Laboratory Practical Manual for Medical Microbiology and Parasitology

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Laboratory Practical Manual for Medical Microbiology and Parasitology

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 Laboratory Practical Manual for Medical Microbiology and Parasitology

NWADIOHA Samuel Iheanacho, (MB BCH; MSc; FMCPath; FIIA)

Professor of Medical Microbiology & Parasitology,
Consultant Clinical Microbiologist & Infectious Disease Physician,
Laboratory Consultant @ Maternity & Children Hospital, Hail Kingdom of Saudi
Arabia.
Formerly, A Staff of Benue State University, Makurdi. Nigeria.
Email: [email protected]

ODIMAYO Michael Simidele, (MBBS, PGDE, FMCPath, FIIA)

Professor of Microbial Pathology.
Consultant Clinical Microbiologist & Infectious Disease Physician.
Department of Microbial Pathology,
University of Medical Sciences.
Ondo State. Nigeria.
Email: [email protected]

ABAYOMI Fadeyi, (BSc, MBBS, MSc, FMCPath)

Professor of Medical Microbiology & Parasitology.
Consultant Clinical Microbiologist & Infectious Disease Physician.
College of Health Sciences,
University of Ilorin.
Ilorin. Nigeria.
Email: [email protected]

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 Laboratory Practical Manual for Medical Microbiology and Parasitology

Laboratory Practical Manual for

Medical Microbiology
and Parasitology

Author(s) : NWADIOHA Samuel Iheanacho

ODIMAYO Michael Simidele
ABAYOMI Fadeyi

ISBN : 978-93-94174-63-4

Page(s) : 110

Published Year : 2024

Published by : Excellent Publishers

No. 38/48, Second street, Ellappa Nagar
Kanchipuram – 631501, Tamilnadu, India.
Cell +91-9842641794
[email protected]
www.excellentpublishers.com

Disclaimer :

The author is solely responsible for the contents of the book in this volume in any manner,
Errors, if any are purely unintentional and readers are requested to communicate such errors to
the authors to discrepancies in futures.

Note: No part of this publication may be reproduced or transmitted in any form or by any
means, electronic or mechanical, including photocopying, recording, or any information storage
and retrieval system, without permission in writing from the publisher.

Copyright 2024 Excellent Publishers, All Rights Reserved

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 Laboratory Practical Manual for Medical Microbiology and Parasitology

PREFACE
A complementary text to practical guides in bacteriology, parasitology, virology,
and immunology. It addresses some fundamental questions on the subjects. The
book proves invaluable to postgraduate students in medical microbiology,
medical specialist doctors in medical microbiology, and medical students.

It is a product of practice and teaching of medical microbiology. The purpose is

intended for a sound understanding of the subject, as well as preparing the
students to the gradual evolution into automation of medical microbiology
practice in Africa.

Good luck! As you get married to this book

Authors

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 Laboratory Practical Manual for Medical Microbiology and Parasitology

Table of Contents
Contents Page No.
Chapter-1 1-13
Introduction to Microbiology
Iheanacho nwadioha
1.1 Introduction 1
1.2 Classification of pathogens 2
1.3 Prokaryotes 2
1.4 Bacteria 2
1.4.1 Gram positive bacteria 3
1.4.2 Gram negative bacteria 3
1.5 Waxy cell walls 3
1.6 Bacteria lacking cell walls 3
1.7 Shapes of Bacteria 3
1.8 Resembles Viruses 3
1.9 Viruses 4
1.10 RNA Viruses
1.10.1 ARBO virus (Arthropod-Borne virus) 4
1.11 ROBO virus (Rodent- Borne virus) 4
1.12 DNA virus 4
1.13 Eukaryotes 5
1.14 Fungi 5
1.14.1 Yeasts e.g. Candida albicans, Cryptococcus neoformans etc. 5
1.14.2 Filamentous fungi or mould; eg. Aspergillus spp, penicillium spp. 5
1.14.3 Dimorphic fungi; eg. Histoplasma spp, Coccidiodes immitis 6
1.15 Fungi are typical eukaryotes with 6
1.16 Parasites 6
1.17 Protozoa 6
1.18 Nemathelminthes (Round Worms) 8
1.19 Intestinal Nematodes 8
1.20 Tissue nematodes 8
1.21 Trematodes (Flukes) 10
1.22 Cestodes 12
1.23 Arthropods 13
1.24 Prions 13
Chapter-2 14-22
Laboratory Safety
Odimayo Simidele
2.1 Bio Safety Issues in the Laboratory 14
2.2 Personal Health and Safety Measures 14

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2.3 Practice of personal hygiene 15

2.4 The Following are Important in Making the Workplace Safe 15
2.5 Risk Groups 17
2.6 Specimens 19
2.7 Spills 20
2.8 Hazardous Chemicals 20
2.9 No Storage in Alphabetical Order 21
2.10 Vapours 21
2.11 Explosives 21
2.12 Chemicals spills 21
2.13 Chemical spills 21
2.14 Fire 22
2.15 Causes 22
2.16 Electricity 22
2.17 Noise 22
Chapter-3 23-32
Specimens in Medical Microbiology
Iheanacho Nwadioha & Fadeyi Abayomi
3.1 Introduction 23
3.2 Fundamentals to be Considered Before Collecting Samples 23
3.3 Transport 23
3.4 Storage 24
3.5 Some Common Laboratory Specimens 24
3.6 Urine 24
3.7 Collection 24
3.8 Transport and storage 24
3.9 Stool Collection 24
3.10 Sputum 25
3.11 Transport and storage 25
3.12 Cerebrospinal Fluid (CSF) 25
3.13 Transport and storage 25
3.14 Swab 25
3.15 Throat Swab 25
3.16 Transport and storage 25
3.17 Aspirates 25
3.18 Transport and storage 26
3.19 Skin Scrapings 26
3.20 Transport 26
3.21 Nail Clippings 26
3.22 Transport 26
3.23 Skin Snip 26

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3.24 Blood Culture 26

3.25 Sample Rejection Criteria 27
3.26 Procedure 27
3.27 Examples of Sample Rejection Criteria 27
3.27.1 Unlabelled or mislabeled samples 27
3.27.2 Duplicate samples 27
3.27.3 Leaky containers 27
3.27.4 Contaminated samples 28
3.27.5 Inappropriate sample sources 28
3.27.6 Delayed transport time and sample processing 28
3.27.7 In General, the following conditions will lead to rejection of a 28
specimen by the UTH Laboratory
3.27.8 Actions for When Samples are Rejected 29
3.28 Instrumentation 30
Chapter-4 33-38
Microscopes and Microscopy
Odimayo Simidele
4.1 Introduction 33
4.2 Microscopes 33
4.3 Types of Microscopes 33
4.4 Microscopy 34
4.5 Parts of a Compound Microscope 35
4.6 Why Perform Microscopic Examination 36
4.7 Using the Microscope 36
4.8 Simple Lens 36
4.9 Compound Microscope 36
4.10 Care when using the microscope 37
4.11 Sample Preparation for Microscopic Examination 37
4.12 Wet Preparation 37
4.13 Hanging Drop 38
4.14 Preparation 38
4.15 Preparation of Smears for Staining 38
4.16 Fixing Smears 38
Chapter-5 39-41
Stains and Staining Techniques in Microbiology
Iheanacho Nwadioha & Fadeyi Abayomi
5.1 Introduction 39
5.2 Staining 39
5.3 Types of Stains 39
5.4 Common stains used in Bacteriology 39
5.5 Negative Staining 39

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5.6 Simple Positive Staining 40

5.7 Common stains used in Bacteriology 40
5.8 Gram staining 40
5.9 Interpretation 40
5.10 Ziehl Neelsen staining 40
5.11 Interpretation 40
5.12 Spore staining 41
5.13 Interpretation 41
5.14 Capsule staining 41
5.15 Interpretation 41
5.16 Further Notes 41
Chapter-6 42-45
Media in Microbiology
Iheanacho Nwadioha & Odimayo Simidele
6.1 Introduction 42
6.2 Aim of Culture 42
6.3 Types of Culture Media 42
6.4 Composition of Media 42
6.5 Classification based on Consistency 43
6.6 Examples of Media Used in Microbiology 43
6.6.1 Selenite F and Tetrathionate Enrichment Broths 43
6.6.2 Deoxycholate Citrate Agar (DCA) 43
6.6.3 Eosin Methylene Blue (EMB) Agar 44
6.6.4 MacConkey (MAC) agar 44
6.6.5 Xylose Lysine Deoxycholate (XLD) agar 44
6.6.6 Salmonella Shigella (SS) agar 44
6.6.7 Kligler Iron Agar (“KIA”) 44
6.6.8 Triple Sugar Iron agar (“TSI) 44
6.6.9 Anaerobic Media 44
6.6.10 Virology/Cell cultures media 45
6.6.11 Parasitology 45
6.6.12 Mycology 45
Chapter-7 46-47
Culture Techniques
Odimayo Simidele
7.1 Aerobic culture 46
7.2 Inoculating Artificial Agar Solid Agar in Petri Dishes 46
7.3 Aim 46
7.4 Procedure 46
7.5 Inoculation of Slopes & Butts 46
7.6 Procedure 46

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7.7 Inoculation of Liquid Media 47

7.8 Anaerobic culture 47
Chapter-8 48-49
Introduction to Anaerobiosis
Iheanacho Nwadioha
8.1 Introduction 48
8.2 Why they are Anaerobes 48
8.3 Media for Isolating Anaerobes 48
8.4 Achieve Anaerobic Culture Condition Using 49
8.5 Components of Anaerobic Chamber 49
Chapter-9 50-53
Cutural Identification of Bacteria
Odimayo Simidele
9.1 Introduction 50
9.2 Haemolysis 50
9.2.1 α-Haemolysis 50
9.2.2 Beta-Haemolysis 50
9.2.3 Gamma-Haemolysis 50
9.2.4 α’ Hemolysis 51
9.3 Swarming 51
9.4 Lactose Fermentation 51
9.5 Pigment Production 51
9.6 Colonial Morphology 51
9.6.1 Size of Colonies 51
9.6.2 Margin 51
9.6.3 Elevation 51
9.6.4 Density/Optical Properties 52
9.6.5 Colour 52
9.6.6 Consistency/Texture (Determined by Touching Colony with 52
Sterile Wire Loop)
9.6.7 Pigments 52
9.6.8 Odour 52
9.7 Growth In Liquid Media 52
9.8 Growth in Cell Cultures 53
Chapter-10 54-57
Biochemical Tests for Identification of Bacteria Isolates
10.1 Indication 54
10.2 Catalase test 54
10.3 Slide coagulase test 54

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Chapter-11 58-60
General Considerations in Identification of Common Bacteria Isolates
11.1 Introduction 58
11.2 Follow the Identification guide below 58
11.3 Characterisation of oxidase-negative isolates 59
Chapter-12 61-64
Antibiotic Susceptibility Testing
12.1 Objectives 61
12.2 Techniques 61
12.3 Dilution 61
12.3.1 Broth dilution method 61
12.4 Diffusion 62
12.4.1 Kirby Bauer method 62
12.5 Kirby Bauer Method of Antibiotic Susceptibility Testing 62
12.6 Stokes Method 62
12.7 Precautions in Kirby Bauer Method 63
12.8 Antimicrobial Gradient Method 63
12.9 Other Antimicrobial Susceptibility Testing Methods 63
12.10 Automated Instrument Systems 63
12.11 Genotypic method 64
12.12 Synergy tests 64
Chapter-13 65-68
Immunodiagnosis in Medical Microbiology
13.1 Introduction 65
13.2 Antigen Detection Methods 65
13.3 Enzyme immunoassay for Hepatitis B 66
13.4 Direct antigen for Cryptococcal meningitis using latex 66
agglutination
13.5 Antibody Detection Methods 66
13.6 Application 67
13.7 Precipitation assays 67
13.8 Neutralization tests 67
13.9 Microscope –Assisted- Labeled- Reagent Technology 67
13.10 Treponema pallidum Haemagglutination Test 68
13.11 Febrile Agglutinin Test for Salmonella 68
13.12 Test of Cell Mediated Immunity 68
Chapter-14 69-94
Laboratory Techniques in Parasitology
14.1 Specimen Collection and Preservation 69
14.2 Blood Specimen 69
14.2.1 Timing 69
14.2.2 Type of Sample 69

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14.2.3 Capillary blood obtained by finger prick 69

