishes and pipettes. Do not overload the

oven. When not used as a sterilizing oven, a hot air

oven can also be used at a lower temperature

(80–100C) to dry routine glassware. Follow care-

fully the manufacturer’s instructions on how to use

and maintain the oven.

OW TO DISPENSE CULTURE MEDIA

Media should be dispensed in a clean draught-free

room. Most ﬂuid media are dispensed into screw-

capped bottles or tubes, and then sterilized by

autoclaving. Sterile media must be dispensed into

sterile petri dishes, tubes or bottles using an aseptic

technique.

Dispensing sterile media into petri dishes

1 Lay out the sterile petri dishes on a level surface.

2 Mix the medium gently by rotating the ﬂask or

bottle. Avoid forming air bubbles. Flame

sterilize the neck of the ﬂask or bottle and

pour 15–20 ml of medium into each dish

(90–100 mm diameter).

Plate 7.3 WTB Binder E 28 model, 60 –230°C hot air oven

with external thermometer and timer and ﬁtted with thermo-

stat and overheat cut-out. Courtesy of WTB Binder.

If air bubbles enter while pouring, rapidly ﬂame

the surface of the medium before gelling occurs.

Rotate the dish on the surface of the bench to

ensure an even layer of agar.

3 When the medium has gelled and cooled, stack

the plates and seal them in plastic bags to

prevent loss of moisture and reduce the risk of

contamination. Do not leave the plates exposed

to bright light especially sunlight.

4 Store at 2–8C.

Note: Agar plates should be of an even depth (not

less than 4 mm) and of a ﬁrm gel. The surface of the

medium should be smooth and free from bubbles.

Dispensing and solidifying high protein media

(inspissation)

High protein media such as Dorset egg medium

and Loefﬂer serum medium are dispensed asepti-

cally in screw-capped bottles and solidiﬁed in a

sloped position at a controlled temperature (75–80

C) for 1–2 hours. Solidiﬁcation of protein media

using heat to coagulate the protein, is called inspis-

sation.

Inspissation can be carried out in an inspissator

(water-jacketed container which allows water vapour

to enter the inspissating area), or in a 75–80C ther-

mostatically controlled water bath or oven (over a

tray of water to prevent drying of the medium). The

bottle tops should be left loose during inspissation

(tighten them later).

To prevent bubbles forming in the medium, the

temperature should be raised slowly and 80C must

not be exceeded.

HOW TOINOCULATE CULTUREMEDIA

Immediately before inoculating a culture medium,

check the medium for visual contamination or any

change in its appearance which may indicate

deterioration of the medium, e.g. darkening in

colour.

When inoculating, or seeding, culture media an

aseptic (sterile) technique must be used. This will:

– prevent contamination of cultures and spe ci-

mens,

– prevent infection of the laboratory worker and

the environment.

Aseptic techniques

● Flame sterilize wire loops, straight wires, and

metal forceps before and after use (see Fig. 7.3).

Whenever possible, use a Bunsen burner with a

54 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.4

protective tube, e.g. Bactiburner to avoid particles

being dispersed when ﬂame sterilizing wire loops

(see p. 62 and p. 170 in Part 1 of the book).

Note: To prevent the release of aerosols, wire

loops must be well made (see later text).

Aerosols can also be released when spreading

inocula on media containing air bubbles.

● Flame the necks of specimen bottles, culture

bottles, and tubes after removing and before

replacing caps, bungs, or plugs.

● When inoculating, do not let the tops or caps of

bottles and tubes touch an unsterile surface. This

can be avoided by holding the top or cap in the

hand as shown in Fig. 7.4.

Fig 7.3 Sterilizing a wire loop in the ﬂame of a Bunsen

burner.

Fig 7.4 Inoculating a bottle of sterile medium. The neck of

the bottle is ﬂamed before and after inoculating the medium.

The cap of the bottle is held in the hand as shown.

● Always use racks to hold tubes and bottles con-

taining specimens or culture media.

● Make slide preparations from specimens after

inoculating the culture media.

● Decontaminate the work bench before starting

the day’s work and after ﬁnishing.

Note: Decontamination of infected material is

described in Part 1 of the book (see subunit 3.4).

● Use a safety cabinet when working with haz-

ardous pathogens (see pp. 64–65 in Part 1).

● Wear protective clothing, wash the hands after

handling infected material, and never mouth-

pipette, eat, drink, or smoke in the laboratory

(see also subunit 3.4 in Part 1).

Making a wire loop

Loops must be made correctly to ensure inocula are

well spread, and to prevent the release of aerosols

from long and springy loops or loops that are not

completely closed. The length of wire from the loop

to the loop holder should be short (6 cm) and the

loop itself should be small (2 mm diameter) and fully

closed.

Reusable loops are usually made of nichrome

(nickel-chromium) wire because it cools quickly, is

not too rigid, and is less expensive than platinum

wire. The thickness of the wire should be of standard

wire gauge (swg) 26 or 27. Disposable plastic loops

are widely available but more expensive to use than

wire loops.

Purchasing ready-made wire loops

Order nickel-chromium wire loop of 2 mm diameter (holding

1/500 ml) and no longer than 60 mm in length. Order as a

complete loop with handle. Alternatively purchase the ready-

made wire loops and wire loop holder with screw-head chuck.

Suppliers include Developing Health Technology (see

Appendix 11). If unable to purchase ready-made, make a loop

as described in the following method.

Method of making a wire loop to ﬁt in a screw-head

chuck wire holder

1 Cut a piece of wire about 125 mm in length (swg

26 or 27). Wind it around a loop holder as

shown in Fig. 7.5.

2 Using a pair of scissors, cut off one arm of the

wire leaving the loop and about 50 mm of wire.

Bend the loop back to make it central using a

pair of forceps.

3 Insert the wire in a loop holder as shown in Fig.

7.5. Make sure the loop is completely closed.

Note: When sterilizing a wire loop, hold it in the blue

part of a Bunsen burner ﬂame (see Fig. 7.3). When

MICROBIOLOGICAL TESTS 55

7.4

the laboratory does not have a piped gas supply, use

a portable burner such as a Labogaz burner. Allow

the loop to cool before using it.

Fig 7.5 How to make a wire loop (see text for method).

Inoculation of media in petri dishes

The technique used to inoculate media in petri

dishes (plates) must provide single colonies for

identiﬁcation. It must also show whether a culture is

pure or mixed, i.e. consisting of a single type of

organism or several different organisms. A pathogen

must be isolated in pure culture before it can be

identiﬁed and tested for antimicrobial sensitivity.

The inoculation of media in petri dishes is

referred to as ‘plating out’ or ‘looping out’. It is not

necessary to use whole plates of media for every

specimen. Considerable savings can be made by

using a half or even a third of a plate (especially

when the medium is a selective one). The area of

medium used must be sufﬁcient to give separate

colonies.

Before inoculating a plate of culture medium, the

surface of the medium must be dried, otherwise

single colonies will not be formed. To do this,

remove the lid of the plate and place this face

upwards on an incubator shelf. Invert the base con-

taining the medium and let it rest at an angle on the

lid. Usually 30–40 minutes incubation at 35–37C

is sufﬁcient time to dry the surface of an agar plate.

Inoculating technique

1 Using a sterile loop or swab of the specimen,

apply the inoculum to a small area of the plate

(the ‘well’) as shown in Fig. 7.6.

2 Flame sterilize the loop. When cool, or using a

second sterile loop, spread the inoculum as

shown in Fig. 7.6 (follow the steps 2 through to

5). This will ensure single colony growth.

Note: A simpliﬁed technique of inoculating plates is

shown in Fig. 7.7. This can be used by medical and

nursing staff when culturing specimens directly from

patients, e.g. urogenital specimens for the isolation

of Neisseria gonorrhoeae. The techniques of inocu-

lating half a plate and a third of a plate of medium

are shown in Fig. 7.8 and Fig. 7.9.

Inoculation of slopes

To inoculate slopes such as Dorset egg medium or

Loefﬂer serum, use a sterile straight wire to streak

the inoculum down the centre of the slope and then

spread the inoculum in a zig-zag pattern as shown

in Fig. 7.10.

To inoculate a slope and butt medium, such as

Kligler iron agar, use a sterile straight wire to stab

into the butt ﬁrst and then use the same wire to

streak the slope in a zig-zag pattern (see Fig. 7.11).

Inoculation of stab media (deeps)

Use a sterile straight wire to inoculate a stab

medium. Stab through the centre of the medium as

shown in Fig. 7.12, taking care to withdraw the wire

along the line of inoculum without making further

stab lines.

Inoculation of ﬂuid media

Broths and other ﬂuid media are inoculated using a

sterile wire loop, straight wire, or Pasteur pipette

depending on whether the inoculum is a colony, a

ﬂuid culture, or a specimen. The inoculation of blood

culture broths is described in subunit 7.14.

When using a wire loop to subculture colonies,

hold the bottle or tube at an angle and rub the loop

against the side of the container below the level of

the ﬂuid.

When using a Pasteur pipette to inoculate a ﬂuid

culture hold the pipette as shown in Fig. 7.13 (box).

How to make Pasteur pipettes in the laboratory is

shown in Fig. 7.13.

56 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.4

Labelling of inoculated media

Using a grease pencil or marker pen, label inocu-

lated media with the date and the patient’s number.

Always label the base of a culture plate, not the lid

(lids can be accidentally switched).

Label a slope on the underside of the medium so

that the wording does not obscure the culture. A

stab culture should be labelled above the level of the

agar.

When a plate is to be incubated anaerobically it

should be marked ‘An O

’ or when in a carbon

dioxide atmosphere it should be marked ‘CO

’.

INCUBATIONOF INOCULATEDMEDIA

Inoculated media should be incubated as soon as

possible. A delay in incubation can affect the viabil-

ity of pathogens especially anaerobes, pneumococci,

meningococci, gonococci, and Haemophilus inﬂuen-

zae. It can also increase the risk of plates becoming

contaminated from small insects and dust.

Uninoculated and inoculated media must be pro-

tected from sunlight.

Microorganisms require incubation at the tem-

perature and in the humidity and gaseous

atmosphere most suited to their metabolism. The

length of time of incubation depends on how long

an organism takes to develop the cultural character-

istics by which it is recognized.

Temperature of incubation

The temperature at which a microorganism grows

best is referred to as its optimum temperature.

The temperature below which growth stops (not

necessarily resulting in death) is called the mini-

mum temperature, and that above which growth

stops and death occurs is called the maximum

temperature.

The temperature selected for routine culturing is

35–37C with most microbiologists recommending

35C in preference to 36 C or 37C. In general, the

growth of microorganisms is more affected by slight

rises above their optimum temperature than by

reductions below it.

Incubators for use in district laboratories

The speciﬁcations of an electric incubator for use in district

microbiological laboratories can be found in subunit 4.9 in

Part 1 of the book. A small, low cost, mains and battery

operated incubator is also described for those laboratories

incubating only a few cultures. Subunit 4.9 also includes

guidelines on the use and care of incubators.

In tropical countries it is possible to grow some

pathogens at local room temperatures, e.g. Vibrio

MICROBIOLOGICAL TESTS 57

7.4

Fig 7.6 Inoculation of a plate of culture

medium to give single colonies

Fig 7.7 Simplified technique of

inoculating a plate of culture medium,

suitable for use in clinics

Fig 7.9 Different ways of inoculating

a third of a plate of culture medium

Fig 7.8 Inoculation of half

a plate of culture medium

Fig 7.10 Inoculation of

an agar slope

Fig 7.11 Inoculation of a butt

and slope. Use a straight line to

inoculate the butt first

Fig 7.12 Inoculation of

a deep (stab)

Fig 7.9 Different ways of inoculating

a third of a plate of culture medium

Fig 7.8 Inoculation of half

a plate of culture medium

58 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.4

1 Etch the glass tubing in

16 cm lengths.

2 With the etch uppermost,

break the tubing.

4 Heat the centre of the rod, turning

it continuously in the hottest part of

the flame.

3 Round off the ends of each

rod in the flame. When cool,

wash and dry.

5 Continue heating until the glass

becomes molten.

7 Etch the centre, and break to give two Pasteur pipettes.

9 For use, hold the pipette as shown.

6 Remove from the flame and pull slowly and steadily. Pull

more quickly if a thin stemmed pipette is required.

8 Using a stick, plug the ends of the

pipettes with non-absorbent

cotton wool. Place in a metal canister

and sterilize in an oven or autoclave.

Note: the glass tubing should be 0.8–0.9 mm thick and have an external diameter of 6–7 mm.

Fig 7.13 How to make a Pasteur pipette

cholerae. Growth, however, tends to be slower and

variations in temperature can affect growth. When

not using an incubator, cultures must be protected

from sunlight, contamination, and drying by being

placed in a container in the dark.

Yersinia enterocolitica grows best at 20–28C

which helps to identify the species. Temperature of

growth is also used in the differentiation of

Mycobacterium species, e.g. no growth is produced

by M. tuberculosis and M. ulcerans at 25C whereas

many opportunistic and saprophytic mycobacteria

grow at this lower temperature.

Humidity

An atmosphere which is too dry can affect the

growth and viability of many pathogens, e.g. gono-

cocci are rapidly killed in dry conditions. Inclusion of

a piece of damp blotting paper in the bottom of a

candle jar, is therefore recommended for the culture

of gonococci.

Gaseous atmosphere

Microorganisms vary in their need for oxygen and

use of it as a means of producing energy.

Depending on its atmospheric requirements, an

organism can be described as:

● An obligatory (strict) aerobe: Requires free

oxygen to survive. An example of an obligatory

aerobe is Pseudomonas aeruginosa.

● A microaerophilic organism: Grows best in the

presence of only a trace of free oxygen. An

example of a microaerophilic organism is

Campylobacter jejuni.

● An obligatory (strict) anaerobe: Survives only in

the absence of oxygen. An example of an obliga-

tory anaerobe is Clostridium tetani.

● A facultative anaerobe: Can live with or without

free oxygen. Examples of facultative anaerobes

are Staphylococcus aureus, Streptococcus pyo-

genes, Escherichia coli.

● A carboxyphilic organism: Requires an atmos-

phere which contains carbon dioxide. An

example of a carboxyphilic organism is Neisseria

meningitidis. Traces of carbon dioxide (5–10%),

however, are thought to help the growth of most

bacteria.

Culturing of anaerobes

An anaerobic atmosphere is essential for the growth

of strict anaerobes such as Clostridium species,

Bacteroides species, and anaerobic streptococci.

Anaerobic incubation also helps to differentiate

pathogens and to isolate facultative anaerobes from

MICROBIOLOGICAL TESTS 59

7.4

specimens containing commensals, e.g. Strepto-

coccus pyogenes from throat swabs. The haemo-

lytic reactions of beta-haemolytic streptococci are

also more pronounced following anaerobic incu-

bation.

There are several techniques for obtaining anaer-

obic conditions. Those which are more suited for

district microbiology laboratories include the use of:

● Commercially produced sachets containing

oxygen removing chemicals. These recently

developed safe technologies do not produce

hydrogen and therefore do not require a catalyst,

i.e. they are non-gas generating systems.

● Copper coated steel wool to remove oxygen.

● Reducing agents in culture media.

Commercially produced oxygen-removing

systems

Examples of systems that produce anaerobic con-

ditions by using chemicals that absorb oxygen are

the Anaerocult system produced by Merck/BDH

and the AneroGen system produced by Oxoid (see

Appendix 11). Whereas the oxygen-removing

sachets produced by Oxoid can only be used in

anaerobic jars, Merck produces a range of sachets

that can be used to incubate a single culture plate (in

a sealed plastic bag), up to four plates, and plates in

anaerobic jars.

Merck Anaerocult anaerobic system

The sachets contain a mixture of iron powder, citric acid,

sodium carbonate, and kieselguhr. Addition of a small volume

of water activates the chemicals. Oxygen is rapidly removed

leaving anaerobic conditions. Some carbon dioxide is

produced. The following sachets are available:

– Anaerocult P (order No. 1.13807.0001) for the anaerobic

incubation of one petri dish. Each pack contains

25 sachets and 25 foil bags (petri dish with sachet is

placed inside a foil bag). No anaerobic jar is needed.

Reusable clips (Anaeroclips) to seal the bags are required.

These are available in packs of 25 (order No.

1.14226.0001).

– Anaerocult A mini (order No. 1.01611.0001) for the

anaerobic incubation of up to 4 petri dishes. Each pack

contains 25 sachets and 25 foil bags (anaerobic jar is not

needed). As for Anaerocult P, sealing clips are required

(see above text).

– Anaerocult A (order No. 1.13829.0001), for use in 2.5 litre

capacity anaerobic jars. Each pack contains 10 sachets.

No clips are needed.

Control of Anaerocultsystem: Use of the anaerobic indicator

strip, Anaerotest is recommended. Each pack contains

50 strips.

Oxoid AneroGensystem

The active oxygen-removing component in AneroGensachets

is ascorbic acid. The sachets are designed for use in 2.5 litre

and 3.5 litre capacity anaerobic jars. The paper sachet is

placed in the jar immediately before it is closed. No water is

needed to activate the chemical. Within 30 minutes of closing

the jar, the oxygen level is reduced to below 1% (carbon

dioxide level is 9–13%).

Two types of AneroGensachet are available:

– AN 3.5 sachets (order No. ANO35A) for use with 3.5 litre

anaerobic jars. Each pack contains 10 sachets.

– AN 2.5 sachets (order No. CN025A) for use with 2.5 litre

jars. Each pack contains 10 sachets.

Control of the AneroGen system

Use of an anaerobic control indicator (resazurin) in the jar is

recommended (order No. BRO55B) to ensure anaerobic con-

ditions have been produced).

Use of copper coated steel wool to remove

oxygen

This is a simple method of obtaining anaerobiasis

when it is not possible to obtain the commercially

produced oxygen-removing sachets. It can be

adapted for incubating single plates or several plates.

The plates can be incubated in a plastic bag provid-

ing it is airtight. The system uses steel wool which is

activated immediately before use by being dipped in

acidiﬁed copper sulphate solution. The metallic

copper on the surface of the iron rapidly absorbs

oxygen. Anaerobic conditions are obtained more

rapidly by removing some of the air from the bag

before it is sealed. A source of carbon dioxide is

added and also an indicator to check for anaero-

biasis. The method of use is as follows:

1 Place the inoculated plates in an undamaged

strong plastic bag no larger than the size

required. Support the bag on a tray or other

rigid sheet.

2 Place in the bag an open tube or bottle contain-

ing equal volumes of magnesium carbonate and

sodium bicarbonate for the release of carbon

dioxide. About 1.5 g of each chemical is required

for a bag measuring about 250  180 mm. The

chemicals can be pre-mixed and stored dry in

screw-cap bottles ready for use. Alternatively use

half an Alka-Seltzer tablet.

3 Prepare the steel wool as follows:

– Take a loose pad of about 3–5 g of steel

wool (grade 0 or 1). This is sufﬁcient for a

bag measuring about 250  180 mm.

– Dip the steel wool for a few seconds in acidi-

ﬁed copper sulphate solution (see below)

until the wool appears dark grey with no

more than a trace of copper colour. Drain,

and place in an open dish.

60 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.4

Note: Some batches of steel wool are greasy

and resistcoating with the copper (i.e. do not

become dark grey). Ifthis happens, wash the

steel wool in detergent, rinse well, andshake

to remove the water. When de greased, the

steelwool will rapidly become coppercoate d.

Preparation of the acidiﬁed copper sulphate solution

The solution requires renewing every week.

Prepare by mixing:

Copper sulphate, 10% w/v solution . . . . . . . . . . 5 ml

Tween 80 (or Lissapol), 10% w/v . . . . . . . . . . . . 5 ml

Sulphuric acid, 2 mol/1* . . . . . . . . . . . . . . . . . . . . 3 ml

Distilled water. . . . . . . . . . . . . . . . . . . . . . . . to 200 ml

*Prepare by adding 11 ml of concentrated acid to

89 ml of water.

Caution: NEVER add the water to the acid because

sulphuric acid is hygroscopic. Handle the acid with

great care because it is highly corrosive.

4 Add 5 ml of water to the magnesium carbonate

and sodium bicarbonate. When using an Alka-

Seltzert ablet, moisten the tablet with a few drops

of water. Carbon dioxide will be slowly released.

5 Enclose an anaerobic strip indicator in the an-

aerobic bag to check for anaerobiasis (buy

commercially from Oxoid or Merck as described

previously).

Note: Complete anaerobic conditions are usually

obtained within 4 hours of sealing the bag.

6 Just before sealing the anaerobic bag, use a

piece of rubber tubing and syringe to draw out

some of the air.

7 Seal the bag with Sellotape or other reliable

adhesive tape, and incubate the bag on its tray at

35–37C. Providing a transparent bag has been

used, the cultures can be viewed for growth

without needing to open the bag.

Use of reducing agents in culture media

Examples of media that contain reducing agents

include:

– Thioglycollate broth which is used mainly to

culture anaerobes in blood (see subunit 7.14).

The medium contains the reducing agent

sodium thioglycollate and the indicator methyl-

ene blue or resazurin to show that the medium

is reduced. Preparation of the medium is

described in Appendix 1.

– Cooked meat medium which is use d to culture

Clostridium and Bacteroides species. The anaer-

obes grow at the bottom of the medium among

themeat particleswhich containeffe ctivereducing

substances. Themedium shows saccharolytic and

proteolytic reactions and also gas production. Its

preparationis described in Appendix 1.

A simple way of providing anaerobic conditions in

litmus milk medium, peptone water media, and

broths is by using an iron strip (25  3 mm sheet

iron) or an iron nail to remove the oxygen. The strip

or nail is ﬂame sterilized and while still hot it is

dropped into the medium. When the medium has

cooled, it can be inoculated.

Anaerobic container

With the availability of oxygen-removing systems to

produce anaerobic conditions, an anaerobic jar with

pressure gauge, valves, etc is no longer needed. An

acrylic air-tight container such as that supplied by

BD Diagnostics (see Appendix 11) and shown in

Plate 7.4 is suitable.

Availability

Two sizes of container are available from BD Diagnostics:

– 15 plate container, code 260671

– 30 plate container, code 260672

Each container has a lid with easy to close latches which give

a secure airtight seal. A removeable rack is available for

holding culture plates. To obtain anaerobic conditions inside

the container, a BD GasPak EZ anaerobic container sachet

code 260678, is used. To obtain a carbon dioxide enriched

atmosphere, a GasPak EZsachet code 260679 is needed. A

Campylobactercontainer sachet is also available, code 260680.

An anaerobic indicator is required when incubating anaerobi-

cally, code 271051.

MICROBIOLOGICAL TESTS 61

7.4

BD pouch systems

BD Diagnostics also supply a pouch system for incu-

bating anaerobically up to 2 culture plates (see Plate

7.4) or up to 4 culture plates. Each resealable pouch

is supplied with a gas-generating sachet and anaer-

obic indicator strip. Pouches are also available for

providing a CO

enriched atmosphere and con-

ditions for incubating Campylobacter.

Plate 7.4 Left: BD GasPak container system. Right: BD

GasPakpouch system.

Courtesy BD Diagnostics.

Plate 7.5 Systems for culturing in carbon dioxide. The jar

can be used with chemicals which provide a carbon dioxide

(CO

) enriched atmosphere and the tin with a candle to

provide CO

conditions (see text). Reproduced from

Laboratory diagnosis of sexually transmitted diseases,WHO

1999,with the permission of the World Health Organization.

Culturing in carbon dioxide

A carbon dioxide enriched atmosphere is required

for the growth of Neisseria gonorrhoeae, Neisseria

meningitidis, Brucella species, and Streptococcus

pneumoniae. Commercially available carbon dioxide

gas-generating systems are available (see previous

text). When such a system is not available, a simple

way of providing a carbon dioxide enriched atmos-

phere is to enclose the inoculated plates (inverted) in

an airtight jar or tin with a lighted candle.* As the

candle burns, the oxygen content is reduced leaving

a carbon dioxide content of 3–5% by the time the

candle is extinguished. The screw-cap jar or tin

should not be more than half ﬁlled. For the culture

of Neisseria organisms, place a piece of damp ﬁlter

paper or blotting paper in the bottom of the con-

tainer to provide a moist atmosphere.

*Important: It is necessary to use a white wax smoke-

less candle or nite-light to avoid the release of fumes

which may be bactericidal or interfere with the

growth of bacteria. When it is not possible to obtain

a good quality candle, carbon dioxide can be gen-

erated chemically in a jar or tin by reacting sodium

bicarbonate with tartaric acid or citric acid (see

below).

Generation of carbon dioxide from chemicals

To obtain a 10% carbon dioxide atmosphere in a jar of about

3 litre capacity, mix 0.7 g sodium bicarbonate with 1.7 g

tartaric acid (or 2.4 g citric acid). Immediately before closing

the jar, moisten the chemicals with water. Alternatively,

use an Alka-Seltzer tablet (moistened with a few drops of

water).

Note: The above described chemical method of pro-

viding a carbon dioxide enriched atmosphere is not

suitable for the culture of campylobacters. For the

growth of these microaerophilic organisms a candle

jar is required or the use of special commercially

produced sachets such as those manufactured by

BD Diagnostics (see previous text).

REPORTING CULTURES

The manner of reporting cultures depends on

whether the specimen has been taken from a site

which is normally sterile or from a site with a normal

microbial ﬂora.

Sites normally sterile: Identify and report all

bacteria isolated as far as their genera, and if helpful,

identify the actual species. Specimens received from

sterile sites include blood, bone marrow, cere-

brospinal ﬂuid, pleural and peritoneal ﬂuids, and

urine.

Note: Urine is regarded as being collected from a site which is

sterile (the urinary tract), even though the urethra is not

sterile and a mid-stream specimen may become contaminated

with organisms as it is being passed.

When isolating organisms from sites that are

normally sterile, it is necessary to use culture media

which will support the growth of a wide range of

organisms and often an enriched medium (see

beginning of this subunit).

Sites having a microbial ﬂora: Interpretation of

cultures is more difﬁcult and requires a knowledge

of the patient’s clinical condition to judge whether an

isolate of a pathogen is the cause of a patient’s

illness. The laboratory report should indicate those

organisms for which isolation techniques have been

performed. For example, when no pathogens have

been isolated from a faecal specimen cultured on a

selective medium, the report should state ‘No

Salmonella, Shigella or cholera organisms isolated’,

not ‘No pathogen isolated’ or ‘Normal bacterial ﬂora

only’.

Specimens received from sites having a normal bac-

terial ﬂora include faeces, sputum, skin, throat, and

62 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.4–7.5

nose swabs, vaginal, cervical, and urethral swabs.

When attempting to isolate organisms from speci-

mens collected from sites having a normal microbial

ﬂora, it is quicker, more economical, and cultures are

easier to read when selective media are used.

Itis not helpfulin a reportto list allthe normal ﬂora

bacteria that have been isolate d. It can also be con-

fusingto report isolatessemiquantitatively, e.g. ‘heavy

growthof . .. ’. The numberof organisms that grow is

affected by many variables, including the culture

medium used, conditions under which a specimen

is kept, and length of time before a specimen is

cultured. It is best simply to state that a particular

pathogen hasb een isolated.

Preliminary reports

Although the full procedure for identifying an

organism must be carried through, it can be useful

in some situations to issue a preliminary report, e.g.

a Gram report of a cerebrospinal ﬂuid, or a wet

preparation containing organisms suggestive of

V. cholerae. Such preliminary reporting can help a

medical ofﬁcer in the treatment of a patient or the

need to introduce isolation and control measures

when a highly infectious pathogen is indicated.

7.5 Biochemical tests

to identify bacteria

While several commercial systems for identifying

bacteria are available, these are often difﬁcult to

obtain or too expensive to use in developing coun-

tries. This subunit includes a range of conventional

biochemical tests and tablet identiﬁcation tests which

most district laboratories will be able to perform.

The following tests are described in this subunit:

Test Purpose

 Beta-glucuronidase To identify E. coli

 Bile solubility To differentiate

S. pneumoniae from

other alpha-haemolytic

streptococci

 Catalase To differentiate

staphylococci from

streptococci

 Citrate utilization To differentiate

enterobacteria

 Coagulase To identify S. aureus

 DNA-ase To help identify S. aureus

 Indole To differentiate Gram

negative rods, particularly

E. coli

 Litmus milk To help identify

decolorization Enterococcus and some

clostridia

 Lysine decarboxylase To assist in the

identiﬁcation of

salmonellae and shigellae

 Oxidase To help identify Neisseria,

Pasteurella, Vibrio,

Pseudomonas

 Urease To help identify Proteus,

Morganella, Y. enterocoli-

tica, H. pylori

Biochemical tests to screen for Salmonella and

Shigella in faecal specimens

Kligler iron agar (KIA) and Rosco enzyme tests

to screen for Salmonella and Shigella in faecal

specimens are described in subunit 7.11.

Carbohydrate fermentation tests

Peptone water sugars and Rosco sugar fermentation

tablets to identify bacteria by their fermentation

reactions are described in subsequent subunits

under speciﬁc organisms.

PYR test

The pyrrolidonyl aminopeptidase (PYR) test to

identify S. pyogenes is described in subunit 7.18.2.

Rosco diagnostic tablets for microbial

identiﬁcation

The diagnostic tablets (Diatabs) developed by Rosco

Diagnostica are microbial identiﬁcation tests in stable

tablet form. Some tablets are double test tablets, i.e.

a single tablet is used to test for two reactions, e.g.

indole combined with beta-glucuronidase (PGUA).

The tablets have a long shelf-life (2–4 y) and good

stability in tropical climates. They are available in

vials of 25 or 50 tablets. The Rosco identiﬁcation

tablets are more economical to use than most com-

mercially produced diagnostic identiﬁcation discs,

strips, stick tests, and some conventional biochemical

tests that use expensive dehydrated media. The fol-

lowing are some of the Rosco tablet tests described

in this publication:

MICROBIOLOGICAL TESTS 63

7.5.1

Carbohydrate tests Oxidase

Citrate Pyrrolidonyl aminopeptidase

Aesculin hydrolysis (PYR)

Factors X, V, XV Urease

Glutamyl aminopeptidase Voges-Proskauer (VP)

Hippurate hydrolysis

The full range of Rosco identiﬁcation tablets can be found in

the 2005, 6th edition Diagnostic tablets for bacterial identiﬁ-

cation, available from Rosco Diagnostica (see Appendix 11).

The Rosco tablets are easy to use. For most tests a

tablet is simply added to a saline suspension of the

test organism in a small test tube and the test incu-

bated at 35–37C. For many tests, reactions can be

read the same day after a few hours incubation.

Separate reagents are required to read the reactions

of some tests (supplied by Rosco).

Note: Rosco Diagnostica also produces low cost,

stable (3–4 year shelf-life at room temperature),

antimicrobial susceptibility testing tablets. These are

described in subunit 7.16.

7.5.1 Bile solubility test

This helps to differentiate S. pneumoniae, which is

soluble in bile and bile salts, from other alpha-

haemolytic streptococci (viridans streptococci) which

are insoluble.

Principle

A heavy inoculum of the test organism is emulsiﬁed in physi-

ological saline and the bile salt sodium deoxycholate is added.

This dissolves S.pneumoniae as shown by a clearing of the tur-

bidity within 10–15 minutes. Viridans and other streptococci

are not dissolved and therefore there is no clearing of the tur-

bidity.

Required

— Sodium deoxycholate, Reagent No. 74

100 g/l (10% w/v)

— Physiological saline (sodium chloride, 8.5 g/l)

Tube method

Although the bile solubility test can be performed by

testing colonies directly on a culture plate or on

a slide (see subunit 7.18.4), a tube technique is

recommended because the results are easier to

read.

1 Emulsify several colonies of the test organism in

a tube containing 2 ml sterile physiological

saline, to give a turbid suspension.

L-Arabinose Indole

Beta-lactamase Lysine decarboxylase (LDC)

Beta-galactosidase (ONPG) Nitrate reduction

Beta-glucuronidase (PGUA) Ornithine decarboxylase (ODC)

2 Divide the organism suspension between two

tubes.

3 To one tube, add 2 drops of the sodium deoxy-

cholate reagent and mix.

4 To the other tube (negative control), add 2 drops

of sterile distilled water and mix.

5 Leave both tubes for 10–15 minutes at 35–

37C.

6 Look for a clearing of turbidity in the tube con-

taining the sodium deoxycholate, as shown in

Plate 7.6.

Results

Clearing of turbidity . . . . . . . . . . . . . . . . . Probably

S. pneumoniae

No clearing of turbidity . . . . Organism is probably

not S. pneumoniae

There should be no clearing of turbidity in the

negative control tube to which distilled water was

added.

Note: Some strains of S. pneumoniae are not dis-

solved by bile salts, and very occasionally some

strains of viridans streptococci give a positive test.

64 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.5.1–7.5.2

7.5.2 Catalase test

This test is used to differentiate those bacteria that

produce the enzyme catalase, such as staphylococci,

from non-catalase producing bacteria such as strep-

tococci.

Principle

Catalase acts as a catalyst in the breakdown of hydrogen

peroxide to oxygen and water. An organism is tested for

catalase production by bringing it into contact with hydrogen

peroxide. Bubbles of oxygen are released if the organism is a

catalase producer. The culture should not be more than 24

hours old.

Plate 7.6 Bile solubility test. Left: Shows saline suspension of

test organism without bile salt added. Centre: Shows clearing

of turbidity after adding bile salt, indicating that the organism

is probably S.pneumoniae. Right: Shows a negative test.

Controls

Bile solubility positive control: Streptococcus

pneumoniae

Bile solubility negative control: Enterococcus

faecalis

Plate 7.7 Catalase tube test. Right: Shows a positive test.

Left: Shows a negative test. Courtesy of AH Westley.

Required

Hydrogen peroxide, 3% H

(10 volume solution)

Method

1 Pour 2–3 ml of the hydrogen peroxide solution

into a test tube.

2 Using a sterile wooden stick or a glass rod (not a

nichrome wire loop), remove several colonies of

the test organism and immerse in the hydrogen

peroxide solution.

Important: Care must be taken when testing an

organism cultured on a medium containing

blood because catalase is present in red cells. If

any of the blood agar is removed with the

organism, a false positive reaction may occur.

3 Look for immediate bubbling as shown in Plate

7.7.

Results

Active bubbling . . . . . . . . . . . . Positive catalase test

No bubbles . . . . . . . . . . . . . . Negative catalase test

Caution: Performing the test on a slide is not rec-

ommended because of the risk of contamination

from active bubbling. When the rapid slide tech-

nique is used, the hydrogen peroxide solution

should be added to the organism suspension after

placing the slide in a petri dish. The dish should then

be covered immediately, and the preparation

observed for bubbling through the lid.

Controls

Positive catalase control: Staphylococcus species

Negative catalase control: Streptococcus species

7.5.3 Citrate utilization

test

This test is one of several techniques used occasion-

ally to assist in the identiﬁcation of enterobacteria.

The test is based on the ability of an organism to use

citrate as its only source of carbon.

Ways of performing a citrate test

● Using a Rosco citrate identiﬁcation tablet. This is

the most economical method when only a few

tests are performed. The tablets have a long

shelf-life and good stability in tropical climates.