14.2.4 Venous blood obtained by venipuncture 70
14.3 Stool Specimen 70
14.4 Serum/Plasma Specimens 71
14.4.1 Specimen Requirements 71
14.4.2 Serum for all tests 72
14.5 Other Specimens 72
14.6 Tissue Specimens 72
14.7 Stool Parasitology 72
14.8 Stool Specimen processing 72
14.9 Macroscopic examination 72
14.10 Specimen Collection 73
14.11 Appearance 73
14.12 Microscopic Examination 73
14.13 Possible Pathogens 73
14.14 Stool Concentration Techniques 73
14.15 Flotation Technique 73
14.16 Sedimentation Technique 74
14.17 Specific staining reactions 75
14.18 Urine 76
14.19 Sample Collection 76
14.20 Microscopic Examination 77
14.21 Report the Appearance of Urine 77
14.22 Blood 77
14.23 Sample Collection 77
14.24 Wet Blood Preparation 77
14.25 Making Films 77
14.25.1 Fresh film preparation and examination 77
14.25.2 Thick Film 78
14.25.3 Thin Blood Film 78
14.26 Staining 79
14.26.1 Leishmans (Thin films) 79
14.26.2 Giemsa 79
14.26.2.1 Thin films 79
14.26.2.2 Thick Film 79
14.26.3 Fields Stain 79
14.26.3.1 Thin Film 79
14.26.3.2 Thick Film 80
14.27 Blood Specimen 80
14.28 Preparing Blood films 80
14.28.1 Thick films 80

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14.28.2 Thin films 81

14.29 Staining of Blood films with Giemsa 81
14.29.1 Thin films 81
14.29.2 Thick film 82
14.30 Special Procedures for Detecting Microfilariae 82
14.31 Blood microfilariae 82
14.31.1 Centrifugation (Knott’s technique) 82
14.31.2 Filtration 82
14.32 Microscopic Examination 83
14.33 Assessment of Malaria Parasite density 86
14.33.1 Thick Blood Film 86
14.33.1.1 Double counter technique 86
14.33.1.2 Earle and Perez method 86
14.33.2 Thin Blood Film 87
14.33.2.1 Percentage of parasitaemia 87
14.34 Skin Snip 87
14.35 Other Clinical Specimens 87
14.36 Isolation of Leishmania Organisms 87
14.37 Sputum Specimens 87
14.38 Induced Sputum and Bronchoalveolar Lavage (Bal) For 88
Pneumocystis jirovecii
14.39 Aspirates 88
14.40 Other Specimens 89
14.41 Vaginal Swabs for Detection and Susceptibility of 89
Trichomonas
14.42 Cellulose Tape or Swube Tube Procedure for Demonstration 89
of Pinworm Eggs
14.43 Urine Specimens 89
14.44 Biochemical Diagnosis 90
14.45 Advantages of isoenzyme diagnosis 90
14.46 Disadvantages of isoenzyme diagnosis 90
14.47 Immunodiagnosis 90
14.47.1 Antibody Based Methods 90
14.47.2 Antigen based tests 91
14.48 DNA Based Tests 91
14.49 Culture 92
14.50 Malaria Culture 92
14.51 Leishmanial culture 93
14.52 Trypanosomiasis culture 93
14.53 Entamoeba histolytica 93
14.54 Strongyloides stercoralis/ hookworms 93

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14.55 Animal Innoculation 94

Chapter-15 95-96
Laboratory Techniques in Mycology
Iheanacho Nwadioha
15.1 Specimens 95
15.2 Direct Microscopic Examination 95
15.3 Wet Preparation 95
15.4 Unstained Wet Mount 95
15.5 KOH Preparation 95
15.6 Staining: Calcoflour White – KOH Stain 95
15.7 Gram Stain 96
15.8 Indian Ink 96
15.9 Germ Tube Test 96
15.10 Culture 96
15.11 Lactophenol Cotton Blue Mount 96
15.12 Other Identification Techniques 96
15.13 Antifungal Susceptibility Testing 96
Chapter-16 97-105
Laboratory Techniques in Medical Virology
Odimayo Simidele
16.1 Specimen Collection, Transport and Storage 97
16.1.1 Specimen collection 97
16.1.2 Timing of Collection 98
16.1.3 Transport Conditions 99
16.1.4 Processing of Specimens 100
16.2 Specimen Processing 100
16.3 Virus Detection Methods 100
16.4 Cytology and Histology 100
16.5 Electron Microscopy (EM) 100
16.6 Immunodiagnosis (Antigen-antibody detection) 101
16.6.1 Neutralization Test 101
16.6.2 Haemagglutination 102
16.6.3 Complement Fixation 102
16.6.4 Precipitation Test 102
16.6.5 Immunofluorescent antibody tests 102
16.6.6 Gel diffusion 102
16.6.7 Radial haemolysis 102
16.6.8 ELISA 102
16.6.9 Complement fixation Test 103
16.6.10 Haemagglutination 103
16.7 Molecular Biology Techniques 103

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16.7.1 Probing 103

16.7.2 Polymerase Chain Reaction (PCR) 103
16.7.3 Electropherotyping 103
16.8 Viral Culture 103
16.9 Anti Viral Susceptibility Testing 104
16.10 Phenotypic assays 104
16.11 Genotypic assay 104
16.12 Further Reading 105

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Chapter 1
Introduction to Microbiology
Iheanacho nwadioha

1.1 Introduction

Antonie van Leeuwenhoek (1632–1723) was one of the first people to observe
microorganisms, using a microscope of his own design, and made one of the most
important contributions to biology. Robert Hooke was the first to use a microscope to
observe living things. Hooke's 1665 book, Micrographia, contained descriptions of plant
cells.

Lazzaro Spallanzani (1729–1799) found that boiling broth would sterilise it and kill any
microorganisms in it. He also found that new microorganisms could settle only in a
broth if the broth was exposed to the air.

Louis Pasteur (1822–1895) expanded upon Spallanzani's findings. He demonstrated that

the living organisms that grew in such broths came from outside, as spores on dust,
rather than spontaneously generated within the broth. Thus, Pasteur dealt the death
blow to the theory of spontaneous generation and supported germ theory instead.
In1860-1885, Louis Pasteur invented pasteurization and developed vaccines for rabies
and anthrax supporting the germ theory

Ferdinand Julius Cohn (January 24, 1828 – June 25, 1898) was a German biologist. His
classification of bacteria into four groups based on shape (sphericals, short rods,
threads, and spirals) is still in use today.

In 1876, Robert Koch (1843–1910), the father of modern medical microbiology

established that microbes can cause disease, that microbial agent is responsible for a
particular disease- ‘Koch’s postulates’. He found that the blood of cattle who were
infected with anthrax always had large numbers of Bacillus anthracis.

Medical Microbiology is a branch of medicine that includes five sciences, namely,

bacteriology, virology, parasitology, immunology and mycology. The medical
microbiology laboratory is involved in various functions including diagnosis of disease
conditions, the identification of microorganisms involved in infectious disease
processes, assisting the clinicians in providing effective management of patients, etc.

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Work of the Microbiology Laboratory includes, Microbiologic examination of Specimen,

Consultations on the investigation and management of patients with infection problem,
Control of hospital Infections and Teaching and Research.
A Medical Microbiologist is a specialist in medicine who

 Provides clinical consultations on the investigation, diagnosis and treatment of

patients suffering from infectious diseases
 Establishes and directs infection control programs across public health care,
infectious disease prevention and epidemiology
 Provides the scientific and administrative direction of a clinical microbiology
 Is also involved in teaching at all levels, and in research which includes the
development of vaccine

1.2 Classification of pathogens

Pathogens are animate and inanimate objects capable of inducing a disease process.
Pathogenic microorganisms that have the ability to cause disease include bacteria,
fungi, viruses, and protozoa.
1.3 Prokaryotes
 Organisms with absence of a nuclear membrane
 DNA is not physically separated from cytoplasm
 relatively small size,
 order of 1 µm in diameter
 Bacteria
 Can replicate/ cell metabolism independently

1.4 Bacteria

 Gram's Stain is a widely used method of

staining bacteria as an aid to their
identification. It was originally devised
by Hans Christian Joachim Gram, a
Danish doctor.
 Gram's stain differentiates between two
major cell wall types.
 Bacterial species with walls containing
small amounts of peptidoglycan are
Gram-negative
 Bacteria with walls containing relatively
large amounts of peptidoglycan are
Gram-positive.
Figure. 1.1

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1.4.1 Gram positive bacteria

 Cocci e.g. Staphylococcus, Streptococcus,

Enterococcus etc.
 Bacilli e.g. Corynebacterium, Listeria,
Lactobacillus, Clostridium, Bacillus etc.

1.4.2 Gram negative bacteria

 Cocci e.g. Neisseria, Moraxella etc.

 Bacilli e.g. Proteus, Escherichiacoli,
Klebsiella, Salmonella, Pseudomonas spps.
etc.

Figure. 1.2
1.5 Waxy cell walls

Not all bacteria can be stained by Gram's method, the best-known exception belong to
the genus Mycobacterium. Identified by Ziehl Nelseen stain.

1.6 Bacteria lacking cell walls

Four types of bacteria with deficient cell walls are

 Mycoplasma species
 L-forms
 Spheroplasts
 Protoplasts

1.7 Shapes of Bacteria

 Cocci (Singular coccus)

 Bacilli (rods) (Singular: rod, bacillus)
 Vibrios (Singular: vibrio)
 Spirilla (Singular: spirillum)
 Spirochaetes (Singular: spirochaete)

1.8 Resembles Viruses

 Rickettsiae,
 Chlamydiae

1.9 Viruses

 the smallest infectious agents

 Size ranging from about 20 nm to about 300 nm in diameter

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 Genome ;one kind of nucleic acid (RNA or DNA but never both)
 entire infectious unit is termed a virion.

The nucleic acid is encased in a protein shell, which may be surrounded by a lipid-
containing membrane. The entire infectious unit is termed a virion.

There are DNA or RNA viruses

1.10 RNA viruses

 Orthomyxovirus e.g. influenza virus

 Paramyxovirus e.g. mumps virus, measles
virus, parainfluenza virus.
 Retrovirus e.g. HIV I & II, HTLV
 Corona virus e.g. SARS, SARSCoV-2
 Picornavirus e.g. hepatitis A, Polio, Rhino
 Reovirus e.g. Rota
 Calicivirus e.g. Hepatitis E, Norwalk

Figure. 1.3

1.10.1 ARBO virus (Arthropod-Borne virus)

 Alphavirus e.g. O’nyong-nyong
 Bunyavirus e.g. Crimea-Congo
 Flavivirus e.g. Yellow fever,
 Hepatitis C, Dengue

1.11 ROBO virus (Rodent- Borne virus)

 Arenavirus e.g. Lassa

 Filovirus e.g. Ebola

1.12 DNA virus

 Hepadnavirus e.g. hepatitis B virus.

 Herpes virus e.g. herpes simplex virus (HHV -1 and -2), Varicella- zoster(HHV-3),
Ebstein Bar(HHV-4), CMV(HHV-5), Kaposi(HHV-8).
 Papovavirus e.g. papilloma, warts
 Parvovirus e.g. B19
 Deltavirus e.g. hepatitis D
 Adenovirus e.g. adenovirus

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1.13 Eukaryotes

 organisms containing a membrane-bound nucleus

 specialized membrane bound organelles such as Mitochondria
 relatively larger in size
 Algae, Protozoa, Fungi and Slime moulds.
 Can replicate/ cell metabolism independently

1.14 Fungi

Fungi are non-photosynthetic eukaryotic organisms that absorb nutrients from their
environment. Saprophytes/Pathogens appear in yeast or in molds (hyphae) forms.

1.14.1. Yeasts e.g. Candida albicans, Cryptococcus neoformans etc.

 Yeasts: Unicellular Organisms which reproduce

by budding (blastoconidia formation) or by
binary fission.
 Continuation of the budding process can
produce a chain of elongated yeast cells called
pseudohyphae.
 Yeasts are larger than bacteria and are commonly
spherical to egg shaped
 Usually prefer warmer temperatures
 Grow at 37°C
 Form a creamy opaque or pasty colonies on
culture media.
 Grow rapidly within 24-48 hrs.
Figure. 1.4 Yeast Form

1.14.2 Filamentous fungi or mould; eg. Aspergillus spp, penicillium spp.

 Moulds: They have branching tubular structures

called hyphae.
 Grow at 25°C
 Form a mass of intertwined network of hyphae
called mycelia on culture media.
 Vegetative (in substrate) & aerial (above) mycelia
 Colonies are fluffy, cottony, woolly, or powdery.
 Grow slowly requiring about 6 weeks.

Figure. 1.5 Mould form

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1.14.3 Dimorphic fungi; eg. Histoplasma spp, Coccidiodes immitis

 Dimorphic Fungi: Exist as Yeast (or spherules) at 35-37°C in tissues and as Moulds at
25°C in the environment.
 Conversion to the yeast form appears to be essential for pathogenicity.
 Identified by morphological or biochemical characteristics and the appearance of
their fruiting bodies.
 Blastomyces dermatitidis forms hyphae in vitro and yeasts in tissue.