● Using Simmon’s citrate agar but the dehydrated

medium is only available in 500 g pack size from

manufacturers. After being opened the medium

does not have good stability in tropical climates.

Citrate utilization using a Rosco citrate tablet

Citrate identiﬁcation tablets (code 56511) are avail-

able from Rosco Diagnostica (see Appendix 11) in a

vial of 50 tablets.

1 Prepare a dense bacterial suspension of the test

organism in 0.25 ml sterile physiological saline in

small tube.

2 Add a citrate tablet and stopper the tube.

3 Incubate overnight at 35–37C.

Results

Red colour . . . . . . . . . . . . . . . . . Positive citrate test

Yellow-orange colour . . . . . . . . Negative citrate test

Controls

A positive citrate test reaction is obtained with

Klebsiella pneumoniae and a negative reaction with

Escherichia coli.

MICROBIOLOGICAL TESTS 65

7.5.3–7.5.4

Citrate method using Simmon’s citrate agar

1 Prepare slopes of the medium in bijou bottles as

recommended by the manufacturer (store at

2–8C).

2 Using a sterile straight wire, ﬁrst streak the slope

with a saline suspension of the test organism and

then stab the butt.

3 Incubate at 35C for 48 hours. Look for a bright

blue colour in the medium.

Results

Bright blue . . . . . . . . . . . . . . . . . Positive citrate test

No change in colour . . . . . . . . Negative citrate test

of medium

Controls

As described above.

7.5.4 Coagulase test

This test is used to identify S. aureus which produces

the enzyme coagulase.

Principle

Coagulase causes plasma to clot by converting ﬁbrinogen to

ﬁbrin. Two types of coagulase are produced by most strains of

S.aureus:

 Free coagulase which converts ﬁbrinogen to ﬁbrin by acti-

vating a coagulase-reacting factor present in plasma. Free

coagulase is detected by clotting in the tube test.

 Bound coagulase (clumping factor) which converts

ﬁbrinogen directly to ﬁbrin without requiring a coagulase-

reacting factor. It can be detected by the clumping of

bacterial cells in the rapid slide test.

A tube test must always b e performed when the

result of a slide test is not clear, or when the slide test

is negative and Staphylococcus has been isolated

from a serious infection. A tube test may be

required to detect some MRSA (methicillin resistant

S. aureus) strains although some commercially

available latex test kits to differentiate coagulase

positive and coagulase negative staphylococci,

overcome this. Before performing a coagulase test,

examine a Gram stained smear to conﬁrm that the

organism is a Gram positive coccus.

Required

EDTA anticoagulated human plasma (preferably

pooled and previously HIV and hepatitis tested) or

rabbit plasma. The plasma should be allowed to

warm to room temperature before being used.

Plasma: Oxalate or heparin plasma can also be used. Do not

use citrated plasma because citrate-utilizing bacteria e.g.

enterococci, Pseudomonas and Serratia may cause clotting of

the plasma (in tube test). Occasionally, human plasma may

contain inhibitory substances which can interfere with coagu-

lase testing. It is therefore essential to test the plasma using a

known coagulase positive S.aureus. The plasma can be stored

frozen in amounts ready for use.

Slide test method (detects bound coagulase)

1 Place a drop of distilled water on each end of a

slide or on two separate slides.

2 Emulsify a colony of the test organism (pre-

viously checked by Gram staining) in each of the

drops to make two thick suspensions.

Note: Colonies from a mannitol salt agar culture

are not suitable for coagulase testing. The

organism must ﬁrst be cultured on nutrient agar

or blood agar.

3 Add a loopful (not more) of plasma to one of the

suspensions, and mix gently. Look for clumping

of the organisms within 10 seconds, as shown in

Plate 7.8.

No plasma is added to the second suspension.

This is used to differentiate any granular appear-

ance of the organism from true coagulase

clumping.

Results

Clumping within 10 secs . . . . . . . . . . . . . S. aureus

No clumping within 10 secs . . . . . . . . . . No b ound

coagulase

Note: Virulent strains of Yersinia pestis are also coagulase

positive.

66 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.5.4

Controls

Positive coagulase control: Staphylococcus aureus

Negative coagulase control: Escherichia coli or

Staphylococcus epidermidis

Tube test method (detects free coagulase)

1 Take three small test tubes and label:

T  Test organism (18–24 h broth culture)*

Pos  Positive control (18–24 h S. aureus broth

culture)*

Neg  Negative control (sterile broth)*

*Nutrient broth is suitable (see No. 63). Do not use

glucose broth.

2 Pipette 0.2 ml of plasma into each tube.

3 Add 0.8 ml of the test broth culture to tube T.

Add 0.8 ml of the S. aureus culture to the tube

labelled ‘Pos’.

Add 0.8 ml of sterile broth to the tube labelled

‘Neg’.

Plate 7.8 Coagulase slide test. Upper: Positive agglutination

of S.aureus. Lower: Saline preparation to check for granular-

ity of the strain.

Plate 7.9 Tube coagulase test. Upper: S. aureus positive

control. Centre: A positive test indicating the organism is

S.aureus. Lower: A negative control. Courtesy of AH Westley.

4 After mixing gently, incubate the three tubes at

35–37C. Examine for clotting after 1 hour (see

Plate7.9).If noclotting hasoccurre d,examine after

3hours. If thetest is stillnegative, leave the tubeat

roomtemperature overnight and examine again.

Note: When looking for clotting, tilt each tube

gently.

Results

Clotting of tube contents or . . . . . . . . . . . S. aureus

ﬁbrin clot in tube

No clotting or ﬁbrin clot . . . . . . . . . . . Negative test

Note: There should be no clotting in the negative

control tube.

Commercially produced agglutination tests to

identify S. aureus

Several latex agglutination test kits have been devel-

oped to identify S. aureus based on the detection of

clumping factor, and, or protein A. These tests are

discussed in 7.18.1.

7.5.5 DNA-ase test

This test is used to help in the identiﬁcation of

S. aureus which produces deoxyribonuclease (DNA-

ase) enzymes. The DNA-ase test is particularly useful

when plasma is not available to perform a coagulase

test or when the results of a coagulase test are

difﬁcult to interpret.

Principle

Deoxyribonuclease hydrolyzes deoxyribonucleic acid (DNA).

The test organism is cultured on a medium which contains

DNA. After overnight incubation, the colonies are tested

for DNA-ase production by ﬂooding the plate with a weak

hydrochloric acid solution. The acid precipitates unhydro-

lyzed DNA. DNA-ase-producing colonies are therefore

surrounded by clear areas due to DNA hydrolysis.

Required

— DNA-ase agar plate No. 33

Up to six organisms may be tested on the

same plate.

— Hydrochloric acid Reagent No. 43

1 mol/1 (1 N)

Method

1 Dividea DNA-ase plate into therequired number

of strips by marking the underside of the plate.

2 Using a sterile loop or swab, spot-inoculate the

test and control organisms. Make sure each test

area is labelled clearly.

3 Incubate the plate at 35–37C overnight.

4 Cover the surface of the plate with 1 mol/l

hydrochloric acid solution. Tip off the excess

acid.

5 Look for clearing around the colonies within 5

minutesof adding the acid, asshown in Plate 7.10.

Results

Clearing around the colonies . . . . . . . . . . DNA-ase

positive strain

MICROBIOLOGICAL TESTS 67

7.5.5–7.5.6

No clearing around the colonies . . . . . . . . DNA-ase

negative strain

Note: Some methicillin resistant S. aureus (MRSA)

strains give a negative DNA-ase test. Some coagu-

lase negative staphylococci are weakly positive.

Also, S. pyogenes, Moraxella and Serratia species

frequently give a positive DNA-ase test.

Controls

Positive DNA-ase control: Staphylococcus aureus

Negative DNA-ase control: Staphylococcus

epidermidis

7.5.6 Indole test

Testing for indole production is important in the

identiﬁcation of enterobacteria. Most strains of

E. coli, P. vulgaris, P. rettgeri, M. morganii, and

Providencia species break down the amino acid

tryptophan with the release of indole.

Principle

The test organism is cultured in a medium which contains

tryptophan. Indole production is detected by Kovac’s or

Ehrlich’s reagent which contains 4 (p)-dimethylamino-

benzaldehyde. This reacts with the indole to produce a red

coloured compound. Kovac’s reagent is recommended in

preference to Ehrlich’s reagent for the detection of indole

from enterobacteria.

Plate 7.10 Deoxyribonuclease (DNA-ase) test. Numbers

493, 465, and 475 are positive tests indicating that the

organisms are S.aureus. A positive S. aureus control strain is

shown in top left section. Courtesy of RW Davies.

Ways of performing an indole test

An indole test can be performed:

● As a single test using tryptone water and Kovac’s

reagent.

● As a combined beta-glucuronidase-indole test

using a Rosco PGUA/Indole identiﬁcation tablet

and Kovac’s reagent. This is useful when identi-

fying E. coli.

● As a combined lysine decarboxylase-indole test

using a Rosco LDC/Indole identiﬁcation tablet.

This is useful in helping to identify salmonellae

and shigellae (see Chart 7.8 in subunit 7.11).

Detecting indole using tryptone water

1 Inoculate the test organism in a bijou bottle con-

taining 3 ml of sterile tryptone water.

2 Incubate at 35–37C for up to 48 h.

3 Test for indole by adding 0.5 ml of Kovac’s

reagent. Shake gently. Examine for a red colour

in the surface layer within 10 minutes.

Kovac’s indole reagent: This can be economically pur-

chased as a ready-made reagent from Merck (see

Appendix 11). It is available in a 100 ml bottle, code

1.09293.0100 or in a 30 ml dropper bottle, code

1.11350.0001.It has a longshelf-life when stored at 4–8 C.

Results

Red surface layer . . . . . . . . . . . . Positive indole test

No red surface layer . . . . . . . . Ne gative indole test

Note:A positive indole testis shown in colourPlate 2.

Detecting indole using Rosco PGUA/Indole

tablet

PGUA/Indole tablets (code 59011) are available

from ROSCO Diagnostica (see Appendix 11), in

a vial of 50 tablets. They have a long shelf life

(3–4 y).

1 Prepare a dense suspension of the test organism

in 0.25 ml physiological saline in a small tube.

2 Add a PGUA/Indole tablet and close the

tube. Incubate at 35–37C for 3–4 hours (or

overnight).

3 First read the beta-glucuronidase (PGUA)

reaction:

Results

Yellow colour . . . . . . . . . . . . Positive PGUA test

Colourless . . . . . . . . . . . . . Negative PGUA test

4 Add 3 drops of Kovac’s reagent (Rosco code

920–31 or other Kovac’s reagent) and shake.

5 Wait 3 minutes before reading the indole

reaction. Examine the colour of the surface layer.

68 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.5.6–7.5.7

Results

Red surface layer . . . . . . . . . Positive indole test

Yellow surface layer . . . . . . Negative indole test

Note: About 94% of E. coli strains are PGUA

positive and 99% are indole positive.

Detecting indole using Rosco LDC/Indole

tablet

LDC/Indole tablets (code 58411) are available from

Rosco Diagnostica (see Appendix 11) in a vial of

50 tablets. They have a long shelf-life (3–4 y).

1 Prepare a dense suspension of the test

organism in 0.25 ml physiological saline in a

small tube.

2 Add an LDC/ Indole tablet. Add 3 drops of

parafﬁn oil* and close the tube.

*The oil overlayer provides the anaerobic conditions

required for the LDC reaction.

3 Incubate at 35–37C for 3–4 hours (or

overnight).

4 First read the lysine decarboxylase (LDC)

reaction:

Results

Blue/violet colour* . . . . . . . . . . Positive LDC test

Yellow,green or grey colour .. Negative LDC test

*If examining after overnight incubation, a positive test is

indicated by a strongblue or violet colour.

5 Add 3 drops of Kovac’s reagent (Rosco code

920–31 or other Kovac’s reagent) and shake.

6 Wait 3 minutes before reading the indole

reaction. Examine the colour of the surface layer.

Results

Red surface layer . . . . . . . . . Positive indole test

Yellow surface layer . . . . . . Negative indole test

7.5.7 Litmus milk

decolorization test

This test is a rapid inexpensive technique to assist in

the identiﬁcation of enterococci. It is based on the

ability of most strains of Enterococcus species to

reduce litmus milk by enzyme action as shown by

decolorization of the litmus.

Note: Enterococci can also be identiﬁed using an

aesculin hydrolysis test (see later text).

Principle

A heavy inoculum of the test organism is incubated for up to

4 hours in a tube containing litmus milk. Reduction of the

litmus milk is indicated by a change in colour of the medium

from mauve to white or pale yellow.

Required

Litmus milk medium No. 50

Method

1 Using a sterile loop, inoculate 0.5 ml of sterile

litmus milk medium with the test organism.

Important: A heavy inoculum of the test

organism must be used. Scraping the loop

three times across an area of heavy growth is

recommended.

2 Incubate at 35–37C for up to 4 hours, examin-

ing at half hour intervals for a reduction reaction

as shown by a change in colour from mauve to

white or pale yellow (compare with the positive

control.) The reaction is shown in colour Plate 5.

Note: The incubation time should not be more

than 4 hours because some strains of viridans

streptococci will reduce litmus milk with pro-

longed incubation.

Results

White or pale yellow-pink colour. . . . Suggestive of

Enterococcus

No change or a pink colour . . . . . . . . Probably not

Enterococcus

Controls

Positive control: Enterococcus species

Negative control: Viridans streptococci

Note: The work of Schierl and Blazevic* demonstrated that

up to 83% of Enterococcus could be identiﬁed by the rapid

litmus milk reduction test. No false positive reactions were

observed. A negative result can be checked by culturing the

organism in aesculin broth and examining daily for up to 7

days for aesculin hydrolysis as shown by a blackening in the

medium, Enterococci hydrolyze aesculin.

*Schierl E. A., Blazevic D. J. Rapid identiﬁcation of entero-

cocci by reduction of litmus milk. Journal of Clinical

Microbiology, 14, 2, pp. 227–228, 1981.

Aesculin hydrolysis test to identify enterococci

This test can be economically performed using a

Rosco bile aesculin tablet (Bile esculin 404–21). The

tablets are available from Rosco Diagnostica (see

Appendix 11) in a vial of 25 tablets. They have good

stability (3–4 y).

MICROBIOLOGICAL TESTS 69

7.5.7–7.5.8

The test can be performed by placing a tablet on

a blood agar plate inoculated with the test organism

and incubating it at 35–37C overnight. A positive

test is indicated by the tablet and colonies around it

turning black/grey. A negative test is shown by the

tablet remaining white and no change in colour of

the colonies. A zone of inhibition may appear

around the tablet.

Alternatively, the test can be performed by

making a dense suspension of the test organism in

0.25 ml of physiological saline in a small tube,

adding a tablet, and incubating at 35–37C for 4

hours (or overnight). A positive reaction is shown by

a black/grey colour in the medium.

Note: An aesculin hydrolysis can also be performed

by incubating the test organism on bile aesculin agar

but this medium is expensive.

7.5.8 Oxidase test*

*Cytochrome oxidase test

The oxidase test is used to assist in the identiﬁcation

of Pseudomonas, Neisseria, Vibrio, Brucella, and

Pasteurella species, all of which produce the enzyme

cytochrome oxidase.

Principle

A piece of ﬁlter paper is soaked with a few drops of oxidase

reagent. A colony of the test organism is then smeared on the

ﬁlter paper. Alternatively an oxidase reagent strip can be used

(see later text). When the organism is oxidase-producing, the

phenylenediamine in the reagent will be oxidized to a deep

purple colour.

Occasionally the test is performed by ﬂooding the culture

plate with oxidase reagent but this technique is not rec-

ommended for routine use because the reagent rapidly kills

bacteria. It can however be useful when attempting to isolate

N.gonorrhoeae colonies from mixed cultures in the absence of

a selective medium. The oxidase positive colonies must be

removed and subcultured within 30 seconds of ﬂooding the

plate.

Important: Acidity inhibits oxidase enzyme activity,

therefore theoxidase test must not be performed on

coloniesthat produce fermentation on carbohydrate-

containing media suchas TCBS or MacConkey agar.

Subinoculation on nutrient agar is required before

the oxidase test c an be performed. Colonies tested

from a medium that contains nitrate may give unre-

liable oxidase test results.

Required

Oxidase reagent freshly Reagent No. 64

prepared or use an oxidase

reagent strip (see below)

Note: Fresh oxidase reagent is easily oxidized. When

oxidized it appears blue and must not be used.

Stable oxidase reagent strips

These can be purchased from Merck (see Appendix 11) in a

pack of 50 strips (code 1.13300.0001). The strips have a 5 year

shelf-life when stored at 2–8C.

Method (fresh reagent)

1 Place a piece of ﬁlter paper in a clean petri dish

and add 2 or 3 drops of freshly prepared oxidase

reagent.

2 Using a pie ce of stick or glass rod (not an

oxidized wire loop), remove a colony of the test

organism and smear it on the ﬁlter paper.

3 Look for the development of a blue-purple

colour within a few seconds as shown in colour

Plate 3.

Results

Blue-purple colour . . . . . . . . . . Positive oxidase test

(within 10 seconds)

No blue-purple colour . . . . . Negative oxidase test

(within 10 seconds)

Note: Ignore any blue-purple colour that develops

after 10 seconds.

Method using an oxidase reagent strip

1 Moisten the strip with a drop of sterile water.

2 Using a piece of stick or glass rod (not an oxidized wire

loop) remove a colony of the test organism and rub it on

the strip.

3 Look for a red-purple colour within 20 seconds.

Red-purple colour . . . . . . . . positive oxidase test.

Note: When using a Merck reagent strip, follow exactly the

manufacturer’s instructions on how to perform the test.

Controls

Positive oxidase control: Pseudomonas aeruginosa

Negative oxidase control: Escherichia coli

7.5.9 Urease test

Testing for urease enzyme activity is important in

differentiating enterobacteria. Proteus strains are

70 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.5.8–7.5.9

strong urease producers. Y. enterocoliticaalso shows

urease activity (weakly at 35–37C). Salmonellae

and shigellae do not produce urease.

Principle

The test organism is cultured in a medium which contains urea

and the indicator phenol red. When the strain is urease-

producing, the enzyme will break down the urea (by hydroly-

sis) to give ammonia and carbon dioxide. With the release of

ammonia, the medium becomes alkaline as shown by a change

in colour of the indicator to pink-red.

Ways of performing a urease test

● Using a Rosco urease identiﬁcation tablet.

● Using modiﬁed Christensen’s urea broth.

Urease test using a Rosco urease tablet

Urease identiﬁcation tablets are available from Rosco

Diagnostica (code 57511) in a vial of 50 tablets. The

tablets have a long shelf-life (3–4 y).

1 Prepare a dense ‘milky’ suspension of the test

organism in 0.25 ml physiological saline in a

small tube.

2 Add a urease tablet, close the tube and incubate

at 35–37C (preferably in a water bath for a

quicker result) for up to 4 hours or overnight.

Proteus and M. morganiiorganism give a positive

reaction within 4 hours.

Results

Red/purple colour . . . . . . . . Positive urease test

Yellow/orange . . . . . . . . . . Negative urease test

Urease test using Christensen’s (modiﬁed)

urea broth

1 Inoculate heavily the test organism in a bijou

bottle containing 3 ml sterile Christensen’s

modiﬁed urea broth (for preparation, see

Appendix 1).

2 Incubate at 35–37C for 3–12 h (preferably in a

water bath for a quicker result).

3 Look for a pink colour in the medium as shown

in colour Plate 4.

Results

Pink colour . . . . . . . . . . . . . . Positive urease test

No pink colour . . . . . . . . . Negative urease test

7.6 Examination of sputum

Possible pathogens

 BACTERIA

Gram positive Gram negative

Streptococcus pneumoniae Haemophilus inﬂuenzae

Staphylococcus aureus Klebsiella pneumoniae

Streptococcus pyogenes Pseudomonas aeruginosa

Proteus species

Yersina pestis

Moraxella catarrhalis

Also Mycobacterium tuberculosis, Mycoplasma

pneumoniae, and Legionella pneumophila.

 FUNGI AND ACTINOMYCETES

Pneumocystis jiroveci, Blastomyces dermatitidis,

Histoplasma capsulatum, Aspergillus species, Candida

albicans, Cryptococcus neoformans, and Nocardia

species.

 PARASITES

Paragonimus species (describe d in subunit 5.13 in

Part 1 of the book).

Notes on pathogens

● Infection of the lungs with M. tuberculosis causes pul-

monary tuberculosis. In developing countries tuberculosis

(pulmonary and disseminated) is increasing in incidence

and becoming more difﬁcult to control (see also subunit

7.18.28). Poverty, the HIV pandemic, movement of dis-

placed people, and emergence of multidrug-resistant

strains of M. tuberculosis are major factors contributing

to the spread of tuberculosis. Susceptibility to M. tuber-

culosis and other mycobacteria is increased with HIV

infection and tuberculosis also progresses more rapidly in

those with HIV disease. Tuberculosis is the commonest

HIV-related disease in developing countries, causing

about 30% of deaths in AIDS patients. The development

of tuberculosis in HIV co-infected persons accelerates

progression to full-blown AIDS.

Tuberculosis is difﬁcult to diagnose by sputum exam-

ination in those co-infected with HIV when there is

marked immunosuppression resulting in diffuse inﬁltra-

tion without cavitation. When immune responses are less

suppressed, typical pulmonary caseating granulomas and

cavities form, and AFB can usually be detected in sputum.

● S. pneumoniae and H. inﬂuenzae are the commonest

causes of acute respiratory tract infections in tropical

countries. S. pneumoniae causes lobar pneumonia and

bronchopneumonia in young children (especially when

malnourished), in those co-infected with HIV (major

HIV-related pathogen), the elderly, the bed-ridden and

other debilitated persons.

● S. aureus, S. pyogenes, and H. inﬂuenzae are often sec-

ondary invaders in patients with inﬂuenza virus

pneumonia. H. inﬂuenzae is associated with acute and

chronic bronchitis and chest infections in post-surgical

MICROBIOLOGICAL TESTS 71

7.6

patients and the elderly. S.aureus can produce a severe

pneumonia (with a tendency to form abscesses),

especially in children and following inﬂuenza.

● P. aeruginosa is more commonly found in patients with

chronic lung cavities or as a complication of treatment

with immunosuppressive drugs.

● K. pneumoniae may be found with E. coli and yeasts as a

complication of antibiotic therapy.

● Moraxella catarrhalis (formerly Branhamella catarrhalis)

can cause upper and lower respiratory tract infections,

mostly in adults with pre-existing respiratory disease and

those with immunodeﬁciency.

● Y.pestis (highly infectious) can be found in the sputum of

patients with pneumonic plague. The specimen may

contain blood.

● M. pneumoniae causes primary atypical pneumonia.

● L. pneumophila causes Legionnaire’s disease, a severe

and often fatal form of pneumonia.

● P. jiroveci is an opportunistic fungal pathogen, causing

pneumonia in those with immunosuppression. It is a

common pathogen in HIV infected young children in

developing countries (see subunit 7.18.55).

Note: Several of the other fungi and actinomycetes listed

are also associated with disease in those with immuno-

suppression.

● Eosinophils can be found in the sputum of patients with

allergic respiratory conditions such as asthma.

Commensals

Sputum as it is being collected passes through the

pharynx and the mouth. It therefore becomes con-

taminated with small numbers of commensal

organisms from the upper respiratory tract and

mouth. These include:

Gram positive Gram negative

Staphylococcus aureus Neisseria spe cies

Staphylococcus Moraxella catarrhalis

epidermidis (S. albus) Haemophilus inﬂuenzae

Viridans streptococci Fusobacteria

Streptococcus pneumoniae Coliforms

Enterococci

Micrococci

Lactobacilli

Diphtheroids

Yeast-like fungi

COLLECTION AND TRANSPORT OF S PUTUM

Sputum for microbiological investigation is collected

and transported as follows:

In a hospital with a microbiology laboratory

1 Give the patient a clean (need not be sterile), dry,

wide-necked, leak-proof container, and request

him or her to cough deeply to produce a sputum

specimen.

Caution: When a sputum specimen is being

collected, adequate safety precautions must

be taken to prevent the spread of infectious

organisms and to avoid contaminating the

outside of the container. Use a phenol-containing

disinfectant to wipe the outside of the container

after collecting the specimen.

Important: The specimen must be sputum, not

saliva. Sputum is best collected in the morning

soon after the patient wakes and before any

mouth-wash is used. When pulmonary tubercu-

losis is suspected, up to three specimens may

need to be examined to detect AFB.

Mucopus aspirated from the nasopharynx

When it is not possible to obtain sputum from children

with suspected pneumonia or bronchopneumonia,

pathogens can often be isolated from mucopus aspirated

from the nasopharynx.

2 Lab el the container, and complete a request

form as described in subunit 7.1.

3 When pneumonia or bronchopneumonia is sus-

pected, deliver the sputum to the laboratory with

as little delay as possible because organisms such

as S. pneumoniae and H. inﬂuenzae require

culturing as soon as possible.

Note: Specimens for the isolation of S. pneu-

moniae and H. inﬂuenzae must never be

refrigerated.

When Pneumocystis pneumonia is suspected: Collect

broncho-alveolar lavage (BAL) or induced sputum as

described in subunit 7.18.52.

When pneumonic plague is suspected: Deliver the

sputum to the laboratory as soon as possible. Make

sure the specimen is marked HIGH RISK.

In a health centre for dispatch to a

microbiology laboratory

1 Collect the sputum in a container supplied by the

microbiology laboratory (see previous text).

Follow the technique and observe the precau-

tions mentioned under the hospital collection of

sputum.

2 To ensure the survival of pathogens such as

S. pneumoniae and H. inﬂuenzae, transfer a

purulent part of the sputum to a cotton-wool

swab, and insert it in a container of Amies trans-

port medium (see No. 11). Label the container

using a lead pencil.

Amies medium will help the pathogens to survive and

avoid the overgrowth of fast-multiplying commensals.

3 Send the sputum specimen and swab with a

72 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.6

request form to reach the microbiology

laboratory within 6 hours. Instructions regarding

the packaging of specimens can be found at the

end of subunit 7.1.

LABORATORY EXAMINATION

OF SPUTUM

Specimens for immediate attention: When

pneumonia or bronchopneumonia is suspected, the

sputum must be cultured as soon as possible

because organisms such as H. inﬂuenzae and

S. pneumoniae do not survive well in specimens.

Caution: Whenever possible, sputum specimens

should be examined in a biological safety cabinet

(described in subunit 3.3 in Part 1 of the book).

1 Describ e the appearance of the specimen

Describe whether the sputum is:

Purulent: Green-looking, mostly pus

Mucopurulent: Green-looking with pus and mucus

Mucoid: Mostly mucus

Mucosalivary: Mucus with a small amount of saliva

When the sputum contains blood, this must also be

reported.

Unsuitable sputum specimens: When the sputum is mostly

saliva, report the specimen as ‘Unsuitable for microbiological

investigation’ and request another specimen.

Note: Before culturing sputum, many laboratories examine a

wet preparation or Field’s stained smear microscopically for

cells. When large numbers of squamous epithelial cells (often

covered with bacteria) are present and only a few or no pus or

macrophage cells, this indicates that the specimen is unsuit-

able for culturing.

2 Examine the spe cimen microscopically

Gram smear

Using a piece of stick, transfer a purulent part of the

sputum to a glass slide, and make a thin smear.

Allow the smear to air-dry in a safe place. Fix as

described in subunit 7.3.2, and stain by the Gram

technique (see subunit 7.3.4).

Examine the smear for pus cells and predominant

bacteria. Look especially among the pus cells for:

● Gram positive diplococci (capsulated) that could

be S. pneumoniae (se e colour Plate 28). The

Day 1

organisms, however, are not often seen and can

be difﬁcult to differentiate from the normal

microbial ﬂora.

● Gram positive cocci in groups that could be

S. aureus (see colour Plate 24), but not often

seen.

● Gram negative rods and cocco-bacilli that

could be H. inﬂuenza (see colour Plate 48),

particularly when these are the predominant

organisms.

● Gram negative capsulated rods that could be

K. pneumoniae, but not often seen.

● Gram negative diplococci in and between pus

cells that could be M. catarrhalis (see Notes on

Pathogen).

Gram stained smears of sputum must be reported

with caution. Cocci, diplococci, streptococci, and rods

may be seen in normal sputum because these

organisms form part of the normal microbial ﬂora of

the upper respiratory tract.

Note: When pus cells are present but no bacteria are

seen in a Gram stained smear, this may indicate the

presence of microorganisms such as M. tuberculosis,

Chlamydophila pneumoniae, Mycoplasma pneumo-

niae, Legionella pneumophilia or viruses.

Ziehl-Neelsen smear to detect AFB

Studies have shown that the chances of detecting

AFB in sputum smears are signiﬁcantly increased

when sputum is ﬁrst treated with 5% v\v sodium

hypochlorite (NaOC1), i.e. bleach, followed by cen-

trifugation or overnight sedimentation (see subunit

7.18.28). Because NaOC1 kills M. tuberculosis, the

NaOC1 concentration technique is also safer for lab-

oratory staff. NaOC1 treated sputum cannot be

used for culture.

Sodium hypochlorite centrifugation technique to

concentrate AFB

1 Transfer 1–2 ml of sputum (particularly that

which contains any yellow caseous material) to a

screw-cap Universal b ottle or other container of

15–20 ml capacity.

Caution: Open specimen containers with care

and at arms length to avoid inhaling infectious

aerosols. When available, handle the specimen

inside a safety cabinet.

2 Add an equal volume of concentrated sodium

hypochlorite (bleach) solution and mix well.

Sodium hypochlorite (NaOC1) solution: This is widely

available as bleach for domestic and laundering purposes,

MICROBIOLOGICAL TESTS 73

7.6

e.g. Domestos, Jik, Presept, Chloros, eau de Javel and

other trade names. These domestic bleach solutions gen-

erally contain about 5% available chlorine and should be

used undiluted in the NaOC1 technique to concentrate

AFB.

Caution: Bleach is corrosive and toxic when ingested or

inhaled. It also has an irritating vapour and therefore it

should be used in a well-ventilated place. Store it out of

direct sunlight in a cool place, away from acids,methanol

and oxidizing chemicals. When in contact with acids,

sodium hypochlorite liberates toxic gas.

3 Leave at room temperature for 10–15 minutes,

shaking at intervals to break down the mucus in

the sputum.

4 Add ab out 8 ml of distilled water, or when

unavailable use boiled ﬁltered rain water. Mix

well.

5 Centrifuge at 3000 g for 15 minutes or at

250–1000 g for 20 minutes.

Note: When a centrifuge is not equipped to take

Universal containers, divide the specimen

between two conical tubes (which can be

capped).

When centrifugation is not possible, leave the

NaOC1 treated sputum to sediment overnight

(see following text).

6 Using a glass Pasteur pipette or plastic bulb

pipette, remove and discard the supernatant

ﬂuid. Mix the sediment. When two tubes

have been used, combine the two sediments.

Transfer a drop of the well-mixed sediment to a

clean scratch-free glass slide. Spread the

sediment to make a thin preparation and allow to

air-dry.

7 Heat-ﬁx the smear and stain it using the Ziehl-

Neelsen technique (see subunit 7.3.5). Examine

it microscopically for AFB. M. tuberculosis in

Ziehl-Neelsen stained sputum smears is shown

in colour Plates 56 and 57.

Note: Up to three specimens may need to be

examined to detect M. tuberculosis in sputum. One

specimen should be collected as an early morning

sputum (see previous text).

Concentration of AFB in NaOC1 treated sputum following

overnight sedimentation

When a centrifuge is not available or there is no mains

electricity, a study in Ethiopia* has shown that overnight

sedimentation of NaOC1 treated sputum can also increase the

sensitivity of smear examination. In the study, 8.5% of

patients were AFB positive by direct smear examination,

25.5% following overnight sedimentation of NaOC1

treated sputum, and 38% when the treated sputa were

centrifuged.

*Gebre-Selassie, S. Evaluation of the concentration sputum

smear technique for the laboratory diagnosis of pulmonary

tuberculosis. Tropical Doctor, 33, July, 2003.

ADDITIONAL

Saline preparation when paragonimiasis is

suspected

Transfer a small amount of sputum, especially that

which is brown-coloured and stringy, to a slide. Add

a drop of physiological saline, mix, and cover with a

cover glass.

Using the 10objective with the condenser iris

diaphragm closed sufﬁciently to give good contrast,

examine the preparation for Paragonimus eggs.

Paragonimus eggs are described and illustrate d in

subunit 5.13 in Part 1 of the book.

Note:Clinical and laboratory features that help to differenti-

ate pulmonary tuberculosis from paragonimiasis are discussed

in a WHO document: Paragonimiasis and tuberculosis–

Diagnostic confusion, 1994, WHO/TUB/94.181 and

WHO/HPE/94.3, available from WHO, 1211 Geneva,

Switzerland–27.

Eosin preparation when an allergic condition

requires investigation

Transfer a small amount of sputum to a slide. Add a

small drop of alkaline eosin solution (Reagent No.

35), mix, and cover with a cover glass. Using the

10 and 40 objectives with the condenser iris

closed sufﬁciently to give good contrast, examine the

preparation for eosinophils*.

*Eosinophils can be easily differentiated from pus cells

because they contain bright red-staining granules and a bi-

lobed nucleus. Free eosinophilic granules may be seen in the

preparation and occasionally elongated refractile Charcot

Leyden crystals (formed from the material of dead

eosinophils).

Large numbers of eosinophils in sputum can also be found

with paragonimiasis.

Potassium hydroxide (KOH) preparation

when Aspergillus infection is suspected

Transfer a small amount of sputum to a glass slide.

Add a drop of potassium hydroxide solution

(Reagent No. 69), mix, and cover with a cover

glass.

Examine the preparation for fungi using the 10

and 40 objectives with the condenser iris

diaphragm closed sufﬁciently to give good

contrast:

Aspergillus: The appearance of fungal hyphae

typical of Aspergillus species is described in subunit

7.18.49. A Gram stained preparation is shown in

colour Plate 70.

74 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.6

Giemsa and toluidine blue 0 preparations

when Pneumocystis infection is suspected

(AIDS patients)

Making smears from broncho-alveolar lavage (BAL)

or induced sputum and the examination of stained

smears for P. jirovecicysts and intracystic b odies are

described in subunit 7.18.52.

Giemsa stained preparation when

histoplasmosis is suspected

Examination of Giemsa stained smears for intracel-

lular Histoplasma yeast cells is describe d in subunit

7.18.43.

Giemsa or Wayson stained preparation

when pneumonic plague is suspected

Fix the sputum smear with methanol for 5 minutes.

Stain using Giemsa technique (see subunit 7.3.10)

or rapid Wayson’s technique (see 7.3.8). Bipolar

staining of Y. pestis is described in subunit 7.18.22

and shown in colour Plate 54.

Caution: Y. perstisis highly infectious (Hazard Group

3 pathogen), therefore handle specimens with great

care. Laboratory-acquired infections can occur fol-

lowing accidental inoculation or inhalation of the

organisms. Minimize procedures that create aerosols

and whenever possible carry out procedures in a

safety cabinet (see pp. 61–65 in Part 1 of the book).

3 Culture the spe cimen

To obtain as pure a culture as possible of a respira-

tory pathogen it is necessary to reduce the number

of commensals inoculated. Ways of reducing com-

mensal numbers include washing the sputum free

from saliva or liquefying and diluting it. The tech-

nique using saline-washed sputum is described. The

dilution technique requires a liquefying agent such

as dithiothreitol (Sputolysin, Sputasol) which is

expensive and unstable.

Blood agar and chocolate agar

– Wash a purulent part of the sputum in about

5 ml of sterile physiological saline.

– Inoculate the washed sputum on plates of:

● Blood agar, see No. 16

● Chocolate (heated blood) agar, see No. 16

Inoculation technique to reduce commensal numbers:

Using the technique described in subunit 7.4 (to inoculate

a whole plate of agar), ﬂame the loop in between each

spread. This will help to obtain a pure growth of the

pathogen in the areas of the 3rd and 4th spread.

– Add an optochin disc to the blood agar plate

within the area of 2nd spread. This will help to

identify S. pneumoniae (see subunit 7.18.4).

MICROBIOLOGICAL TESTS 75

7.6

Summary of Microbiological Examination of Sputum

ADDITIONAL INVESTIGATIONS

1 Describe  Report whether specimen:

Specimen

– purulent, mucopurulent,

mucoid, salivary

– contains blood

2 Examine  Gram smear: For pus  Giemsa smear:When pneumonic

Microscopically

cells and bacteria plague or histoplasmosis is

suspected

 Zn smear: For AFB  KOH preparation: When

Aspergillusinfection is suspected

 Toluidine blue-O and Giemsa

smears: When Pneumocystis

pneumonia is suspected

 Eosin preparation: When an

allergic condition requires

investigation

 Saline preparation: When

paragonimiasis is suspected

3 Culture  Blood agar  Culture for M. tuberculosis

Specimen

– Add an optochin disc (In Reference Laboratory)

– Incubate aerobically See text

 Chocolate agar

– Incubate in CO

Day 1

4 Examine and  Blood and chocolate  Test H. inﬂuenzae for beta-

Report Cultures

agar cultures lactamase production

Report signiﬁcant growth of:

 Antimicrobial susceptibility tests

S.pneumoniae as required

H.inﬂuenzae

S.aureus

Less commonly found

pathogens:

K.pneumoniae, P. aeruginosa,

M.catarrhalis, S. pyogenes,

Proteus,C. albicans

Key: Zn  Ziehl-Neelsen, KOH  Potassium hydroxide, CO

 Carbon dioxide

Day 2 and Onwards

Availability: Optochin discs can be purchased economi-

callyfrom Mast Diagnostics (see Appendix 11) in vials of

100discs,code D-42. Theyhave a shelf-lifeof about2 years.

– Incubate the blood agar plate aerobically and the

chocolate agar plate in a carbon dioxide

enriched atmosphere (see subunit 7.4).

ADDITIONAL

Culture of sputum for M. tuberculosis

Culturing and susceptibility testing of M. tuberculosis

are usually undertaken in a Tuberculosis Reference

Laboratory, mainly for surveillance purposes, to

determine levels of drug resistance, and to manage

treatment failures and relapses (see also subunit

7.18.28).

Culture of sputum when pneumonic plague

is suspected

Isolation of Y. pestisis describe d in subunit 7.18.22.

4 Examine and report the cultures

Blood agar and chocolate agar cultures

Look especially for a signiﬁcant growth of:

 Streptococcus pneumoniae sensitive to optochin,

see subunit 7.18.4

 Haemophilus inﬂuenzae, see subunit 7.18.24

 Staphylococcus aureus, see subunit 7.18.1

Less frequently isolated pathogens

Klebsiella pneumoniae, see subunit 7.18.17

Pseudomonas aeruginosa, see subunit 7.18.20

Moraxella catarrhalis, see end of subunit 7.18.24

Streptococcus pyogenes, see subunit 7.18.2

Proteus species, see subunit 7.18.18

Candida albicans, see subunit 7.18.47

Antimicrobial susceptibility testing

Susceptibility tests should be performed only when

the amount of cultural growth of a pathogen is sig-

niﬁcant. Strains of S. pneumoniae should be tested

on blood agar for susceptibility to penicillin, tetracy-

cline, and erythromycin. Penicillin susceptibility is

best determined using an oxacillin 1 g disc. A zone

size less than 20 mm indicates reduced susceptibil-

ity. H. inﬂuenzae strains should be tested for beta-

lactamase production (see end of subunit 7.16) and

susceptibility to ampicillin, tetracycline, and co-tri-

moxazole. Susceptibility testing of S. aureus strains is

described in subunit 7.16.

Day 2 and Onwards

76 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.6–7.7

7.7 Examination of throat

and mouth specimens

Possible pathogens

 BACTERIA

Gram positive Gram negative

Streptococcus pyogenes Vincent’s organisms

Corynebacterium

diphtheriae

Corynebacterium ulcerans

 VIRUSES

Respiratoryviruses, enteroviruses andherpes simplex

virus type 1.

 FUNGI

Candida albicans and other yeasts.

Note: Pathogens in the upper respiratory tract such

as Bordetella pertussis, Streptococcus pneumoniae,

and Neisseria meningitidis, are usually more success-

fully isolated from naso-pharyngeal secretions

collected by aspiration.

Notes on pathogens

● S. pyogenes, Lanceﬁeld Group A beta-haemolytic

Streptococcusis the commonest cause of bacterial pharyn-

gitis (sore throat), especially in young children. Its

association with rheumatic heart disease and importance

in developing countries are discussed in subunit 7.18.2.

The term scarlet fever is used when streptococcal pharyn-

gitis is accompanied by a characteristic skin rash.

● C. diphtheriae produces a powerful and often fatal

exotoxin and therefore when diphtheria is suspected, the

patient is treated immediately with antitoxin. The role of

the laboratory is to conﬁrm the clinical diagnosis.

● C. albicans infection of the mouth (oral thrush) is

common in those with HIV disease. It may also affect

young children, those who have been treated with anti-

biotics over a long period, and occasionally diabetic

patients and those with other systemic diseases.

● Infection with Vincent’s organisms (Borrelia vincenti in

association with Gram negative anaerobic fusiform

bacilli) causes Vincent’s angina (Vincent’s gingivitis), an

ulcerative tonsilitis with tissue necrosis.

Commensals

Gram positive Gram negative

Viridans streptococci Moraxella catarrhalis

Non-haemolytic streptococci Neisseria pharyngitidis

Fusobacteria

Streptococcus pneumoniae Coliforms

Staphylococcus epidermidis Bacteroides species

Micrococci Haemophilus inﬂuenzae

Lactobacilli (mostly non-capsulate

Diphtheroids strains)

Also various spirochaetes, actinomycetes, aerobic

and anaerobic spore-bearers, and yeasts.

COLLECTION AND TRANSPORT OF TH ROAT AND

MOUTH SWABS

Whenever possible throat and mouth swabs should

be collected by a medical ofﬁcer or experienced

nurse.

In a hospital with a microbiology laboratory

1 In a good light and using the handle of a spoon

to depress the tongue, examine the inside of the

mouth. Look for inﬂammation, and the presence

of any membrane, exudate, or pus.

– With diphtheria, a greyish-yellow membrane (later

becoming greyish green-black and smelly) can often

be seen extending forwards over the soft palate and

backwards onto the pharyngeal wall.

– With a streptococcal sore throat, the tonsils are

inﬂamed and often covered in yellow spots.

– With C. albicans infection, patches of white exudate

may be seen attached in places to the mucous

membrane of the mouth.

– With Vincent’s angina, there is ulceration of the

mouth, throat, or lips. Viral tonsillitis can also cause

ulceration of the tonsils.

2 Swab the affected area using a sterile cotton-

wool swab. Taking care not to contaminate the

swab with saliva, return it to its sterile container.

Important: For 8 hours before swabbing, the

patient must not be treated with antibiotics or

antiseptic mouth-washes (gargles).

Caution: It can be dangerous to swab the throat

of a child with acute haemophilus epiglottitis

because this may cause a spasm that can

obstruct the child’s airway. Blood for culture

should be collected instead.

3 Within two hours of collection, deliver the swab

with a completed request form to the laboratory.

In a health centre for dispatch to a

microbiology laboratory

1 Using a sterile swab (supplied in a tube of silica

gel by the microbiology laboratory), collect a

specimen from the infected area as described

under the hospital collection of throat swabs.

2 Taking care not to contaminate the swab, return

it to its tube. Seal with adhesive tape and label

the tube.

MICROBIOLOGICAL TESTS 77

7.7

3 Send the swab with a completed request form to

reach the microbiology laboratory within 3 days.

Instructions regarding the packaging and

dispatch of specimens can be found at the end

of subunit 7.1.

Transport of swabs in tubes containing silica gel

It has been shown that S.pyogenes will remain viable for at

least 3 days (at ambient temperatures) on swabs stored in

tubes containing 3–5 g of dessicated silica gel.

Note:Other systems for transporting specimens to be investi-

gated for S.pyogenes are described in the WHO publication

Laboratory diagnosis of group A streptococcal infections.

LABORATORY EXAMINATION OF

THROAT and MOUTH SPECIMENS

1 Culture the spe cimen

Blood agar

– Inoculate the swab on a plate of blood agar (see

No. 16). Use the loop to make also a few stabs in

the agar (well area). Colonies of S. pyogenes

growing below the surface will show more

distinct zones of haemolysis because of the

anaerobic conditions provided.

– When a swab is received in silica gel (e.g. from a

health centre), moisten it ﬁrst with sterile nutrient

broth and then inoculate the plate.

– Add a 0.05 unit bacitracin disc (Reagent No. 15)

to the plate. This will help in the identiﬁcation of

S. pyogenes (see subunit 7.18.2). Some workers

also add a co-trimoxazole disc (as used for

susceptibility testing) which prevents the growth

of other bacteria, making it easier to see beta-

haemolytic S. pyogenes colonies.

– Incubate the plate preferably anaerobically or,

when this is not possible, in a carbon dioxide

enriched atmosphere overnight at 35–37C.

Candle jar incubation will detect most beta-

haemolytic streptococci.

Note: Beta-haemolytic streptococci produce

larger zones of haemolysis when incubated

anaerobically. A minority of Group A

Streptococcus strains will only grow anaerobically.

Immunological detection of S.pyogenes antigen in specimens

Immunochromatographic tests, dip sticks and other simple to

perform technologies have been developed to detect

S. pyogenes antigen directly in specimens. These tests are

described in subunit 7.18.2.

Day 1

ADDITIONAL

Culture of specimen when diphtheria is

suspected

When diphtheria is suspected and culture is speciﬁ-

cally requested, inoculate the swab on Tinsdale

medium or tellurite blood agar (see subunit 7.18.7).

Incubate the plate aerobically at 35–37C for up to

48 hours, examining for growth after overnight

incubation.

Note: The isolation of Bordetella pertussis from

nasopharyngeal secretions is described in subunit

7.18.25.

2 Examine the spe cimen microscopically

Gram smear

Make an evenly spread smear of the specimen on a

slide. Allow the smear to air-dry in a safe place. Fix

as described in subunit 7.3.2, and stain by the Gram

technique (see subunit 7.3.4). Use dilute carbol

fuchsin (1 in 10 dilution) as the counterstain in pref-

erence to safranin or neutral red (stains Vincent’s

organisms better).

Examine the smear for pus cells and Vincent’s

organisms:

Vincent’s organisms: These are seen as Gram

negative spirochaetes (B. vincenti) and Gram

negative fusiform rods as shown in colour Plate 65.

Other bacteria: No attempt should be made to

report routinely other bacteria in a Gram stained

smear from a throat swab because the throat

contains a wide variety of commensals that cannot

be distinguished morphologically from pathogens.

When thrush is suspected, look for Gram positive

Candida yeast cells (see colour Plate 72).

ADDITIONAL

Albert stained smear when diphtheria is

suspected

Prepare the smear as described previously under

Gram smear. Fix with alcohol (see subunit 7.3.2) and

stain by the Albert staining technique (see subunit

7.3.9). Examine the smear for bacteria that could be

C. diphtheriae.

Look for pleomorphic rods containing dark-staining

volutin granules as shown in colour Plate 32. The

pleomorphic rods tend to join together at angles

giving the appearance of Chinese letters.

Pleomorphism and granule formation are best seen

in smears from a Loefﬂer serum or Dorset egg

78 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.7

medium culture. Smears directly from specimens

may not show these features.

Volutin granules

Although the presence of volutin granules is characteristic of

C. diphtheriae, especially when the organisms are pleomor-

phic, some virulent strains of the gravisbiovar (biotype) may

contain few or no volutin granules. It is also possible for com-

mensal diphtheroids to contain volutin granules but the

commensals are not pleomorphic like C.diphtheriae.

When C. diphtheriae is cultured on tellurite blood agar

and modiﬁed Tinsdale medium, granule formation is usually

restricted.

Note: In Gram stained smears, C. diphtheriae stains

variably and weakly Gram positive, whereas com-

mensal diphtheroids appear strongly Gram positive.

3 Examine and report the cultures

Blood agar culture

Look for beta-haemolytic colonies that could be

Streptococcus pyogenes (Lanceﬁeld Group A

Streptococcus). Most strains are sensitive to baci-

tracin as shown in colour Plate 26. However,

bacitracin sensitivity cannot be completely relied on

to identify S. pyogenes. The organism should be

tested serologically to conﬁrm that it belongs to

Lanceﬁeld Group A or tested biochemically using

the PYR test (see following text).

Lanceﬁeld grouping of beta-haemolytic streptococci

Beta-haemolytic streptococci are grouped by their speciﬁc cell

wall polysaccharide antigens (C substance) using speciﬁc anti-

serum. S. pyogenes belongs to Group A. Simple to use latex

and co-agglutination slide test kits are available for grouping

beta-haemolytic streptococci. Details of these can be found in

subunit 7.18.2.

PYR test for the presumptive identiﬁcation of S.pyogenes

S. pyogenes produces the enzyme pyrrolidonyl peptidase

which is able to break down the substrate L-pyrrolidonyl-

beta-naphthylamide (PYR). The reaction is detected using an

aminopeptidase reagent. Details of the test and its availability

in a simple to use tablet form can be found in subunit 7.18.2.

Isolation and identiﬁcation of C. diphtheriae

The cultural features of C. diphtheriae on Tinsdale

medium andtellurite blood agar (TBA) are described

in subunit 7.18.7.When colonies suspe cted of being

C. diphtheriae are isolated, identify as follows:

– Examine a Gram stained smear for variable

staining pleomorphic rods as shown in colour

Plate 33.

– Subinoculate two slopes of Dorset egg medium

(see No. 34) or Loefﬂer serum agar (see No. 52).

Incubate at 35–37C for 6 hours or until

sufﬁcient growth is obtained.

Day 2 and Onwards

Examine an Albert stained smear of the subcul-

ture for pleomorphic rods containing volutin

granules (see colour Plate 32). Examine a Gram

stained smear to check that the subculture is a

pure growth.

– Identify the isolate biochemically as described in

subunit 7.18.7.

– Using the growth from the other subculture, test

the strain for toxin production using the Elek pre-

cipitation technique as described in subunit

7.18. 7.

Antimicrobial susceptibility testing

WHO in its publication Basic Laboratory Procedures

MICROBIOLOGICAL TESTS 79

7.7

in Clinical Bacteriology

advises that routine suscep-

tibility tests on throat or pharyngeal isolates are most

often not required, and may even be misleading.

The major pathogens involved in bacterial pharyn-

gitis are S. pyogenes and C. diphtheriae.

Benzylpenicillin and erythromycin are considered as

the antibiotics of choice to treat both types of infec-

tion. In cases of diphtheria, treatment with antitoxin

is also indicated.

REFERENCES

1 Laboratory diagnosis of group A streptococcal infections,

WHO, 1996. ISBN 9241544953. Obtainable from WHO

Publications, 1211 Geneva, 27-Switzerland.

2 Basic laboratory procedures in clinical bacteriology,

WHO, 2nd edition, 2003.

Summary of Microbiological Examination of Throat and Mouth Swabs

ADDITIONAL INVESTIGATIONS

1 Culture  Blood agar  MTM or TBA: When diphtheria

Specimen

– Add a bacitracin disc suspected

– Incubate, preferably

anaerobically (or in CO

)

2 Examine  Gram smear  Giemsa or Wayson’s smear:

Microscopically

Look for: When diphtheria suspected

– Pus cells and Gram

negative Vincent’s organisms

– Gram positive pleomorphic

rods when diphtheria

suspected

– Gram positive yeast cells

when thrush suspected

Day 1

3 Examine and  Blood agar culture  MTM or TBA cultures

Report Cultures

Look for beta-haemolytic Examine for growth of

streptococci, sensitive to C.diphtheriae

bacitracin.

Identify as S. pyogenes

Lanceﬁeld group

PYR test

Key: MTM  Modiﬁed Tinsdale medium, TBA  Tellurite blood agar

Day 2 and Onwards

7.8 Examination of pus, ulcer

material and skin specimens

Note: The collection and examination of effusions, i.e.

synovial, pleural, pericardial and ascitic ﬂuids are described in

subunit 7.9.

PUS

Possible pathogens*

*It is impossible to list all the pathogens that may be found in

pus. Those listed are the more commonly isolated pathogenes

from wounds, abscesses, burns, and draining sinuses.

 BACTERIA

Gram positive Gram negative

Staphylococcus aureus Pseudonomas aeruginosa

Streptococcus pyogenes Proteusspecies

Enterococcus species Escherichia coli

Anaerobic streptococci Bacteriodes spe cies

Other streptococci Klebsiella species

Clostridium perfringens Pasteurella species

and other clostridia

Actinomycetes

Actinomyces israeli

Also Mycobacterium tuberculosis

 FUNGI

Histoplasma c. duboisii, see 7.18.43

Candida albicans, see 7.18.47

Fungi that cause mycetoma are described in

subunit 7.18.41.

 PARASITES

Entamoeba histolytica

(in pus aspirated from an amoebic liver abscess),

see subunit 5.14.1 in Part 1 of the book

Commensals

Any commensal organisms found in pus are usually

those that have contaminated the specimen from

skin, clothing, soil, or from the air if an open wound.

ULCER MATERIALAND S KIN SPECIMENS

Possible pathogens

 BACTERIA

Gram positive Gram negative

Staphylococcus aureus Escherichia coli

Streptococcus pyogenes Proteus

Enterococcus species Pseudomonas aeruginosa

Anaerobic streptococci Yersinia pestis

Erysipelothrix rhusiopathiae Vincent’s organisms

Bacillus anthracis

80 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.8

Also Mycobacterium leprae, Mycobacterium ulcerans,

Treponema carateum, and Treponema pertenue.

Note: Pathogens that may be found in specimens

from genital ulcers are described in subunit 7.10.

 VIRUSES

Poxviruses and herpes viruses

 FUNGI

Dermatophytes (ringworm fungi) see 7.18.38

Malassezia furfur, see 7.18.39

Fungi that cause chromoblastomycosis, see

7.18.40

Candida albicans, see 7.18.47

 PARASITES

Leishmania species*

Onchocerca volvulus*

Dracunculus medinensis*

*These parasites are described in Part 1 of the book.

Commensals

Commensal organisms that may be found on the

skin include:

Gram positive Gram negative

Staphylococci Escherichia coli

Micrococci and other coliforms

Anaerobic cocci

Viridans streptococci

Enterococci

Diphtheroids

Propionibacterium acnes

Notes on pathogens

● S.aureus is the commonest pathogen isolated from subcu-

taneous abscesses and skin wounds. It also causes

impetigo (small pustules that form yellow crusty sores,

usually around the mouth). Penicillin and methicillin

resistant strains of S. aureus are common causes of

hospital-acquired wound infections.

● P. aeruginosa is associated with infected burns and

hospital-acquired infections.

● E. coli, Proteus species, P. aeruginosa, and Bacteroides

species are the pathogens most frequently isolated from

abdominal abscesses and wounds. Pus containing

Bacteroides species has a very unpleasant smell (as also

pus containing other anaerobes).

● C. perfringens is found mainly in deep wounds where

anaerobic conditions exist. The toxins produced cause

putrefactive decay of the infected tissue with gas produc-

tion. The death and decay of tissue by C.perfringens is

called gas gangrene (see subunit 7.18.9).

● Chronic leg ulceration is common in those with sickle cell

disease. The commonest pathogens isolated are

S. aureus, P. aeruginosa, S. pyogenes, and Bacteroides

species.

● M. tuberculosis is associated with ‘cold’ abscesses.

● Actinomycetes (ﬁlamentous bacteria) and several species

of fungi cause mycetoma (see subunit 7.18.41). Specimens

of pus from the draining sinuses contain granules, exam-

ination of which helps to differentiate whether the

mycetoma is bacterial (treatable) or fungal (less easily

treated).

● A. israeli and other species of Actinomyces cause actino-

mycosis (see 7.18.31). Small yellow granules can be found

in pus from a draining sinus (often in the neck).

● Vincent’s organisms (Borrelia vincenti with Gram

negative anaerobic fusiform bacilli) are associated with

tropical ulcer (see p. 228). The ulcer is commonly found

on the leg, often of malnourished persons, especially

children. Staphylococci and streptococci are frequently

secondary invaders.

● B. anthracis causes anthrax, with the cutaneous form of

the disease producing a pustule usually on the hand or

arm (see 7.18.6). The ﬂuid from the pustule is highly infec-

tious.

● Y. pestis causes plague (see 7.18.22). The disease is

referred to as bubonic plague when the organism infects a

lymph gland and produces a painful swelling referred to

as a bubo. The organism can be found in the ﬂuid aspi-

rated from the bubo and in the surrounding inﬂamed

tissue. The organism is highly infectious.

● M. leprae can be found in skin smears in lepromatous

leprosy and occasionally also in borderline forms of the

disease (see 7.18.30).

● M. ulcerans causes M. ulcerans disease (buruli ulcer). The

countries in which the disease occurs are listed in 7.18.29.

● E. rhusiopathiae causes erysipeloid, a rare inﬂammatory

skin condition, usually affecting the ﬁnger or hand of

those handling meat, poultry, or ﬁsh.

● T. carateum causes pinta (see 7.18.32). It is found in

southern Mexico, Central America and Columbia.

● T. pertenue causes yaws (see 7.18.32). It is found in

tropical Africa (especially in West Africa), Central

America, and also in parts of South-east Asia.

● Skin diseases (bacterial and fungal) are common in those

with HIV diseases. Bacterial infections include recurrent

infections caused by S.aureus and S. pyogenes.

COLLECTION AND TRANSPORT OF PU S, ULCER

MATERIAL

, SKIN SPECIMENS

Specimens should be collected by a medical ofﬁcer

or an experienced nurse. Pus from an abscess is best

collected at the time the abscess is incised and

drained, or after it has ruptured naturally. When col-

lecting pus from abscesses, wounds, or other sites,

special care should be taken to avoid contaminating

the specimen with commensal organisms from the

skin. As far as possible, a specimen from a wound

should be collected before an antiseptic dressing is

applied.

In a hospital with a microbiology laboratory

1 Using a sterile technique, aspirate or collect from

a drainage tube up to 5 ml of pus. Transfer to a

leak-proof sterile container.

MICROBIOLOGICAL TESTS 81

7.8

When pus is not being discharged, use a sterile

cotton-wool swab to collect a sample from the

infected site. Immerse the swab in a container of

Amies transport medium (see No. 11).

2 Label the specimen and as soon as possible

deliver it with a completed request form to the

laboratory.

When mycetoma is suspected: Obtain a specimen

from a draining sinus tract using a sterile hypoder-

mic needle to lift up the crusty surface over the sinus

opening. This method of specimen collection has the

advantages that the pus obtained is usually free

from secondary organisms and the draining

granules can usually be seen clearly and removed

for microscopical examination. Transfer the pus to a

sterile container.

When tuberculosis is suspected: Aspirate a sample of

the pus and transfer it to a sterile container.

When the tissue is deeply ulcerated and necrotic (full

of dead cells): Aspirate a sample of infected material

from the side wall of the ulcer using a sterile needle

and syringe. Transfer to a sterile container.

Fluid from pustules, buboes, and blisters: Aspirate a

specimen using a sterile needle and syringe. Transfer

to a sterile container.

Serous ﬂuid from skin ulcers, papillomas, or papules,

that may contain treponemes: Collect a drop of the

exudate directly on a clean cover glass and invert it

on a clean slide. Immediately deliver the specimen to

the laboratory for examination by dark-ﬁeld

microscopy.

Skin specimens for ringworm fungi: Collect and

examine as described in subunit 7.18.38.

Skin smears for M. leprae: Collect and examine as

described in subunit 7.18.30.

Caution: Specimens from patients with suspected

plague or anthrax are highly infectious. Label such

specimens HIGH RISK and handle them with care.

In a health centre for dispatch to a

microbiology laboratory

1 Collect the specimen using a sterile cotton-wool

swab. Insert it in a container of Amies transport

medium (see No. 11), breaking off the swab stick

to allow the bottle top to be replaced tightly.

When the material is aspirated ﬂuid from a

pustule, transfer the ﬂuid to a sterile, leak-proof

container. Stopper, and seal in a leak-proof

plastic or metal container.

Note: It is not possible to transport exudate from

a suspected treponemal ulcer because the tre-

ponemes remain motile for only a short time.

2 Make a smear of the material on a clean slide (for

Gram staining) and allow to air-dry in a safe

place. Heat-ﬁx the smear (see subunit 7.3.2).

Caution: Do not make a smear for transporting

when the specimen is from a patient with sus-

pected anthrax or bubonic plague.

3 Send the specimens with a completed request

form to reach the microbiology laboratory within

6 hours. Instructions regarding the packaging

and transport of specimens can be found at the

end of subunit 7.1.

LABORATORY EXAMINATION OF

PUS, ULCER AND SKIN SPECIMENS

1 Describ e the appearance of the specimen

When from a patient with suspected mycetoma or

actinomycosis, report the appearance of the

specimen and whether it contains granules.

Detection of granules

White, yellow, brown, red, or black granules of varying size,

shape, and consistency may be found in pus draining from

sinuses in mycetoma (actinomycetic or fungal) and in actino-

mycosis. The granules are colonies of organisms.

To free the granules from the pus, shake a portion of the

specimen (or dressing) in sterile distilled water. Wait for a few

minutes (to allow the granules to settle), remove the super-

natant ﬂuid, and transfer a few of the granules to a slide. A

hand magnifying lens may be required to see clearly the small

granules.

Note: Identiﬁcation of organisms that cause

mycetoma is described in subunit 7.18.41 and actin-

omyosis in subunit 7.18.31.

2 Examine the spe cimen microscopically

Note: When a swab has been used to collect the

pus, inoculate the culture media ﬁrst before using

the swab to make smears.

Gram smear

Make an evenly spread smear of the specimen on a

Day 1

82 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.8

slide. Allow the smear to air-dry in a safe place. Fix

as described in subunit 7.3.2, and stain by the Gram

technique (see subunit 7.3.4).

Examine the smear for bacteria among the pus cells

using the 40 and 100objectives. Look especially

for:

● Gram positive cocci that could be S. aureus (see

colour Plate 24) or streptococci that could be

S. pyogenes or other beta-haemolytic strepto-

cocci, anaerobic streptococci, or enterococci (see

colour Plate 25).

● Gram negative rods that could be Proteus

species, E. colior other coliforms, P. aeruginosa or

Bacteroides species.

● Gram positive large rods with square ends that

could be C. perfringens or B. anthracis (see colour

Plate 34).

● Large numbers of pleomorphic bacteria (strepto-

cocci, Gram positive and Gram negative rods of

various size and fusiform bacteria), associated

with anaerobic infections.

● Gram positive yeast cells with pseudohyphae,

suggestive of Candida albicans (see colour Plate

72).

● Vincent’s organisms if tropical ulcer is suspected.

These appear as Gram negative spirochaetes

(B. vincenti) and Gram negative fusiform rods

(see colour Plate 65).

ADDITIONAL

Ziehl-Neelsen smear when tuberculosis or

M. ulcerans disease is suspected

Make a smear of the specimen as describ ed under

Gram smear and allow to air-dry in a safe place. Fix

and stain by the Ziehl-Neelsen technique as

described in subunit 7.3.5. Examine the smear for

acid fast bacilli (AFB) using the 100objective. The

appearance ofAF Bin a Ziehl-Neelsen stained smear

is shown in colour Plate 56. M. tuberculosis is

described in subunit 7.18.28 and M. ulcerans in

subunit 7.18.29.

Note: Staining smears for M. leprae is described in

7.18.30.

Giemsa or Wayson’s stained smear when

bubonic plague is suspected (see also subunit

7.18.22)

Make an evenly spread smear of the specimen on a

slide and allow to air-dry in a safe place. Fix with

methanol for 5 minutes and stain by the Giemsa

technique (see subunit 7.3.10) or by the rapid

Wayson technique (see subunit 7.3.8). Look for

Gram negative bipolar stained organisms. Y. pestis

organisms are shown in colour Plate 54 and

described in subunit 7.18.22.

Caution: Y. pestisis highly infe ctious.

Polychrome Loefﬂer methylene blue smear

when cutaneous anthrax is suspected

Make an evenly spread smear of the specimen on a

slide and allow to air-dry in a safe place. Fix the

smear by covering it with potassium permanganate

40 g/l solution (Reagent No. 71) for 10 minutes.

Wash off with water, and stain as described in

subunit 7.3.7.

Examine the smear for chains of large blue-stained

rods surrounded by mauve stained capsules charac-

teristic of B. anthracis as shown in colour Plate 55

(McFadyean’s reaction). See also subunit 7.18.6.

Caution: B. anthracis is highly infectious.

Examination by dark-ﬁeld microscopy to

detect treponemes

The examination of a specimen by dark-ﬁeld

microscopy for motile treponemes when yaws or

pinta is suspected, is the same as that describ ed

for syphilis (see subunit 7.10). T. pertenue and

T.carateum are identicalin morphology to T.pallidum

as shown in Plate 60.

Potassium hydroxide preparation when

ringworm or other superﬁcial fungal

infection is suspected

The examination of a potassium hydroxide prep-

aration for the detection of ringworm fungi is

described in subunit 7.18.38 and M. furfur in 7.18.39.

Examination of preparations for fungi that cause

chromoblastomycosis is described in subunit 7.18.40.

3 Culture the spe cimen

Blood agar MacConkey agar, cooked meat

medium (or thioglycollate broth)

– Inoculate the specimen:

● On blood agar (see No. 16) to isolate S. aureus

and streptococci. Add a bacitracin disc if strepto-

cocci are seen in the Gram smear.

● On MacConkey agar (see No. 54) to isolate

Gram negative rods.

● Into cooked meat medium (see No. 27) or thio-

glycollate broth (see No. 80).

Cooked meat medium:This is an enrichment medium for

aerobes and anaerobes. The glucose in the medium helps

to produce a rapid growth of anaerobes (at the bottom of

the medium).

MICROBIOLOGICAL TESTS 83

7.8

– Incubate the inoculated blood agar plate at

35–37Cin a carbon dioxideatmosphere (candle

jar) and the MacConkey agar plate aerobically.

Incubate the inoculated cooked meat medium at

35–37C for up to 72 hours.Subculture at 24 h,

and if indicated at 48 h and 72 h.

Anaerobic culture

When an anaerobic infection is suspected (specimen

is often foul-smelling), or the Gram smear shows an

‘anaerobic mixed ﬂora’, inoculate a second blood

agar plate and incubate it anaerobically (see subunit

7.4) for up to 48 hours. The anaerobic blood agar

plate may be made selective by adding neomycin to

it (see No. 16). At a ﬁnal neomycin concentration of

50–70 g/ml, the majority of facultative anaerobic

Gram negative rods will be inhibited. To aid detec-

tion of anaerobes, a metronidazole disc (5 µg) may

be added to the anerobic blood plates as the

majority of anaerobes show a zone of inhibition,

whereas aerobes grow up to the disc.

ADDITIONAL

Culture of specimen when bubonic plague is

suspected

The Central Public Health Laboratory should be

notiﬁed at the earliest opportunity when plague is

suspected. Whenever possible, isolation of Y. pestis

should be undertaken in this laboratory. Blood or a

bubo aspirate should be sent for culturing together

with a full case history and the report of the micro-

scopical examination, i.e. whether bipolar stained

organisms were seen (see subunit 7.18.22).

Culture of specimen when infection with

M. tuberculosis or M. ulcerans is suspected

The facilities of a specialist tuberculosis laboratory

are required for the isolation, identiﬁcation and

susceptibility testing of M. tuberculosis, M. ulcerans,

and other mycobacteria.

4 Examine and report the cultures

Blood agar and MacConkey agar cultures

Look especially for colonies that could be:

 Staphylococcus aureus (see subunit 7.18.1)

 Streptococcus pyogenes (see 7.18.2)

 Pseudomonas aeruginosa (see 7.18.20)

 Proteus species (see 7.18.18)

 Escherichia coli (see 7.18.14)

 Enterococcus species (see 7.18.5)

 Klebsiella species (see 7.18.17)

Day 2 and Onwards

84 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.8

Summary of Microbiological Examination of Pus, Ulcer Material and

Skin Specimens

ADDITIONAL INVESTIGATIONS

1 Describe  Look for granules: When

Specimen

mycetoma or actinomycosis is

suspected

2 Culture  Blood agar  Culture for M. tuberculosis or

Specimen

Incubate aerobically M.ulcerans

 MacConkey agar

Requires facilities of a

Incubate aerobically

Tuberculosis Reference

 Cooked meat medium

Laboratory

Subculture at 24 h, 48 h, and

72 h as indicated

 Neomycin blood agar when

anaerobic infection is suspected

Incubate anaerobically up to 48 h

3 Examine  Gram smear  Ziehl-Neelsen smear:

Microscopically

For pus cells and bacteria When tuberculosis or

M.ulcerans disease is suspected

 KOH preparation:

When a fungal or actinomycete

infection is suspected

 Giemsa or Wayson’s smear:

When bubonic plague is

suspected

 Polychrome methylene blue:

When cutaneous anthrax is

suspected

 Dark-ﬁeld microscopy:

To detect treponemes when

yaws or pinta is suspected

Day 1

4 Examine and  Blood agar and MacConkey  Antimicrobial susceptibility tests

Report Cultures agar cultures As indicated

Look particularly for:

S.aureus

S.pyogenes

P.aeruginosa

Proteus species

E.coli

Day 2 and Onwards

Enterococcus species

Klebsiella species

Anaerobes:

C.perfringens

Bacteroides fragilis group

Peptostreptococcusspecies

Anaerobic blood agar culture and cooked

meat culture

Look for growth that could be Clostridium perfrin-

gens, Bacteroides fragilis group, or Peptostrepto-

coccus species.