1.15 Fungi are typical eukaryotes with

 a multi-layered rigid cell wall containing CHITIN (glucose and mannose polymers),
chitosan, glucan, mannan, and others
 a cell membrane containing glycoproteins, lipids and ergosterol (which replaces
cholesterol).
 Nucleus is bound by a nuclear membrane. Inside the nucleus are several diploid
chromosomes.

1.16 Parasites

 organisms that take up their abodes, temporarily or permanently, on or within other

living organisms for the purpose of procuring food and shelter.
 Helminths, protozoa, Arthropods
 Protozoa (Simple, single celled micro-organisms consisting of a nucleus and
cytoplasm). unicellular parasites.
 Platyhelminths (flat worms): Flukes (trematodes) and Tapeworms (cestodes)
 Nemathelminths (Round worms): Cylindrical worms (nematodes)
 Arthropoda

 Ectoparasite – Lives on the outer surface or superficial tissues of its host (e.g fleas) –
Infestation.
 Endoparasite- Lives within its hosts (e.g malaria parasite)-Infection.
 Facultative parasites –Leads both free and parasitic existence
 Obligative parasite –Completely dependent on the host

1.17 Protozoa

 Amoebas e.g. Entamoeba histolytica, Entamoeba coli

 Flagellates eg. Gardia lamblia, Trichomonas vaginalis
 Ciliates eg. Balantidium coli
 Coccidians eg. Plasmodium spp
 Microsporidians eg. Enterocytozoonspp

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Figure. 1.6 Flagellates

Figure. 1.7 Amobae

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1.18 Nemathelminthes (Round Worms)

 Non-segmented cylindrical worms that taper at both ends.

 Possess a shiny cuticle (skin).
 The digestive system is a simple tube which begins with mouth and ends in an anus
 Sexes are separate with the male worms being smaller than the females.
 In some male there is a testis.
 Female worms possess (according to species) one or two tubular ovaries which lead
to a uterus or uteri.
 Females are either viviparous (produce larvae) or oviparous (lay eggs).
 Nematodes, which infect humans, live in the tissues or intestinal tract. Mainly insect
vectors transmit tissue nematodes.
 Most of the medically important intestinal nematodes are soil transmitted (i.e spread
by faecal pollution of soil)

1.19 Intestinal Nematodes

Ascaris lumbricoides, Hookworms (Ancylostoma doudenale, Necator americanus), Trichuris

trichiuria, Strongyloides stercoralis, Capillaria philippinensis, Trichostrongylus spp.

1.20 Tissue nematodes

 Filarialworms (Wuchereria bancrofti, Brugia malayi, Loa loa, Onchocerca volvolus)

 Trichinella spiralis, Drancuculus medinensis, Anisakis spp
 Angiostongylus cantonensis/ A. costaricensis
 Cutaneous larva migrans (Ancylostoma braziliensis/ A. caninum)
 Visceral larva migrans (Toxocara canis)

Figure. 1.8

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Figure. 1.9

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Figure. 1.10

1.21 Trematodes (Flukes)

 Unsegmented, mostly flat leaf-like worms (except schistosomes)

 Attach to their host by means of suckers. (oral sucker & ventral sucker)
 No body cavity.
 Digestive system consists of a mouth and oesophagus. There is no anus.
 Excretory system is composed of excretory cells called flame cells, collecting
tubules, and an excretory pore.
 With the exception of schistosomes, trematodes are hermaphroditic.
 Most trematodes eggs are operculated (with lids).

Blood Flukes (Schistosoma haematobium, S. mansoni, S. japanicum)

Liver Flukes (Clonorchis sinensis, Opisthorchis felineus, Fasciola hepatica)

Lung Flukes (Paragonimus westermani)

Intestinal Flukes (Fasciolopsis buski)

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Figure. 1.11

Figure. 1.12 Operulated egg Figure. 1.13 Fasciola gigantica

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1.22 Cestodes

 Body is tape-like and is made up of a head (scolex) and many proglottids

(segments). The head attaches the tapeworm to its host by suckers and in some
species hooks also.
 Proglottids are formed from behind the head. The proglottids which contain eggs
are known as gravid segments.
 Tape worms are hermaphroditic with male and
female reproductive organs being found in each
mature proglottid.
 In most species, the eggs are released when a
gravid segment becomes detached and ruptures.
 There is no mouth or digestive system. (obtains
nutrients through its body surface)
 There is a simple excretory system.

 Taenia saginata (beef tapeworms)

 Taenia solium (sheep tapeworms)
 Diphlo bothrium latum (fish tapeworms)
 Hymeno lepsis nana (dwarf tapeworms)
 Echinococcus granulosis (Hydatid cyst disease)
 Echinococcus multicularis (Alveolar cyst disease)

Figure. 1.14

Figure. 1.15

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1.23 Arthropods

 Insecta (6legs, 3body segments, wings): mosquitoes, flies, bugs, lice, fleas
 Arachnida (8 legs, no wings,): mites, ticks, spider, scorpions
 Lice (body louse): pediculosis
 Mites (Sarcoptes scabiei): Scabies
 Fleas (Tunga penetrans): Tungiasis
 Tumbufly (Cordylobia anthropophaga): furuncular myiasis
 Mosquitoes: malaria, Arbovirus dses (yellow fever, dengue fever), filariasis
(Wuchereria bancrofti, Brugia malayi)
 Triatome (kissing) bugs: Chagas’ disease
 Body lice: Trench fever (Bartonella quintana), Epidemic relapsing fever (Borrelia
recurrentis), Epidemic typhus (Rickettsie prowazekii)
 Hard ticks: Babesiosis, Rocky mountain spotted fever (Rickettsia rickettsii), Lyme
ds (Borrelia burgdorferi)

1.24 Prions

 unconventional, transmissible agents

 proteinaceous infectious agents.
 Slow but progressive infection
 characteristic neuropathologic changes occur, but no inflammatory or immune
response is elicited
 Kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease,
fatal familial insomnia, Bovine spongiform encephalopathy (BSE) and Scrapie

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Chapter 2
Laboratory Safety
Odimayo Simidele
2.1 Bio Safety Issues in the Laboratory

These measures and concepts developed to protect all persons and animals who interact
with the laboratory. Primarily aims at protecting laboratory staff and animals.
However, biosafety issues transcend the medical laboratory. Examples of these are
Bhopal industrial in India and Chernobyl in the USSR.

Biosafety is about reducing accidents and mitigating the effects of such in animals and
humans. The scope of biosafety practice includes infections, chemical accident, ionising
radiations, fire, spills, acoustic pollution, light ray damages etc.

Measures in the laboratory that aim at reducing laboratory acquired infections are
dependent on the following factors:

 Risk index group

 biosafety levels

These factors determine the scope of equipment, therapeutic and preventive measures
that used in the laboratory. These factors ultimately determine the laboratory design.

Universal safety precautions are the gold standard for laboratory safety irrespective of
biohazards. These universal precautions are:

Prevention of eating and drinking in the laboratory

 Avoidance of foods storage in refrigerator and/or freezers meant for storing

reagents and specimens
 Hand washing between procedures
 Face and eye protection by masks and goggles
 Protective clothing – Gowns, coats, positive pressure suits.

2.2 Personal Health and Safety Measures

Personal health and safety measures include:

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 practice of personal hygiene.

 wearing of protective clothing.
 protective inoculations and medical examinations.

2.3 Practice of personal hygiene

 Laboratory staff must practice a high standard of personal hygiene. This includes:
 Washing hands and arms with soap and water before attending outpatients,
visiting wards, after handling specimens and infected material, when leaving the
laboratory, and at the end of the day’s work. When running water supplies are
interrupted, a tip type water container suspended over the sink should be used for
handwashing, not a basin of water.
 Frequent handwashing is the most effective action a laboratory worker can take to
avoid laboratory-acquired infection.
 Covering any cuts, insect bites, open sores, or wounds on the hands or other
exposed parts of the body with a water-proof adhesive dressing. Irritating Insect
bites should be treated.
 Wearing closed shoes and not walking barefoot.
 Not eating, drinking, chewing gum, smoking, or applying cosmetics in any part of
the laboratory and not sitting on laboratory benches.
 Note: Food or drink should never be stored in a laboratory refrigerator.

2.4 The Following are Important in Making the Workplace Safe

Laboratory premise that is structurally sound and in good repair with a reliable water
supply and a safe plumbing and waste disposal system. Drainage from sinks must be
closed and connected to a septic tank or to a deep pit. Note: If there is a shortage of
piped water, provision must be made for the storage of water, e.g. collection of rain
water in storage tanks. It is not safe for a laboratory to function without an adequate
water supply.

Adequate floor and bench space and storage areas. The overall size of the laboratory
must be appropriate for the workload, staff numbers, storage and equipment
requirements.

Well constructed floor with a surface that is nonslip, impermeable to liquids, and
resistant to those chemicals used in the laboratory. It should be bevelled to the wall and
the entire floor should be accessible for washing. The floor must not be waxed or
covered with matting. Floor drains are recommended.

Walls that are smooth, free from cracks, impermeable to liquids, and painted with
washable light coloured paint.

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When practical, a door at each end of the laboratory so that laboratory staff will not be
trapped should a fire break out. Doors should open outwards and exit routes must
never be obstructed. Where fitted, internal doors should be self closing and contain
upper viewing panes.
External doors must be fitted with secure locks.

Adequate ventilation supplied by wall vents and windows that can be opened. The
windows should not face the prevailing winds to avoid excessive dust entering the
laboratory in the dry season and the wind interfering with work activities. Windows
should be fitted with sun blinds and insect proof screens, and when indicated secure
window bars.

Sectioning of the laboratory into separate rooms or working areas. The area where
blood samples are collected from patients must be away from the testing area of the
laboratory. Seating should be provided for patients outside the laboratory. The
specimen reception area must be equipped with a table or hatchway which has a
surface that is impervious, washable, and resistant to disinfectants. There should also be
a First Aid area in the laboratory containing a First Aid box, eyewash bottle and fire
blanket.

Bench surfaces that are without cracks, impervious, washable, and resistant to the
disinfectants and chemicals used in the laboratory. Benches, shelving, and cupboards
need to be well constructed and kept free of insect and rodent infestation. Benches
should be kept as clear as possible to provide maximum working area and facilitate
cleaning.

Suitable storage facilities, including a ventilated locked store for the storage of
chemicals and expensive equipment.

Where required, a gas supply that is piped into the laboratory with the gas cylinder
stored in an outside weatherproof, well-ventilated locked store.

A staff room that is separate from the working area where refreshments can be taken
and personal food and other belongings stored safely. Wall pegs should be provided in
the laboratory on which to hang protective clothing. Near to the staff room there should
be a separate room with toilet and hand-washing facilities.

There should be separate toilet facilities for patients.

A handbasin with running water, preferably sited near the door. Whenever possible,
taps should be operated by wrist levers or foot pedals. Bars of soap should be provided,
not soap dispensers.

Ideally paper towels should be used. If this is not possible small cloth hand towels that

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are laundered daily should be provided.

Provision of protective safety cabinets and fume cupboards as required and when
feasible.
Safe electricity supply with sufficient wall electric points to avoid the use of adaptors
and extension leads.

Fire extinguishers sited at accessible points. These need to be of the dry chemical type.

Several buckets of sand and a fire blanket are also required.

As good illumination as possible. Low energy tube lights are recommended. Window
screens must be fitted to protect from direct sunlight and glare but these should not
make the working areas too dark.

2.5 Risk Groups

RG1- Unlikely to cause human or animal disease

RG2- Moderate individual risk, low community risk. Laboratory exposures lead to
serious infection. Effective treatment and preventive measures are available. The risk of
spread is limited. (H. influenza, Strept pneumonia).

RG3- High individual and low community risk, can cause serious human/ animal
disease, does not spread, readily while effective prevention and treatment modalities
are available. (TB, tetanus)

RG4- High individual and community risk, causes human and animal disease. It is
readily transmitted directly or indirectly. Effective treatment and preventive measures
are not available. CJD, Avian flu, Lassa fever.

It is against this background that facilities are designed and equipped to contain the
infections according to the risk group. While the infectious group are classified
according to risk group the laboratory facilities by Biosafety level (BS) 1,2,3,4

Facilities are developed based on a composite design of features, constructions,

containment facilities, equipment, practices and operational procedures. These allow for
working with agents irrespective of groups. Classification of micro-organisms into RGs
is country specific.