 C. perfringens: Grows rapidly in cooked meat

medium with hydrogen sulphide gas produc-

tion (gas bubbles in turbid medium) and

reddening but no decomposition of the meat

(saccharolytic reaction). On anaerobic blood

agar, colonies are usually seen after 48 h incu-

bation. Most strains produce a double zone of

haemolysis (inner zone of clear haemolysis,

outer zone of partial haemolysis) as shown in

colour Plate 36. C. perfringens is describe d in

subunit 7.18.9.

 B. fragilis: Grows in cooked meat medium pro-

ducing decomposition with blackening of the

meat (foul-smelling proteolytic reaction). On

anaerobic blood agar, non-haemolytic grey

colonies (Gram negative pleomorphic rods) are

seen, usually within 48 hours. B. fragilis group is

described in subunit 7.18.27.

 Peptostreptococcus: Grows in cooked meat

medium with the production of large amounts of

hydrogen sulphide gas. On anaerobic blood

agar, Peptostreptococcus produces small non-

haemolytic white colonies (Gram positive cocci)

after 48 h incubation. They are resistant to

metronidazole (5 g disc).

Conﬁrming organisms are anaerobes

When there is a mixed growth and colonies appear on the

anaerobic plate that are not present on the aerobic plate,

conﬁrm that the organisms are anaerobes by subinoculating

the colonies on three plates of blood agar and incubating one

aerobically, one anaerobically, and the third in a carbon

dioxide atmosphere (candle jar).

Subculture of cooked meat medium

Subculture the cooked meat broth after overnight

incubation and when indicated also at 48 h and

72 h, when the routine plate cultures are sterile or

the organisms isolated do not correspond to those

seen in the original Gram smear. Subculture as

described previously.

Antimicrobial susceptibility testing

Susceptibility testing may be required for S. aureus,

enterobacteria and non-fermentative Gram negative

rods. Only routinely used antibiotics should be

tested. New and expensive antibiotics should only

be tested on special request or when the isolate is

resistant to other drugs.

Anaerobic pathogens*: Susceptibility tests should not

routinely be performed on anaerobic bacteria by the

disc diffusion technique. Most anaerobic infections

are caused by penicillin-susceptible bacteria with the

exception of infections originating in the intestinal

tract or vagina. Such infections generally contain

B. fragilis which produces beta-lactamase and is

resistant to penicillins, ampicillins and most

cephalosporins. Such infections can be treated with

clindamycin, metronidazole or chloramphenicol.

Aminoglycosides have no activity against anaerobes

but they are often used for the treatment of patients

who have mixed infections.

*Information from: Basic laboratory procedures in clinical

bacteriology, WHO, 2nd edition, 2003.

7.9 Examination of effusions

An effusion is ﬂuid which collects in a body cavity or

joint. Fluid which collects due to an inﬂammatory

process is referred to as an exudate and that which

forms due to a non-inﬂammatory condition is

referred to as a transudate. When the effusion is an

exudate, it is important to investigate whether the

inﬂammatory process is an infective one (septic) or

caused by a non-infective process, e.g. malignancy.

When the ﬂuid is a transudate, no further microbio-

logical testing is required.

Effusions sent to the laboratory for investigation

include:

Fluid Origin

Synovial From a joint

Pleural From the pleural cavity

(Space between the lungs

and the inner chest wall)

Pericardial From the pericardial sac

(Membranous sac

surrounding the heart)

Ascitic (peritoneal) From the peritoneal

(abdominal) cavity

Hydrocele Usually from the sac

surrounding the testes

MICROBIOLOGICAL TESTS 85

7.8–7.9

Possible pathogens

SYNOVIAL FLUID

(Synovitis and Infective arthritis)

Gram positive Gram negative

Staphylococcus aureus Neisseria gonorrhoeae

Streptococcus pyogenes Neisseria meningitidis

Streptococcus pneumoniae Haemophilus inﬂuenzae

Anaerobic streptococci Brucella species

Actinomycetes Salmonella serovars

Escherichia coli

Pseudomonas aeruginosa

Proteus

Bacteroides

Also Mycobacterium tuberculosis.

PLEURAL AND PERICAR DIAL FLUIDS

(Empyema and Purulent Pericarditis)

Gram positive Gram negative

Staphylococcus aureus Haemophilus inﬂuenzae

Streptococcus pneumoniae Bacteroides

Streptococcus pyogenes Pseudomonas aeruginosa

Actinomycetes Klebsiella strains

Other enterobacteria

Also Mycobacterium tuberculosis, fungi, and viruses

especially coxsackie B virus.

ASCITIC FLUID

(Ascites and Peritonitis)

Gram positive Gram negative

Enterococcus species Escherichia coli

Streptococcus pneumoniae Klebsiellastrains

Staphylococcus aureus Other enterobacteria

Streptococcus pyogenes Pseudomonas aeruginosa

Streptococcus agalactiae Bacteroides

Viridans streptococci

Clostridium perfringens

Also Mycobacterium tuberculosis and Candida species.

HYDROCELE FLUID

Occasionally Wuchereria bancrofti microﬁlariae and

rarely Brugiaspecies c an be found in hydrocele ﬂuid.

These parasites are described in Part 1 of the book.

Notes on causes of effusions

● Synovitis means inﬂammation of the synovial membrane

(lining of a joint capsule). It can be caused by bacteria,

rheumatic disorder, or injury. Infective synovitis is usually

secondary to bacteraemia. Patients with existing or

previous joint disorders are most at risk.

● Inﬂammation of a joint is called arthritis. The term

polyarthritis is used when many joints are affected.

Arthritis can be caused by bacteria (infective arthritis),

rheumatoid arthritis, gout and pseudogout, osteoarthritis

86 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.9

(cartilage and bone disease), spondylarthritic disorders,

and ﬁlariasis. Arthritis may also precede hepatitis and

accompany other viral diseases including rubella, infec-

tious mononucleosis, and arborviral infections.

In gonococcal arthritis (usually following gonococcal

bacteraemia), gonococci are difﬁcult to ﬁnd because the

infection tends to remain in the synovial membrane.

Reiter’s syndrome (one of the spondylarthritic disor-

ders) affects young men. It involves inﬂammatory

arthritis, urethritis and conjunctivitis. It is often a compli-

cation of non-speciﬁc urethritis or follows bacterial

dysentery. The term reactive arthritis is used when arthri-

tis alone develops following urethritis or bacterial

dysentery.

‘Tropical arthritis’ is a non-speciﬁc but distinctive

form of arthritis, thought to have an immunological basis

perhaps in association with a virus or rickettsia.

● The term pleural effusion is used to describe a non-

purulent serous effusion which sometimes forms in

pneumonia, tuberculosis, malignant disease, or pul-

monary infarction (embolism). It may also occur with

systemic lupus erythematosus, lymphoma, rheumatoid

disease, or amoebic liver abscess. The commonest cause

of pericardial effusion in developing countries is pericar-

dial tuberculosis (often HIV-related).

Empyema is used to describe a purulent pleural

effusion when pus is found in the pleural space. It can

occur with pneumonia, tuberculosis, infection of a

haemothorax (blood in the pleural cavity), or rupture of

an abscess through the diaphragm.

Note: The transfer of ﬂuid (transudate) into the pleural

cavity is called hydrothorax. It occurs in cardiac failure,

nephrotic syndrome, severe malnutrition, and advanced

cirrhosis. The collapse of a lung brought about by air in

the pleural space is called pneumothorax.

● Causes of acute pericarditis other than infecting micro-

organisms, include myocardial infarction, rheumatic

fever, malignant disease, systemic lupus erythematosus,

uraemia, and trauma.

● Peritonitis means inﬂammation of the peritoneum, which

is the serous membrane that lines the peritoneal cavity.

Ascites refers to the accumulation of ﬂuid in the peri-

toneal cavity causing abdominal swelling.

Peritonitis can be caused by the rupture of an abdom-

inal organ, or as a complication of bacteraemia. Causes of

ascites include tuberculosis, advanced schistosomiasis,

portal hypertension, cardiac failure, and malignancy

especially of the ovary, stomach, colon, and liver. A

chylous ascites can develop as a complication of advanced

ﬁlariasis.

Commensals

The small amounts of ﬂuid which surround the joints

and can be found in the pleural cavity, pericardial

sac, and peritoneal cavity, have no normal microbial

ﬂora.

COLLECTION AND TRANSPORT OF EFFUSIONS

Collection of synovial, pleural, pericardial, peritoneal,

or hydrocele ﬂuid is carried out by a medical ofﬁcer.

In a hospital with a microbiology laboratory

1 After aspiration, aseptically dispense the ﬂuid as

follows:

– 2–3 ml into a dry, sterile, screw-cap tube or

bottle to observe for clotting.

– 9 ml into a screw-cap tube or bottle which

contains 1 ml of sterile tri-sodium citrate (see

No. 73). Mix the ﬂuid with the anticoagu-

lant.*

*Tri-sodium citrate prevents clotting, especially of

exudates. The sterile citrated sample can be used to

estimate cell numbers, protein concentration, and for

microscopy and culture.

2 Label, and as soon as possible deliver the

samples with a completed request form to the

laboratory.

In a health centre for dispatch to the

laboratory

1 After aspiration, aseptically dispense the ﬂuid as

follows:

– 5 ml into a bottle of sterile thioglycollate

broth (see No. 80) and mix.

– 9 ml into a screw-cap tube or bottle which

contains 1 ml of sterile tri-sodium citrate (see

No. 73). Mix the ﬂuid with the anticoagulant.

– If any ﬂuid remains, dispense into a dry,

sterile, screw-cap tube or bottle, and observe

for clotting.

2 Label each cont ainer with the date and the

patient’s name, number, and health centre.

3 Send the samples with a completed request form

to reach the microbiology laboratory within a few

hours. The inoculated thioglycollate broth should

be kept in a warm environment, but not over

37C or in direct sunlight.

LABORATORY EXAMINATION OF

EFFUSIONS

1 Describ e the appearance of the specimen

Report:

– Colour of the effusion

– Whether it is clear, cloudy, or purulent (like pus)

Day 1

MICROBIOLOGICAL TESTS 87

7.9

– Whether it contains blood

– Whether it is clotted (sample without anti-coagu-

lant)

Purulent effusion: When the specimen is pus or

markedly cloudy, examine and report a Gram

stained smear as soon as possible. Proceed to

examine the specimen as for pus (described in

subunit 7.8).

Blood-stained effusion: Culture the specimen

and examine a Gram stained smear (see following

text).

Note: When the specimen is not pus or a blood-

stained effusion, transfer about 1 ml of the

well-mixed citrated sample to a separate tube or

bottle (need not be sterile). Use this to estimate cell

numbers and protein concentration (see later text).

This will avoid contaminating the remainder of the

sample which may be required for culture. When

the non-citrated sample does not contain clots, this

should be used for the cell count and protein in pref-

erence to the citrated sample.

2 Examine the ﬂuid for cells

Estimate the number of white cells in the ﬂuid using

the technique described for c.s.f. (see subunit 7.13).

Report whether the cells are mainly polymorpho-

nuclear neutrophils (pus cells) or lymphocytes.

Tuberculous effusions contain mainly lymphocytes

and often plasma cells (it is rare to ﬁnd AFB in the

ﬂuid).

Note: A transudate may contain a few cells, whereas

an exudate usually contains many cells.

3 Estimate the protein

Dilute the ﬂuid 1 in 100 in physiological saline (0.1

ml effusion mixed with 9.9 ml saline). Estimate the

total protein using the technique described for mea-

suring total protein in c.s.f. (see subunit 6.11 in Part

1 of the book). Multiply the result by 100.

Note: A transudate usually contains less than 30 g/l

(3 g/dl) of protein whereas an exudate contains

more than 30 g/l.

Note: An exudative pleural effusion containing lym-

phocytes with no organisms seen in the Gram smear

is found with tuberculosis.

The following table summarizes the results of a few

tests which can be performed in district laboratories

to differentiate transudates from exudates.

4 Culture the spe cimen

Culture the ﬂuid when it contains more than a few

white cells and more than 30 g/l of protein, or when

it appears blood-stained (if pus, process it as

described in subunit 7.8).

Centrifuge the citrated sample in a sterile tube at

high speed for about 20 minutes to sediment the

bacteria. Remove the supernatant ﬂuid (do not

discard) and resuspend the sediment. Culture the

sediment as follows:

Chocolate agar, blood agar and MacConkey

agar

● Inoculate the sediment on chocolate (heated

blood) agar, blood agar, and MacConkey agar

(see Appendix 1).

● Incubate the chocolate agar plate in a carbon

dioxide enriched atmosphere at 35–37C for up

to 48 hours (see subunit 7.4), checking for

growth after overnight incubation.

Incubate the blood agar plate and MacConkey

agar plate aerobically at 35–37C for up to 72

hours, examining for growth after overnight

incubation.

ADDITIONAL

Culture of specimen when tuberculosis is

suspected

Isolation, identiﬁcation, and sensitivity testing of

M. tuberculosis and other mycob acteria require the

facilities of a Tuberculosis Reference Laboratory.

5 Examine the spe cimen microscopically

Gram smear

Make a thin evenly spread smear of a purulent

effusion or sediment from a centrifuged non-

purulent sample (use the citrated specimen). When

88 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.9

dry, ﬁx the smear with methanol for 2 minutes, and

stain it by the Gram technique (see subunit 7.3.4).

Examine the smear for pus cells and bacteria using

the 40 and 100obje ctives.

Look especially for:

● Gram positive cocci that could be S. aureus.

● Gram positive streptococci that could be

S. pyogenes, or possibly enterococci.

● Gram positive diplococci or short chains that

could be pneumococci.

● Gram negative rods that could be enterobacteria,

Pseudomonas, or H. inﬂuenzae especially if the

rods are pleomorphic.

● Gram negative intracellular diplococci that could

be gonococci when the ﬂuid is from a joint.

● Gram positive branching threads that could be

actinomycetes.

Ziehl-Neelsen smear

Make a smear on a slide using several drops of

sediment from the centrifuged ﬂuid. Fix the dried

smear and stain by the Ziehl-Neelsen technique as

described in subunit 7.3.5. AFB are usually few

and therefore a careful search of the smear is

required. Frequently, no AFB can be found.

The appearance of M. tuberculosis in a Ziehl-

Neelsen stained smear is shown in colour Plate 56.

Fluorochrome smear

Tubercle bacilli in effusions can rapidly be detected in a ﬂuo-

rochrome stained smear examined by ﬂuorescence

microscopy. The ﬂuorescing rods can be detected using the

40 objective. The auramine staining technique is described

in subunit 7.3.6.

Note: The commonest cause of pericardial effusion in devel-

oping countries is pericardial tuberculosis (PCTB) often

linked to HIV infection.

ADDITIONAL

Wet preparation to detect crystals when

gout or pseudogout is suspected (usually in

men)

When the ﬂuid is from a joint, transfer a drop of the

sediment from the centrifuged ﬂuid (citrated

sample) to a slide, and cover with a cover glass.

Examine the preparation using the 10 and 40

objectives with the condenser iris closed sufﬁciently

to give maximum contrast. Look for colourless,

extracellular and intracellular (inside white cells)

crystals:

– Monosodium urate crystals are needle-like in

form and measure 8–10 m in length. They can

be found in effusions from patients with gout.

– Calcium pyrophosphate crystals measure up to

Transudate* Exudate

Appearance Clear, pale yellow Purulent, cloudy,

or blood-stained

Clotting Does not clot Often clots

Cells Few cells Purulent: Many

cells, mostly

neutrophils

Non-purulent:

Few or many

cells, mostly

lymphocytes

Protein Less than 30 g/l More than 30 g/l

*When the sample is a transudate there is no need to examine

it further.

MICROBIOLOGICAL TESTS 89

7.9

Summary of Microbiological Examination of Effusions

1 Describe  Describe colour and whether:

Specimen

– clear, cloudy, or purulent

– blood stained

– contains clots (non-citrated sample)

IF PUS: Examine as described in subunit 7.8

IF BLOOD STAINED: Proceed to step 4

2 Examine  Estimate cell numbers

for Cells

 Report % of cells that are:

– neutrophils

– lymphocytes

3 Protein  Report total protein in g/l

TRANSUDATE: Clear unclotted ﬂuid with few cells and protein below 30 g/l: No need to

test further.

EXUDATE: When cloudy ﬂuid with more than a few cells and protein over 30 g/l:

Proceed to steps 4 and 5

Note: When the ﬂuid contains many pus cells, examine as described for

Pus in subunit 7.8

4 Culture  Blood agar  Culture for M. tuberculosis

Specimen

Incubate aerobically Requires facilities of a

 Chocolate agar

Tuberculosis Reference

Incubate in CO

Laboratory

 MacConkey agar

Incubate aerobically

5 Examine  Gram smear  Wet preparation for crystals:

Microscopically Look for pus cells and When gout or pseudogout is

bacteria suspected (joint ﬂuid only)

 Ziehl-Neelsen  Cytology smear:

Look for AFB When malignancy is suspected

Day 1

6 Examine and  Blood, chocolate, MacConkey  Antibiotic susceptibility test

Report Cultures

agar cultures As required

Look particularly for:

S.aureus

S.pyogenes

S.pneumoniae

Day 2 and Onwards

H.inﬂuenzae

Neisseria species

Enterobacteria

P.aeruginosa

ADDITIONAL INVESTIGATIONS

25 m in length, are rod-shaped, and may have

a line running through them. They can be found

in effusions from patients with pseudogout.

Gout: High serum urate levels are usually found in patients

with gout. Normal levels are found in patients with pseudo-

gout.

Cytology smear when malignancy is

suspected

Make two thin smears of effusion sediment and

while still wet, ﬁx the smears in a container of 95%

v/v ethanol for 20 minutes. Send the smears to a

Cytology Laboratory for special staining and exam-

ination for malignant cells.

6 Examine and report the cultures

Chocolate agar, blood agar, and MacConkey

agar cultures

Look especially for colonies that could be:

 Staphylococcus aureus, see subunit 7.18.1

 Streptococcus pyogenes, see subunit 7.18.2

 Streptococcus pneumoniae, see subunit 7.18.4

 Haemophilus inﬂuenzae, see subunit 7.18.24

 Enterobacteria, see subunit 7.18.14.

 Pseudomonas aeruginosa, see subunit 7.18.20

 Neisseria species, see subunits 7.18.12 and 7.18.13

7.10 Examination of

urogenital specimens

Possible pathogens

URETHRAL SWABS

Neisseria gonorrhoeae, Chlamydia trachomatis

(serovars D-K), and occasionally Ureaplasma,

Mycoplasma, and Trichomonas vaginalis.

CERVICAL SWABS

From non-puerperal women: Neisseria gonor-

rhoeae, Chlamydia trachomatis (serovars D-K),

Streptococcus pyogenes, herpes simplex virus.

From women with puerperal sepsis or septic

abortion: Streptococcus pyogenes, other beta-

haemolytic streptococci, Staphylococcus aureus,

Enterococcus species, anaerobic cocci, Clostridium

Day 2 and Onwards

90 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.9–7.10

perfringens, Bacteroides, Proteus, Escherichia coli and

other coliforms, Listeria monocytogenes.

VAGINAL SWABS

Vaginal discharge may be due to infection of the

vagina or infection of the cervix or uterus. Pathogens

causing vaginal infections include Trichomonas vagi-

nalis, Candidaspecies, and Gardnerella vaginalis with

anaerobes.

GENITAL ULCER SPECIMENS

Treponema pallidum, Haemophilus ducreyi, Klebsiella

(Calymmatobacterium) granulomatis, Chlamydia tra-

chomatis (serovars L1, L2, L3), herpes simplex virus.

Note: HIV infection is described in subunit 7.18.55.

Notes on pathogens

● N. gonorrhoeae causes gonorrhoea. Infections in men are

associated with urethral discharge and painful urination

(dysuria). In symptomatic men, gonococcal urethritis can

be diagnosed presumptively in up to 95% of patients by

examining a Gram stained smear of pus cells (in urine

sediment or urethral discharge) for intracellular Gram

negative diplococci. Culture is indicated when the disease

is suspected but organisms cannot be found or when treat-

ment fails.

In women, N.gonorrhoeae causes cervicitis and ure-

thritis. A presumptive diagnosis from a Gram smear has

both a low sensitivity and low speciﬁcity with intracellular

Gram negative diplococci being detected in only 40–60%

of infected women. Many infections are asymptomatic.

Cultural techniques (or specialist antigen tests, see

7.18.13) are required to diagnose urogenital gonorrhoea

in women (culture is about 90% sensitive). Untreated

gonococcal infections in women can lead to pelvic inﬂam-

matory disease (PID), ectopic pregnancy and other

complications. Co-infection with other sexually transmit-

ted pathogens is common. Pregnant women with

gonorrhoea can pass infection to their newborn infant

causing gonococcal conjunctivitis (ophthalmia neona-

torum).

Antimicrobial resistance is shown by many strains of

N.gonorrhoeae, e.g. to penicillin, tetracycline, spectino-

mycin, and more recently to ﬂuroquinoles.

● Chlamydia trachomatis has a high prevalence worldwide.

Sexually transmitted C. trachomatis serovars D-K cause

urogenital infections and serovars L1–L3 cause lym-

phogranuloma venereum (LGV). Sensitive and speciﬁc

antigen tests have been developed for diagnosing chlamy-

dial infections (see subunit 7.18.37).

In men, C. trachomatis is a common cause (up to

40%) of non-gonococcal urethritis (NGU). Species of

Mycoplasma and Ureaplasma can also cause urethritis.,

Many Chlamydiainfections are asymptomatic. In women,

60% or more of urogenital chlamydial infections are

asymptomatic. The syndromic diagnosis of chlamydial

infection (and also gonorrhoea) in women is difﬁcult and

often leads to overtreatment. Untreated Chlamydiainfec-

tions are associated with serious complications including

PID, ectopic pregnancy, and infertility, see also subunit

7.18.37.

In LGV, C. trachomatis infects lymph nodes in the

groin and surrounding tissues. A small ulcer forms at the

site of infection followed by inﬂammation and painful

swelling of the lymph glands (buboes). LGV is endemic in

many tropical and subtropical countries. It is mainly diag-

nosed clinically.

● T.pallidum causes syphilis. The early and late forms of the

disease are described in subunit 7.18.32. In primary and

secondary syphilis, motile T.pallidum spirochaetes can be

detected in serous ﬂuid from lesions, examined by dark-

ﬁeld microscopy. T. pallidum cannot be isolated by

cultural techniques.

About 3–5 weeks following infection (1–2 weeks

after the appearance of the genital chancre), antibodies

can be found in the patient’s serum. Non-speciﬁc antibody

tests and speciﬁc treponemal antibody tests are used to

diagnose syphilis (see subunit 7.18.32). It is particularly

important to screen pregnant women for infectious

syphilis because T.pallidum can cause abortion, prema-

ture delivery, still-birth, and infection of the newborn

(congenital syphilis).

● H. ducreyi causes chancroid, or soft sore. It is a common

cause of genital ulceration in tropical countries. The

ulcers are painful, shallow and tend to be ragged. Often

there is also painful swelling of the inguinal lymph nodes.

The organism is difﬁcult to detect microscopically in

specimens and H. ducreyi is not easily cultured (see

subunit 7.18.25). Chancroid is becoming increasingly difﬁ-

cult and expensive to treat due to the resistance of

H.ducreyi to commonly available antimicrobials.

● K. granulomatis causes a genital ulcerative condition

called granuloma inguinale, also known as donovanosis. It

is particularly prevalent in India, Papua New Guinea,

Vietnam, South Africa, Zambia, Zimbabwe, Brazil and

other parts of South America. Ulceration of the genitalia

and surrounding skin can be extensive but unlike LGV,

the lymph glands are less involved. The disease is usually

diagnosed by ﬁnding intracellular Donovan bodies in

Giemsa stained smears from infected tissue.

● Inﬂammatory sexually transmitted infections (STIs) e.g.

gonorrhoea, Chlamydia infection, and STIs that cause

ulceration, e.g. syphilis, chancroid, LGV, genital herpes,

granuloma inguinale, facilitate the transmission of HIV

and increase susceptibility to HIV infection. Bacterial

STIs may also enhance the survival and replication of

HIV in the urogenital tract. Treatment of bacterial STI

has been shown to reduce the incidence of HIV transmis-

sion (42% reduction reported from a study in Tanzania

following introduction of syndromic STD treatment).

HIV co-infection may result in some STIs being more

severe (e.g. ulceration), progressing more rapidly with

earlier complications (e.g. PID, neurosyphilis), and

patients responding less well to treatment with relapses.

● T. vaginalis is a ﬂagellate protozoan parasite. It causes

trichomoniasis with a purulent vaginal discharge in

women and occasionally a nonpurulent urethral discharge

in men. Most infections (about 80%) can be diagnosed

microscopically.

● C. albicans is a yeast fungus. It is described in subunit

7.18.47. Vaginitis caused by Candida species produces a

white odourless discharge. Vaginal candidiasis (vaginal

thrush, or moniliasis) is especially common during preg-

nancy and may also occur when using oral contraceptives,

as a complication of diabetes mellitus, or after prolonged

antimicrobial treatment. It is usually diagnosed micro-

MICROBIOLOGICAL TESTS 91

7.10

scopically. Culture is not recommended as the presence of

small numbers of yeast cells is a normal ﬁnding.

● G. vaginalis (with anaerobes, including Bacteroides,

Peptostreptococcus, Mobiluncus) is a common cause of

bacterial vaginosis, a non-inﬂammatory infection of the

vagina which alters the normal lactobacillary microbial

ﬂora. It produces a thin greyish-white discharge with a

characteristic ammoniacal ﬁshy odour (intensiﬁed by

adding a few drops of 10% KOH) and higher than normal

pH. Clue cells can be seen in Gram-stained smears.

Commensals

Urethral swabs: Diphtheroids, Acinetobacter

species, and enterobacteria. Skin commensals (see

subunit 7.8) may also be present.

Cervical swabs: The cervix is normally sterile.

Vaginal swabs from puberty to menopause

(acid pH in vagina): Lactobacilli, anaerobic or

microaerophilic streptococci, Clostridium species,

Bacteroides. Acinetobacter species, fusobacteria,

G. vaginalis, mycoplasma, and small numbers of

diphtheroids and yeasts.

Vaginal swabs after menopause (alkaline pH):

Diphtheroids, micrococci, S. epidermidis, viridans

streptococci, enterobacteria, C. albicans and other

yeasts.

COLLECTION AND TRANSPORT OF UROGENITAL

SPECIMENS

Urogenital specimens should be collected by a

medical ofﬁcer or an experienced nurse.

Amies medium (see No. 11) is the most efﬁcient

medium for transporting urethral, cervical, and

vaginal swabs. Specimens should be transported in

a cool box.

Collection of urethral discharge from male

patients

1 Cleanse around the urethral opening using a

swab moistened with sterile physiological saline.

2 Gently massage the urethra from above down-

wards. Using a swab, collect a sample of

discharge. Make a smear of the discharge on a

microscope slide by gently rolling the swab on

the slide. This will avoid damaging pus cells

which contain the bacteria.

Note: Very few pus cells may be present if the

patient has recently passed urine. Allow 2–4

hours after urination before collecting a

specimen.

3 When culture is indicated (see previous test),

collect a sample of pus on a sterile cotton-wool

swab. If possible, before inserting the swab in a

container of Amies transport medium, inoculate

a plate of culture medium (see later text).

4 Label the specimens and deliver them to the lab-

oratory as soon as possible. Inoculated culture

plates must be incubated within 30 minutes.

Isolation of N.gonorrhoeae from urine

In acute urethritis, it is often possible to detect N.gonorrhoeae

in pus cells passed in urine, especially the ﬁrst voided urine of

the day (centrifuged to sediment the pus cells).

Note: A rectal swab is also required from homosex-

ual patients. A selective medium is required to

isolate N. gonorrhoeae from a rectal specimen.

Collection of cervical specimens from female

patients

A specimen collected from the endocervical canal is

recommended for the isolation of N. gonorrhoeae

by culture. Use a sterile vaginal speculum to

examine the cervix and collect the specimen.

1 Moisten the speculum with sterile warm water,

and insert it into the vagina.

Note: Do not lubricate the speculum with a gel

that may be bactericidal.

2 Cleanse the cervix using a swab moistened with

sterile physiological saline.

3 Pass a sterile cotton-wool swab 20–30 mm into

the endocervical canal and gently rotate the

swab against the endocervical wall to obtain a

specimen.

4 When gonorrhoea is suspected, before inserting

the swab in Amies transport medium, if possible

inoculate a plate of culture medium (see later

text).

5 Label the specimens and deliver to the labora-

tory as soon as possible. Inoculated culture plates

must be incubated within 30 minutes.

Note: Women may also asymptomatically carry

N. gonorrhoeae in the rectum and can transmit the

pathogen to consorts.

Collection of vaginal discharge to detect

T. vaginalis, C. albicans and G. vaginalis

Two preparations are required:

● Wet preparation to detect motile T. vaginalis: Use

a sterile swab to collect a specimen from the

vagina. Transfer a sample of the exudate to a

microscope slide. Add a drop of physiological

saline* and mix. Cover with a cover glass. Label

and deliver to the laboratory for immediate

examination (see later text).

*Use only a sterile saline solution or one that is checked

daily by the laboratory to exclude contaminating motile

organisms which can be mistaken for T. vaginalis. In

tropical climates it is easy for saline solutions to become

contaminated.

92 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.10

● Dry smear for Gram staining to detect Candida

and examine for clue cells

Although yeast cells can be seen in an unstained

wet preparation, the Gram positive cells and

pseudohyphae of C. albicans are more easily

seen in a Gram stained smear. Use a sterile swab

to collect a specimen from the vagina. Transfer a

sample of the exudate to a microscope slide and

spread it to make a thin smear. Allow the smear

to air-dry, protected from insects and dust. Label

and deliver to the laboratory with the wet prep-

aration.

Appearance and pH of vaginal discharge in Candida,

Trichomonas, and Gardnerella infections:

T.vaginalis: Yellow-green purulent discharge with pH over

C.albicans: White odourless discharge with pH below 5*

G.vaginalis: Grey, offensive, smelling thin discharge with

pH over 5* (Fishy ammoniacal smell becomes

more intense after adding a few drops of 10%

potassium hydroxide).

*The normal reaction of vaginal discharge (puberty to

menopause) is pH 3.0–3.5. The pH can be measured using

Whatman pH papers (see p. 174 in Part 1 of the book)

Collection of specimen to detect T. pallidum

Todetect motile T. pallidum spirochaetes, a specimen

must be collected before antibiotic treatment.

1 Wearing protective rubber gloves, cleanse

around the ulcer (chancre) using a swab moist-

ened with physiological saline. Remove any scab

which may be present.

Caution: T. pallidumspirochaetes are highly infec-

tious.

2 Gently squeeze the lesion to obtain serous ﬂuid.

Collect a drop on a cover glass and invert it on a

microscope slide.

Note:The cover glass and slide must be completely clean.

3 Immediately deliver the preparation to the lab-

oratory for examination by dark-ﬁeld microscopy

(see later text).

Collection of specimen to detect

K. granulomatis

1 Cleanse around the ulcerated area using a swab

moistened with physiological saline.

2 Pinch off a small piece of tissue from the edge or

base of a lesion. Crush this between two micro-

scope slides.*

*Technique recommended by Richens.

3 Label the slides and deliver them to the labora-

tory as soon as possible. When a delay is

anticipated, ﬁx the smears with absolute

methanol (methyl alcohol) for 1–2 minutes.

MICROBIOLOGICAL TESTS 93

7.10

STIs which can be investigated in district laboratories

● Gonorrhoea: In symptomatic males, using Gram smear to detect Gram negative diplococci in pus

cells present in urethral discharge or ﬁrst voided urine.

Note: Pus cells without intracellular diplococci indicate non-gonococcal urethritis.

● Syphilis: By detecting motile spirochaetes in serous ﬂuid from genital chancre or skin lesion examined

by dark-ﬁeld microscopy. Reagin and speciﬁc treponemal antibody tests are used to diagnose syphilis

serologically and screen pregnant women for infection.

● Trichomoniasis: By detecting motile T. vaginalistrophozoites in fresh wet vaginal preparations.

● Candidiasis: By detecting yeast cells and pseudohyphae in wet vaginal preparations or Gram stained

smears.

● G. vaginalis bacterial vaginosis: By examining Gram stained smears of vaginal discharge (ﬁshy odour,

watery, non-inﬂammatory, pH over 5) for epithelial cells with adhering polymorphic bacteria (clue

cells).

● Granuloma inguinale (donovanosis): By detecting intracellular bipolar stained cocco-bacilli (K. granu-

lomatis) in Giemsa or RapiDiff stained preparations of ulcer material.

● Chlamydia: When rapid antigen test is available (see subunit 7.18.37).

In district laboratory with culture facilities or specialist sexually transmitted diseases (STD) laboratory:

● Gonorrhoea: Particularly in women with suspected urogenital infection, using a selective enriched

medium to isolate N. gonorrhoeae.

HIV: The laboratory diagnosis of HIV infection is described in subunit 7.18.55.

Laboratory diagnosis of other STIs in specialist laboratories

The following STIs require expensive technologies or the facilities of a specialist laboratory for their

diagnosis:

● Urogenital Chlamydia infections (C. trachomatis serovars D–K)

Usually diagnosed by tissue culture or immunologically (e.g. ELISA, IFAT). PCR technologies have also

been developed. Simple to perform rapid antigen tests are available (see subunit 7.18.37).

● Chancroid: Usually diagnosed culturally by isolating H. ducreyi using a selective enriched

medium.

● LGV (C. trachomatis serovars L1–L3): Diagnosed immunologically or by tissue culture.

● Genital herpes (HHV-1, HHV-2) infection: Can be diagnosed immunologically or by tissue culture.

Uterine curettings (scrapings) for histological examination

Immediately after collection, place the curettings in a con-

tainer of formol saline ﬁxative (Reagent No. 38). Use about

ten times the volume of ﬁxative to specimen.

Label, and send with a request form to a Histology

Laboratory. Instructions regarding the packaging and mailing

of pathological specimens can be found in subunit 7.1.

Cervical smear to be examined for malignant cells

A smear to be examined for malignant cells must be spread

thinly and evenly on a slide and while still wet, immersed in a

container of alcohol ﬁxative (Reagent No. 8) for at least 30

minutes. Remove the smear, and allow to air-dry. Send with a

request form to a Cytology Laboratory.

LABORATORY EXAMINATION OF

UROGENITAL SPECIMENS

1 Culture the spe cimen

Modiﬁed New York City (MNYC) or Thayer

Martin medium

– Inoculate the specimen on MNYC medium (see

No. 58) or other selective enriched culture

medium suitable for isolating N. gonorrhoeae

from urogenital specimens such as Thayer

Martin medium (see No. 79).

Note: When using MNYC medium, colonies can be tested

directly for beta-lactamase production and utilization of

carbohydrates, whereas colonies from Thayer Martin

medium require subculturing ﬁrst.

– With as little delay as possible, incubate the inoc-

ulated plate in a moist* carbon dioxide enriched

atmosphere (see subunit 7.4) at 35–37C for up

to 48 h, examining for growth after overnight

incubation.

*Place a damp piece of ﬁlter paper in the bottom of the

candle jar.

ADDITIONAL

Blood agar (aerobic and anaerobic),

MacConkey agar, and cooked meat medium

when puerperal sepsis or septic abortion is

suspected

– Inoculate the specimen on two plates of blood

agar (see No. 16) and incubate one anaerobically

and the other aerobically at 35–37C overnight.

– Inoculate the specimen on MacConkey agar (see

No. 54) and incubate the plate aerobically at

35–37C overnight.

– Inoculate the specimen in cooked meat medium

(see No. 27) and incubate at 35–37C, subcul-

turing as indicated at 24 h, 48 h and 72 h.

2 Examine the spe cimen microscopically

Gram smear

Fix the smear with methanol (see subunit 7.3.2), and

stain by the Gram technique (see subunit 7.3.4).

Using the 40 and 100 objectives, examine the

smear for pus cells and bacteria.

Day 1

94 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.10

Smear from a patient with suspected gonorrhoea

Look for pus cells containing Gram negative diplo-

cocci that could be N. gonorrhoeae (see colour Plate

43). When the pus cells have been damaged, the

organisms may be seen lying outside the pus cells

(extracellular).

Presumptive diagnosis of gonorrhoea from a Gram smear

Whenintracellular Gram negativediplococci are seen in auro-

genitalsmear, a presumptive diagnosis of gonorrhoea can be

made.Such a diagnosis is often possible for male patients but

moredifﬁcult for female patients (see Notes on pathogens).

Non-gonococcal urethritis

Chlamydia trachomatisis a common cause of non-gonococcal

urethritis (NGU), particularly in men (see subunit 7.18.37). A

presumptive diagnosis of NGU can be made when a urethral

smear contains 5 or more pus cells and no intracellular Gram

negative diplococci (or more than 15 pus cells in a ﬁrst voided

urine specimen from a male patient).

Vaginal smear from a patient with suspected bacterial

vaginosis or candidiasis

Look especially for:

● Large Gram positive yeast cells and pseudohy-

phae that could be C. albicans or other Candida

species (see colour Plate 72).

● Clue cells, i.e. epithelial cells with adhering Gram

negative short bacilli and Gram variable cocco-

bacilli that could be G. vaginalis and anaerobes

(see colour Plate 45). The margins of the epithe-

lial cells are often obscured. With bacterial

vaginosis, there are few or no pus cells and lac-

tobacilli are usually absent. When clue cells

predominate, report the smear as ‘Clue cells

seen, suggestive of bacterial vaginosis’.

Smear from a patient with suspected puerperal sepsis

or septic abortion

Look especially among pus cells for:

● Large Gram positive rods with straight ends, that

could be C. perfringens (see colour Plate 34).

● Gram positive streptococci, that could be S. pyo-

genes or other beta-haemolytic streptococci (see

colour Plate 25).

● Gram positive cocci resembling S. aureus (see

colour Plate 24).

● Gram negative rods, that could be Bacteroides or

coliforms.

Wet (saline) preparation to detect T. vaginalis

To detect motile T. vaginalistrophozoites, the prep-

aration must be examined as soon as possible after

the specimen is collected. Examine the preparation

using the 10 and 40 obje ctives, with the con-

denser iris diaphragm closed sufﬁciently to give good

contrast. The preparation must not be too thick.

T. vaginalis trophozoites are a little larger than pus

cells, measuring 10–20 m in diameter. They are

round or oval in shape and move by means of an

undulating membrane and ﬂagella. There are 4

anterior fragella and a ﬁfth ﬂagellum forms an undu-

lating membrane. An axostyle protrudes from the

end of the organism. A trophozoite is shown in Plate

7.15 in subunit 7.12.

A careful search is often necessary to detect the ﬂa-

gellates among the pus cells. Movement is often

slight (on the same spot) and not progressional.

Note: When more than 10 minutes have passed

since the collection of the specimen, motility can

often be increased by incubating the preparation at

35–37C for a few minutes (in a petri dish contain-

ing a damp piece of cotton-wool). When the

organisms are not found, culture in Diamond or

Feinberg medium should be considered (the organ-

isms can be detected after 2–4 days incubation).

Acridine orange (ﬂuorochrome) stained preparation to detect

T.vaginalis, yeast cells, and clue cells

When facilities for ﬂuorescence microscopy are available,

examination of an acridine orange stained vaginal smear is

recommended because T.vaginalis, yeast cells, and clue cells

can be rapidly detected. The acridine orange ﬂuorescence

technique is described in subunit 7.3.11 and stained tri-

chomonads are shown in colour Plate 44.

ADDITIONAL

Dark-ﬁeld preparation to detect motile

T. pallidum

A preparation for the detection of motile trepo-

nemes must be examined as soon as possible after

the specimen is collected (within 15 minutes) and

before the patient has been treated (or self-treated)

with antibiotics. Handle the preparation with care

because the organisms are highly infectious.

Examine the preparation by dark-ﬁeld

microscopy using the 10and 40 objectives (see

pp. 122–123 in Part 1 of the book). A good light

source is essential.

Use of a dark-ﬁeld stop to obtain dark-ﬁeld microscopy

To detect T.pallidum spirochaetes it is not necessary to use an

expensive dark-ﬁeld condenser. The spirochaetes can be seen

using the 40objective and therefore a dark-ﬁeld stop for this

objective positioned in the ﬁlter holder below the condenser

of a standard microscope can be used (see pp. 122–123 in Part

1 of the book).

The preparation must be sufﬁciently thin to obtain

good dark-ﬁeld. Remove excess ﬂuid by pressing a

sheet of blotting paper on top of the preparation.

T. pallidum: Look for brightly illuminated, thin,

delicate, tightly wound spirochaetes, measuring

6–15 m long with 8–14 evenly sized coils (see

colour Plate 60). They have a bending and slowly

MICROBIOLOGICAL TESTS 95

7.10

rotating motility and may be seen lengthening and

shortening. The spirochaetes of T. pallidum require

differentiation from saprophytic genital spirochaetes.

These are of variable size, thicker than T. pallidum,

have fewer coils and a different motility.

Giemsa stained preparation to detect

K. granulomatis

Fix the smear(s) with methanol as described in

subunit 7.3.2 and stain by the Giemsa technique

(see 7.3.10) or RapiDiff technique. Using the 10

and 40 objectives, examine the smear for

macrophage cells containing K. granulomatis coc-

cobacilli, also referred to as Donovan bodies.

Use the 100 objective to examine the cocco-

bacilli for bipolar staining. The organisms are often

described as having the appearance of closed safety

pins (see colour Plate 46).

Antigen test to detect Chlamydia: Details can

be found in subunit 7.18.37.

3 Examine and report the cultures

MNYC and Thayer Martin cultures

N. gonorrhoeae produces small raised, grey shiny

colonies on MNYC medium (see colour Plate 42)

and Thayer Martin medium (see colour Plate 41)

after overnight incubation.

– Perform an oxidase test (see subunit 7.5.8).

Neisseriae are strongly oxidase positive.

– Gram stain the colonies. N. gonorrhoeae appears

as a Gram negative coccus.

Note:The accuracy of a diagnosis of gonorrhoea based on

the isolation of oxidase-positive Gram negative cocci

from a selective medium is as high as 99% from urethral

and cervical sites. Conﬁrmation of the diagnosis is by

biochemically testing and serotyping the isolate.

– Test the colonies for beta-lactamase production

as described at the end of subunit 7.16.

ADDITIONAL

Blood agar and MacConkey agar cultures

Look for colonies that could be:

 Streptococcus pyogenes or other beta-haemolytic

streptococci, see subunit 7.18.2

 Staphylococcus aureus, see subunit 7.18.1

 Clostridium perfringens, see subunit 7.18.9

 Proteus species, see subunit 7.18.18

 Enterococcus, see subunit 7.18.5

 Escherichia coli, see subunit 7.18.14

Day 2 and Onwards

96 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.10

Summary of Microbiological Examination of Urogenital Specimens

ADDITIONAL INVESTIGATIONS

1 Culture  MNYC medium or When puerperal or septic abortion

Specimen

Thayer-Martin medium is suspected:

– Incubate in CO

 Blood agar (2 plates)

(moist environment)

– Incubate aerobically

– Incubate anaerobically

 MacConkey agar

– Incubate aerobically

 Cooked meat medium

– Incubate overnight.

Subculture as indicated at

24 h, 48 h, 72 h.

2 Examine  Gram smear  Dark-ﬁeld: When syphilis is

Microscopically

– Urethral: Intracellular suspected

Gram negative diplococci

 Giemsa smear:

– Vaginal: Yeast cells

When K.granulomatis infection

(candidiasis) Clue cells

(donovanosis) is suspected

(bacterial vaginosis)

 Cervical smear(s) sent to

– Vaginal/cervical: Pus cells

histology/cytology laboratory:

and bacteria associated with

When malignancy is suspected

puerperal sepsis and septic

abortion

 Wet preparation

Motile T.vaginalis

Day 1

3 Examine and  MNYC plate or Thayer-Martin  Blood agar, MacConkey agar

Report Cultures

plate plates

– Examine for N. gonorrhoeae – Look especially for:

Colonies resembling

S.pyogenes

N. gonorrhoeae:

S.aureus

● Oxidase test

C.perfringens

● Gram stain colonies

Proteus

● Beta-lactamase test

Enterococcus

E.coli

Bacteroides

– Antimicrobial susceptibility

tests as required

Day 2 and Onwards

Note: Other bacteria that can b e isolated

from cervical swabs taken from patients with puer-

peral sepsis or septic abortion are listed at the

beginning of this subunit under ‘Possible

Pathogens’.

Important: When the plate cultures are sterile but

pus cells were seen in the original Gram smear, or

the organisms that have grown overnight do not

resemble morphologically those seen in the Gram

smear, subculture the cooked meat medium and

reincubate the original blood agar plates for a

further 24 hours. Organisms such as Bacteroidesare

slow-growing.

REFERENCE

1 Richens J. Donovanosis (granuloma inguinale). Chapter

7.7 in Sexually transmitted infections and AIDS in the

tropics(see Further information).

FURTHER INFORMATION

World Health Organization and Reproductive Health and

Research. Sexually transmitted and other reproductive

tract infections – A guide to essential practice. WHO, Geneva,

2005.

Van Dyck E, Meheus AZ, Piot P. Laboratory diagnosis of

sexually transmitted diseases. WHO, 1999. ISBN 92 4 154501 1.

Available from WHO Publications, 1211 Geneva, 27-

Switzerland. An excellent colour illustrated text for

laboratory personnel.

Arya UP, Hart CA.Sexually transmitted infections and AIDS

in the tropics. CAB1 Publishing, 1998. ISBN 085199 2625.

Available from CAB1 Publishing, Wallingford, Oxon OX10

8DE, UK.

Professor RC Ballard. Syndromic case management of STDs

in Africa.Flow charts and treatment options. Available from

the National Reference Centre for STDs, South African

Institute for Medical Research, PO Box 1038, Johannesburg,

2000, South Africa.

HIV/AIDS: Readers are referred to the end of subunit

7.18.55.

7.11 Examination of faecal

specimens

Possible pathogens

 BACTERIA

Gram positive Gram negative

Clostridium perfringens Shigella species

types A and C Salmonella serovars

Clostridium difﬁcile Campylobacter species

Bacillus cereus (toxin) Yersinia enterocolitica

MICROBIOLOGICAL TESTS 97

7.10– 7.1 1

Staphylococcus aureus Escherichia coli

(toxin) (ETEC, EIEC, EPEC, VTEC)

Vibrio cholerae 01, 0139

Other Vibrio species

Aeromonas species

Also Mycobacterium tuberculosis

 VIRUSES

Mainly rotaviruses and occasionally Norwalk

agent, adenoviruses, astrovirus, calcivirus and

coronavirus.

 PARASITES

Entamoeba histolytica, Giardia lamblia, intestinal

coccidia (Isospora, Cryptosporidium, Cyclospora)

and other protozoan enteric pathogens are

described in subunit 5.4 in Part 1 of the book.

Important helminth enteric pathogens are listed

on pp. 208–209 in Part 1 and described in

subunit 5.5.

Notes on pathogens

● Acute infective diarrhoea and gastroenteritis (diarrhoea

with vomiting) are major causes of ill health and pre-

mature death in developing countries in situations

where water supplies are contaminated and sanitation

is poor. Loss of water and electrolytes from the body

can lead to severe dehydration which if untreated can be

rapidly fatal in young children, especially those that are

malnourished, hypoglycaemic, and generally in poor

health.

Invasiveorganisms such as shigellae, campylobacters,

some salmonellae, and E. histolytica are associated with

dysentery (passing of blood and mucus in stools).

Organisms such as rotaviruses, V. cholerae, and entero-

toxigenicE. coli, cause watery (secretory) diarrhoea.

Diarrhoea may also be caused by intestinal worms,

post-infective tropical malabsorption, lactase deﬁciency,

antibiotic or other drug therapy which alters the normal

intestinal ﬂora, and from dietary causes including gluten

intolerance.

Diarrhoea is also associated with HIV disease,

malaria, severe malnutrition, pneumonia, hepatitis, cir-

rhosis of the liver, inﬂammation of the pancreas,

tuberculosis of the intestine, colitis, previous surgery of

the bowel, and malignant diseases of the intestinal tract.

● Shigella species: S. dysenteriae, S. ﬂexneri, S. boydii, and

S. sonnei are described in subunit 7.18.15. Dysentery

caused by shigellae is referred to as bacillary dysentery or

shigellosis. WHO estimates that Shigellaspecies cause at

least 50% of the cases of bloody diarrhoea in young

children in developing countries. S.dysenteriae serotype 1

(Sd 1) is particularly virulent, causing endemic and

epidemic dysentery with high death rates. It is highly

infectious. Resistance to commonly available antimicro-

bials is an increasing problem, particularly in dysentery

caused by Sd 1.

● Salmonella organisms are described in subunit 7.18.16.

S.Typhi and S. Paratyphi cause enteric fever (typhoid and

paratyphoid) which is endemic in many tropical and

developing countries. Other salmonellae cause food-

poisoning and bacteraemia.

● Campylobacter species are described in subunit 7.18.21.

C. jejuni and C. coli are common causes of enteritis in

young children in developing countries.

● V. cholerae serogroups (serovars) 01 and 0139 cause

endemic and epidemic cholera. In recent years, epidemic

cholera caused by V.cholerae 01 (biotype El Tor) has

spread from Asia and Indonesia to many African coun-

tries, Far East, South Paciﬁc, South America, and Mexico.

Epidemic cholera caused by the serotype 0139, emerged

in Bengal in 1992 and spread rapidly to other parts of

India, Bangladesh, Pakistan, Thailand, Nepal, Malaysia,

Burma, Saudi Arabia, and China.

The severe dehydration, vomiting, abdominal pain,

and acidosis associated with cholera are due to the action

of an exotoxin produced by the organism (cholera toxin)

which causes water and electrolytes to ﬂow into the bowel

lumen (see subunit 7.18.19). In severe infections, typical

‘rice water’ stools containing many vibrios are passed con-

tinuously, necessitating urgent ﬂuid replacement therapy

to prevent collapse and death. V.cholera 01 El Tor is

becoming increasingly resistant to commonly available

antimicrobials and V.cholerae0139 is also becoming resis-

tant to some antimicrobials.

● V. parahaemolyticus has been reported as causing food-

poisoning (through contaminated seafood) in many parts

of the world including Africa, Asia, America, and

Europe.

● Strains of E. coli recognized as causing diarrhoeal disease

include enterotoxigenic E. coli (ETEC), enteropatho-

genic E. coli (EPEC), enteroinvasive E. coli (EIEC) and

verotoxigenic E. coli (VTEC), also referred to as

enterohaemorrhagic E. coli (EHEC). These and other

diarrhoea causing E. coli strains are described in

subunit 7.18.14.

● Y. enterocolitica has been reported as causing gastroen-

teritis in Africa, Japan, Europe, and Canada. The

organism is invasive and some strains are toxigenic.

Identiﬁcation of the organism is described at the end of

subunit 7.18.22.

● C. perfringens type A causes food-poisoning by secreting

enterotoxin in the intestine during sporulation. Alpha-

toxin is the main lethal toxin produced (see subunit

7.18.9).

C.perfringens type C is associated with severe jejuni-

tis (enteritis necroticans) which is referred to in Papua

New Guinea as pigbel. It is a cause of death in young

children especially in Papua New Guinea (Highlands),

China, the Solomon Islands, Bangladesh, and some parts

of East Africa. Infection is by the ingestion of contami-

nated pig meat. A lethal beta-toxin is produced (see

subunit 7.18.9).

● C. difﬁcile causes antimicrobial associated diarrhoea and

sometimes pseudomembranous colitis, a rare and

occasionally fatal condition. Investigation of the disease

requires the facilities of a specialist microbiology labora-

tory.

● S.aureus food-poisoning is caused by the ingestion of pre-

formed toxin in contaminated food (often dairy

products). Occasionally, staphylococcal enterocolitis is a

complication of broad-spectrum antibiotic therapy.

● B.cereus food-poisoning is caused by the ingestion of pre-

formed toxin usually in rice or other cereals which have

been cooked and then stored for several days in warm

temperatures.

98 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.11

● Rotavirus is the commonest cause of acute secretory diar-

rhoea in young children (6 months–3 years). Diarrhoea is

the result of loss of extracellular ﬂuid, due to impaired

absorption. Diarrhoea and vomiting can lead to severe

dehydration. Most infections are accompanied by fever.

● Persistent diarrhoea, often leading to diarrhoea-wasting

syndrome (‘slim disease’) is common in AIDS. It is

thought to be due in part to opportunistic protozoal

pathogens such as Cryptosporidium, Cyclospora,Isospora

and microsporidia. Bacterial infections associated with

diarrhoea in HIV/AIDS patients include Salmonella,

Campylobacter, Shigellaand mycobacteria.

Commensals

The normal microbial ﬂora of the gastrointestinal

tract is greatly inﬂuenced by diet. Microorganisms

which may form part of this normal ﬂora include:

– Coliform bacilli and species of Proteus,

Pseudomonas, Clostridium, Bacteroides, Entero-

coccus, and lactobacilli.

– Also Mycoplasma, Candida species and a variety

of protozoa and viruses.

COLLECTION AND TRANSPORT OF FAECES

Faeces for microbiological examination should be

collected during the acute stage of diarrhoea.

In a hospital with a microbiology laboratory

1 Give the patient a clean, dry, disinfectant-free

bedpan or suitable wide-necked container in

which to pass a specimen. The container need

not be sterile. Ask the patient to avoid contami-

nating the faeces with urine.

2 Transfer a portion (about a spoonful) of the

specimen, especially that which contains mucus,

pus, or blood, into a clean, dry, leakproof con-

tainer.

Worms and tapeworm segments: When the specimen

contains worms or tapeworm segments, transfer these to

a separate container and send them to the laboratory for

identiﬁcation.

3 Write on the request form the colour of the

specimen and whether it is formed, semiformed,

unformed, or ﬂuid. Report also if blood, mucus,

worms, or tapeworm segments are present.

4 Label the specimen and send it with a request

form to reach the laboratory within 1 hour (if a

delay longer than 1 hour is anticipated, collect

the specimen in Cary-Blair medium, see later

text).

Rectal swabs: Only when it is not possible to obtain faeces,

collect a specimen using a cotton wool swab. Insert the swab

in the rectum for about 10 seconds. Care should be taken to

avoid unnecessary contamination of the specimen with

bacteria from the anal skin.

Important: When the specimen contains blood or

amoebic dysentery is suspected, deliver it to the

laboratory as soon as possible. A fresh specimen is

required to demonstrate actively motile amoebae

and also to isolate shigellae.

In a health centre for transport to a

microbiology laboratory

1 Request a specimen from the patient as

described previously under the hospital collec-

tion of faeces.

Note: Leaves, cardboard boxes, plastic bags are

not suitable for the collection of faeces.

2 Transfer a portion of the faeces to a cotton wool

swab. Insert the swab in a container of sterile

Cary-Blair transport medium (see No. 22),

breaking off the swab stick to allow the bottle top

to be replaced tightly.

Salmonella serovars, Shigella, Vibrio and Yersinia

species survive well in Cary-Blair medium for up

to48 hours, andCampylobacter for upto 6 hours.

Note: Merthiolate iodine formaldehyde (MIF) solution

must not be used because MIF kills living organisms. MIF

is used as a ﬁxative for protozoal parasites.

When cholera is suspected: Transfer about 1 ml of

specimen into 10 ml of sterile alkaline peptone

water (see No. 10) and label. The specimen

should reach the Microbiology Laboratory within

8 hours of collection.

3 Write a description of the specimen on the

request form (see previous text).

Note: When worms or tapeworm segments are

present, transfer these (using forceps) to a container

of physiological saline and send to the laboratory for

identiﬁcation.

Instructions regarding the packaging and dispatch of

specimens can be found at the end of subunit 7.1.

LABORATORY EXAMINATION OF

FAECES

Role of microbiological laboratory in investigating infective

diarrhoeal disease

With most patients, diarrhoea is self-limiting and can be

treated with rehydration and other supportive therapy

without the need for antimicrobials and microbiological inves-

tigations. The microbiological examination of faecal

specimens is mainly undertaken:

MICROBIOLOGICAL TESTS 99

7.11

 To investigate outbreaks of dysentery (mainly shigel-

losis), cholera, and other acute bacterial infective

diarrhoeal disease of public health concern.

 To assist the central public health laboratory in the sur-

veillance of endemic shigellosis and salmonellosis

(including susceptibility of pathogens to antimicrobials).

 To diagnose symptomatic amoebic dysentery, giardiasis

and other locally important intestinal parasitic infections.

Recommended reading: Mundy C, Shears P. Diarrhoeal

disease outbreak investigation and surveillance. Diagnostics

in Africa, Africa Health, pp. x–xiii, September, 1994.

1 Describ e the appearance of the specimen

– Colour of the specimen.

– Whether it is formed, semiformed, unformed or

ﬂuid.

– Presence of blood, mucus or pus

– Presence of worms, e.g. Enterobius vermicularis,

Ascaris lumbricoides, or tapeworm segments e.g.

Taenia species.

Day 1

Appearance of faecal specimens in some diseases

Appearance Possible Cause

Unformed, containing pus ● Shigellosis

and mucus mixed with blood ● E1EC dysentery

● Campylobacter enteritis

Unformed with blood and ● Amoebic dysentery

mucus (acid pH)

Unformed or semiformed, ● Schistosomiasis

often with blood and mucus

Bloody diarrhoea ● EHEC 0157 infection

(without pus cells) (haemorrhagic colitis)

Watery stools ● ETEC, EPEC diarrhoea

● Cryptosporidiosis

● Rotavirus enteritis

Rice water stools with ● Cholera

mucous ﬂakes

Unformed or watery and ● Salmonella infection

sometimes with blood, mucus,

and pus

Unformed, pale coloured, ● Giardiasis

frothy, unpleasant smelling ● Other conditions that

stools that ﬂoat on water cause malabsorption,

(high fat content) e.g. post-infective

tropical malabsorption

Fluid stools (containing ● Lactase deﬁciency

lactose with pH below 6)

Unformed or semiformed ● Melaena (gastrointestinal

black stools bleeding)

(positive occult blood) ● Hookworm disease

● Iron therapy

Normal faeces: Appear brown and formed or semi-

formed. Infant faeces are yellow-green and

semiformed.

2 Examine the spe cimen microscopically

Saline and eosin preparations to detect

E. histolytica and other parasites

– Place a drop of fresh physiological saline on one

end of a slide and a drop of eosin stain (Reagent

No. 36) on the other. Using a piece of stick or

wire loop, mix a small amount of fresh specimen

(especially mucus and blood) with each drop.

Cover each preparation with a cover glass.

Important: The eosin preparation must not be

too thick otherwise it will not be possible to see

amoebae or cysts.

– Examine the preparations using the 10 and

40 obje ctives with the condenser iris closed

sufﬁciently to give good contrast.

– Look especially for motile E. histolytica tropho-

zoites containing red cells, motile G. lamblia

trophozoites, motile Strongyloides larvae, and the

eggs and cysts of parasitic pathogens.

Note: The microscopical appearance of E. histolytica,

G. lamblia and other protozoal parasites are

described and illustrated in subunit 5.4 in Part 1 of

the book and Strongyloides larvae and the eggs of

helminths in subunit 5.5 in Part 1. Faecal concen-

tration techniques are described in subunit 5.3.

ADDITIONAL

Methylene blue preparation to detect faecal

leucocytes when the specimen is unformed

– Place a drop of methylene blue stain (Reagent

No. 51) on a slide. Mix a small amount of

specimen with the stain, and cover with a cover

glass.

– Examine the preparation for faecal leucocytes

using the 40 objective with the condenser iris

closed sufﬁciently to give good contrast.

– Report also the presence of red blood cells (RBC)

as these are often present with pus cells in

inﬂammatory invasive diarrhoeal disease (see fol-

lowing text).

Faecal leucocytes (WBCs): Look for mononu-

clear cells and polymorphonuclear cells (pus cells).

Mononuclear cells contain a nucleus which is not

100 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.11

lobed whereas polymorphonuclear cells contain a

nucleus which has two or more lobes (see colour

Plate 6). Sometimes the cells are too damaged to be

recognized (do not attempt to identify).

Pus cells are associated with bacteria that cause

inﬂammation of the large intestine (see following

text). Often red cells are also found. Mononuclear

cells are found mainly in typhoid and in some para-

sitic infections, including amoebic dysentery.

Causes of inﬂammatory diarrhoeal disease

Shigellaspecies

Campylobacterspecies

Salmonella(non-typhoid serovars)

E.histolytica

EIEC

Less common:

B.coli (see 5.4 in Part 1)

Y.enterocolitica

C.difﬁcile

C.perfringens (causing pigbel)

Aeromonasspecies

Basic fuchsinsmear to detect campylobacters

Prepare when the specimen is unformed and, or,

contains mucus, pus, or blood and is from a child

under 2 y.

– Make a thin smear of the specimen on a slide.

When dry, gently heat-ﬁx. Stain by covering the

smear with 10 g/l basic fuchsin* for 10–20

seconds. Wash well with water and allow to air-

dry.

*Dissolve 1 g basic fuchsin in 100 ml of water, and ﬁlter.

– Examine the smear for campylobacters using the

100  oil immersion objective.

Campylobacter organisms: Look for abundant small,

delicate, spiral curved bacteria (often likened to gull

wings), S-shapes, and short spirochaetal forms as

shown in colour Plate 15.

Note: Examination of stained faecal smears for campylobac-

ters has been shown to be a sensitive method for the

presumptive diagnosis of campylobacter enteritis. Culture of

Campylobacterspecies is described in subunit 7.18.21.

Motility test and Gram stained smear when

cholera is suspected

Examine an alkaline peptone water culture (sample

from the surface of the culture) for vibrios showing

a rapid and darting motility. The preparation is best

examined using dark-ﬁeld microscopy but the

vibrios can also be seen using transmitted light.

Techniques for detecting motile bacteria are

described in subunit 7.3. Experience is required to

identify the characteristic motility of V. cholerae.

Examine also a Gram-stained smear of the culture

Note; Blood can also be found in the stools of patients with

haemorrhoids, ulcerative colitis, or tumours of the intestinal

tract.

for Gram negative vibrios (use 1 in 10 dilution of

carbol fuchsin as the counterstain instead of neutral

red), see colour plate 7.

Use of V. cholerae 0 (group 1) antiserum to immobilize

vibrios

Cholera can be caused by serogroups 01 and 0139 and there-

fore a negative test cannot exclude cholera when V.cholerae0

group 1 antiserum only is used in an immobilization test.

Some workers have also found the test to be unreliable

because the vibrios can be immobilized by preservative in the

antiserum.

Antigen detection: A rapid, simple to perform

dipstick test to detect V. cholerae 01 and 0139 in

faeces has been developed. The test is described in

subunit 7.18.19.

3 Culture the spe cimen

When the specimen is formed or semiformed, make

a thick suspension of it in about 1 ml of sterile

peptone water.

Xylose lysine deoxycholate (XLD) agar

– Inoculate a loopful of fresh emulsiﬁed faeces or a

ﬂuid specimen on XLD agar (see No. 90).

– Incubate the XLD agar plate aerobically at

35–37°C overnight.

XLD agar:This selective medium is recommended for the iso-

lation of salmonellae and particularly shigellae from faecal

specimens. It contains the indicator phenol red which is red at

an alkaline pH (medium is pH 7.4) and yellow at an acid pH.

Shigellae form pink-red colonies because they do not

ferment xylose, lactose, or sucrose (except some S. sonnei

strains).

Salmonellae also form pink-red colonies even though

they ferment xylose with acid production. This is because they

break down the amino acid lysine which gives an alkaline

reaction. Hydrogen sulphide (H

S) producing salmonellae

form red colonies with black centres.

Some Proteusstrains and Edwardsiella species form pink-

red colonies with black centres. Escherichia coli, Enterobacter

species, and some other enterobacteria produce yellow

colonies due to carbohydrate fermentation.

Note: Some workers also recommend the use of a less selec-

tive medium such as MacConkey agar in addition to XLD

agar.

ADDITIONAL

Alkaline peptone water and TCBS agar when

cholera is suspected

– Inoculate several loopfuls of specimen in alkaline

(pH 8.6) peptone water (see No. 10), and

incubate at 35–37°C for 5–8 hours.

Note: Prior enrichment in alkaline peptone water is not

necessary if the specimen is likely to contain large

numbers of vibrios (e.g. in acute cholera). Alkaline

peptone water is a useful transport medium for

V.cholerae.

MICROBIOLOGICAL TESTS 101

7.11

– Subculture several loopfuls of the peptone water

culture (taken from the surface) on thiosulphate

citrate bile-salt sucrose (TCBS) agar (see No. 81).

Incubate aerobically at 35–37°C overnight.

TCBS medium: The choice of TCBS agar as a primary selec-

tive medium for isolating V.cholerae and other Vibrio species

is discussed in subunit 7.18.19.

Sorbitol MacConkey agar, when an outbreak

of E. coli 0157 is suspected

– Inoculate a loopful of specimen on sorbitol

MacConkey agar (see No. 77).

– Incubate the plate aerobically at 35–37°C

overnight.

Sorbitol MacConkey agar

This MacConkey medium contains the carbohydrate sorbitol

instead of lactose. E.coli 0157 produces colourless colonies on

the medium because it does not ferment sorbitol. Most other

E.coli strains and other enterobacteria ferment sorbitol, pro-

ducing pink colonies. Sorbitol MacConkey agar is therefore a

useful way of screening for E.coli 0157 (reported as having a

speciﬁcity of 85% and sensitivity of 100%).

Culture of Campylobacter

This is described in subunit 7.18.21.

Investigation of food-poisoning caused by

clostridia, S. aureus and B. cereus

For the isolation of pathogens and, or, toxins that

cause clostridial, staphylococcal and Bacillus cereus

food-poisoning, readers are referred to Collins and

Lyne’s Microbiological Methods, and other microbi-

ology textbooks (see Recommended Books listed on

p. 379).

4 Examine and report the cultures

XLD agar culture

Look for colonies that could be Shigella or

Salmonella. Shigella and H

S negative strains of

Salmonella produce 1–2 mm diameter red colonies

on XLD agar. Red colonies with black centres are

produced by H

S positive salmonellae, e.g. strains of

S. Typhimurium.

Proteus, Providencia and Pseudomonas organisms may also

produce red colonies on XLD agar. Some Proteus strains

are also H

S producing and form red colonies with black

centres.

Note: Salmonella and Shigella XLD cultures are

shown in colour Plates 11 and 12.

On MacConkey agar, shigellae, and salmonellae

and other non-lactose fermenting organisms,

produce colourless colonies. E. coli and other lactose-

fermenting organisms produce pink colonies.

Day 2 and Onwards

Identiﬁcation of suspect Salmonella and Shigella

isolates

Perform a urease test using urea broth or a Rosco

urease identiﬁcation tablet as described in subunit

7.5.9.

A positive urease test within 2–4 h indicates that the

organism is probably Proteus. No further tests are

required.

When the urease test is negativeat 4 hours, proce ed

as follows:

1 Perform indole and lysine decarboxylase (LDC)

tests as described in subunit 7.5.6.

2 Inoculate a tube of Kligler iron agar (see No. 45).

Use a sterile straight wire, stab ﬁrst the butt and

then streak the slope. Close the tube with a

loose-ﬁtting cap and incubate at 35–37°C

overnight.

Results

LDC Shigella are LDC negative.

Salmonella serovars are LDC positive

except S. Paratyphi A which is LDC

negative.

Indole S. sonneiis indole negative. Other

shigellae give variable indole reactions

(see subunit 7.18.15)

Salmonellaserovars are indole negative.

KIA Salmonella and Shigella organisms

produce a pink-red slope and yellow

butt. Many salmonellae also produce

blackening due to hydrogen sulphide

production and cracks in the medium

due to gas production from glucose

fermentation. Salmonella Typhi

produces only a small amount of

blackening and no cracks in the

medium. KIA reactions are shown in

colour Plate 13.

Note: The features of Salmonella serovars are

described in subunit 7.18.16 and Shigella species in

subunit 7.18.15.

Serological identiﬁcation of salmonellae and

shigellae

1 Test serologically any isolate giving reactions sug-

gestive of Salmonella or Shigella, using a slide

technique.

Note: The antisera required to identify Shigella species

are listed in subunit 7.18.15 and Salmonella serovars in

subunit 7.18.16. When antisera are not available, send

102 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.11

isolates of the organism (on nutrient agar) to the Regional

Public Health Laboratory for serotyping.

2 Emulsify a small amount of growth from the KIA

culture in a loopful of physiological saline on a

slide. Mix by tilting the slide backwards and

forwards for about 30 seconds. Examine for

agglutination against a dark background. When

there is agglutination (autoagglutination), the

strain is unsuitable for serological testing. A

nutrient agar culture should be sent for further

testing to a Reference Laboratory.

3 When there is no autoagglutination add one

loopful of test antiserum, and mix. Examine for

agglutination. A positive test will show strong

clear agglutination within 1 minute.

Interference by K antigens:Surface (K) antigens possessed by

some salmonellae and shigellae, for example the Vi antigen of

SalmonellaTyphi, can interfere with O antigen testing. When

this is suspected (e.g. positive Vi agglutination, but no agglu-

tination with O Group 1 antiserum), heat a saline suspension

of the organism in a container of boiling water for 30 minutes.

This will inactivate the surface antigens and enable O antigen

testing to be performed. Retest the heat-treated organisms

when the suspension has cooled.

TCBS agar culture

V. cholerae is sucrose fermenting and therefore

produces yellow 2–3 mm in diameter shiny colonies

on TCBS agar with a yellow colour in the medium,

as shown in colour Plate 8.

Note: With prolonged incubation (48 h or more) the

colonies may become green.

V. ﬂuvialis is also sucrose fermenting and may

occasionally be isolated as a pathogen from faecal

specimens (see subunit 7.18.19).

Vibrio parahaemolyticus is non-sucrose fermenting

and therefore produces green-blue 2–3 mm in

diameter colonies on TCBS agar, as shown in colour

Plate 10. V. mimicusalso produces non-sucrose fer-

menting green-blue colonies and is sometimes

isolated (see subunit 7.18.19).