Factors determining the classification of microbes

Pathogenicity

Mode of transmission /host range dependent on herd immunity

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 Density
 Movement of host population (pandemic)
 Vectors
 Environmental hygiene
Preventive measures

 Prophylaxis
 Vaccination
 Antisera- Passive immunisation
 Sanitary measures- food, water, hygiene
 Control of animal reservoirs e.g. arthropod vectors

Availability of effective treatment

 Passive immunisation
 Post exposure vaccination
 Antimicrobials
 Antivirals
 Antibiotic chemotherapy
 Drug resistance and effective treatment

Table. 2.1 Classification of laboratories by Biosafety level (BSL) is based on the

equipment provided in such laboratories

Risk Biosafety Lab Lab Practice Safety Equipment

group Level type/Purpose
1. 1 Basic teaching, Good None Open bench
Research microbiological work
techniques(GMT)
2. 2 Primary health GMT+ Protective Open bench+
diagnostic clothing+ Biological safety
services Biohazard signs, cabinets(BSC) for
directional airflow potential activities
3. 3 Special Level 2+ special BSC ± Primary
diagnostic clothing controlled devices
services and access +directional
research airflow
4. 4 Dangerous Level 3+ airlock Class III BSC+
pathogen entry Positive Pressure
units Shower exit suits+ Class II BSC
Special waste double ended
disposal autoclave through
the wall filtered air

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Table. 2.2 Biosafety requirements summary

Biosafety Level lab

Provision 1 2 3 4
Isolation of NO NO NO Yes
laboratory
Room sealable NO NO NO Yes
for
decontamination
Ventilation NO NO NO Yes
Inward flow
Controlled
ventilation
system
HEPA filtered
air exhaust
Double door NO NO NO Yes
entry
Air entry NO NO NO Yes
Anteroom NO NO NO No
Effluent NO NO NO Yes
treatment
Autoclave NO Desired NO Yes
On site NO NO NO Yes
In laboratory NO NO NO Yes
room NO NO NO Yes
Double ended NO NO NO Yes
Biological safety Yes
NO desired NO
cabinets
Personnel safety
monitoring
Personnel NO NO NO Yes
capability
monitoring

2.6 Specimens

Specimens with limited information

Safety precautions (Universal precaution)

1. Barrier gloves, gowns, eye protection

2. Basic containment in BSL
3. Transport specimen in accordance with national international rules

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 Check and collect data on the data on the patient

 Epidemiological data(mortality and mortality)
 Suspected route of transmission outbreak investigation data
 Geographical origin of specimen
 No damages
 Reduce infection risk by increasing the efficiency of packages- This varies with
specialty
 Package should conform with UN regulations for the transport of dangerous goods
concerned agencies are: ICAO, ADGR, UNCEDG
 Package should be in 3 layers viz:
1. Primary receptacle
2. Secondary receptacle---watertight, leak proof packaging
3. Third layer Data forms/Letters of information

2.7 Spills

Heavy duty, gloves, protective clothing, face and eye protection, Cover spills with cloth
or paper towels. Apply disinfectant over spillage in concentric circles from outwards
towards the centre.

After an interval of 30mins, clear away materials. Remove broken glass and sharps with
dust pan and stiff.

Contaminated materials should be disposed of in leak proof puncture proof containers.

Inform authority

2.8 Hazardous Chemicals

These are additional hazards found in the laboratory (P32)

Routes of exposure

 Inhalation
 Contact
 Ingestions
 Needle sticks
 Through broken skin

Chemicals for daily use are to be kept in the laboratory. Bulk stocks are to be kept in
special buildings.

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2.9 No Storage in Alphabetical Order

2.10 Vapours

Vapours cause drowsiness, lack of coordination, and increased proneness to accidents.

Contact with liquid chemicals cause skin damage, the severity of which depends on the
degree of corrosive of the chemical.

2.11 Explosives

Azides+ Cu(Pb) →Explosion

Aged ethers
Perchloric Acid on wood work, brickwork, fabric...... Explosion
Picric/ Picrates +Heat
Picrates + Impact

2.12 Chemicals spills

1. Protective clothing-heavy duty, rubber gloves overshoes boots.

2. Equipment –Forceps for picking up glass fragments, mops, cloths paper, towels,
buckets
3. Pour soda ash for neutralizing acids and corrosives
4. Sand mops up alkalis
5. Use non-flammable detergents

2.13 Chemical spills

Actions to be taken

1. Notify Officer in-charge

2. Evacuate non essential personnel
3. Attend to contaminated persons
4. Extinguish flames if possible turn off gas supply, switch off electrical equipment
5. Avoid breathing from spilled material
6. Establish exhaust ventilation if safe
7. Secure necessary items

A. Compressed gas cylinders and liquefied gas cylinders securely fixed to the
wall/solid bench. Transport cylinders capped on trolleys. Store bulk containers in
facility away from the lab where it is labeled and locked. Keep away from radiators,
open flames, heat sources, and sparkling electrical equipment. Keep away from
sunlight
B. Do not incinerate small single use cylinders

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2.14 Fire
Warnings On doors, rooms,
Routes
Instructions

2.15 Causes

 Electrical current overloading

 Poor electrical maintenance porous cables
 Long gas tubing, long gas leads
 Equipment not for laboratory environment
 Open flames
 Deteriorated gas tubing
 Improper segregation of incompatible chemicals
 Sparkling equipment near flammable sprays
 Inadequate improper ventilation
Table. 2.3 Fire Extinguishers

Type For Not for

Water Paper, wood fabric Electric fire, flammable liquids burning
Carbon Flammable liquids, gases, Re-usable equipment but produces
Dioxide alkali, metal, electrical fires difficult residues
Foam Flammable liquids Electrical fires

2.16 Electricity

 Install
 Circuit breakers
 Earth fault interrupters
 3-prong plugs
 All these must conform to National electrical standards/codes

2.17 Noise

 Has an insidious effect usually associated with Laser systems, animal houses
 Action regular noise measurement
 Hearing protection
 Appoint a Safety officer
 Ensure biosafety, biosecurity, technical compliances
 Carry out safety audits
 Check out
 Security, vandalism etc

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Chapter 3
Specimens in Medical Microbiology
Iheanacho Nwadioha & Fadeyi Abayomi
3.1 Introduction

Specimen collection, transportation and storage are very important considerations in

the laboratory as the quality of lab results depend on the quality of the specimens and
their condition on arrival to the lab. The correct type of specimen should be collected at
the right time and transported to the lab immediately or a transport medium used to
preserve them. Each specimen should be labelled with the patient’s name, date and time
of collection, hospital number, and ward or health centre. Specimens should be
collected in sterile, easy to open, dry leak proof containers free from all traces of
contaminants.

3.2 Fundamentals to be Considered Before Collecting Samples

a) Materials must be from the actual site of infection.

b) Aim at no contamination from adjacent tissues, organs or secretions.
c) Optimal time for collection of specimens must be established to provide the best
chance of recovering causative agents.
d) Sufficient quantity of specimen must be obtained.
e) Appropriate collection devices and specimen containers bust be used eg wide-
mouthed bottles for sputum samples.
f) Aim at collection of specimen before administration of antibiotics.

3.3 Transport
 Aim at maintaining sample in as near its original state as possible.
 Transport sample to the laboratory as soon as possible after collection.
 Common Transport media for transporting samples to lab and maintaining viability
of organism include:
 Stuart medium,
 Cary-Blair medium,
 Sodium Borate solution
 Amies medium
 Thioglycollate broth
 Sucrose-Phosphate-Glutamate (SPG)

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3.4 Storage

If delay is expected, you are to store the specimen appropriately by:

i. Refrigeration( 4 -6 oC) eg urine samples

ii. Addition of boric acid eg urine samples
iii. Freezing At -20oC; -70oC e.g. serum for antibody/ antigen assays.

3.5 Some Common Laboratory Specimens

Various clinical specimens that can be submitted to the microbiology laboratory for
isolation and identification of infectious causative agents and collection procedure
include;

3.6 Urine

The first urine passed at the beginning of the day is the most concentrated and most
suitable for microbiological study and therefore should be sent to the laboratory as soon
as possible.

Types of urine specimen include:

 fore urine (the first part of the urine flow),

 mid-stream urine (the middle part of the urine flow),
 hind urine ( the terminal part of the urine flow),
 supra pubic urine, and
 Catheter urine.

3.7 Collection

To obtain a ‘clean- catch’ specimen, wash the hands and clean the area around the
urethral opening, allow the area to dry and collect the urine, with the labia held apart in
females.

3.8 Transport and storage

If immediate delivery is not possible, urine specimen can be stored at 4-60C for not more
than 2 hours. Boric acid preservative should be used for longer storage period.

3.9 Stool Collection

Transfer a portion of the stool specimen that contains mucus, blood or pus into a clean
dry leak proof container.

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Transport and storage: Send to the lab within one hour. Transport in Cary-Blair’s
medium, if a delay is anticipated.

3.10 Sputum

Sputum is produced by coughing deeply into a wide necked leak proof container and is
best collected in the morning before the mouth is washed.

3.11 Transport and storage

Transport to the lab with as little delay as possible. To prevent the death of fastidious
organisms, transfer a portion to a cotton swab and insert in an Amie’s transport
medium.

3.12 Cerebrospinal Fluid (CSF)

This is collected aseptically by a medical officer from the arachnoid space between the
fourth and fifth lumbar vertebrae into a dry sterile container.

3.13 Transport and storage

CSF specimens are delivered immediately to the lab and so should always be collected
in the hospital with the needed laboratory services.

3.14 Swab

Used for throat swabs, High Vaginal Swabs, Endocervical swab, Wound swabs etc.

3.15 Throat Swab

In good light and using the handle of a spoon to depress the tongue, examine the inside
of the mouth and swab the affected area using a sterile cotton swab. Take care not to
contaminate the swab with saliva.

3.16 Transport and storage

Transport to the laboratory within two hours of collection. To prevent deterioration of

organisms, use a sterile swab containing silica gel and seal the tube. (viable for at least 3
days).

3.17 Aspirates

Include pleural, pericardial, ascitic (peritoneal) effusions, and synovial fluid. Aspiration
is done by a medical officer. Dispense into dry, sterile screw cap tubes, one plain, and
the other containing sterile tri- sodium citrate.

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3.18 Transport and storage

Transport 5ml into a bottle containing sterile thioglycollate broth and the other into a tri
sodium citrate containing bottle.

3.19 Skin Scrapings

Clean the affected area with 70% ethanol, collect skin scrapes. Using a blunt scalpel
blade, scrape the surface up to the margin of lesions onto a clean piece of paper.

3.20 Transport

Send to the laboratory in paper packages labelled with the patients name and hospital
number.

3.21 Nail Clippings

After cleaning with 70% ethanol, using a sterile scissors, take clippings of the infected
part of the nail into a clean piece of paper.

3.22 Transport

Send to the laboratory in paper packages labelled with the patients’ name and hospital
number.

3.23 Skin Snip

Used for investigation of filariasis. The skin over the shoulder blade is sterilized with
alcohol. A needle is used to lift up the skin gently and the skin is cut with the scalpel
blade.

3.24 Blood Culture

Used for investigation of septicaemia/bacteraemia. Label the blood culture bottle with
appropriate information. Be sure this information matches that on request form.
Disinfect the rubber septum of the blood culture bottle with a 70% alcohol swab. Allow
it to air dry. Identify a good vein, wipe the skin with a 70% alcohol swab and then
povidone-iodine. Allow to dry. If the vein is palpated again, repeat the skin
disinfection.

Insert the needle into the vein with the bevel of the needle face up and withdraw
desired amount of blood and release the tourniquet. Place a sterile cotton ball or gauze
over the insertion site while holding the needle in place. Withdraw the needle and have
the patient hold the cotton ball or gauze firmly in place until the wound has stopped
bleeding. Cover the insertion site with an adhesive bandage.

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Immediately (within 1 minute) inoculate (add 1-2 ml of blood into 18-19 ml of blood
culture broth for children And 5-10 ml of blood into 40-45 ml of blood culture broth for
adult. The acceptable broth dilution factor for blood culture specimen is 10. Ratio of
blood to broth is 1:10 to 1:5 for adults.

Introduce blood sample into the blood culture medium using a new needle after
disinfecting the top of the bottle with 70% alcohol.

After inoculation, mix gently several times by rotation and transport to a microbiology
laboratory immediately.

Note: Blood culture bottles should be stored at 4°C when not in use and pre-warmed to
room temperature (25°C) or 37°C before inoculation.

3.25 Sample Rejection Criteria

Policy that defines conditions that would render a specimen unacceptable for
processing in Microbiology

3.26 Procedure

All rejected specimens are documented in the lab computer with date, time and the
physician notified.

3.27 Examples of Sample Rejection Criteria

3.27.1 Unlabelled or mislabeled samples

3.27.2 Duplicate samples

Most duplicate samples received on the same day are unacceptable and should not be
processed. Exceptions include blood culture samples, cerebral spinal fluid (CSF), tissue,
and sterile body fluids (excluding urine).

 If it has been verified by the person collecting the sample that two samples received
at the same time are identical, for the purpose of testing, these samples may be
combined and processed as one.
 If duplicate samples are received at different times on the same day, notify the
patient’s physician or nurse, and document. If it is acceptable not to process the
sample, report “Duplicate sample: test not performed”, and note the reference
number of the sample that was processed.