Selectivity of TCBS

Very occasionally, Aeromonas species and enterococci

produce small yellow colonies on TCBS agar (see colour Plate

9). Proteus strains may produce yellow or yellow-green

colonies with black centres, and some Pseudomonas strains

form small green colonies.

Identiﬁcation of a suspect V. cholerae isolate

1 Examine a Gram stained smear of the culture for

Gram negative vibrios as shown in colour Plate 7.

The organisms may appear less curved after

culture.

2 Subculture the organism on a slope of nutrient

agar (use a heavy inoculum), and incubate for

4–6 hours.

3 Perform an oxidase test on the nutrient agar

culture (see subunit 7.5.8).

Note:It is not possible to perform an oxidase test directly

from a TCBS culture because the acid produced by the

sucrose fermenting colonies will inhibit the oxidase

reaction. Subculturing to nutrient agar is also required to

perform serological tests reliably.

Note: When the oxidase test is positive, presume the

isolate to be V. cholerae. It must be tested serologi-

cally (using nutrient agar culture) to conﬁrm the

organism is V. cholerae 01 or 0139.

MICROBIOLOGICAL TESTS 103

7.11

Antisera to identify V. cholerae 01 and 0139

In the investigation of a cholera epidemic, a prompt

diagnosis is required. With the rapid spread of

V. cholerae 0139, many laboratories will ne ed to use

both V. cholerae 01 antiserum and V. cholerae 0139

(Bengal) antiserum to identify isolates. There is no

need to differentiate between classical and El Tor

biovars (most outbreaks of V. cholerae01 are caused

by the El Tor biovar), nor between Ogawa and Inaba

serotypes (see subunit 7.18.19).

Note: Biochemical tests that can be used to differ-

entiate V. cholerae from other Vibrio species are

described in subunit 7.18.19.

Chart 7.8 Tests used to identify presumptively shigellae and salmonellae

KIA Medium Reactions

Motility Indole LDC Slope Butt Black Cracks

S) (Gas)

SHIGELLAE

Shigella dysenteriae  d  RY

Shigella ﬂexneri  d  RY

Shigella boydii  d  RY

Shigella sonnei RY

SALMONELLAE

Salmonella Paratyphi A RY



Salmonella Paratyphi B RY

Salmonella Paratyphi C RY



Salmonella Typhi RY

Weak

Other Salmonella

serovars 

RY

Key: KIA  Kligler iron agar, LDC  Lysine decarboxylase, d  different strains give different results,

R  Red-pink (alkaline reaction), Y  Yellow (acid reaction).

Notes

1 Some strains of serotype 6 produce gas. 2 Serotypes 13 and 14 produce gas. 3 About 12% of strains

produce H

S weakly. 4 A minority of strains do not produce H

S. 5 Salmonella Pullorum and Salmonella

Gallinarum are non-motile. 6 A minority of strains do not produce H

The reactions of salmonellae and shigellae compared with other enterobacteria are summarized in

Chart 7.10 in subunit 7.18.15.

104 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.11

Summary of the Microbiological Examination of Faecal Specimens

ADDITIONAL INVESTIGATIONS

Day 1

1 Describe

Specimen

 Report

– Colour

– Whether formed, semi-

formed, unformed, ﬂuid

– Presence of blood, mucus,

pus

– Presence of worms

2 Examine

Microscopically

 Saline and eosin:

To detect parasites

 Methylene blue:

To detect WBCs when the

specimen is unformed

 Basic fuchsin smear:

When Campylobacter enteritis

is suspected

 Motility test and Gram smear:

From alkaline peptone water

culture when cholera is

suspected

3 Culture

Specimen

 XLD agar

Incubate aerobically

 MacConkey agar

Incubate aerobically

 Alkaline peptone water and

TCBS agar:

When cholera is suspected

 Sorbitol MacConkey agar:

When infection with VTEC

0157 is suspected

Day 2 and Onwards

 Examine and

Report Cultures

 XLD and MacConkey agar

cultures

Look for: Salmonella and

Shigella:

– Exclude Proteus using

urease test

– Identify presumptively:

● LDC and indole

● Motility

● KIA

– Identify serologically

– Perform susceptibility

testing on Shigella isolates

 Examine TCBS culture

– Look for colonies that could

be V.cholerae

– Gram stain colonies

– Subculture on nutrient agar

– Perform oxidase test

– Identify serologically

 Examine sorbitol MacConkey

agar culture

– Look for colourless colonies

that could be VTEC 0157

– Perform 0157 latex

agglutination test

Sorbitol MacConkey agar culture

E. coli 0157 strains are non-sorbitol fermenting, pro-

ducing colourless colonies on sorbitol MacConkey

agar. Most other E. coli strains ferment sorbitol,

forming pink colonies.

Isolates suspected of being E. coli 0157 require 0

serotyping using a rapid latex agglutination test or

other technique to detect 0157 antigen. When the

test is positive, suspect the organism to be verotoxi-

genic E. coli 0157 strain if investigating an outbreak

of haemorrhagic colitis (bloody diarrhoea without

pus) often in association with haemolytic uraemic

syndrome (HUS). Conﬁrmation that the organism is

a vero-toxin (VT) producing E. coli 0157 strain

requires the facilities of a Microbiology Reference

Laboratory able to test for VT.

Availability of latex agglutination test kits to detect E. coli

0157 antigen

Manufacturers and suppliers of latex agglutination tests to

detect E.coli 0157 antigen include Pro-lab Diagnostics: 50 test

kit, code PL070, and Oxoid: 100 test kit, code DR0620M. The

shelf-life of most test kits is about 18 months. Details of

manufacturers can be found in Appendix 11.

Note: Further information on E. coli 0157 can be

found in subunit 7.18.14.

7.12 Examination of urine

Possible pathogens

 BACTERIA

Gram positive Gram negative

Staphylococcus Escherichia coli

saprophyticus Proteus species

Haemolytic streptococci Pseudomonas

aeruginosa

Klebsiella strains

*Salmonella Typhi

*Salmonella Paratyphi

*Neisseria gonorrhoeae

*These species are not primarily pathogens of the

urinary tract, but may be found in urine.

Also Mycobacterium tuberculosis, Leptospira

interrogans, Chlamydia, Mycoplasma and Candida

species.

 PARASITES

Schistosoma haematobium, Trichomonas vagi-

nalis, and occasionally Enterobius vermicularis,

Wuchereria bancrofti and Onchocerca volvulus.

MICROBIOLOGICAL TESTS 105

7.11– 7.12

Finding intestinal parasites in urine indicates

faecal contamination.

Note: S. haematobium, E. vermicularis, W. ban-

crofti and O. volvulus are describe d in Part 1 of

the book and T. vaginalisin subunit 7.10.

Notes on pathogens

● The presence of bacteria in urine is called bacteriuria. It is

usually regarded as signiﬁcant when the urine contains 10

organisms or more per ml (10

/1) in pure culture.

Infection of the bladder is called cystitis. It causes fre-

quency, dysuria (pain on passing urine), suprapubic pain,

sometimes haematuria and usually pyuria (increased

number of pus cells in urine). The term acute urethral

syndrome, (dysuria-pyuria) is used to describe acute

cystitis accompanied by pyuria but in which no bacteria

are detected by routine culture.

Infection of the kidney is called pyelonephritis. It

causes loin pain, pyuria, rigors, fever, and often bacter-

aemia. Risk of infection is increased when there is urine

retention due to the bladder not emptying completely, or

when urinary ﬂow is obstructed due to renal stones,

urinary schistosomiasis, enlarged prostate (commonest

cause of recurring UTI in men), or tumour. Persistent or

recurrent urinary tract infection (UTI) can lead to renal

failure.

● Urinary tract infections occur more frequently in women

than men due to the shortness of the female urethra.

Symptomatic and asymptomatic UTI is common in preg-

nancy. Undetected, untreated, asymptomatic bacteriuria

can lead to pyelonephritis later in pregnancy or during

puerperium.

● E. coli is the commonest urinary pathogen causing

60–90% of infections. Some strains are more invasive, e.g.

capsulated strains are able to resist phagocytosis, other

strains are more adhesive.

● UTIs caused by Pseudomonas, Proteus, Klebsiella species

and S.aureus, are associated with hospital-acquired infec-

tions, often following catheterization or gynaecological

surgery. Proteus infections are also associated with renal

stones.

● S. saprophyticus infections are usually found in sexually

active young women.

● Infection of the anterior urinary tract (urethritis) is

mainly caused by N. gonorrhoeae (especially in men),

staphylococci, streptococci, and chlamydiae.

● Candida urinary infection is usually found in diabetic

patients and those with immunosuppression.

● M. tuberculosis is usually carried in the blood to the

kidney from another site of infection. It is often suspected

in a patient with chronic fever when there is pyuria but

the routine culture is sterile.

Pyuria with a negative urine culture may also be

foundwhen there isinfection with Chlamydia trachomatis,

Ureaplasma,orN. gonorrhoeae, or when a patient has

takenantimicrobials.

● S. Typhi and S. Paratyphi can be found in the urine of

about 25% of patients with enteric fever from the third

week of infection. Excretion of bacteria is not associated

with pyuria.

Typhoid carriers may excrete S. Typhi in their urine

for many years. Carriers are common in schistosomiasis

endemic areas.

Commensals

The bladder and urinary tract are normally sterile.

The urethra however may contain a few commen-

sals and also the perineum (wide variety of Gram

positive and Gram negative organisms) which can

contaminate urine when it is being collected.

With female patients, the urine may become

contaminated with organisms from the vagina.

Vaginal contamination is often indicated by the

presence of epithelial cells (moderate to many) and

a mixed bacterial ﬂora.

Most urine specimens will contain fewer than 10

contaminating organisms per ml providing the urine

has been collected with care to minimize contami-

nation and the specimen is examined soon after

collection before the commensals have had time to

multiply signiﬁcantly.

OLLECTION AND TRANSPORT OF U RINE

Whenever possible, the ﬁrst urine passed by the

patient at the beginning of the day should be sent

for examination. This specimen is the most concen-

trated and therefore the most suitable for culture,

microscopy, and biochemical analysis.

Midstream urine (MSU) for microbiological examin-

ation is collected as follows:

In a hospital with a microbiology laboratory

1 Give the patient a sterile, dry, wide-necked, leak-

proof container and request a 10–20 ml

specimen.

Important: Explain to the patient the need to

collect the urine with as little contamination as

possible, i.e. a ‘clean-catch’ specimen.

Female patients: Wash the hands. Cleanse the

area around the urethral opening with clean

water, dry the area with a sterile gauze pad, and

collect the urine with the labia held apart.

Male patients: Wash the hands before collect-

ing a specimen (middle of the urine ﬂow).

Note: When a patient is in renal failure or a

young child, it may not be possible to obtain

more than a few millilitres of urine.

2 Label the container with the date, the name and

number of the patient, and the time of collection.

As soon as possible, deliver the specimen with a

request form to the laboratory.

When immediate delivery to the laboratory is not

possible, refrigerate the urine at 4–6°C. When a

delay in delivery of more than 2 hours is antici-

pated, add boric acid preservative to the urine

106 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.12

(see ‘Collection in a health centre’). Specimens

containing boric acid need not be refrigerated.

Deterioration of urine

The following changes occur when unpreserved urine is left at

room temperature:

● Any bacteria in the urine will multiply so that the bac-

terial count will be unreliable. When the organisms are

urease-producing, the ammonia released will increase the

pH of the specimen which will result in the destruction of

cells and casts. Bacteria will also break down any glucose

which may be present.

● When white cells, red cells, and casts are present, these

will begin to lyze especially in a concentrated specimen.

● The concentration of protein in the urine will be altered.

When bilirubin is present this may be oxidized to

biliverdin which will not be detected. Likewise, urobilino-

gen will not be detected because it will be oxidized to

urobilin.

In a health centre for dispatch to a

microbiology laboratory

1 Give the patient a sterile, dry, leak-proof con-

tainer with instructions on how to collect a

clean-catch MSU (see previous text).

2 Add a measured amount of boric acid powder

(0.1 g/10 ml of urine) to preserve the specimen,

and mix well.

Note: A simple way of measuring routinely the

boric acid is to use a small narrow tube (precipi-

tin tube), marked to hold 0.2 g of the chemical

(sufﬁcient for 20 ml urine). This ‘measuring-tube’

can be kept attached to the neck of the boric

acid container by an elastic band.

Boric acid preservative

At a concentration of 10 g/l (1% w/v), bacteria remain

viable without multiplying. White cells, red cells, and casts

are also well preserved, and there is no interference in the

measurement of urinary protein and glucose. Boric acid

has been shown to be inhibitory to some enterococci and

Pseudomonasstrains.

Important: Urine for culture must not be pre-

served with a bactericidal chemical such as

thymol, bleach, hydrochloric acid, acetic acid, or

chloroform.

3 Label the container, and send the urine with a

request form to reach the Microbiology

Laboratory within 48 hours. When possible

examine the urine microscopically in the Health

Centre for bacteria and pus cells to screen for

urinary infection.

Dipslides

Commercially prepared Dipslidesconsist of media-coated dis-

posable plastic slide-spoons. Inoculation is by immersing the

slide-spoon in a container of urine or by allowing a ﬂow of

urine to pass over the medium. They are used to avoid the

overgrowth of commensals when there is likely to be a delay

in a specimen reaching the laboratory. Dipslides, however, are

expensive, have a shelf-life of only about 4 months from

manufacture, and it may not be possible to separate a true

pathogen for susceptibility testing when contaminating organ-

isms are also present but not obvious in a heavy growth.

Preserving urines using boric acid (see previous text) is less

expensive and also enables a urine to be examined micro-

scopically.

Collection of urine when renal tuberculosis is

suspected

The specimen will need to be tested in a

Tuberculosis Reference Laboratory. The testing lab-

oratory should provide written instructions on the

collection of urine for the isolation of M. tuberculosis.

LABORATORY EXAMINATION OF

URINE

1 Describ e the appearance of the specimen

Report:

– Colour of specimen

– Whether it is clear or cloudy (turbid)

Day 1

MICROBIOLOGICAL TESTS 107

7.12

Normal freshly passed urine is clear and pale yellow

to yellow depending on concentration (see also

urine biochemical tests, described in subunit 6.11 in

Part I of the book).

Note: When left to stand, a cloudiness may develop

due to the precipitation of urates in an acid urine or

phosphates and carbonates in an alkaline urine.

Urates may give the urine a pink-orange colour.

2 Examine the specimens microscopically

Urine is examined microscopically as a wet prep-

aration to detect:

● signiﬁcant pyuria, i.e. WBCs in excess of 10

cells/l (10

/1) of urine

● red cells

● casts

● yeast cells

● T. vaginalismotile trophozoites

● S. haematobium eggs

● bacteria (providing the urine is freshly collected)

To diagnose urinary schistosomiasis microscopically,

and to detect casts when few in number, examin-

ation of a sediment from centrifuge d urine is

required to concentrate the eggs and casts. Because

concentration techniques are noteasily st andardized

it is difﬁcult to estimate white cell numbers to estab-

lish whether there is signiﬁcant pyuria (pus cell

numbers above normal).In practice most district lab-

oratories report the numbers of white cells in

centrifuged urine as few, moderate number, or

many (see later text) with only moderate numbers

and many being regarded as signiﬁcant when inves-

tigating UTI. Examination of a Gram stained smear

providesadditional useful information (see later text).

Value of examining uncentrifuged urine

Detecting bacteria in uncentrifuged (fresh) urine indicates

urinary infection, i.e. bacteriuria in excess of 10

/ml. Pyuria

can be quantiﬁed by counting WBCs or estimating numbers

by examining a drop of urine on a slide (1 WBC per low

power ﬁeld corresponds to 3 cells per l). Alternatively, when

an inverted microscope is available, 60 l of urine can be

examined in a ﬂat-bottom well of a microtitration plate and

cell numbers calculated using a simple formula. Most labora-

tories that examine large numbers of urine specimens use an

inverted microscope technique to screen for signiﬁcant pyuria

and whether a specimen requires culturing.

Note: To examine a drop of unstained uncentrifuged urine on

a microscope slide by transmitted light microscopy, requires

careful focussing and adequate closing of the condenser iris to

provide good contrast.

Appearance Possible Cause

Cloudy ● Bacterial urinary infection

Urine usually has

an unpleasant smell

and contains WBCs

Red and cloudy ● Urinary schistosomiasis

Due to red cells ● Bacterial infection

Brown and cloudy ● Malaria haemoglobinuria

Due to haemoglobin ● Other conditions that

cause intravascular

haemolysis

Yellow-brown, or ● Acute viral hepatitis

green-brown ● Obstructive jaundice

Due to bilirubin

Yellow-orange ● Haemolysis

Due to urobilin, ● Hepatocellular jaundice

i.e. oxidized

urobilinogen

Milky-white ● Bancroftian ﬁlariasis

Due to chyle

Note: Other changes in the colour of urine can be

caused by the ingestion of certain foods, herbs, and

drugs especially vitamins.

Preparation and examination of a wet

preparation

1 Aseptically transfer about 10 ml of well mixed

urine to a labelled conical tube.

2 Centrifuge at 500–1000 g for 5 minutes. Pour

the supernatant ﬂuid (by completely inverting

the tube) into a second container not the original

one. This can be used for biochemical tests to

avoid contaminating the original urine which

may need to be cultured (depending on the

ﬁndings of the microscopical examination).

3 Remix the sediment by tapping the bottom of

the tube. Transfer one drop of the well-mixed

sediment to a slide and cover with cover glass.

Note: Do not discard the remaining sediment

because this may be needed to prepare a Gram

smear if WBCs and, or, bacteria are seen in the

wet preparation.

4 Examine the preparation microscopically using

the 10and 40 objective with the condenser

iris closed sufﬁciently to give good contrast.

Report the following:

Bacteria (report only when the urine is freshly

passed): Usually seen as rods, but sometimes cocci

or streptococci (see Plate 7.13). Bacteriuria is usually

accompanied by pyuria (pus cells in urine).

Note: In a urinary infection, protein and nitrite are often

found in the urine (see later text). With E.coli infections, the

urine is markedly acid. An alkaline urine is found with

Proteusinfections.

White cells (pus cells): These are round, 10–15

m in diameter, cells that contain granules as shown

in Plate 7.11. In urinary infections they are often

found in clumps. In urine sediments, white blood

cells (WBC) are usually reported as:

Few: Up to 10 WBCs/HPF (high power ﬁeld, i.e.

using 40 objective)

Moderate number: 11–40/HPF

Many: More than 40 WBC/HPF

108 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.12

Note:A few pus cells are normally excreted in urine. Pyuria is

usually regarded as signiﬁcant when moderate or many pus

cells are present, i.e. more than 10 WBC/l (see previous

text). Bacteriuria without pyuria may occur in diabetes,

enteric fever, bacterial endocarditis, or when the urine

contains many contaminating organisms.

Pyuria with a sterile routine culture may be found with

renal tuberculosis, gonococcal urethritis, C.trachomatis infec-

tion, and leptospirosis, or when a patient with urinary

infection has been treated with antimicrobials.

Red cells: These are smaller and more refractile

than white cells (see Plate 7.11). They have a deﬁnite

outline and contain no granules. When the urine is

isotonic, they have a ringed appearance as shown in

Fig. 7.15. They are usually reported as few, moderate

or many in number per high power ﬁeld.

Note: When the urine is hypertonic, i.e. more concentrated

than the ﬂuid inside the red cells, ﬂuid will be drawn out of the

cells and they will appear smaller than normal and often

crenated (spiky) as shown in Fig. 7.15.

When haematuria is due to glomerulonephritis, the red

cells often vary in size and shape (dysmorphic).

In sickle cell disease, sickled red cells can sometimes be

seen in the urine.

Fig 7.14 Pus cells, see also Plate 7.11

Fig 7.15 Red cells, see also Plate 7.11

Enlarged Normal size Crenated

Haematuria (red cells in urine) may be found in

urinary schistosomiasis (usually with proteinuria),

bacterial infections, acute glomerulonephritis

(inﬂammation of the glomeruli of the kidneys), sickle

cell disease, leptospirosis, infective endocarditis,

calculi (stones) in the urinary tract, malignancy of the

urinary tract, and haemorrhagic conditions.

Note: The ﬁnding of red cells in the urine of women

may be due to menstruation.

Casts: These can usually be seen with the 10

objective provided the condenser iris is closed sufﬁ-

ciently to give good contrast. They consist of

solidiﬁed protein and are cylindrical in shape

because they are formed in the kidney tubules. The

following casts can be found in urine:

– Hyaline casts, which are colourless and empty

(see Plate 7.12). They are associated with damage

to the glomerular ﬁlter membrane. A few may be

MICROBIOLOGICAL TESTS 109

7.12

Plate 7.11 Urine sediment showing pus cells (larger granu-

lated cells) and red cells as seen with the 40 objective.

Courtesy of Professor DK Banerjee.

Plate 7.12 Hyaline cast in urine as seen with the 40 objec-

tive. Courtesy of M Amphlett.

Plate 7.15 Yeast cells and Trichomonas vaginalis in urine

sediment as seen with the 40 objective. Courtesy of

M Amphlett.

Plate 7.16 Yeast cells and pseudohyphae of Candida

albicans in urine sediment as seen with the 40 objective.

Courtesy of M Amphlett.

Plate 7.13 Large cellular cast, pus cells, red cells, and

bacteria (bacilli in background) in urine sediment as seen with

the 40objective.

Plate 7.14 Epithelial cells, red cells and occasional pus cell in

urine sediment as seen with the 10 objective.

Plate 7.17 Egg of Schistosoma haematobium and red cells in

urine sediment as seen with the 40 objective.

Plate 7.18 Spermatozoa and occasional pus cell in urine

sediment as seen with the 40 objective.

seen following strenuous exercise or during

fever.

– Waxy casts, which are hyaline casts that have

remained in the kidney tubules a long time. They

are thicker and denser than hyaline casts, often

appear indented or twisted, and may be yellow

in colour (see Fig. 7.17). They usually indicate

tubular damage and can sometimes be seen in

renal failure.

– Cellular casts, which contain white cells or red

cells (see Plate 7.13). Red cell casts appear orange

red. They indicate haemorrhage into the renal

tubules or glomerular bleeding. White cell casts

are found when there is inﬂammation of the

kidney pelvis or tubules. Yellow-brown pig-

mented casts may be seen in the urine of

jaundiced patients.

– Granular casts, which contain irre gular sized

granules originating from degenerate cells and

protein. They are also associated with renal

damage.

110 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.12

Yeast cells: These can be differentiated from red

cells by their oval shape and some of the yeasts

usually show single budding (see Plate 7.16). If in

doubt, run a drop of dilute acetic acid under the

cover glass. Red cells will be haemolyzed by the

acid, but not yeast cells.

Note: Glove powder in urine also resembles yeasts. It can be

distinguished by adding a drop of iodine (as used in Gram

stain). Glove powder granules (starch), turn blue-black.

Yeast cells are usually reported as few, moderate,

or many per HPF. They can be seen in the urine of

women with vaginal candidiasis, and occasionally in

specimens from diabetics and those with immuno-

suppression.

Trichomonas vaginalis: Found in the urine of

women with acute vaginitis (occasionally seen in the

urine of men). The trichomonads are a little larger

than white cells and are usually easily detected in

fresh urine because they are motile. They move by

ﬂagella and an undulating membrane (see Plate

7.15) .

Eggs of S. haematobium: Recognized by their

large size (about 145  55 m) and spine at one

end (see Plate 7.17 and colour Plate 5.40 in Part 1 of

the book). The urine will contain red cells and

protein. Urinary schistosomiasis is described in

subunit 5.6 in Part 1 of the book.

Other parasites that may be found in urine

● Veryoccasionally the microﬁlariaeof Wuchereriabancrofti

canbe foundin urine. Thishappens when aurogenital lym-

phatic vessel ruptures. The urine appears milky-white or

reddish-pink(chyle mixed with blood). The microﬁlariae

are large (225– 300  10 m), motile, and sheathed. No

nucleiarepresent in thetail (featurelooked for ina Giemsa

stainedpreparation). W. bancrofti microﬁlariae areshown

incolour Plate5.58b, c in subunit5.11 in Part 1of the book.

● Microﬁlariae of Onchocerca volvulus may be found in the

urine in onchocerciasis, especially in heavy infections.

The larvae are large (280–330  7 m), unsheathed, with

a slightly enlarged head-end and a tail which is sharply

pointed and contains no nuclei (see colour Plate 5.62 in

subunit 5.12 in Part 1 of the book).

Fig 7.17 Different casts which may be found in urine.

Cellular casts Granular casts

Hyaline casts Waxy casts

Epithelial cells: These are easily seen with the 10

objective (see Plate 7.14). They are nucleated and

vary in size and shape. They are usually reported as

few, moderate, or many in number per low power

(10 objective) ﬁeld. It is normal to ﬁnd a few

epithelial cells in urine. When seen in large numbers,

however, they usually indicate inﬂammation of the

urinary tract or vaginal contamination of the

specimen.

Fig 7.16 Epithelial cells

● Occasionally the eggs of Enterobius vermicularis are

found in urine, especially from young girls when the eggs

are washed off the external genitalia when urine is being

passed (see Plate 5.38 in subunit 5.5 in Part 1 of the book).

MICROBIOLOGICAL TESTS 111

7.12

six-sides (see Fig. 7.18). They are soluble in 30%

v/v hydrochloric acid (unlike uric acid crystals

which they may resemble). They can be found in

cystinuria, a rare congenital metabolic disorder in

which cystine is excreted in the urine.

– Cholesterol crystals, which look like rectangles

with cut-out corners (see Fig. 7.18). They are

insoluble in acids and alkalis but soluble in ether,

ethanol, and chloroform. They are rarely found

except in severe kidney disease or when a lym-

phatic vessel has ruptured into the renal pelvis.

– Tyrosine crystals, which are yellow or dark-

coloured and look like needles massed together

(see Fig. 7.18). They are insoluble in ethanol,

ether, and acetone. They are occasionally found

in severe liver disease.

Other crystals found in urine

● Occasionally sulphonamide crystals (see Fig. 7.18) are

found in the urine of patients being treated with

sulphonamides. When deposited in the urinary tract they

can cause haematuria and other complications.

● Triple phosphate crystals are occasionally found in

alkaline urine (see Fig. 7.18). They have little or no

clinical signiﬁcance.

● Calciumoxalate crystals arefrequently seen (seeFig. 7.18).

When found in freshly passed urine they may

indicatecalculi in the urinary tract.

● Uric acid crystals are yellow or pink-brown. They can

sometimes be found with calculi.

Spermatozoa: Occasionally found in the urine of

men, they can be easily recognized by their head

and long thread-like tail (see Plate 7.18). They may be

motile in fresh urine.

Contaminants which can be found in urine

These include cotton ﬁbres, starch granules, oil droplets,

pollen grains, moulds, single-celled plants (diatoms) and

debris from dirty slides or containers.

Examination of a Gram stained smear

Prepare and examine a Gram stained smear of the

urine when bacteria and, or white cells are seen in

the wet preparation.

– Transfer a drop of the urine sediment to a slide

and spread it to make a thin smear. Allow to air-

dry, protected from insects and dust. Heat ﬁx or

methanol ﬁx the smear (see subunit 7.3.2) and

stain it by the Gram technique as described in

subunit 7.3.4.

– Examine the smear ﬁrst with the 40 objective

to see the distribution of material, and then with

the oil immersion objective. Look especially for

bacteria associated with urinary infections,

Crystals

These have a characteristic refractile appearance.

Normal urine contains many chemicals from which

crystals can form, and therefore the ﬁnding of most

crystals has little importance. Crystals should be

looked for in fresh urine when calculi (stones) in the

urinary tract are suspected. Crystals which may be

found in rare disorders include:

– Cystine crystals, which are recognized by their

Fig 7.18 Different crystals which may be found in urine.

Cysteine crystals Cholesterol crystals

Sulphonamide crystals Tyrosine crystals

Calcium oxalate crystals Triple phosphate crystals

especially Gram negative rods. Occasionally

Grampositive cocci andstreptococci mayb eseen.

Note: Usually only a single type of organism is

present in uncomplicated acute urinary infections.

More than one type of organism is often seen in

chronic and recurring infections. Vaginal contami-

nation of the specimen is indicated by a mixed

bacterial ﬂora (including Gram positive rods) and

often the presence of epithelial cells.

Neisseria gonorrhoeae in urine

In male patients with acute urethritis, it is often

possible to make a presumptive diagnosis of gonor-

rhoea by ﬁnding Gram negative intracellular

diplococci in pus cells passed in urine (see colour

Plate 43) and subunit 7.10.

3 Test the specimen biochemically

Biochemical tests which are helpful in investigating

UTI include:

 Protein

 Nitrite

 Leukocyte esterase

Protein

Most laboratories test urine routinely for protein

using sulphosalicylic acid reagent or a protein

reagent strip test, as described on pp. 371–372 in

Part 1 of the book. Proteinuria is found in most bac-

terial urinary tract infections. Other causes include

glomerulonephritis, nephrotic syndrome, eclampsia,

urinary schistosomiasis, hypertension, severe febrile

illnessses, HIV associated renal disease and treat-

ment with nephrotoxic antiretroviral drugs.

Nitrite

Urinary pathogens, e.g. E. coli (commonest cause of

UTI), Proteus species, and Klebsiella species, are able

to reduce the nitrate normally present in urine to

nitrite. This can be detected by the Greiss test or a

nitrite reagent strip test (see pp. 380–381 in Part 1

of the book), providing the organisms are present in

the urine in sufﬁcient concentration. When ﬁrst

morning urine is tested, about 80–90% of UTI

caused by nitrate-reducing pathogens can be

detected. The test is negative when the infection is

caused by pathogens that do not reduce nitrate such

as Enterococcus faecalis, Pseudomonas species,

Staphylococcus species and Candida organisms, or

when as previously mentioned the bacteria are too

few in the urine. Occasionally the nitrite test is

negative because nitrate is lacking in the urine due

to the person being on a diet lacking in vegetables.

112 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.12

Greiss test to screen for UTI in pregnancy

A Greiss test to detect nitrite reducing pathogens such as

E.coli, together with a protein test and the visual examination

of urine for cloudiness, are useful ways of screening for UTI

in pregnancy in antenatal clinics.

The visual inspection of urine and a test for protein are

routinely performed in most antenatal clinics. A simple Greiss

test can be performed as follows:

Greiss test using a dry reagent

1 Transfer 0.5–1.0 g (pea-size) of well mixed dry Greiss

reagent* to the well of a white porcelain tile or to a small

test tube.

*Prepare by mixing together 6.2 g alpha-naphthylamine,

1 g sulphanilic acid, 25 g citric acid. Store in a cool dry

place. Renew about every 3 months.

2 Moisten the reagent with a drop of urine (ﬁrst morning

urine). The urine should be tested within about 1 hour of

being collected.

3 Look for the immediate development of a pink-red

colour.

Pink-red colour .. . Positive nitrite test

Leukocyte esterase (LE)

This enzyme is speciﬁc for polymorphonuclear neu-

trophils (pus cells). It detects the enzyme from both

active and lyzed WBCs. LE testing is an alternative

method of detecting pyuria when it is not possible to

examine fresh urine microscopically for white cells or

when the urine is not fresh and likely to contain

mostly lyzed WBCs.

LE can be detected using a reagent strip test

such as the Combur 2 Test LN (Roche strip) which

detects both nitrite and leukocytes (LE) or a multi-

test reagent strip with an area for leukocyte

detection (see p. 385 in Part 1 of the book).

False negative strip test results can occur when

theurine contains boric acid or excessive amounts of

protein(500 mg/100ml) or glucose (2 g/100ml)

(see also p. 381 in Part 1).

Other biochemical urine tests:Details of tests to detect other

biochemical substances in urine, e.g. glucose, ketones, biliru-

bin, urobilinogen, and haemoglobin, can be found in subunit

6.11 in Part 1 of the book. The use and control of urine

reagent strip tests are also covered in this subunit.

4 Culture the spe cimen

It is not necessary to culture urine which is micro-

scopically and biochemically normal, except when

screening for asymptomatic bacteriuria in preg-

nancy. Culture is required when the urine contains

bacteria (as indicated by the Gram smear), cells,

casts, protein, nitrite, or has a markedly alkaline or

acid reaction.

Estimating bacterial numbers

It is necessary to estimate the approximate number

of bacteria in urine because normal specimens may

contain small numbers of contaminating organisms,

usually less than 10000 (10

) per ml of urine. Urine

from a person with an untreated acute urinary infec-

tion usually contains 100000 (10

) or more bacteria

per ml.

The approximate number of bacteria per ml of

urine, can be estimated by using a calibrated loop or

a measured piece of ﬁlter paper. Both methods are

based on accepting that a single colony represents

one organism. For example, if an inoculum of ml

produces 20 colonies, the number of organisms

represented in ml of urine is 20, or 10000 in

1 ml (500  20).

The calibrated loop method using quarter plates

of culture media (see later text) is recommended

because it is inexpensive, simple to perform, and

provides individual colonies that are easier to

identify and remove for antimicrobial susceptibility

testing.

Cystine lactose electrolyte-deﬁcient (CLED)

agar

– Mix the urine (freshly collected clean-catch

specimen) by rotating the container.

– Using a sterile calibrated wire loop, e.g. one

that holds ml (0.002 ml), inoculate a loopful

of urine on a quarter plate of CLED agar (see

No. 30). If microscopy shows many bacteria, use

a half plate of medium.

– Incubate the plate aerobically at 35–37°C

overnight.

Cystine lactose electrolyte-deﬁcient (CLED) agar is widely

used by laboratories to isolate urinary pathogens because it

gives consistent results and allows the growth of both Gram

negative and Gram positive pathogens. (The indicator in

CLED agar is bromothymol blue and therefore lactose

fermenting colonies appear yellow). The medium is elect-

rolyte-deﬁcient to prevent the swarming of Proteusspecies.

4 Examine and report the cultures

CLED agar culture

Look especially for colonies that could be:

 Escherichia coli (perform indole and beta-gluca-

ronidase tests for rapid identiﬁcation, see subunit

7.5.6)

 Proteus species, see subunit 7.18.18

 Pseudomonas aeruginosa, see subunit 7.18.20

 Klebsiella strains, see subunit 7.18.17

 Staphylococcus aureus, see subunit 7.18.1

 Staphylococcus saprophyticus, see subunit 7.18.1

 Enterococcus faecalis, see subunit 7.18.5

Day 2 and Onwards



500



500



500

MICROBIOLOGICAL TESTS 113

7.12

Appearance of some urinary pathogens on CLED agar

● E. coli: Yellow (lactose-fermenting) opaque colonies

often with slightly deeper coloured centre.

● Klebsiella species: Large mucoid yellow or yellow-white

colonies.

● Proteus species: Transluscent blue-grey colonies.

● P.aeruginosa: Green colonies with rough periphery (char-

acteristic colour).

● E. faecalis: Small yellow colonies.

● S.aureus: Deep yellow colonies of uniform colour.