3.27.3 Leaky containers

 A sample is unacceptable when the outside of the container is grossly contaminated

with the sample. In this case, request a new sample.

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 If the container is leaking, analyze the sample only if the sample is not compromised
and if the leakage will not contaminate any laboratory equipment during the testing
process.

3.27.4 Contaminated samples

 Contaminated samples are unacceptable. Types of contamination include when

another type of sample is mixed with the desired sample. For example, a urine
sample should not contain stool, and vice versa.
 In the case of a contaminated sample, request a new sample.

3.27.5 Inappropriate sample sources

Samples that do not conform to the type of sample needed for the requested test(s) are
unacceptable.

 For example:
o Do not process saliva in place of sputum.
o 24–hour urine samples are unacceptable for routine bacterial cultures.
o The type of anti-coagulant for a blood sample (or the absence of an anti-
coagulant) must be appropriate for the type of blood test.
 If an incorrect or inappropriate sample type is received, request a new sample and
specify the proper sample for the test requested.

3.27.6 Delayed transport time and sample processing

 Ideally, all samples should be less than 2 hours old when received.
 Appropriate transport media and detailed instructions should be available for
samples transported to laboratories.
 If the time between sample collection and receipt is too long for a valid test to be
performed, with respect to sample requirements for the requested test(s), request a
new sample.
 If a sample was received after prolonged delay but is not rejected by the laboratory,
document it and indicate the length of time after collection that the sample was
received.

3.27.7 In General, the following conditions will lead to rejection of a specimen by the
UTH Laboratory

 Specimen label or request form that does not meet the minimum data set required by
the UTH Laboratory.
 Specimen label and request form with mismatched details.
 Illegible details that cannot be deciphered even after seeking a second opinion from
appropriately qualified staff.

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 Leaked specimen
 No ward details
 Unlabeled specimen container
 Insufficient specimen
 Specimen container past expiry date
 Wrong specimen container
 Specimen inappropriately handled with respect to temperature, timing, or storage
requirements.
 haemolysed specimen
 Lipaemic specimen: cloudy or milky serum sometimes due to the patient’s diet may
be rejected depending on the test requested.

3.27.8 Actions for When Samples are Rejected

 If the unacceptable sample can be replaced, notify the requesting healthcare provider.
 Document the reason for the sample unacceptability and request another sample.
 Do not discard the sample until the patient’s healthcare provider has confirmed that
another can be collected.
 If the patient has already been started on antimicrobial therapy or if a repeat sample
cannot be collected, this must be documented.

If a repeat sample is not available, document the problem and proceed with the test if
possible.

 Rejected specimens and request forms are still assigned a laboratory number for
record purposes. The reason for rejection is indicated and a report printed and
dispatched to the destination ward indicated on the request form. The rejected
specimen will be disposed of by the laboratory.

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3.28 Instrumentation

The Followings are common instrument used in Medical Microbiology laboratory:

Identify each item and label them. Find out the uses and functional principle of each.

Slide Rack Staining rack Wire loops with holders

Centrifuge Incubator

Biosafety Cabinet or Inoculation unit Coupling jar, measuring cylinder,

conical flask, beaker

Figure. 3.1

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Distiller Vortex Mixer Autoclave Hot Air Oven

Autoclave Incubator Centrifuge Electrophoretic

System

Laboratory Timer Measuring Cylinder, Vortex Mixer Magnetic Stirrer

Beaker, Conical
Flask

Beaker, Conical Coupling Jar Distiller Electronic

Flask, Coupling Jar Weighing Scale
Figure. 3.2

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Binocular light microscope Inverted Microscope

Figure. 3.3

Inoculation bench Pippette Universal bottle Bijou bottle

Tripod stand Culture Plates specimen bottles Stains (in kidney

dish)
Figure. 3.4

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Chapter 4
Microscopes and Microscopy
Odimayo Simidele
4.1 Introduction

Most samples in Medical Microbiology laboratory have to be examined macroscopically

and microscopically in the course of processing. Macroscopic examination involves
specimen assessment with unaided eyes. This is often done to document the colour of
samples, presence of blood or mucus or parasites or filamentous fungus among other
relevant features in the specimen. Microscopic examination requires a microscope. This
study session introduces the students to some of the basic concepts they need to know
on microscopy

4.2 Microscopes

These are instruments used in the laboratory to view objects not visible to the naked
eye. Microscopy is the science of observing small objects using a microscope. These
scientific instruments come in different power magnifications.

Extensive and significant use was first documented by Antony Van Leuwenhoek (1675),
a Dutchman who used simple biconvex lens to magnify microorganisms. He found
many organisms in water, pond, saliva and intestinal contents of healthy subjects and
called them animalcules. He described them in such a great detail that is hardly
surpassed up till today. He depicted them in drawings as spheres, rods and spiral
filaments (spirochaetes).

4.3 Types of Microscopes

Microscopes can conveniently be divided into 2 broad groups i.e. light microscopes and
electron microscopes. A light microscope, also known as optical microscope uses visible
light to magnify images of objects. Electron microscopes use electrons instead of light to
magnify objects and allow a much higher resolution.

There are two types of electron microscopes namely Transmission Electron Microscope
(TEM) and Scanning Electron Microscope (SEM).

There are two types of light microscope: simple microscopes and compound
microscopes.

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A simple microscope uses a lens to magnify an image, while compound microscope

uses two systems of lenses to enlarge an image. The two systems of lenses used by a
compound microscope are the objective and the eye piece lenses. These lenses combine
to give a much higher resolution than the simple microscope. Compound light
microscopes can be monocular, binocular or trinocular depending on the number of
ocular viewpoints.

Some of the several modifications of compound light microscopes are the binocular
compound light microscope, inverted microscope, dark field microscope, phase contrast
microscope, digital microscopes and stereo microscopes.

Binocular compound light microscopes have two ocular view segments and are most
common.

A digital microscope has a computerised camera device (CCD) attached to it and

connected to a computer. These microscopes may or may not have eyepiece viewing
ports. The CCD is commonly connected to the third ocular segment of a trinocular
microscope.

A stereo microscope uses two objective lenses that are set at slightly different angles,
giving the viewer a three-dimensional picture of the object being looked at.

Dark field; dark field condenser replaces the light condenser for refractile organisms eg.
Spirochaetes.

Phase contrast; the phase contrast condenser replaces the light condenser and causes a
shift in the light passing through transparent specimen thereby causing a contrast
/amplitude in the image.

Fluorescent; UV light or LED replaces the light source and causes fluorescent materials
to fluoresce.

Inverted microscope: has the light source and condenser on top pointing down while
the objective lens system is below the stage pointing up. It is used commonly in cell
culture laboratories. It was invented in 1850 by Lawrence Smith.

An electron microscope (EM) uses electron rays as source of energy. It is composed of

an Electron Source, which generates a stream of electrons that accelerate toward the
specimen using a positive electrical potential. These interactions and effects are detected
and transformed into an image. EM has the highest image magnification capability.

4.4 Microscopy

The science of observing small objects using a microscope involves magnification which

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reflects the power of the microscope.

4.5 Parts of a Compound Microscope

 Eye piece lens; the lens at the top that you look through. Usually 10x or 15x power.
 Tube; connects the eyepiece to the objective lenses.
 Arm/ limb; supports the tube and connects it to the base
 Base; bottom of the microscope, used for support.
 Light source/ illuminator; this could be an electrical bulb or a mirror reflecting sun
rays into the microscope for
illumination.
 Stage; this is the flat platform
where you place your slides.
Stage clips holds the slide in
place.
 Revolving nose piece; this is
the part that holds two or
more objective lenses and
can be rotated to change
power.
 Objective lens; usually three
or four in number with a
nominal power of 4x, 10x,
40x, and 100x. The 100x
objective is used with
immersion oil. The shortest
lens is the lowest power and
the longest one is the lens
with greatest power.
 Colour coding of Objective
lenses: white 40X
magnification
 Red 10X magnification
 Yellow 40X magnification
 Green 100X magnification
Figure. 4.1
 Rack stop; an adjustment that determines how close the objective can get to the slide.
 Condenser lens; it is focus knob located in the lower left of the microscope body. It

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focuses the light onto the specimen.

 Diaphragm or iris; it is found under the stage. It is used to vary the amount of light
projected unto the slide.
 Fine and coarse focus knob; used for fine and coarse adjustments.

4.6 Why Perform Microscopic Examination

a) Validates quality of specimen

b) For presumptive diagnosis eg direct gram stain of specimen
c) Detection of specific organism may guide choice of media for culture
d) Quality control comparison with isolates recovered at culture.

4.7 Using the Microscope

4.8 Simple Lens

To examine a non-transparent object such as insect with a simple lens, it is better to

stand with one’s back to light; a transparent object, such as a section or a tube of fluid is
better examined facing the light.

i Hold the lens as close to your eye as convenient and retain in this position.
ii Move the object towards and away from the head and lens in order to bring it into
focus and view it from different angles.
iii. Use the simple lens to view and examine the parts of parasites provided.

4.9 Compound Microscope

1 Make sure that both the surface of the stage and the underside of specimen are dry
and clean.
2 Place the specimen on the stage in the slide holder.
3 Focus the specimen with the 10x objective.
4 Focus the condenser (should be within 1mm of its topmost position) and leave it in
this position for all objectives.
5 If the microscope is not fitted with a pre-centred condenser, check and centralize the
condenser.
6 Examine the slide provided with the 10x objective and obtain the best image by:
-closing the iris about two thirds,
-adjust the lamp brightness control to give good illumination with the minimum of
glare.
7 Use the mechanical stage to examine the specimen systematically.
8 Examine the specimen with the ‘40x objective’. Obtain the best image by:
- Opening the iris more,
- Increasing the illumination
9 To examine the specimen with the 100x objective. Move the 40x objective to the side,

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place a drop of immersion oil on the specimen and bring the 100x objective into
position. Obtain the best image by:
- Opening the iris fully,
- Further increase the illumination.
NB: To prevent damage to the 100x objective lens, move the objective to one side before
removing the specimen.

4.10 Care when using the microscope

1. Do not force any mechanism e.g. iris lever, focusing controls or mechanical stage
control. These mechanisms are delicate and need to be handled with care.
2. Make sure the surface of the stage and the underside of specimen are dry and clean
before inserting the specimen on the slide holder.
3. Always move the 100X objective to one side when inserting and removing
specimens to prevent scratching of the front lens of the objective.
4. Before beginning to examine a specimen, the microscope is checked to ensure that:
i. The objectives are clean and free from immersion oil
ii. The eyepieces are free from dust
iii. The sub-stage condenser is racked up until its top surface is 1-2 mm below the
object on the slide.
5. If the microscope has to be moved, it should be lifted by the upright limb with
support of the base.
6. Do not keep the illuminator on when not in use.
7. Always clean the objective lens before and after using the microscope.
8. Protect from dust. Cover with transparent nylon or cloth when not in use.
9. The oil immersion objectives must be cleaned each day after use with ‘lens tissue’
and xylene, alcohol must not be used as the cement that unites component lenses
may be soluble in alcohol.
10. Before switching on the microscope turn the lamp brilliance control to its lowest
setting, then increase it to its lowest setting, then increase it to about three- quarter
of its power.

4.11 Sample Preparation for Microscopic Examination

 Unstained specimen e.g wet preparation which could be saline or iodine

preparation, hanging drop, KOH preparation (potassium hydroxide).
 Stained specimen e.g Gram stain, Ziehl Nelseen Stain, flagellar stain

4.12 Wet Preparation

Wet preparation is used mainly to examine specimens and cultures for motile bacteria,
to examine CSF for yeast cell eg. Cryptococcus neoformans using Indian ink, etc.

Place a small drop of bacteria suspension on a slide and cover with a cover slip.

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Examine the preparation microscopically using 10x and 40x objectives. The iris
diaphragm of the condenser should be sufficiently closed to give good contrast.

4.13 Hanging Drop

This is another method of examining live material and is particularly suitable for
observing motility in bacteria.

4.14 Preparation

Using a hollow ground slide or plasticin, make provision for hanging-drop.

i Clean a cover slip.
ii Using sterile wire loop, place one small drop of material in the centre of cover slip.
iii Using a swab stick place a small drop of liquid paraffin on each corner of the cover
slip.
iv Avoid contact between oil and specimen.
v Invert the clean hollow-ground or prepared slide over the specimen so that the well
covers the specimen drop and the cover slip adheres to the slide by virtue of surface
tension.
vi Quickly and smoothly turn over the slide. The specimen drop should be hanging, if
not, remove the cover slip and start again.
vii Examine the preparation by first focusing the edge of the specimen drop using low
power (reduce light). Then switch to dry high (increase light). Observe “Brownian
movement” and distinguish this from true motility in bacteria.

4.15 Preparation of Smears for Staining

Grease free slides should be used and well labelled.

Liquid sample; using a sterile wire loop or Pasteur pipette, transfer one or two drops of
the of the sample to a slide to make a smear which should be spread evenly covering an
area of about 15-20 mm diameter on a slide. Sterile saline can be used to dilute thick
suspensions. Allow to air dry in a safe place protected from sunlight, dust and insects.
Solid culture; first place a drop of sterile saline or distilled water on a glass slide, with a
sterile straight wire or wire loop, touch a colony of the organism on the agar plate and
emulsify. Allow it to air dry.

4.16 Fixing Smears

The purpose of fixation is to preserve microorganisms and to prevent smears from

being washed from slides during staining. Fixation can be done by heating, using
alcohol or other chemicals. Heat fixation; rapidly pass an air dried slide through the
flame of a Bunsen burner. Allow the smear to cool before continuing with staining or
until the alcohol evaporates. Alcohol fixation; it is far less damaging than heat fixing.
Fix with one or two drops of 70% alcohol and leave for two minutes.

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Chapter 5
Stains and Staining Techniques
in Microbiology
Iheanacho Nwadioha & Fadeyi Abayomi

5.1 Introduction

Aim: To achieve contrast thereby highlighting the microorganisms and differentiating

them from one another and from background material and debris.

5.2 Staining

Staining can be positive or negative. When the organism is stained, it is positive,

however, when only the background is stained but the organisms are not stained, then
it is negative staining. Negative staining is of use more in electron microscopy of
viruses.

5.3 Types of Stains

a. simple stains eg. Loeffler’s methylene blue.

b. Differential stains eg Gram stain, Acid-Fast stain,
c. Special stains eg. capsule stain, spore staining, flagellar stain.

5.4 Common stains used in Bacteriology

5.5 Negative Staining

Practice negative staining as follows: place a drop of nigrosin or Indian-ink on a clean

slide, mix with one loopful of saliva or light bacteria suspension and make a smear of
the mixture using the blood film technique. Allow to air dry. Examine with oil
immersion lens. Observe that micro-organisms are not stained by this procedure but
they stand out white against the blue-grey background.

A similar effect can be obtained by placing a cover slip over a drop of nigrosin-sample
mixture. This is termed a wet negative stained preparation.

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5.6 Simple Positive Staining

Take a clean slide, flame to remove grease, cool, and mark slide into 2 sections with a
grease pencil. Number sections 1and 2 and in each section spread one loopful of
specimen fluid. Dry and fix the smears in the flame and allow cooling. Stain by flooding
with Loefler’s methylene Blue for 3minutes. Rinse with water. Clean the back of the
slide with filter paper and place in a slanting position to air dry. Examine with oil
immersion lens.

5.7 Common stains used in Bacteriology

5.8 Gram staining

Make a thin smear, allow to air dry, Heat fix by passing slide 3-4 times through the
flame of a Bunsen burner, Cover smear with a primary stain (crystal violet) for 45-60s,
Rinse with water, Cover smear with mordant (Lugol’s iodine) for 45-60s, rinse again,
decolourise with acetone for 1-3s (a longer duration is required if alcohol is used for
decolourization), rinse and counterstain with safranin or dilute carboh Fuschin or
neutral red for 1-2mins, rinse, place in a slanting position to air dry. Examine with 40x
objective lens to see the staining and distribution, then under oil-immersion lens.

5.9 Interpretation

Gram positive organism stains purple

while gram negative organisms stain
pink or red

Figure. 5.1 Gram reactions

5.10 Ziehl Neelsen staining

Make a thin smear from the clinical sample, Allow to air dry. Heat fix or alcohol fix.
Cover the smear with strong carbol fuchsin. Heat from underneath with alcohol lamp
until vapour starts to rise. Do not allow to boil. Leave hot steaming carbol fuchsin for 5
mins, rinse, decolourise with 25% H2SO4 for 3 mins. Counterstain with 0.3% methylene
blue for 1 min. Rinse and allow to air dry and view under 100x (oil immersion) objective
lens.

5.11 Interpretation

Acid fast organisms stain red while background stain blue (if methylene blue counter
stain is used) or green (if malachite green counter stain is used).

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5.12 Spore staining

Make a smear of the bacteria and fix the slide. Place on a staining rack. Flood the slide
with 5% malachite green. Apply flame underneath until steam appears and leave for
one minute. Do not allow to boil. Wash under tap water. Flood the slide with 0.5%
safranin and leave to act for 30 secs. Rinse under running water, air dry and examine
using 100x objective with immersion oil.

5.13 Interpretation

Spores stain pale- green while vegetative bacteria stain brownish red.

5.14 Capsule staining

Prepare a smear, and air dry. Do not heat fix. Flood the smear with crystal violet for 1
minute. Rinse with water, air-dry and observe with oil immersion objective.

5.15 Interpretation

The capsule appears unstained while the whole organism is stained purple.

5.16 Further Notes

Fixed and stained preparations can be stored if required for revision purposes. But all
immersion oil must be first washed off with XYLENE (xylol) and the slide stored dry.

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Chapter 6
Media in Microbiology
Iheanacho Nwadioha & Odimayo Simidele
6.1 Introduction

Culture media is an artificial way of recovering disease causative agents in the lab. It
must provide all nutritional support required for growth of the organism.

6.2 Aim of Culture

1. To demonstrate the presence of microorganisms causing diseases in specimens.

2. To test the sensitivity of isolated pathogens to antibiotics.

6.3 Types of Culture Media

1. Basic media – it is a simple media that will support the growth of organisms do not
need any special requirement for growth. Examples are: nutrient agar, peptone
water
2. Selective media– these are solid media that contain substances that allow the growth
of one organism while inhibiting the growth of others. E.g. Salmonella-shigella agar,
Thayer Martins medium, MacConkey agar
3. Enrichment media- it is a fluid selective media. Eg. Selenite F broth for Salmonella.
4. Enriched media – they contain certain growth factors required for growth of
fastidious organisms. e.g. blood agar, chocolate agar for Streptococcus, Neisseria etc.
5. Differential media- these media contain dyes or other substances for easy
differentiation of organisms e.g. MacConkey for lactose and non-lactose fermenters,
Mannitol Salt agar.
6. Identification media- these are used to aid in the identification of microorganisms e.g.
Triple Sugar Iron (TSI), citrate agar.
7. Transport media- they are semisolid media containing substances to prevent the
overgrowth of commensals and ensure the survival and recovery of pathogens. Eg.
Cary – Blair, Amie’s, Stuart media.

6.4 Composition of Media

Synthetic media contain chemically definable organic and inorganic compounds. The
composition is usually as follows;

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 Distilled Water,
 Sucrose (Source of carbon and energy),
 K2HPO4 (pH buffer and source of phosphorus and potassium),
 (NH4)2HPO4 (pH buffer and source of nitrogen and phosphorus) etc.

Complex or non-synthetic media contain at least one ingredient that is not chemically
definable usually of plant, yeast or animal extract. It consists of the following;

 Water,
 Peptone (Source of nitrogen, sulphur and phosphorus)
 Beef extract and yeast extract (source of vitamins and other growth factors),
 Glucose (source of carbon and energy),
 Sodium chloride etc.

6.5 Classification based on Consistency

Solid: They are solidified by the incorporation of a gelling agent like agar or gelatin.
They are mainly used in petri dishes as plate cultures or in bottles or tubes as slants or
deeps. The purpose of using solid media is to isolate discreet colonies of each organisms
present in a specimen eg. blood agar.

Semi-solid: They require a little amount of agar and are used mainly for motility &
biochemical tests and as transport media. eg Stuart transport medium.

Liquid: Used mainly as enrichment media for organisms that are likely to be few. Eg
Thioglycollate broth for blood culture, alkaline peptone water.

6.6 Examples of Media Used in Microbiology

6.6.1 Selenite F and Tetrathionate Enrichment Broths

These media permit growth of coliforms. Hence large inocula may be obtained from
stool specimens after 1-2gm gram of stool have been added to 9-10ml for broth and
incubated for 24 hours. Subcultures can be made on suitable plating media such as
MacConkey agar.

6.6.2 Deoxycholate Citrate Agar (DCA)

This medium is used for enumeration of coliforms in water and milk, and for isolation
of enteric pathogens It contains lactose as the fermentable carbohydrate. Growths of
gram positive bacteria are inhibited in the medium. Colonies of lactose fermenters
appear red or pink while non-lactose fermenters appear colourless on the medium.

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6.6.3 Eosin Methylene Blue (EMB) Agar

This medium contains lactose as the fermentable carbohydrate. Most gram-positive

bacteria are inhibited while colonies of Klebsiella spp is usually pink, Escherichia coli is
metallic green and other pathogens colourless on the medium. It is extensively used as a
differential but non-selective medium for cultivation of all the enteric gram negative
bacilli.

6.6.4 MacConkey (MAC) agar

This is intended particularly for isolation of enteric pathogens; it contains lactose as the
fermentable carbohydrate, and bile salts and crystal violet to inhibit gram positive
bacteria. Colonies of lactose fermenters appear pink and non-lactose fermenters appear
colourless on the medium.

6.6.5 Xylose Lysine Deoxycholate (XLD) agar

This selective medium is recommended for the isolation of Salmonella and particularly,
shigellae from faecal specimens. It contains phenol red as indicator. Shigella and
Salmonella form pink red colonies due to resultant alkaline milieu. Some Proteus strains
and Edwardsiella species form pink-red colonies with black centres. E. coli, Enterobacter
species produce yellow colonies due to carbohydrate fermentation.

6.6.6 Salmonella Shigella (SS) agar

This selective medium was developed for use in the selective isolation of Salmonella and
Shigella. However it is more selective for Salmonella.

6.6.7 Kligler Iron Agar (“KIA”)

This medium contains 1% lactose, 0.2% glucose, phenol red, and ferrous sulphate. The
coli-aerogenes group of bacteria gives acid throughout the slant and gas in the butt, the
paratyphoid group gives acid and gas in the butt and typhoid and dysentery group
gives acid only in the butt. Sulphide fermenters, which include most salmonellae, turn
the line of stab black.

6.6.8 Triple Sugar Iron agar (“TSI)

This is similar to KIA but in additional contains 1% sucrose. Hence most strains of
Proteus, as well as coliforms, give an “acid throughout reaction”.

6.6.9 Anaerobic Media

Robertson cooked meat medium, thioglycollate broth blood agar, chocolate agar,
Nutrient agar, Mueller Hinton agar

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6.6.10 Virology/Cell cultures media

HeLa, HEp-2 cells, primary monkey kidney cells

6.6.11 Parasitology

Nelson’s medium, Cysteine-Peptone-Liver-Maltose (CPLM), Buffered Charcoal Yeast

Extract (BCYE)

6.6.12 Mycology

Birdseed agar; Cornmeal agar; Sabouraud Dextrose Agar.

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Chapter 7
Culture Techniques
Odimayo Simidele
7.1 Aerobic culture

7.2 Inoculating Artificial Agar Solid Agar in Petri Dishes

7.3 Aim
 To get discrete colonies for identification.
 Shows if culture is pure or mixed

7.4 Procedure

 Dry the surface of the culture medium in

hot air oven.
 Using a sterile wire loop (if liquid) or
swab stick used in taking the specimen,
apply the inoculum to a small area of the
plate (primary inoculums site or ‘the
well’)
 Flame sterilizes the wire loop, allow it to
cool and then, spread inoculum from the
well through four quadrants using a
back and forth motion while turning
plate at 90oc angle.
Figure. 7.1
7.5 Inoculation of Slopes & Butts
7.6 Procedure

 If the slope/ slant alone is to be inoculated, using

straight wire, streak inoculums down the centre of
the slope. Then Spread in a zigzag pattern.
 If a slant and butt of a medium (eg KIA) are to be
inoculated, stab into butt first using the straight
wire loop and with the same wire, streak along
slope in a zig- zag manner.
Figure. 7.2

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 If the butt of a medium alone is to be inoculated,

using a sterile straight wire, stab through the
centre of the medium. Withdraw wire gently
along the line of inoculum without making
further stab lines.

Figure. 7.3

The following are important in achieving satisfactory plating of media:

 Ensure that the surface of the medium is dry and free from visible
contamination.
 Flame the wire loop (loop and shank) to redness, and allow sufficient time for the
loop to cool in air before inoculation or touching the inoculum. If in doubt about
the coolness of the wire loop, test by using it to touch the margin of the un-
inoculated plate.
 Carry out inoculation and plating procedures as rapidly as possible in order to
minimize the degree of exposure of the medium to contamination from the air.
 Cover the base plate with lid as soon as plating is completed.

7.7 Inoculation of Liquid Media

A sterile wire loop or Pasteur’s pipette can be used to inoculate.

If sub-culturing from solid medium, wire loop or straight wire can be used to pick
colonies. If a fluid is to be inoculated, use a pipette.