● S. saprophyticus and other coagulase negative staphylo-

cocci: Yellow to white colonies.

The appearances of urinary pathogens on CLED

agar are shown in colour Plates 18 and 19.

Note: Contaminating organisms usually produce a

few colonies of mixed growth. Most urinary infec-

tions show growth of a single type of organism

although mixed infections can occur especially in

chronic infections or following catheterization or

gynaecological surgery.

Reporting bacterial numbers

Count the approximate number of colonies.

Estimate the number of bacteria, i.e. colony-forming

units (CFU) per ml of urine. Report the bacterial

count as:

● Less than 10000 organisms/ml (10

/ml), not

signiﬁcant.

● 10000–100000/ml (10

–10

/ml), doubtful

signiﬁcance (suggest repeat specimen)

● More than 100000/ml (10

/ml), signiﬁcant

bacteriuria.

Example

If 25 E. coli colonies are counted and a ml loop

was used, the approximate number of CFU per ml

of urine: 500  25  12 5 0 0

Such a count would be reported as:

10000–100000 E. coli/ml

Interpretation of bacterial counts: A bacterial count of 10

organisms/ml or more from a fresh ‘clean-catch’ urine

specimen, indicates a urinary infection. A count of 10

–10

/ml,

could mean infection or contamination. A repeat specimen is

indicated. A count of less than 10

/ml is nearly always due to

contamination unless the urine was cultured after antimicro-

bial treatment had been started. It is important, however, to

interpret culture counts in relation to the patient’s clinical

condition. UTIs with lower culture counts are often obtained

from catheterized patients or those with urinary obstruction.

Antimicrobial susceptibility testing

Perform susceptibility testing on urines with signiﬁ-

cant bacteriuria, particularly from patients with a

history of recurring UTI. Cultures from patients with

a primary uncomplicated UTI may not require a

susceptibility test.



500

114 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.12

Summary of the Microbiological Examination of Urine

Day 1

1 Describe

Appearance

 Describe

– Colour

– Whether clear or cloudy

2 Examine

Microscopically

 Wet preparation

Report:

– WBCs (pus cells)

– Red cells

– Casts

– Yeast cells

– T. vaginalis ﬂagellates

– S. haematobium eggs

– Bacteria (fresh urine only)

– Crystals of importance

 Gram smear: When bacteria or

WBCs (pus cells) are seen in

wet preparation.

3 Test

Biochemically

 Tests to help diagnose UTI

– Protein

– Nitrite (Greiss test)

– Leukocyte esterase (when

microscopy for WBCs not

possible)

 Glucose, ketones, bilirubin,

urobilinogen:

As indicated

Day 2 and Onwards

5 Examine and

Report Cultures

 CLED culture

Look particularly for:

E.coli (common cause UTI)

Proteus species

P.aeruginosa

Klebsiella

E.faecalis

S.aureus

S.saprophyticus

 Antimicrobial susceptibility

testing:

As indicated

4 Culture

Specimen

 CLED agar

When bacteria and, or pus

cells are present:

– Inoculate CLED medium

– Incubate aerobically

Report bacterial numbers:

● Less than 10

/ml, not signiﬁcant

● 10

–10

/ml, doubtful signiﬁcance

● More than 10

/ml, signiﬁcant bacteriuria.

ADDITIONAL INVESTIGATIONS

TESTINGURINE FOR HCG (PREG NANCY TESTING)

Human chorionic gonadotrophin (hCG) is a glyco-

protein hormone produced by placental cells soon

after the fertilized ovum is implanted in the uterine

wall. It stimulates the secretion of progesterone by

the ovary. Progesterone maintains the uterus during

the pregnancy and prevents any further release of

eggs from the ovary.

Laboratory pregnancy tests are based on the

detection of rapidly rising levels of hCG in urine or

serum. The amount of hormone excreted in the

urine is almost the same as that found in the blood.

At about the time of the ﬁrst menstrual period, hCG

concentrations in urine and serum are about 100

mIU/ml and double in concentration every 1–2

days. Most pregnancies do not require early conﬁr-

mation by the laboratory.

Medical reasons for requesting a pregnancy test

Include:

– investigation of a suspected ectopic pregnancy,

threatened abortion, or a trophoblastic tumour

such as hydatidiform mole or choriocarcinoma.

– checking whether a woman of child-bearing age

is pregnant before carrying out a medical or

surgical investigation, X-ray, or drug treatment

that could be harmful to the embryo.

In most of these hCG situations a rapid qualitative

hCG test result is adequate. Occasionally it is import-

ant to measure the level of hCG e.g. in the

investigation of trophoblastic tumour when the con-

centration of hCG is high and therefore a

quantitative test will be required (usually carried out

in a specialist laboratory).

Qualitative tests to detect hCG in urine

Most of the recently developed immunochromato-

graphic (IC) pregnancy tests have both high

sensitivity and speciﬁcity. When ectopic pregnancy is

suspected it is important to use a sensitive test i.e.

one that is capable of detecting hCG levels below

100 mIU/ml. Compared with normal pregnancy, the

hCG level is lower in ectopic pregnancy.

Most IC card and strip tests are able to detect 50

mIU/ml or even 25 mIU/ml of hCG. Latex slide

tests, however, are generally less sensitive, usually

becoming positive only when the hCG level is over

500 mIU/ml.

Immunochromatographic (IC) pregnancy tests

Most manufacturers of IC pregnancy tests (see Chart 7.2

on p. 16) use a monoclonal antibody dye conjugate and

polyclonal solid phase antibodies to detect hCG in specimens.

Some tests can be used to detect hCG in both serum and

urine.

MICROBIOLOGICAL TESTS 115

7.12

The specimen is applied to an absorbent pad. The

antibody-dye conjugate binds to the hCG in the specimen

forming an hCG antibody-antigen complex. This complex

migrates by capillary action to the reaction zone where it

binds to the anti-hCG antibody, producing a coloured band

(usually pink-rose colour). In a negative test, no coloured

band is produced. Unbound conjugate binds to the reagent in

the control zone, producing a coloured band, indicating a cor-

rectly performed test. A positive pregnancy test is therefore

shown by coloured bands appearing in both the control and

test zones (see Fig 7.19). A negative test is shown by a

coloured band appearing only in the control zone. Most IC

tests are rapid, providing results within a few minutes.

Urine specimen

Most manufacturers of urine pregnancy tests rec-

ommend testing the ﬁrst morning urine because this

will contain the highest level of hCG. The specimen

must be collected in a clean container which is free

from all traces of detergent and preservative. When

the specimen cannot be tested immediately it

should be refrigerated but for not more than 24

hours. The urine and test device must be at room

temperature before performing the test.

When the urine is cloudy it should be ﬁltered or

centrifuged and the supernatant ﬂuid used.

Specimens that contain excessive bacterial contami-

nation or large amounts of protein or blood, are not

usually suitable for hCG testing.

It is important to follow exactly the manufac-

turer’s instructions regarding the specimen, method

of performing the test, reading of test and control,

storage conditions for the test kit and its expiry date.

The shelf-life of most IC tests is about 2 years. Many

IC pregnancy test kits can be stored at room tem-

perature.

Fig 7.19 Example of a dipstick IC pregnancy test. The strip

is dipped in the urine and the result is read after 5 minutes.

Courtesy of Bioline.

Urine

Control band

Test band

NEGATIVE

Only one band

POSITIVE

Two bands

One step hCG pregnancy test

7.13 Examination of

cerebrospinal ﬂuid (c.s.f.)

Possible pathogens

 BACTERIA

Gram positive

Streptococcus pneumoniae

Streptococcus agalactiae

(Group B)*

Listeria monocytogenes*

Streptococcus suis**

*Mainly isolated from neonates (see also Notes on pathogens).

**S. suis is a pathogen of pigs. It is an important cause of

meningitis in Vietnam, China, and elsewhere among those

living in close proximity to pigs.

Also Mycobacterium tuberculosis and Treponema

pallidum.

Note: Bacteria may also be found in the c.s.f. when

there is a brain abscess, e.g. Bacteroides species and

other anaerobes.

 VIRUSES

Particularly coxsackieviruses, echovirus, and

arboviruses. Also, herpes simplex 2 virus, vari-

cella zoster virus, and lymphocytic chorio-

meningitis virus (LCM). Rarely polioviruses may

be isolated from cerebrospinal ﬂuid.

 FUNGI

Cryptococcus neoformans (mainly in AIDS

patients) and less commonly Aspergillus species.

 PARASITES

Trypanosomaspecies and Naegleria fowleri. Rarely

the larvae of Angiostrongylus cantonensis and

Diroﬁlariaimmitis (c.s.f.usually containseosinophils).

AlsoToxoplasma gondii(mainly in AIDS patients).

Notes on pathogens

● Inﬂammation of the meninges (membranes that cover the

brain and spinal cord) is called meningitis. Pathogens

reach the meninges in the blood stream or occasionally by

spreading from nearby sites such as the middle ear or

nasal sinuses. Fever, headache, neck stiffness, and intoler-

ance of light are typical symptoms of acute bacterial

meningitis. In children, vomiting, convulsions and

lethargy are common. A haemorrhagic rash is associated

with meningococcal meningitis.

116 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.13

Meningitis is described as:

– Pyogenic (purulent), when the c.s.f. contains mainly

polymorphonuclear neutrophils (pus cells), as in

acute meningitis caused by N. meningitidis, H. inﬂ-

uenzae, and S.pneumoniae. Pus cells are also found in

the c.s.f. in acute amoebic meningo-encephalitis.

– Lymphocytic, when the c.s.f. contains mainly lympho-

cytes, as in meningitis caused by viruses,

M.tuberculosis, and C. neoformans. Lymphocytes are

also found in the c.s.f. in trypanosomiasis meningo-

encephalitis, and neurosyphilis.

● In developing countries, meningitis epidemics are usually

caused by N.meningitidis serogroups A and C and only

occasionally by group B and other serogroups. Outbreaks

are common in sub-Saharan Africa (meningitis belt) with

most group A meningococcal meningitis epidemics

occurring in the hot dry season (see also subunit 7.18.12).

In recent years meningococcal meningitis has risen to

epidemic proportions in some countries of South

America, the Middle East, and Asia. Rarely, epidemic

meningitis is caused by S.pneumoniae but endemic pneu-

mococcal meningitis is common and has a high fatality

rate.

● In developing countries, neonatal meningitis is caused

mainly by S. pneumoniae (about one third of cases),

Salmonella serovars and other enterobacteria, N. menin-

gitidis, and H.inﬂuenzae. Streptococcus agalactiae (Group

B) is a rare cause.

● Haemophilus meningitis occurs mainly in infants and

young children below 5 y with a high incidence below 2 y.

● C. neoformans is mainly an opportunistic pathogen,

causing life-threatening meningo-encephalitis in those

with AIDS and other conditions associated with immuno-

suppression. In parts of sub-saharan Africa and other

areas of high HIV prevalence, cryptococcosis has been

reported in up to 30% of AIDS patients.

● Syphilitic meningitis may occur in secondary syphilis but

it is usually a complication of late syphilis (see subunit

7.18.32).

● N.fowleri causes primary amoebic meningoencephalitis, a

rare and usually fatal disease (see pp. 299–300 in Part 1 of

the book).

Commensals: Cerebrospinal ﬂuid has no normal

microbial ﬂora.

OLLECTIONAND TRANSPORTOF CSF

Cerebrospinal ﬂuid must be collected by an experi-

enced medical ofﬁcer or health worker. It must be

collected aseptically to prevent organisms b eing

introduced into the central nervous system.

The ﬂuid is usually collected from the arachnoid

space. A sterile wide-bore needle is inserted

between the fourth and ﬁfth lumbar vertebrae and

the c.s.f. is allowed to drip into a dry sterile container.

A ventricular puncture is sometimes performed to

collect c.s.f. from infants.

Gram negative

Neisseria meningitidis

Haemophilus

inﬂuenzae type b

Escherichia coli*

Pseudomonas

aeruginosa*

Proteus species*

Salmonella serovars

Flavobacterium

meningosepticum*

Collection of c.s.f. from patient with suspected trypanosomia-

sis

When the c.s.f. is to be examined for trypanosomes, it is

usually collected after treatment to kill the trypanosomes in

the blood has been started. This will avoid the accidental

introduction of the parasites into the central nervous system

should the lumbar puncture be traumatic (bloody).

In a hospital with a microbiology laboratory

IMPORTANT: Advize the laboratory before per-

forming a lumbar puncture so that staff are

prepared to receive and examine the specimen

immediately.

Note: A delay in examining c.s.f. reduces the

chances of isolating a pathogen. It will also result in

a lower cell count due to WBCs being lyzed, and to

a falsely low glucose value due to glycolysis. When

trypanosomes are present, they will be difﬁcult to

ﬁnd because they are rapidly lyzed once the c.s.f.

has been withdrawn.

Collection of c.s.f.

1 Take two sterile, dry, screw-capped containers

and label one No. 1 (ﬁrst sample collected, to be

used for culture), and the other No. 2 (second

sample collected, to be used for other investi-

gations).

2 Collect about 1 ml of c.s.f. in container No. 1 and

about 2–3 ml in container No. 2.

3 Immediately deliver the samples with a request

form to the laboratory.

In a health centre

Patients with suspected meningitis usually receive

emergency treatment in a health centre and are

transferred to the nearest hospital for laboratory

investigations and care.

LABORATORY EXAMINATION OF

C.S.F.

Important: Cerebrospinal ﬂuid must be examined

without delay (see previous text), and the results of

tests reported to the medical ofﬁcer as soon as they

become available, especially a Gram smear report.

The ﬂuid should be handled with special care

because a lumbar puncture is required to collect the

specimen.

Day 1

MICROBIOLOGICAL TESTS 117

7.13

1 Report the appearance of the c.s.f.

As soon as the c.s.f. reaches the laboratory, note its

appearance. Report whether the ﬂuid:

– is clear, slightly turbid, cloudy or deﬁnitely

purulent (looking like pus),

– contains blood,

– contains clots.

Normal c.s.f. Appears clear and colourless.

Purulent or cloudy c.s.f.Indic ates presence of pus

cells, suggestive of acute pyogenic bacterial menin-

gitis.

Blood in c.s.f. This may be due to a traumatic

(bloody) lumbar puncture or less commonly to

haemorrhage in the central nervous system. When

due to a traumatic lumbar puncture, sample No. 1

will usually contain more blood than sample No. 2.

Following a subarachnoid haemorrhage, the ﬂuid

may appear xanthrochromic, i.e. yellow-red (seen

after centrifuging).

Clots in c.s.f. Indicates a high protein concentration

with increased ﬁbrinogen, as can occur with

pyogenic meningitis or when there is spinal con-

striction.

2 Test the c.s.f.

Depending on the appearance of the c.s.f., proceed

as follows:

Purulent or cloudy c.s.f.

Suspect pyogenic meningitis and test the c.s.f. as

follows:

 Immediately make and examine a Gram stained

smear for bacteria and polymorphonuclear neu-

trophils (pus cells). Issue the report without delay.

 Culture the c.s.f.

Slightly cloudy or clear c.s.f.

Test the c.s.f. as follows:

 Perform a cell count and note whether there is an

increase in white cells and whether the cells are

mainly pus cells or lymphocytes.

 When cells predominantly pus cells:

– Examine a Gram stained smear for bacteria.

– Examine a wet preparation (sediment from

centrifuged c.s.f.) for motile amoebae which

could be Naegleria (rare).

– Culture the c.s.f.

 When cells predominantly lymphocytes: This

could indicate viral meningitis, tuberculous

meningitis, cryptococcal meningitis, trypanosomi-

asis encephalitis, or other condition in which

lymphocyte numbers in the c.s.f. are increased

(see Chart 7.9). Perform the following tests:

– Measure the concentration of protein or

perform a Pandy’s test. The c.s.f. protein is

raised in most forms of meningitis and

meningoencephalitis.

– Measure the concentration of glucose. This is

helpful in differentiating viral meningitis in

which the c.s.f. glucose is usually normal

from tuberculous meningitis and other con-

ditions in which the c.s.f. glucose is reduced

(see Chart 7.9).

– Examine a wet preparation for encapsulated

yeast cells that could be C. neoformans.

– Examine a wet preparation for trypanosomes

and a Giemsa stained smear for morula

(Mott) cells when late stage trypanosomiasis

is suspected.

Report the c.s.f. as ‘Normal’: when it appears

clear, contains no more than 5 WBC  10

/1, and

the protein concentration is not raised (or Pandy’s

test is negative).

Note: A c.s.f. begins to appear turbid when it

contains about 200 WBC  10

/1.

GRAM SMEAR

A Gram smear is required when the c.s.f. contains

pus cells (neutrophils). It should be the ﬁrst investi-

gation to be performed and reported when the c.s.f.

appears purulent or cloudy (suggestive of acute

pyogenic meningitis). A Gram smear may also

provide useful information when a c.s.f. is unsuitable

for cell counting or biochemical testing (e.g. when it

is heavily blood stained or contains clots).

Making a smear of c.s.f. for Gram staining

1 Mix No. 2 sample c.s.f. and centrifuge most of it

at approximately 1000 g for 5–10 minutes (leave

a small amount of uncentrifuged c.s.f. for a cell

count should this be required).

Purulent c.s.f.Do not centrifuge a purulent ﬂuid. A smear

for Gram staining is best prepared from the uncentrifuged

c.s.f.

2 Transfer the supernatant ﬂuid to another tube (to

be used for glucose and protein tests should

these be required).

3 Mix the sediment. Transfer several drops of the

sediment to a slide, but do not make the prep-

aration too thick because this will make it difﬁcult

118 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.13

to decolorize adequately. Allow the preparation

to air-dry in a safe place.

4 Alcohol-ﬁx the preparation as described in

subunit 7.3.2 and stain it by the Gram technique

(see subunit 7.3.4).

Examining a c.s.f. Gram smear

Examine the smear microscopically for pus cells and

bacteria using the 40 and 100 objectives.

Pus cells: Report as many, moderate number, or

few. Pus cells will be found mainly in pyogenic

bacterial meningitis and in amoebic-meningo-

encephalitis (rare).

Bacteria: Look in well stained (not too thick) areas

for:

 Gram negative intracellular diplococci that could

be N. meningitidis (see colour Plate 38).

 Gram positive diplococci or short streptococci,

that could be S. pneumoniae. It is often possible

to see the capsules as unstained areas around

the bacteria (see colour Plate 28).

 Gram negative rods, possibly H. inﬂuenzae,

especially if ﬁlamentous or other polymorphic

forms are seen (see colour Plate 48).

Gram negative rods could also be E. coli or other

coliforms, especially when the c.s.f. is from a

newborn infant.

 Unevenly stained irregular in size yeast cells

(some showing budding), suggestive of C. neo-

formans (see colour Plate 74). The large capsule

that surrounds the cell does not stain. It is best

seen in an India ink preparation (see later text).

The smear will usually contain lymphocytes.

Important: Advize the medical ofﬁcer immediately

if the Gram smear contains bacteria, pus cells, or

yeast cells (conﬁrmed as capsulated in India ink

preparation).

Acridine orange stained smear to detect bacteria in c.s.f.

When facilities for ﬂuorescence microscopy are available,

examine an acridine orange (A0) stained smear (see subunit

7.3.11). Bacteria especially when few, are more easily

detected in A0 smears. They stain bright orange and cells and

debris stain green or yellow. The organisms can be detected

using the 40objective.

Note: When bacteria and pus cells are seen in the

Gram smear, culture the c.s.f. There is no need to

perform a cell count or measure the protein or

glucose.

When the patient has been given antibiotics (usually

as emergency treatment) it will be more difﬁcult to

detect bacteria in the Gram smear and to isolate

pathogens in culture.

Immunological diagnosis of acute bacterial meningitis

Direct antigen testing of c.s.f. may provide a rapid diagnosis of

acute bacterial meningitis, particularly when the patient has

been treated with antimicrobials and bacteria cannot be

detected in a Gram smear or by culture. Tests are available to

detect N.meningitidis groups A, B, C, Y and W135 (Group B

reagent cross-reacts with E. coli K1 antigen), H. inﬂuenzae

type b, S.pneumoniae, and S. agalactiae.

Manufacturers of latex tests include Bio-Rad

Laboratories, BD Diagnostics, and bioMérieux. Coag-

glutination tests are available from Boule Diagnostics.

Details of manufacturers can be found in Appendix 11.

The tests, however, are expensive and some manufacturers

supply them only in kits containing the full range of tests. Bio-

Rad Laboratories, however, make available the different

antigen tests individually in pack size of 25, e.g. separate test

kits are available for N.meningitidis A, and N. meningitidis C

(useful in determining whether an epidemic is caused

by serogroup A or C), S. pneumoniae and H. inﬂuenzae

type b.

CULTURINGC.S.F.

Culture the c.s.f. when bacteria are seen in the Gram

smear and, or, cells are present, or the protein con-

centration is raised.

Use c.s.f. sample No. 1. When the c.s.f. is clear or

slightly cloudy, centrifuge the sample in a sterile

capped tube for about 15 minutes, and use the

sediment to inoculate the culture media.

Important: Cerebrospinal ﬂuid must be cultured as

soon as possible after collection. When a delay is

unavoidable, the ﬂuid should be kept at 35–37°C

(not refrigerated).

Chocolate (heated blood) agar and blood

agar

– Inoculate the specimen on chocolate agar and

blood agar (see No. 16). When Gram positive

diplococci are seen in the Gram smear, add an

optochin disc to the blood agar plate to assist in

the identiﬁcation of S. pneumoniae.

– Incubate both plates in a carbon dioxide

enriched atmosphere at 35–37°C for up to 48

hours, checking for growth after overnight incu-

bation.

When patient is a newborn infant: Inoculate the

specimen also on MacConkey agar. Incubate aero-

bically at 35–37°C overnight.

CELL COUNT

A white cell count with an indication whether the

cells are pus cells or lymphocytes, is required when

the c.s.f. appears slightly cloudy or clear or when the

Gram smear does not indicate pyogenic bacterial

meningitis.

MICROBIOLOGICAL TESTS 119

7.13

Note: Samples that are heavily blood stained or

contain clots are unsuitable for cell counting. Make

a Gram smear and report the presence of pus cells

and bacteria as previously described.

Method

To identify whether white cells in the c.s.f. are poly-

morphonuclear neutrophils (pus cells) or lympho-

cytes, dilute the c.s.f. in a ﬂuid which stains the cells.

Istonic 0.1% toluidine blue is recommended

because it stains lymphocytes and the nuclei of pus

cells blue. C. neoformans yeast cells stain pink. Red

cells remain unstained. The motility of trypanosomes

is not affected by the dye. When toluidine blue is

unavailable, isotonic methylene blue can be used

which will also stain the nuclei of leucocytes. If pre-

ferred, leucocytes can be differentiated by

examining a Leishman, Giemsa or rapid Field’s

stained smear (sediment from centrifuged c.s.f.) after

counting the cells.

1 Mix the c.s.f. (sample No. 2 uncentrifuged c.s.f.).

Dilute the ﬂuid 1 in 2, i.e. mix 1 drop of c.s.f. with

1 drop of toluidine blue diluting ﬂuid (Reagent

No. 84).*

*The drops must be of equal volume, therefore use

Pasteur pipettes of the same bore size for both ﬂuids and

hold the pipettes vertically when dispensing the drops.

2 Assemble a modiﬁed Fuchs-Rosenthal ruled

counting chamber,* making sure the chamber

and cover glass are completely clean.

*When unavailable, an improved Neubauer (preferably

Bright-Line) chamber can be used. A Fuchs-Rosenthal

chamber is recommended because it has twice the depth

(0.2 mm) and is more suitable for counting WBCs in c.s.f.

3 Using a ﬁne bore Pasteur pipette or capillary

tube, carefully ﬁll the counting chamber with the

well-mixed diluted c.s.f. The ﬂuid must not

overﬂow into the channels on each side of the

chamber.

4 Wait about 2 minutes for the cells to settle.

Count the cells microscopically.

5 Focus the cells and rulings using the 10objec-

tive with the condenser iris closed sufﬁciently to

give good contrast. Before starting the count, use

the 40 objective to check that the cells are

white cells and not red cells (unstained smaller

cells without a nucleus) and note whether the

white cells are mainly polymorphonuclear neu-

trophils (with lobed nucleus) or lymphocytes. If a

mixture of both, estimate approximately the per-

centage of each type of cell.

When yeast cells are seen, examine an India ink

preparation (see later text).

Note: When red cells are seen, mention this in

the report. When many red cells are present, the

c.s.f. is unsuitable for WBC cell counting.

6 Count the cells in 5 of the large squares as

shown in Fig. 7.20.

Note: When the cells are too many to count, dilute the

c.s.f. 1 in 10 (1 drop c.s.f. mixed with 9 drops of diluting

ﬂuid), reﬁll the chamber and count the cells. See later text

for calculation factor to use.

120 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.13

 Improved Neubauer chamber: Multiply cells

counted in 4 squares by 25. Report number of

cells per litre of c.s.f. (see above text).

Normal c.s.f.: Contains up to 5  10

cells/litre

(higher in neonates).

When no WBCs are seen, report the count as: Below

5 cells  10

/1.

IOCHEMICAL TESTING OF C.S.F.

Biochemical c.s.f. tests which may be required

include the measurement of protein and glucose.

Note: When the Gram smear shows organisms and

pus cells, little additional information will be

provided by testing for protein and glucose. When

however no bacteria are seen in the Gram smear

and the cell count is raised, testing for protein and

glucose can help to differentiate those conditions in

which lymphocytes are found in c.s.f., e.g. viral

meningitis (slightly raised protein, normal glucose)

from tuberculous meningitis (high protein, low

glucose).

Measurement of c.s.f. glucose

Glucose must be measured within 20 minutes of the

c.s.f. being withdrawn otherwise a falsely low result

will be obtained due to glycolysis. Use the super-

natant ﬂuid from centrifuged c.s.f. or uncentrifuged

c.s.f. if the sample appears clear.

Glucose can be measured in c.s.f. using a colori-

metric technique or a simpler semiquantitative

technique using Benedict’s reagent. Both techniques

are described in subunit 6.12 in Part 1 of the book.

Fig 7.20 Modiﬁed Fuchs-Rosenthal ruled chamber. Cells are

counted in the squares marked W1, W2, W3, W4, W5.

Multiply the cells counted by 2. Report the

number of cells per litre (1) of c.s.f.

Example (Using 1 in 2 c.s.f. dilution and Fuchs-

Rosenthal chamber)

If 240 cells are counted in 5 squares:

240  2  480

Report as 480  10

cells/1*

*Formerly, 480  10

cells/1 would have been

reported as 480 cells/mm

or 480 cells/l.

When using an Improved Neubauer chamber: Count the

cells in 4 of the large squares as shown in Fig. 7.21.

Multiply the cells counted by 5. Report the number of

cells per litre of c.s.f.

Example

If 64 cells are counted in 4 squares:

64 5  320

Report as 320  10

cells/1.

Calculation factors when using 1 in 10 c.s.f.

dilution

 Fuchs-Rosenthal chamber: Multiply cells counted

in 5 squares by 10. Report number of cells per

litre of c.s.f. (see above text).

Fig 7.21 Improved Neubauer ruled chamber (can be used

when a Fuchs-Rosenthal chamber is not available). Cells are

counted in the squares marked W1, W2, W3, W4.

Normal c.s.f. glucose: This is about half to two thirds

that found in blood i.e. 2.5–4.0 mmol/1 (45–72

mg%).

Raised c.s.f. glucose: Occurs when the blood glucose

level is raised (hyperglycaemia) and sometimes with

encephalitis.

Low c.s.f. glucose: The c.s.f. glucose concentration is

reduced in most forms of meningitis, except viral

meningitis.

In pyogenic bacterial meningitis it is markedly

reduced and may even be undetectable.

Measurement of c.s.f. total protein and

globulin test

Use the supernatant ﬂuid from centrifuged c.s.f. or

uncentrifuged c.s.f. when the sample appears clear.

Total protein can be measured in c.s.f. using a

colorimetric technique or a visual comparative tech-

nique, as described in subunit 6.12 in Part 1 of the

book.

Pandy’s test is a screening test which detects rises in

c.s.f. globulin. It is of value when it is not possible to

measure c.s.f. total protein. The method is described

in subunit 6.12.

Normal c.s.f. protein: Total c.s.f. protein is normally

0.15–0.40 g/l (15–40 mg%). The range for ventricu-

lar ﬂuid is slightly lower. Values up to 1.0 g/l (100

mg%) are normal for newborn infants. Only traces

of globulin are found in normal c.s.f., insufﬁcient to

give a positive Pandy’s test.

Increased c.s.f. total protein with positive Pandy’s test:

Occurs in all forms of meningitis, in amoebic

and trypanosomiasis meningoencephalitis, cerebral

malaria, brain tumours, cerebral injury, spinal cord

compression, poliomyelitis, the Guillain-Barré

syndrome (often the only abnormality), and

polyneuritis. Increases in c.s.f. protein also occur in

diseases which cause changes in plasma proteins

such as myelomatosis.

When the total protein exceeds 2.0 g/l (200 mg%),

the ﬁbrinogen level is usually increased sufﬁciently to

cause the c.s.f. to clot. This may occur in severe

pyogenic meningitis, spinal block, or following

haemorrhage.

Note: In diseases of the nervous system such as multiple scle-

rosis, neurosyphilis, and some connective tissue disorders it is

possible to ﬁnd a positive Pandy’s test for gobulin with only a

slight rise or even normal total protein.

MICROBIOLOGICAL TESTS 121

7.13

ADDITIONALM ICROSCOPICAL INVESTIGATIONS

Ziehl-Neelsen smear when tuberculous

meningitis is suspected

Examine a Ziehl-Neelsen stained c.s.f. smear for acid

fast bacilli (AFB) when tuberculous meningitis is clin-

ically suspected or the c.s.f. contains lymphocytes

and the glucose concentration is low and the protein

raised. AFB, however, are difﬁcult to detect in c.s.f.

The following technique increases the chances of

ﬁnding the bacteria:

1 Centrifuge the c.s.f. at high speed for 20–30

minutes. Remove the supernatant ﬂuid and mix

the sediment. Transfer several drops of the

sediment to a slide, allowing each drop to dry

before adding the next.

2 Fix the dry preparation with methanol (see

subunit 7.3.2) and stain by the Ziehl-Neelsen

technique (see subunit 7.3.5).

3 Examine the smear ﬁrst with 40 objective to

see the distribution of material and then with the

100  objective to detect the AFB. Examine the

entire preparation. The appearance of M. tuber-

culosis in a Ziehl-Neelsen stained smear is shown

in colour Plates 56 and 57.

Fluorochrome smear to detect M. tuberculosisin c.s.f.

When facilities for ﬂuorescence microscopy are available,

examination of an auramine stained smear is a more sensitive

method of detecting AFB in c.s.f. The ﬂuorescing rods can be

detected using the 40objective. The auramine staining tech-

nique and equipment required for ﬂuorescence microscopy

are described in subunit 7.3.6).

India ink preparation when cryptococcal

meningitis is suspected

When cryptococcal meningitis is clinically suspected,

e.g. patient with HIV disease, or when yeast cells are

detected when performing a cell count or examin-

ing a Gram smear, examine an India ink preparation

or a wet preparation by dark-ﬁeld microscopy for

encapsulated yeasts.

1 Centrifuge the c.s.f. for 5–10 minutes. Remove

the supernatant ﬂuid and mix the sediment.

2 Transfer a drop of the sediment to a slide, cover

with a cover glass and examine by dark-ﬁeld

microscopy (see subunit 7.3.1) or add a drop of

India ink (Pelikan black drawing ink is suitable*),

mix and cover with a cover glass.

*When ink is not available, use nigrosin 200g/l (20% w/v)

solution.

Note: Do not make the preparation too thick

otherwise the cells and capsules will not be seen.

3 Examine the preparation using the 40 objec-

tive. Look for oval or round cells, some showing

budding, irregular in size, measuring 2–10 m

in diameter and surrounded by a large unstained

capsule as shown in colour Plate 73. Very

occasionally capsules are absent.

Important: When encapsulated yeasts are detected

in c.s.f., a presumptive diagnosis of cryptococcal

meningitis can be made. The medical ofﬁcer attend-

ing the patient should be notiﬁed immediately.

Further information on C. neoformans can be found

in subunit 7.18.48.

Antigen detection to diagnose cryptococcal meningitis

Soluble antigen can be detected immunologically in both the

serum and c.s.f. of patients infected with C.neoformans, see

subunit 7.18.48.

Wet preparation and Giemsa smear when

trypanosomiasis meningoencephalitis is

suspected

Freshc.s.f. is re quired to detect trypanosomes. About

15 minutes after the ﬂuid is withdrawn, the try-

panosomes begin to lose their motility and are

rapidly lyzed. The trypanosomes are usually few and

therefore a careful search of a wet preparation is

required to detect the motile ﬂagellates.

1 Centrifuge the c.s.f. at about 1000 g for 10

minutes. Remove the supernatant ﬂuid and mix

the sediment.

2 Transfer a drop of sediment to a slide and cover

with a cover glass. Examine for motile try-

panosomes using the 40 objective with the

condenser iris closed sufﬁciently to give good

contrast.

Alternatively examine the preparation by dark-

ﬁeld microscopy (see subunit 7.3.1).

Double centrifugation technique: When a microhaematocrit

centrifuge is available, a more sensitive method of detecting

trypanosomes in c.s.f. is to use a double centrifugation tech-

nique. This involves transferring the sediment from

centrifuged c.s.f. to a capillary tube, and centrifuging it for a

further 1 minute in a microhaematocrit centrifuge. The capil-

lary tube is mounted on a slide and the preparation examined

for motile trypanosomes as described on p. 263 in Part 1 of the

book.

Note: African trypanosomiasis and the investigations

which are used to diagnose late stage disease includ-

ing testing for IgM in c.s.f. are described in subunit

5.8 in Part 1 of the book.

Giemsa smear to detect morula cells

When no trypanosomes are seen in the wet prep-

aration, remove the cover glass and allow the

preparation to air-dry. Fix the smear with methanol

122 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.13

and stain it using Giemsa technique (see subunit

7.3.4) or Field’s rapid stain as used for thin ﬁlms.

Examine the preparation for morula cells (IgM pro-

ducing cells) using the 40 objective. The cells are

easily recognized. They are larger than lymphocytes

with a dark mauve staining nucleus and characteris-

tic vacuoles in their cytoplasm as shown in colour

Plate 5.52 on p. 265 in Part 1 of the book. Finding

morula cells in the c.s.f. of a person with African try-

panosomiasis, indicates central nervous system

involvement.

Wet preparation to detect amoebae

Examine a wet preparation for motile amoebae

when primary amoebic meningoencephalitis is clini-

cally suspected (rare condition caused by N. fowleri)

or the c.s.f. contains pus cells with raised protein and

low glucose, but no bacteria are seen in the Gram

smear. Red cells may also be present.

1 Transfer a drop of uncentrifuged purulent c.s.f. or

a drop of sediment from a centrifuged specimen

to a slide and cover with a cover glass.