Hold bottle containing liquid medium (broth) at right angle. Insert wire loop with
colonies on it rubbing on the side of the bottle just above the fluid level. Touch fluid
gently with the loop and emulsify colonies on the side of bottle in the fluid.

7.8 Anaerobic culture

See chapter on anaerobiosis.

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Chapter 8
Introduction to Anaerobiosis
Iheanacho Nwadioha
8.1 Introduction

Anaerobes are organisms that do not require oxygen for growth and reproduction. They
can be classified as:

1. Obligate anaerobic bacteria: cannot survive the presence of oxygen e.g Bacteroides,
Clostridium species. Strict anaerobes grow in ≤ 0.5% Oxygen e.g Clostridium tetani.
The obligate anaerobes that commonly cause infection can tolerate atmospheric O2
for at least 8 hours and frequently for up to 72 hours.
2. Moderate anaerobes grow in 0.8 to 2.5% O 2
3. Aerotolerant anaerobes grow in ≥ 2.5% O2.
4. Microaerophilic bacteria e.g Campylobacter, Helicobacter can tolerate low Oxygen
concentrations but grow better anaerobically or with > 10% CO 2
5. Facultative anaerobes e.g Escherichia, Klebsiella, Proteus, e.t.c, are organism which can
grow in the presence or absence of oxygen

8.2 Why they are Anaerobes

1. Molecular Oxygen is toxic to them. Substances produced when oxygen is reduced is

toxic to the anaerobic organism.
2. Absence of protective enzymes like superoxide dismutase.

Table. 8.1 Common Anaerobic Infections in Humans

Infections Implicated organism

Brain abscess Bacteriodes spp, Fusobacterium
Eye infection Peptostreptococcus, Clostridium
Intra abdominal infection Bacteriodes, Clostridium perfringes
Gas gangrene Clostridium perfringes, Clostridium novyi
Oral and dental infection Peptostreptococcus, Fusobacterium

8.3 Media for Isolating Anaerobes

 Anaerobic Blood Agar

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 Bacteriodes Bile Esculin agar(BBE)

 Robertson Cooked Meat Medium (RCM)
 Thioglycollate broth
 Kanamycin-Vancomycin-Laked Blood agar (KVLB)

8.4 Achieve Anaerobic Culture Condition Using

1. Anaerobic chamber
2. Anaerobic jars eg Gas Pak
3. Anaerobic Bags or pouches e.g Bio Bag, Gas Pak pouch, Anaerobic pouch
4. Candle Extinction jar

8.5 Components of Anaerobic Chamber

 Catalyst
 Desiccant
 H2 gas – 5-10%
 N2 gas - 80-90%
 CO2 gas – 5-10%
 Chemical Indicator: eg. methylene blue or resazurin.
 Biological Indicator: e.g Pseudomonas

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Chapter 9
Cutural Identification of Bacteria
Odimayo Simidele
9.1 Introduction

Following inoculation of your agar, bacteria growth can be seen on the plate usually
after 24hours of incubation under appropriate incubation conditions. The general
principle is that each organism seeded on the media divides by binary fission several
times to form a colony which becomes large enough to be seen visually.

The characteristic appearance of the colonies of a given bacteria depends on the nature
of the media, length of incubation and the incubation conditions of the agar.
Characteristics observable includes: size, shape, surface appearance, consistency, rate of
growth, presence of haemolysis, swarming, pigment production, change of colour of the
medium (lactose fermentation), mucous production e.t.c, some commonly observable
effects of bacterial growth on a media is discussed with examples.

9.2. Haemolysis

This can be demonstrated on blood agar. This can be alpha (α), beta (β) or gamma (ϒ)
haemolysis.

9.2.1 α-Haemolysis

There is partial haemolysis/ clearing of blood with greenish pigmentation around

colonies on blood agar/chocolate agar. Commonest example is exhibited by
Streptococcal pneumoniae, others such as Enterococcus faecalis, Streptococcus anginosus,
Peptostreptococcus can show other forms of haemolysis apart from α - haemolysis.

9.2.2 Beta-Haemolysis

Shows complete haemolysis/ clearing of blood. There is a zone of complete clearing

around colonies on blood agar; examples of organisms with β-haemolysis are
Streptococcus agalactiae, Streptococcus pyogenes.

9.2.3 Gamma-Haemolysis

There is no zone of clearing around colonies. No change in the medium. e.g

Streptococcus bovis

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9.2.4 α’ Hemolysis

There is a halo of incomplete lysis immediately surrounding the colonies with a zone of
complete haemolysis at the periphery.

9.3 Swarming

Colonies appear as thin film or waves extending on its own beyond points or lines of
inoculation on agar surface due to motility of the organism. Organisms that can swarm
include Proteus mirabilis, Clostridium tetani.

9.4 Lactose Fermentation

Colour change on the plate can be observed in MacConkey (Pink), CLED (yellow)
following growth of lactose fermenters e.g E coli, Klebsiela spp on plate, while non-
lactose fermenters (proteus, pseudomonas) will show no colour change. Colour change
is due to alteration of the pH of the medium and the presence of an indicator for this.
The indicator in MacConkey agar is methyl red.

9.5 Pigment Production

Substances produced by the specie of organism that gives a characteristic colour to the
colonies include pyoverdin by Pseudomonas aeruginosa. This gives its colonies a greenish
coloration. Other pigments produced by pseudomonas include pyorubin (red),
pyocyanin (blue), pyomelanin (Black), e.t.c

9.6 Colonial Morphology

9.6.1 Size of Colonies

Large as seen in Klebsiella spp; Medium as seen in Escherichia coli; Small as seen in
Staphylococcus spp; Pinpoint as seen in Streptococcus spp

9.6.2 Margin

Smooth/ entire e.g Staphylococcus aureus; Filamentous eg Bacillus anthracis; Rough eg

diphtheroids; Irregular eg. Bacillus subtilis; Crenated- eg. Pseudomonas aeruginosae.

9.6.3 Elevation

Raised- E.coli; Convex – staphylococcus aureus; Flat- β-hemolytic streptococcus;

Umblicate- depressed center. Eg Streptococcus pneumoniae; Umbonate- raised or bulging
centre e.g. Serratia marscecens, Pseudomonas.

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9.6.4 Density/Optical Properties

Translucent– β-hemolytic Streptococcus; Opaque- Staphylococcus aureus; Semi-opaque-

Streptococcus agalactiae (Bull’s eye colony); Shiny-Bordetella pertussis (half-pearl),
Klebsiella, E.coli.

9.6.5 Colour

White – coagulase negative Staphylococcus; Gray- Enterococcus; Yellow- Micrococcus,

Neisseria; Buff- diphtheroids, Cream- Staphylococcus spp.

9.6.6 Consistency/Texture (Determined by Touching Colony with Sterile Wire Loop)

Sticky- Staphylococcus aureus; Brittle- Norcadia; Dry- diphtheriods; Mucoid- Klebsiella

pneumoniae.

9.6.7 Pigments

Green – Pseudomonas aeruginosa; Brick red- Serratia marcesens; Blue- kluyvera,

Pseudomonas aeruginosa; Purple- Chromobacterium violaceum; Brown-black- Prevotella
melaninogenica.

9.6.8 Odour

Old sock – Staphylococcus aureus; Fruity / grape-like – Pseudomonas aeruginosa; Putrid-

Proteus mirabilis; Musty basement- Haemophilus influenza; Fresh ploughed field-
Norcardia.

9.7 Growth In Liquid Media

 Streamers
 Puff balls- growth may form puff balls below the surface
 Sediments- some form sediments at the bottom of the tube
 Pellicle- thick growth at the top of the tube
 Turbidity- growth diffuses throughout the tube
 Gas production- usually seen as bubbles
 Scum at side of tube

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Figure. 9.1

9.8 Growth in Cell Cultures

Inclusion bodies, cytopathic effects, etc.

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Chapter 10
Biochemical Tests for Identification
of Bacteria Isolates
10.1 Indication

To identify organism by their utilization of substrates and sugars.

10.2 Catalase test

Take a clean slide. Place two separate drops of hydrogen peroxide on either ends of the
slide. Using a wooden stick, take a sample of your test bacterium into the hydrogen
peroxide drop on one end of the slide and negative control into the hydrogen peroxide
drop on the other end of the slide. Immediate release of burble shows positivity for
catalase.

10.3 Slide coagulase test

Take a clean slide. Place two separate loop-full of saline on the slide and emulsify
suspect growth in each drop. To one drop add one loopfull of plasma. Mix well and
observe for clumping of suspended cells. The second drop is use as a control for
comparison. Please note that some strains clump automatically without the addition of
plasma. In such case the tube test would be used (see demonstration).

Table. 10.1
Test Principle Procedure Interpretation/
Examples
Catalase Used to differentiate Use a wooden stick and INTERPRETATION:
bacteria that transfer the test organism presence of bubbles is a
produces catalase to a tube containing 2-3 positive result.
enzyme. Catalase mls of hydrogen peroxide. Examples:
breaks down OR emulsify the test +ve: Staphylococcus spp
hydrogen peroxide organism with 1 or 2 drops -ve : Streptococcus spp
to oxygen and of hydrogen peroxide on a
water. glass slide.

Citrate Tests for bacteria Use a straight wire to INTERPRETATION:

that utilizes citrate inoculate the citrate slope Bright blue coloration:

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as their sole source and butt. Incubate for 24- +ve. No colour change:
of carbon. 48hrs at 360C. -ve.
Examples: +ve:
Klebsiella -ve: E.coli
Coagulase Used to distinguish Slide test: detects free INTERPRETATION:
coagulase coagulase. Make a dense presence of clot-
producing suspension of the positive result.
staphylococcus from organism using 1-2 drops Examples: +ve: Staph
the non-producing of distilled water on a aureus, Yersinia pestis
species. The free glass slide (Observe for -ve: Staph epidermidis,
and bound auto agglutination). Mix Streptococcus, E.coli,
coagulase form with a loopful of EDTA
fibrin from plasma and observe for
fibrinogen. clumping or agglutination.
Tube method: detects
bound coagulase. Add
0.8ml of test broth to 0.2ml
of plasma. Mix and
incubate at 35-370C for 4
hours.

DNAse Used to identify The DNAse agar is spot INTERPRETATION:

S.aureus which inoculated with the test clearing around the
produces DNAses. and control organisms. colonies- positive result.
DNAse hydrolyses Incubate overnight at 35- Examples: +ve: S. aureus
DNA. The 370C. Cover the surface of -ve: S. epidermidis
hydrochloric acid the plate with 1mol/l
precipitates the hydrochloric acid.
unhydrolysed DNA.
Indole Tests the ability of Inoculate a bijou bottle INTERPRETATION:
the organism to containing peptone water red surface layer-
break down with the test organism. positive.
tryptophan in Incubate at 35-370C for 24- Examples: +ve: E.coli
peptone or tryptone 48 hours. Then add 0.5mls -ve: Klebiella. Spp
water to indole of kovac’s reagent. Shake NB: Kovac’s reagent is
detected by Kovac’s gently and examine for a (p-dimethyl amino
reagent to produce a red colour on the surface benzaldehyde)
red colour. layer.

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Oxidase An oxidase Add 2-3 drops of oxidase INTERPRETATION:

producing organism reagent to a filter paperblue- purple colur- +ve
oxidizes and smear a colony of test
result.
phenlynediamine in organism on it using a Examples: +ve:
the oxidase reagent piece of wooden stick. Neisseria, Pseudomonas,
to a purple colour. Vibrio.
-ve: E.coli
Urease Urease producing Urea agar or broth is INTERPRETATION:
organism will break inoculated with the test pink colour in the
down urea to organisms and incubated medium- positive test.
ammonia and at 35-370C for 12 hours. Examples: +ve: Proteus
carbon dioxide. The -ve: E.coli
ammonia turns the
medium alkaline
which changes
indicator from pink
to red.
CAMP Used to detect the Streak a known beta INTERPRETATION:
test CAMP factor haemolytic S.aureus acrosspresence of arrow-head
produced by a 10% blood agar plate andshaped area of
Streptococcus then inoculate the test haemolysis where the
agalactiae. organism at right angles to
staphylococcus strain
it, not touching the staphmeets the test organism
strain. Incubate overnightis a positive result.
at 350C. Examples: +ve: group B
β- hemolytic
streptococcus
Lactose Lactose fermenters Streak test organism on Examples:+ve:
fermentati produce pink MacConkey or CLED agar Klebsiella, E. coli; -ve:
on colonies on and Incubate overnight at Salmonella, Shigella,
MacConkey agar 35-37 C.
0 Proteus
and yellow on
CLED.
Quellung For bacteria To one drop of broth INTERPRETATION:
test serotyping as a culture or bacteria culture presence of visible
result of capsular emulsified in normal saline capsule is a positive
reaction between on a slide, add 1 drop of result.
the capsular antigen the antiserum and mix Examples: +ve:
and its homologous thoroughly. Place a cover Streptococcus;
antibody which slip and examine using oil -ve: E. coli
cause capsule to immersion objective.
swell.
Methyl To differentiate Grow the organism for INTERPRETATION: A

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Red test between E coli and about 24hrs in glucose- red colour (+ve): E. coli,
enterobacter. phosphate medium. To Yersinia spp, etc. Yellow
(ability to produce 5mls of culture, add 1 drop colour (-ve):
Germ stable acid from of methyl-red (MR) Enterobacter, aerogenes,
tube glucose) solution Klebsiella pneumoniae
etc.
For confirmation of Inoculate 0.5ml of serum
Candida albicans with a yeast colony. INTERPRETATION:
which produces Incubate at 35-370C for 2-3 presence of sprouting
pseudo hyphae. hours. Place a drop on a yeast cell (tube-like)
slide, cover with a cover outgrowth from the
slip and view with 10x and cells (germ tube) is a
40x objective. positive reaction.
Examples: +ve: Candida
albicans
NB: Methyl Red solution: dissolve 0.01g methyl red in 300ml of ethanol and make it up to 500mls with
distilled water.