2 Examinethe preparation using the 10and 40

objectives, with the condenser closed sufﬁciently

to give good contrast. Look for small, clear,

motile,elongate dforms among the pus cells. Use

the 40 objective to identify the amoebae (even

if not ﬁrst se en using the 10 objective, always

examinethe preparation with the 40 objective).

The amoebae often contain vacuoles but not red

cells. Further information on amoebic meningo-

encephalitis can be found on pp. 299–300 in

Part 1 of the book.

Important: When amoebae are seen in the c.s.f.,

immediately notify the medical ofﬁcer attending the

patient. Amoebic meningocephalitis is a rapidly fatal

condition.

Examine and report the cultures

Chocolate agar and blood agar cultures

Look especially for colonies that could be:

 Neisseria meningitidis (growing on chocolate agar

and blood agar, oxidase positive (see subunit

7.18.12 )

 Streptococcus pneumoniae (sensitive to optochin,

see subunit 7.18.4)

 Haemophilus inﬂuenzae (growing only on choco-

late agar, see subunit 7.18.24)

 Cryptococcus neoformans (Gram stain the

colonies), see subunit 7.18.48)

Day 2 and Onwards

MICROBIOLOGICAL TESTS 123

7.13

Summary of the Examination of C.S.F.

Day 1

1 Report

Appearance

 Describe whether c.s.f.

– Clear, slightly turbid,

cloudy, purulent

– Contains blood

– Contains clots

2 Test c.s.f.

Purulent or cloudy c.s.f.

Suspect pyogenic bacterial meningitis

 Gram smear

Report:

– Number of pus cells

– Bacteria

 Culture c.s.f.

– Blood agar and chocolate agar.

Incubate in CO

– If neonate:

Also MacConkey agar.

Incubate aerobically

Slightly turbid or clear c.s.f.

 Perform cell count

Note whether pus cells or lymphs

Day 2 and Onwards

PUS CELLS

 Gram smear

See opposite

 Culture c.s.f.

See opposite

LYMPHS

 Measure protein

 Measure glucose

 Zn: For AFB

 India Ink: For

encapsulated yeasts

ADDITIONAL TESTS

 Wet preparation:

For motile amoebae

 Wet preparation

and Giemsa smear:

For trypanosomes

and morula cells

3 Examine and

Report Cultures

 Chocolate agar and blood

agar cultures

Look particularly for:

N.meningitidis

S.pneumoniae

H.inﬂuenzae (chocolate agar)

 MacConkey agar culture

Look especially for bacteria

that cause neonatal meningitis

ADDITIONAL

 Perform antimicrobial

susceptibility tests as

indicated

 Beta-lactamase test.

H.inﬂuenzae isolates

MacConkey agar culture

Look especially for colonies that could be:

 Escherichiacoli orother coliform,see subunit 7.18.14.

 Streptococcus agalactiae, see subunit 7.18.3.

 Listeria monocytogenes, see subunit 7.18.8.

 Other bacteria that cause neonatal meningitis

(see previous text).

Streptococcus suis identiﬁcation: S. suis is a non-haemolytic

Gram positive coccus, belonging to Lanceﬁeld Group D. It is

CAMP negative, hydrolyzes aesculin and is able to grow on

bile agar.

Antimicrobial susceptibility testing

Testisolatesof S. pneumoniaefor susceptibilityto chlo-

ramphenicol and penicillin (use 1g oxacillin disc).

TestH. inﬂuenzae for beta-lactamase production (see

end of subunit 7.16) and susceptibility to chloram-

phenicol(using chocolate agar).Perform susceptibility

testingon Gram negative rods as indicated.

124 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.13– 7.14

7.14 Culturing blood

Blood culture is required when bacteraemia (septi-

caemia) is suspected.

Bacteraemia

The presence of bacteria in the blood is called bacteraemia. It

is usually pathological although transitory asymptomatic bac-

teraemia can occur during the course of many infections and

following surgical procedures. Bacteraemia occurs in diseases

such as typhoid fever, brucellosis, leptospirosis and endo-

carditis.

Septicaemia

This is a clinical term used to describe severe life-threatening

bacteraemia in which multiplying bacteria release toxins into

the blood stream and trigger the production of cytokines,

causing fever, chills, toxicity, tissue anoxia, reduced blood

pressure, and collapse. Septic shock is usually a complication

Chart7.9 Results of c.s.f. testsin pyogenic bacterial meningitis, other forms of meningitis and meningoencephalitis

Appearance Cells (WBCs) Protein* Glucose* Microscopy/Other Tests

Normal c.s.f. Clear Below 5  10

/1, 0.15 –0.40 g/l 2.5–4.0 mm 0l/l

Colourless

Lymphs

(15–40 mg%) (45–72 mg%)

Pandys: Negative

Pyogenic Purulent Usually many High Very low Gram: Bacteria may

bacterial or cloudy

Pus cells Pandy’s: Positive

be seen resembling:

meningitis ● N. meningitidis

● S. pneumoniae

● H. inﬂuenzae b (below 5 y)

● Coliforms, S.agalactiae,

L.monocytogenes, etc

Viral Clear or Raised Normal or Usually –

meningitis slightly turbid

Lymphs

increased normal

Pandy’s: Neg/Pos

Tuberculous Clear or Raised High Reduced Ziehl-Neelsen: AFB

meningitis slightly turbid

Lymphs Pandy’s: Positive

difﬁcult to ﬁnd

Cryptococcal Clear or Raised Usually Normal or India ink (c.s.f.sediment)

meningitis slightly turbid

Lymphs

increased reduced Encapsulated yeasts

Pandy’s: Positive Gram: Unevenly stained yeasts

of variable size, some budding

Primary amoebic Cloudy Raised Increased Reduced Wet preparation: Motile

meningoencephalitis

Pus cells Pandy’s: Positive

amoebae, often with vacuoles

Trypanosomiasis Clear or Raised High Normal or Wet preparation(c.s.f. sediment):

encephalitis slightly turbid

Lymphs

(IgM: very high) reduced Trypanosomes may be seen

Pandy’s: Positive Giemsa: Morula cells

Syphilitic Usually Raised Normal or Normal or Speciﬁc treponemal antibody

meningitis clear

Lymphs

increased increased test (serum) will be positive

Pandy’s: Positive

c.s.f. VDRL will be positive

*Test may not be required when cause of meningitis is indicated from Gram smear or other microscopical examination.

of septicaemia with Gram negative bacilli, and less frequently,

Gram positive organisms. Prompt treatment is essential.

Bacteraemia usually occurs when pathogens enter

the bloodstreamfrom abscesses, infecte dwounds or

burns, or from areas of localized disease as in pneu-

mococcal pneumonia, meningitis, pyelonephritis,

osteomyelitis,cholangitis, peritonitis, enterocolitis and

puerperal sepsis. There is usually a high white cell

count with neutrophilia, left shift of the neutrophils,

and often toxic granulation (see subunit 8.7).

Possible pathogens isolated from blood

cultures

 BACTERIA

Gram positive Gram negative

Staphylococcus aureus Salmonella Typhi

Viridans streptococci Other Salmonella serovars

Streptococcus pneumoniae Brucella species

Streptococcus pyogenes Haemophilus inﬂuenzae

Enterococcus faecalis Pseudomonas aeruginosa

Clostridium perfringens Klebsiella strains

Anaerobic streptococci Escherichia coli

Proteus species

Bacteroides fragilis

Neisseria meningitidis

Yersinia pestis

Also Mycobacterium tuberculosis (HIV-associated

tuberculosis), Leptospira species, Borrelia species,

rickettsiae, and Bartonella bacilliformis.

 FUNGI

Candidaalbicansand otheryeasts, e.g.Cryptococcus

neoformans, andoccasionally Histoplasma capsula-

tumand other fungithat cause systemic mycoses.

Note: The identiﬁcation of parasites that can be

found in blood (Plasmodium species, Trypanosoma

species, Leishmania species and ﬁlarial parasites) are

described in Part 1 of the book. Leptospira species

are described in subunit 7.18.33, Borrelia species in

7.18.34, rickettsiae in subunit 7.18.35, and B. bacilli-

formis in subunit 7.18.36.

Notes on pathogens

● In typhoid, Salmonella Typhi can be detected in the

blood of 75–90% of patients during the ﬁrst 10 days of

infection and in about 30% of patients during the third

week.

● Salmonella serovars other than Typhi and Paratyphi are

common causes of bacteraemia in children in tropical and

developing countries, particularly those who are anaemic

and malnourished or have malaria.

● Non-typhoid Salmonella (particularly S. Typhimurium

and S. Interitidis) and S. pneumoniae are associated with

recurring bacteraemia in those infected with HIV.

MICROBIOLOGICAL TESTS 125

7.14

● Bacteria that cause neonatal septicaemia include E. coli

and other coliforms, staphylococci, beta-haemolytic

Group B streptococci, and less frequently enterococci,

L.monocytogenes, diphtheroids, and Candida albicans.

● Viridans streptococci are the commonest cause of

subacute infective endocarditis. Other causes include

enterococci, S. epidermidis, H. inﬂuenzae, Coryne-

bacterium species, and very occasionally rickettsiae.

Acute endocarditis is usually caused by Staphylococcus

aureus, Streptococcus pyogenes, and pneumococci.

● Y. pestis can be isolated from the blood in septicaemic

plague. The organism is highly infectious.

● Although motile leptospires can occasionally be found in

blood (by dark-ﬁeld microscopy), leptospirosis is usually

diagnosed serologically (see subunit 7.18.33).

● Brucella species are more likely to be isolated by

blood (or bone marrow) culture in acute brucellosis

during times of fever. Isolation is rare in B.abortus infec-

tions. The organisms are slow-growing. They are highly

infectious.

Commensals

Blood does not have a normal microbial ﬂora.

Common skin contaminants include coagulase-

negative staphylococci, viridans streptococci,

micrococci, and Corynebacterium species.

1 Colle ct blood and inoculate culture media

Whenever possible blood should be collected before

antimicrobial treatment has started. When the

patient has recurring fever, collect the blood as the

temperature begins to rise. For other patients, collect

the blood as soon as possible after receiving the

request. To increase the chances of isolating a

pathogen, it is usually recommended that at least

two specimens (collected at different times) should

be cultured. A strict aseptic technique must be used

to collect the blood (see later text).

Choice of culture media

Media selected for the culture of blood should be

capable of providing the fastest growth and isolation

of as wide a range of pathogens as possible. The

following media are recommended:

● Columbia agar and Columbia broth diphasic

medium with added SPS (sodium polyanethol

sulphonate), also known as Liquoid. SPS prevents

the blood from clotting, neutralizes complement

and other antibacterial substances in fresh blood,

and has some neutralizing effect on polymyxin

B, streptomycin, and gentamicin should these be

present in the blood. SPS also enables a greater

Day 1

volume of blood to be cultured without increas-

ing the volume of broth, i.e. up to 50% of

the total volume of medium. Preparation of

Columbia diphasic medium is described in

Appendix II, No. 26.

Diphasic blood culture medium

A diphasic (Castenada) medium is one that combines an

agar slope with a broth medium (see Plate 7.19). The

blood broth is allowed to run over the slope by tipping the

bottle at regular intervals. Microbial activity can be seen

by growth on the slope (beginning at the broth-agar inter-

face). This avoids the need to subculture from the bottle

and therefore reduces the risk of contaminating the

culture. When brucellosis is suspected, a diphasic medium

is particularly recommended because Brucellaspecies are

slow-growing and also ‘high risk’ category 3 pathogens.

Use of Columbia agar and broth

Tryptone soya (tryptic soy) diphasic medium is often rec-

ommended for culturing blood but organisms such as

S.pneumoniae and N.meningitidis have been shown not to

grow well in this medium. Columbia agar and broth are

recommended for the isolation of these pathogens and

other fastidious organisms. Brucellae, however, grow well

in tryptone soya diphasic medium.

126 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.14

dilute out the blood’s natural bactericidal sub-

stances. The blood should be diluted at least 1 in

10 with broth. Preparation of thioglycollate broth

is described in Appendix II, No. 80.

Isolation of S. Typhi: If wishing to culture for S. Typhi

only, the use of ox-gall medium is recommended

(see subunit 7.18.16).

Commercially produced culture media

A wide range of commercially produced culture media is

available for use with manual and automated blood culture

systems. Manufacturers of media for non-automated use

include Oxoid, Bio-Rad Laboratories, BD Diagnostics and

bioMérieux (see Appendix 11). The media are mostly

tryptone soy or brain heart infusion broth with added growth

factors. Some media are also available with antibiotic

removing resins. A few diphasic media are available, e.g. from

Bio-Rad Laboratories (in triangular bottles), BD Diagnostics

and bioMérieux. The semi-automated Oxoid Signal Blood

culture system recognizes microbial growth in the blood

culture by gas production.

Aseptic blood collection and dispensing

technique

Blood for culture must be collected and dispensed

with great care to avoid contaminating the specimen

and culture medium.

1 Using a pressure cuff, locate a suitable vein in the

arm. Deﬂate the cuff while disinfecting the

venepuncture site.

2 Wearing gloves, thoroughly disinfect the

venepuncture site as follows:

– Using 70% ethanol, cleanse an area about

50 mm in diameter. Allow to air-dry.

– Using 2% tincture of iodine and a circular

action, swab the area beginning at the point

where the needle will enter the vein. Allow

the iodine to dry on the skin for at least 1

minute.

3 Lift back the tape or remove the protective cover

from the top of the culture bottle(s). Wipe the top

of the bottle using an ethanol-ether swab.

Prior inspection of culture media bottles

Do notuse a bottle of culture medium if it shows signs of

contamination, i.e. broth appears turbid. Do not use a

bottle of thioglycollate broth if it appears oxidized, i.e.

more than a third of the top of the medium appears pink

when the indicator in the medium is resazurin, or more

than 20 mm down from the surface of the medium

appears green-blue when the indicator is methylene blue.

When oxidation has occurred, the medium must be

reduced by steaming before it is used.

4 Using a sterile syringe and needle, withdraw

about 20 ml of blood from an adult* or about

Plate 7.19 Right: Diphasic blood culture medium. Left:

Inoculated diphasic medium.

● Thioglycollate broth medium is recommended to

isolate strict anaerobes should an anaerobic

infection be suspected. It consists of nutrient

broth to which is added thioglycollate to provide

the conditions necessary for the growth of anaer-

obes. Because SPS is inhibitory to anaerobic

streptococci, it is not added to this medium,

therefore a sufﬁcient volume of broth must be

used to prevent the blood from clotting and to

2 ml from a young child.

*Note: When an anaerobic culture is not indi-

cated, collect 10–15 ml of blood.

5 Insert the needle through the rubber liner of the

bottle cap and dispense 10–12 ml of blood into

the diphasic culture medium bottle containing

25 ml of broth (see No. 26).

Changing needles: Studies have shown that when dispens-

ing the blood into the culture medium, it is not necessary

to replace the needle with a sterile needle. Not changing

the needle reduces the risk of accidental needle-prick

injury.

When also culturing for anaerobes, dispense

about 5 ml of blood into the thioglycollate

culture medium containing 50 ml of broth (see

No. 80).

Dispense the remaining approximately 2 ml of

blood into a tube or bottle containing ethylene-

diaminetetra acetic acid (EDTA).

EDTA sample: This is collected to perform a total and

differential white cell count, and to examine stained

smears of the plasma buffy coat layer for microorganisms,

when the blood is from a child or a patient with AIDS.

6 Using a fresh ethanol-ether swab, wipe the top of

each culture bottle and replace the tape or pro-

tective cover(s). Without delay, mix the blood

with the broth and mix the blood in the EDTA

container.

Important: The blood must not be allowed to clot

in the culture media because any bacteria will

become trapped in the clot.

7 Clearly label each bottle with the name and

number of the patient, and the date and time of

collection.

8 As soon as possible, incub ate the inoculated

media. Protect the cultures from direct sunlight

until they are incubated.

Diphasic medium

Incubate at 35–37°C for up to 7 days, examin-

ing and subculturing as described later. A longer

incubation period should be allowed when endo-

carditis is suspected. When brucellosis is sus-

pected, loosen the cap of the culture bottle (or

insert a sterile needle in the cap) and incubate in

a carbon dioxide enriched atmosphere (see

subunit 7.4) for up to 4 weeks.

Thioglycollate broth

Incubate at 35–37°C for up to 2 weeks, exam-

ining and subculturing as described later.

MICROBIOLOGICAL TESTS 127

7.14

Culture of blood from neonates

To reduce the risk of contamination, blood from

neonates should be collected from a peripheral vein

not from the umbilical vein. When only a small

amount of blood is obtained, inoculate it into a

bottle of diphasic culture medium. Organisms

causing bacteraemia in young children are usually

present in sufﬁcient concentration to be detected in

small volumes of blood (1–2 ml) of blood).

Examine also a Gram stained smear of the

plasma buffy coat layer, obtained by centrifuging

anticoagulated capillary blood. It is often possible to

make a rapid diagnosis of bacteraemia in infants by

this method.

Plate cultures using lyzed blood to diagnose septicaemia in

young children

Microbiologists in Bangladesh recommend the use of saponin

lyzed blood, plated on blood agar and MacConkey agar as a

sensitive rapid (within 48 h), and inexpensive technique to

diagnose septicaemia in young children.* Blood (2 ml) is col-

lected into a tube containing 0.2 ml of a ﬁlter-sterilized

solution of saponin (2 mg) and SPS (0.8 mg). The lyzed blood

is inoculated directly onto blood agar and MacConkey agar

plates and the plates incubated overnight (blood agar plate in

a candle jar). When the blood is likely to contain antibiotics,

it is ﬁrst centrifuged, the supernatant ﬂuid removed, and the

sediment used to inoculate the plates.

*Further information: Readers should contact the Consultant

Microbiologist, Dhaka Shishu (Children) Hospital, Sher-e-

Bangla Nager, Dhaka 1207, Bangladesh.

2 Examine the spe cimen microscopically

Centrifuge a sample of EDTA anticoagulated venous

blood or heparinized capillary blood and make

smears of the buffy coat layers. Stain as follows:

 Gram smear: To detect Gram positive and

Gram negative bacteria, particularly when the

patient is an infant or young child.

 Ziehl-Neelsen smear: To detect AFB when

the patient has AIDS or suspected HIV disease.

 Giemsa or rapid Field’s smear: To detect

borreliae, or parasites such as trypanosomes,

malaria parasites, and microﬁlariae.

Note: Microﬁlariae and trypanosomes are more easily

detected by their motility using a microhaematocrit con-

centration technique (see Part 1 of the book).

Allow the smears to air-dry, ﬁx with absolute

methanol for 2 minutes and stain by the appropriate

staining technique.

3 Examine and report the cultures

Diphasic culture (Columbia agar and broth)

Using ahand lens, examine twice daily (up to 7 days

or 4 weeks when brucellosis is suspected) for micro-

bialgrowth, indicated bycolonies growingon theagar

slope,usually beginning at the agar-broth interface.

Colonial appearances

Colonies of staphylococci, S. Typhi, brucellae, and

most coliforms can usually be seen easily, whereas

colonies of S. pneumoniae, Neisseria species,

S. pyogenes, and Y. pestis are less easily seen.

Pseudomonas and Proteus species produce a ﬁlm of

growth on the agar.

When growth is present:

– Subculture on blood agar, chocolate agar, and

MacConkey agar.

– Incubate the blood agar and MacConkey agar

plates aerobically and the chocolate agar plate in

a carbon dioxide atmosphere (candle jar).

– Examine a Gram stained smear of the colonies.

Depending on the bacteria seen, test the colonies

further (e.g. for coagulase, catalase, oxidase,

urease, and motility).

● When large Gram positive rods resembling

C. perfringens are seen: Subculture also on

lactose egg yolk milk agar (see No. 47) and

incubate the plate anaerobically (see subunit

7.18.9).

● When motile, urease and oxidase negative Gram

negative rods are isolated: Subculture the

colonies on Kligler iron agar (subunit 7.18.6).

● When catalase positive Gram negative coc-

cobacilli are isolated: Suspect Brucella species and

send the culture (securely) to a microbiology spe-

cialist laboratory for identiﬁcation. Mark the

culture ‘High Risk’.

Blind subculture after overnight incubation

Because some organisms such as Neisseria species

and S. pneumoniae may grow without producing

easily seen colonies, it is advisable to examine a tolu-

idine blue stained smear and subculture onto agar

plates even when no microbial growth is apparent

after overnight incubation.

Important: Always report immediately a

positive blood culture, and send a preliminary

report of the stained smear and other useful test

Day 2 and Onwards

128 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.14

results. When Gram negative rods or staphylococci

are seen in a Gram smear, set up appropriate

susceptibility tests.

Note: When no growth is seen on the slope of the

diphasic culture, wash the broth over the slope

before reincubating the culture (do not allow the

broth to ﬂow into the neck of the bottle).

Thioglycollate broth culture

Examine daily (up to 14 days) for visible signs of bac-

terial growth such as turbidity above the red cell

layer, colonies growing on the surface of the red

cells (‘cotton balls’), haemolysis, gas bubbles, and

clots. Most organisms (not just anaerobes) grow in

thioglycollate broth.

When there are signs of bacterial growth, subculture

the broth (see following text) and examine a tolui-

dine blue stained smear for bacteria.

Toluidine blue stained smear of a broth culture

Bacteria are easier to detect in a broth culture if the prep-

aration is stained using toluidine blue 0.5%w/v (Reagent No.

85) rather than stained by the Gram technique. Make a smear

of the broth, allow to air-dry, ﬁx with absolute methanol for

1–2 minutes and stain with toluidine blue for 30–60 seconds.

Wash off with water, allow the smear to air-dry, and examine

microscopically. When bacteria are detected, examine also a

Gram stained smear to identify whether the organisms are

Gram positive or negative (unless the organisms can be rec-

ognized by their morphology in the toluidine blue

preparation).

Important: When a patient is seriously ill, subculture

the broth (even in the absence of visible bacterial

growth) after overnight incubation, after 48 hours,

and twice weekly for up to 2 weeks.

Subculturing a blood culture broth

A strict aseptic technique must be used to avoid con-

taminating the culture.

1 Using an ethanol-ether swab, cleanse the top of

the bottle. Using a sterile needle and small

syringe, insert the needle through the rubber

liner in the cap, and withdraw about 1 ml of the

broth culture.

2 Inoculate the broth on:

– Blood agar

– Chocolate (heated blood) agar

– MacConkey agar

Incubate the blood agar plate anaerobically for

up to 48 hours, the chocolate agar plate in a

carbon dioxide atmosphere for up to 48 hours,

and the MacConkey agar plate aerobically

overnight.

MICROBIOLOGICAL TESTS 129

7.14

Summary of Blood Culture Procedures

ADDITIONAL

Day 1

1 Collect Blood

Inoculate culture

media

Using an aseptic technique,

dispense:

 10 –12 ml blood into Columbia

agar diphasic medium and

mix. Incubate up to 7 days (4

weeks when brucellosis is

suspected).

 When anaerobic infection is

suspected, dispense 5 ml

blood into thioglycollate broth

and mix.

Incubate up to 14 days.

 2 ml into EDTA and mix.

2 Examine

Microscopically

Prepare buffy coat smears from

EDTA blood:

 Gram smear

 Giemsa smear

 Zn smear: From a patient

with AIDS. Look for AFB.

Day 2 and Onwards

3 Examine and

Report Cultures

After overnight incubation:

 Examine diphasic culture.

Subculture (even when no growth

is seen):

– Blood agar and MacConkey agar.

Incubate aerobically

– Chocolate agar. Incubate in CO

 Examine toluidine blue smear

(diphasic culture).

 Examine thioglycollate culture.

Subculture and examine

microscopically.

Incubate subculture anaerobically.

Note: When there is no growth, wash

slope of diphasic culture. Reincubate

cultures. Subculture as indicated.

Examine subcultures for likely

pathogens

(See beginning of subunit 7.14)

Identify organisms

Perform antimicrobial

susceptibility tests as indicated

When S. Typhi is suspected:

Ox-gall broth is recommended

When brucellosis is suspected:

Tryptic soya diphasic medium

is recommended

3 Swab the top of the culture bottle and reincu-

bate.

Contamination of blood cultures

Contamination can occur when an aseptic technique

is not used at the time the blood is collected or at a

later stage in the laboratory when subculturing.

Frequent contaminants of blood cultures include

commensal staphylococci, micrococci, and diph-

theroids or contaminants from the environment

such as species of Bacillus or Acinetobacter.

Occasionally in immunocompromised patients,

organisms usually considered ‘contaminants’ may be

pathogenic, especially fungi.

Contamination is indicated when an organism is

recovered from only one bottle when it should have

grown in both thioglycollate broth and the diphasic

culture medium or when a mixed microbial ﬂora is

isolated.

Note: An obligatory anaerobe will be isolated from the thio-

glycollate culture only.

Bacteriological investigation of a transfusion

reaction

Severe and often fatal reactions can be caused by

the transfusion of contaminated blood. The bac-

teriological investigation of a transfusion reaction is

as follows:

1 Report whether the blood remaining in the unit

of transfused blood shows any visible signs of

being contaminated such as:

– appearing unusually dark in colour,

– containing small clots,

– the plasma appearing red, or unusually

turbid (examine after centrifuging a sample

of the blood).

Note: Haemolysis does not always occur when

blood is contaminated.

2 Using an aseptic technique, inoculate three

bottles of thioglycollate broth, each with about

4 ml of the well-mixed blood. Mix gently, and

label each bottle with the date and unit number

of the blood.

Incubate one bottle at 35–37°C, one at room

temperature, and refrigerate the other at 4°C for

up to 7 days, examining for growth and subcul-

turing as described under ‘Examining and

reporting of blood cultures’.

3 Perform a motility test (see subunit 7.3.2) and

examine a Gram stained smear of the plasma.

130 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.14– 7.15

Note: Recognition of bacteria in a Gram smear,

especially Gram negative organisms, is often

difﬁcult due to background debris.

Bacteria that may be found in contaminated bank

blood

These are usually organisms that are capable of

growing at room temperature or below. They

include coliforms, Achromobacter species, and

pseudomonads and less commonly Yersinia species.

7.15 Examination of semen

Analysis of semen (seminal ﬂuid) is included in this chapter

because it is often performed in a microbiology laboratory.

The values and clinical notes contained in this subunit have

been referenced from the WHO laboratory manual for the

examination of human semen and semen-cervical mucus inter-

action(see Further information)

When investigating infertility, the basic analysis of

semen (seminal ﬂuid) usually includes:

 Measurement of volume

 Measurement of pH

 Examination of a wet preparation to estimate the

percentage of motile spermatozoa and viable

forms and to look for cells and bacteria.

 Sperm count

 Examination of a stained preparation to estimate

the percentage of spermatozoa with normal

morphology.

Collection and transport of semen

1 Give the person a clean, dry, leak-proof con-

tainer, and request him to collect a specimen of

semen at home following 3–7 days of sexual

abstinence.

Note: When a condom is used to collect the

ﬂuid, this must be well-washed to remove the

powder which coats the rubber. It must be dried

completely before being used.

Coitus interruptus: This method of collection should not

be used because the ﬁrst portion of the ejaculate (often

containing the highest concentration of spermatozoa)

may be lost. Also the acid pH of vaginal ﬂuid can affect

sperm motility and the semen may become contaminated

with cells and bacteria.

2 Ask the person to write his name on the con-

tainer, date and time of collection, period of

abstinence, and to deliver the specimen to the

laboratory within 1 hour after collection.

During transit to the laboratory, the ﬂuid should

be kept as near as possible to body temperature.

This is best achieved by placing the container

inside a plastic bag and transporting it in a

pocket in the person’s clothing.

LABORATORYEXAMINATION OF SEMEN

Caution: Handle semen with care because it may

contain infectious pathogens, e.g. HIV, hepatitis

viruses, herpes viruses.

1 Measure the volume

Normal semen is thick and viscous when ejaculated.

It becomes liqueﬁed usually within 60 minutes due

to a ﬁbrinolysin in the ﬂuid. When liqueﬁed, measure

the volume of ﬂuid in millilitres using a small gradu-

ated cylinder.

Normal specimens: Usually 2 ml or more.

2 Measure the pH

– Using a narrow range pH paper, e.g. pH

6.4–8.0, spread a drop of liqueﬁed semen on the

paper.

– After 30 seconds, record the pH.

pH of normal semen: Should be pH 7.2 or more

within 1 hour of ejaculation. When the pH is over 7.8

this may be due to infection. When the pH is below

7.0 and the semen is found to contain no sperm, this

may indicate dysgenesis (failure to develop) of the

vas deferens, seminal vesicles or epididymis.

3 Estimate the percentage of motile and

viable spermatozoa

Motility

– Place 1 drop (10–15 l)* of well-mixed liqueﬁed

semen on a slide and cover with a 20  20 mm

or 22  22 mm cover glass.

*1 drop falling from a 21 g needle is equivalent to a

volume of 10–15 l.

– Focus the specimen using the 10 objective.

Close the condenser iris sufﬁciently to give good

contrast. Ensure the spermatozoa are evenly dis-

tributed (if not, re-mix the semen and examine a

new preparation).

– Using the 40 objective, examine several ﬁelds

to assess motility, i.e. whether excellent (rapid

and progressive) or weak (slow and non-pro-

gressive). Count a total of 100 spermatozoa, and

note out of the hundred how many are motile.

Record the percentage that are motile and non-

motile.

MICROBIOLOGICAL TESTS 131

7.15

Normal motility: Over 50% of spermatozoa are

motile within 60 minutes of ejaculation.

The spermatozoa remain motile for several hours.

When more than 60% of spermatozoa are non-

motile, examine an eosin preparation to assess

whether the spermatozoa are viable or non-viable

(see following text).

Presence of cells in semen: Report when more than a few leu-

cocytes (pus cells) or red cells are present. When pus cells are

seen, examine a Gram stained smear for bacteria.

Viability

– Mix one drop (10–15 l) of semen with 1 drop

of 0.5% eosin solution* on a slide.

*Dissolve 0.1 g of eosin in 20 ml of fresh physiological

saline.

– After 2 minutes examine the preparation micro-

scopically. Use the 10 objective to focus the

specimen and the 40 obje ctive to count the

percentage of viable and non-viable spermato-

zoa. Viable spermatozoa remain unstained,

non-viable spermatozoa stain red.

Normal viability: 75% or more of spermatozoa

should be viable (unstained). A large proportion of

non-motile but viable spermatozoa may indicate a

structural defect in the ﬂagellum.

4 Perform a sperm count

– Using a graduated tube or small cylinder, dilute

the semen 1 in 20 as follows:

Fill the tube or cylinder to the 1 ml mark with

well-mixed liqueﬁed semen.

Add sodium bicarbonate-formalin diluting ﬂuid

(ReagentNo. 72) tothe 20 mlmark, and mix well.

– Using a Pasteur pipette, ﬁll an Improved

Neubauer ruled chamber with well-mixed

diluted semen. Wait 3–5 minutes for the sper-

matozoa to settle.

– Using the 10objective with the condenser iris

closed sufﬁciently to give good contrast, count the

number of spermatozoa in an area of 2 sq mm,

i.e. 2 large squares.

Note: The total area of an Improved Neubauer and a

Bürker ruled chamber is 9 sq mm, i.e. 9 large squares.

– Calculate the number of spermatozoa in 1 ml of

ﬂuid by multiplying the number counted by

100000.

Normal count: 20  10

spermatozoa/ml or more.

Counts less than 20  10

/ml are associated with

male sterility.

5 Estimate the percentage of spermatozoa

with normal morphology in a stained

preparation

– Make a thin smear of the liqueﬁed well-mixed

semen on a slide. While still wet, ﬁx the smear

with 95% v/v ethanol for 5–10 minutes, and

allow to air-dry.

– Wash the smear with sodium bicarbonate-

formalin solution (Reagent No. 72) to remove

any mucus which may be present. Rinse the

smear with several changes of water.

– Cover the smear with dilute (1 in 20) carbol

fuchsin and allow to stain for 3 minutes. Wash off

the stain with water.

– Counterstain, by covering the smear with dilute

(1 in 20) Loefﬂer’s methylene blue for 2 minutes.

Wash off the stain with water. Drain, and allow

the smear to air-dry.

Staining results

Nucleus of head . . . . . . . . . . . . . . . . . . . . Dark blue

Cytoplasm of head. . . . . . . . . . . . . . . . . . . Pale blue

Middle piece and tail . . . . . . . . . . . . . . . . . Pink-red

Alternative stains: Other staining techniques used to stain

spermatozoa include Giemsa and Papanicolaou.

Morphology of spermatozoa

Examine the preparation for normal and abnormal

spermatozoa using the 40 objective. Use the

100  objective to conﬁrm abnormalities. Count 100

spermatozoa and estimate the percentage showing

normal morphology and the percentage that appear

abnormal.

Note: Laboratory staff who have not been trained to report

stained semen preparations should request the assistance of a

specialist cytology laboratory.

Normal spermatozoa: Measure 50–70 m in

length. Each consists of an oval-shaped head (with

acrosomal cap) which measures 3–5  2–3 m, a

short middle piece, and a long thin tail (at least

45 m in length). In normal semen, at least 50% of

spermatozoa should show normal morphology.

Most specimens contain no more than 20%

abnormal forms.

Abnormal spermatozoa: The following abnor-

malities may be seen:

Head

● Greatly increased or decreased in size.

● Abnormal shape and tapering head (pyriform)

132 DISTRICT LABORATORYPRACTICE IN TROPICAL COUNTRIES

7.15– 7.16

● Acrosomal cap absent or abnormally large.

● Nucleus contains vacuoles or chromatin is

unevenly distributed.

● Two heads.

● Additional residual body, i.e. cytoplasmic droplet.

Middle piece

● Absent or markedly increased in size.

● Appears divided (bifurcated).

● Angled where it meets tail.

Tail

● Absent or markedly reduced in length.

● Double tail.

● Bent or coiled tail.

Note: Abnormal semen ﬁndings should be checked

by examining a further specimen, particularly when

the sperm count is low and the spermatozoa appear

non-viable and abnormal. When the abnormalities

are present in the second semen, further tests are

indicated in a specialist centre.

Further information

WHO laboratory manual for the examination of human semen

and semen-cervical mucus interaction, 4th edition, 1999. ISBN

0521645999. Cambridge University Press.

7.16 Antimicrobial

susceptibility testing

Antimicrobial agents include naturally occurring anti-

biotics, synthetic derivatives of naturally occurring

antibiotics (semi-synthetic antibiotics) and chemical

antimicrobial compounds (chemotherapeutic

agents). Generally, however, the term ‘antibiotic’ is

used to describe antimicrobial agents (usually anti-

bacterial) that can be used to treat infection.

Compared with antibacterial agents, fewer antiviral

and antifungal agents have been developed. Many

antiviral agents have serious side-effects e.g. those

used to treat HIV infection.

Antimicrobial activity

Not all antimicrobials, at the concentration required

to be effective are completely non-toxic to human

cells. Most, however, show sufﬁcient selective toxicity

to be of value in the treatment of microbial diseases.

Antibacterial agents can be grouped by their

mode of action, i.e. their ability to inhibit the syn-

thesis of the cell wall, cell membrane, proteins, and

nucleic acids of bacteria.