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Chapter 11
General Considerations in Identification
of Common Bacteria Isolates
11.1 Introduction

Examine all cultures microscopically in Gram stained preparations. Perform catalase on

all gram positive isolates and slide coagulase test on catalase positive (Staphylococcus
spp) cultures. Record your observations. Observe the types of heamolysis and the results
of Optochin tests on Streptococcus pneumoniae, Streptococcus viridans, and bacitracin tests
on the Streptococcus pyogenes.

Enterobacteriaceae covers those aerobic Gram-negative rods usually found in the

intestinal tract. They are usually divided into two large groups: lactose fermenters and
non-lactose fermenters. All enteric bacilli are similar morphologically; grow readily and
luxuriantly at a wide temperature and pH range on ordinary nutrient agar. They are
relatively resistant to ageing and drying and are widely distributed in nature (sewage,
water, soils, grains, skin, etc).
We will proceed with identification of this common Gram negative rods (Provided on
agar plates) using growth characteristics or colonial morphology and biochemical tests
(see chapter 9 and 10) in the identification.
Let us take a look at identification of the following possible bacteria organisms:
Escherichia coli, Klebsiella sp., Proteus sp., Pseudomonas ssp., Salmonella species, Shigella
flexneri, Vibrio cholerae.

 Examine the cultures provided: Record the colonial morphology of various

organisms. Note swarming, evidence of ammonia production in the urea medium as
colour changed from yellow to purple.
 See demonstration of colonial appearances on different media and biochemical
reaction as discussed in previous practical sessions.

11.2 Follow the Identification guide below

Lactose Fermentation is an important guide in enterobacteriaceae identification. This

can be read on MacConkey agar plate. You should be able to differentiate between
lactose positive and lactose negatives on your MacConkey agar by now. However, the
next step after colonial morphology (plate reading) is gram staining which you are
expected to perform on all isolates.

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Lactose fermenters include Klebsiella, E. coli, Citrobacter, or Enterobacter. Further

identification will be based on table below:

Table. 11.1 Lactose fermenters

Bacteria Motility Indole Citrate Lactose Mucus

fermentation Production
E. coli + + - Early -
Klebsiella - - + Early +
Citrobacter + - + Late -
Enterobacter + - + Early +

Figure. 11.1 Klebsiella on DCA (mucoid Figure. 11.2 E coli on MacConkey agar
colonies)

For the non-Lactose fermenters, follow the following guide for their identification: Test
for the ability to turn oxidase disc from brown to a blue-purple colour (oxidase
positivity).

Oxidase positive organisms will be considered as either Pseudomonas, Vibro, Pasteurella,

Alcaligenes, Aeromonas or Neisseria. The shape of the organism on gram staining, types of
pigment produced, ability to grow at an incubation temperature of 42oC and smell on
growth media will be largely used in the identification among the oxidase positive
gram-negative organisms. Neisseria is gram negative diplococci.

11.3 Characterisation of oxidase-negative isolates

Oxidase negative organisms will be further identified using the group of tests below

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and their interpretation will be based on the table below:

a) Ability to ferment mannitol, sucrose and citrate

b) Motility test
c) Indole production
d) Urease production
e) Lactose and glucose fermentation inside KIA medium.

Organisms to be considered in this group include: Salmonella, Shigella, Proteus, Serratia,

Morganella, Providencia and Yersinia species.

Table. 11.2 Interpretative table for oxidase negative gram negative

non lactose fermenters
Organisms Biochemical tests
Glu Oxi Man Sucr Citr Motil Indol Urea
Salmonella + - + - -2 +* - -
Shigella + - +/- -1 - -* +/- -
Proteus + - - + +/- + +/-4 +
Serratia + - + + + + - +/-
Morganella + - - - - + 3 + +
Providencia + - +/- +/- + + + +/-
Yesinia + - + + - + +/- +slow
Key:
1 =S. sonnei ferments sucrose slowly.
2= Salmonella other than S. typhi and S. paratyphi gives varying reactions.
4= Proteus vulgaris is indole positive while P. mirabilis is indole negative.
*= Important differential characteristics.
Glu = Glucose; Oxi = Oxidase; Man = Mannitol; Sucr = Sucrose; Citr = Citrate;
Motil = Motility; Indol = Indole.

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Chapter 12
Antibiotic Susceptibility Testing
12.1 Objectives

To determine the sensitivity of isolate to various antimicrobial agents. The results of

susceptibility testing have direct influence on the choice of drug for treatment of the
infection.

12.2 Techniques

 Dilution technique
 Diffusion technique

12.3 Dilution

Dilution can be agar or broth based

Dilution techniques measure the Minimum Inhibitory Concentration (MIC) of a drug

and can be used to measure the Minimum Bactericidal Concentration (MBC) of a drug.
It is done by adding dilutions of an antimicrobial agent to a broth or agar medium.

12.3.1 Broth dilution method

 Can be macro-broth / tube or

micro-broth dilution method.
 Make doubling dilutions of the
antibiotic (0, 1, 2, 4, 8 and
16µg/ml) in a tube containing
the liquid growth medium.
 Inoculate with a standardized
bacterial suspension (1-5x
10 CFU/ml).
5

 Incubate overnight at 350C.

 Examine the tube for visible
growth (turbidity).
 The lowest concentration of
antibiotic that prevented visible
growth is the MIC.
Figure. 12.1

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12.4 Diffusion

12.4.1 Kirby Bauer method

Agar diffusion method with disc is a qualitative method to determine if bacteria

sensitivity i.e sensitive, intermediate or resistant.

12.5 Kirby Bauer Method of Antibiotic Susceptibility Testing

 Prepare inoculums by emulsifying 4-5 colonies in broth

 Compare turbidity with 0.5% McFarland standard
 Swab surface of Muller Hinton
agar with suspension
 Place antimicrobial discs on
surface, 20mm apart and 25mm
from edge of petri dish.
 For a 90mm petri dish, place 6
antibiotics disc. For a 120mm
dish, place 9 discs.
 Incubate aerobically at 35oC for
18-24 hours
 Measure zone of inhibition
around each antibiotic disc and
compare with standards in an
interpretative chart.
 Report as Sensitive, Resistant or
Intermediate

Figure. 12.2

12.6 Stokes Method

 Both test and control organisms are

inoculated on the same plate.
 Zone of inhibition of test organism is
compared directly with the control.
 Lack of Standardization is a major pitfall
of this method of testing.

Figure. 12.3

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12.7 Precautions in Kirby Bauer Method

 Mueller Hinton agar is to be used

 pH of agar should be 7.2-7.4
 Depth of media should be 4mm
 Surface of media must be level, not tilted to one side.

12.8 Antimicrobial Gradient Method

The antimicrobial gradient diffusion method uses the principle of establishment of an

antimicrobial concentration gradient in an agar medium as a means of determining
susceptibility. The E-test, an agar diffusion method can also be used to determine the
MIC.

James H. Jorgensen, and Mary Jane Ferraro. Antimicrobial Susceptibility Testing: A Review of General
Principles and Contemporary Practices. Clin Infect Dis. (2009) 49 (11): 1749-1755. doi: 10.1086/647952
Figure. 12.4

12.9 Other Antimicrobial Susceptibility Testing Methods

Minimum Bactericidal Concentration (MBC) tests; this is the lowest concentration of an

antibiotic that will kill a pathogen. To determine the MBC, the dilution representing the
MIC and two or more concentrated dilutions are plated. The MBC is the lowest
concentration that prevents growth of colonies.

Time kill assays; this is used to determine the rate at which an antibiotic/ antiseptic kills
a pathogen. A sample of the product is inoculated with a suspension of the test
organism. After a series of exposure times, the surviving organism is plated out, re-
incubated, counted and expressed as percent per each time point.

12.10 Automated Instrument Systems

This involves using optical detection systems to detect for subtle changes in bacterial
growth. They are used to generate rapid susceptibility results. This Include Vitek 2
system, BD Phoenix automated microbiology systems etc.

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12.11 Genotypic method

Here specific genes that confer antibiotic resistance are detected using polymerase chain
reaction (PCR) and DNA hybridization.

12.12 Synergy tests

Antimicrobial assays in body fluids and serum.

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Chapter 13
Immunodiagnosis in Medical Microbiology
13.1 Introduction

Antigens are substances that evoke the formation of antibodies in an animal.

Antibodies are proteins produced by the body in response to presence of antigens.

Rapid tests have been developed to detect presence of an organism based on the
knowledge of antigenic properties it possesses and antibodies produced by the body
against it. Some bacterial agents are not cultivable in the routine microbiology
laboratory and this characteristic may pose a challenge to diagnosing infections due to
such organisms, hence the need and use of these immunodiagnostic methods.

13.2 Antigen Detection Methods

They are used for early diagnosis of an infectious disease. To identify pathogen in
specimen, or that has been isolated by culture Includes:

1. Particle agglutination tests:- eg. Latex agglutination tests (LA), Co agglutination

tests
2. Precipitin tests: e.g tube and agar precipitin test, Counter Immunoelectrophoresis
(CIE)
3. Microscope- assisted -labeled -Antibody staining: eg. fluorescence-labeled
antibody (FA), Enzyme-labeled Antibody.
4. Solid-phase immunoassays: eg. Enzyme Immunoassay (EIA), Flourescence
Immunoassay (FIA), Radio immunoassay (RIA), Immunochromatographic tests
(IC) and dip stick dot immunoassays.
5. Optical immunoassay

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Table. 13.1 Some Infectious Diseases and Antigen Detection Tests Available to
Detect Them
Infections Tests
BACTERIA: EIA
Grp A streptococcal pharyngitis LA
Meningitis EIA, FA
Chlamydia urethritis FA
Pertussis

FUNGAL: LA, EIA

Cryptococcal meningitis

PARASITIC: FA, EIA

Giardiasis FA, EIA
Cryptosporidiosis FA
Trichomonas vaginalis
VIRAL: EIA
HIV EIA
Hepatitis B virus FA
CMV LA, EIA
Rotavirus gastroenteritis
EIA- Enzyme Immunoassay, LA- Latex Agglutination, FA-Flourescence Antibody

13.3 Enzyme immunoassay for Hepatitis B

EIA is based on the "sandwich principle". The solid phase of the microtiter plate is made
of polystyrene wells coated with mouse monoclonal antibodies specific for HBsAg.
Serum or plasma specimen containing HBsAg when added to the well binds to the
antibody and unbound antigen is washed off. Antibody conjugated to an enzyme (horse
radish peroxidase) is added to capture the bound antigens. After another wash to
remove unbound material, a solution of chromogenic substrate will be added to the
wells and incubated. A colour will develop which is proportional to the amount of
HBsAg bound to Anti-HBs.

13.4 Direct antigen for Cryptococcal meningitis using latex agglutination

Latex particles are coated with cryptococcal antibodies. Reaction between the
cryptococcal antigen in CSF and the latex reagent results in the agglutination of latex
particles which is visible to the naked eye.

13.5 Antibody Detection Methods

Antibody Detection Methods Includes:

Particle agglutination assays (PA): Direct, natural PA; Indirect, carrier PA

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13.6 Application

 Febrile agglutinin test for Salmonella spp

 Weil-Felix test for Rickettsial agents
 Mono-test for detection of heterophile antibodies produced during infectious
mononucleosis due to Epstein Barr Virus.
 Streptozyme test for detection of antibodies to Streptococcus.

13.7 Precipitation assays

 Double Immunodiffusion;
 Counter immune electrophoresis
 Flocculation eg Rapid Plasma Reagin (RPR) test for Treponema palladium antigens.
 Complement fixation tests:

13.8 Neutralization tests

 Virus neutralization
 Antistreptolysin O tests
 Treponema pallidium Immobilization

13.9 Microscope –Assisted- Labeled- Reagent Technology

 Indirect Flourescence Antibody tests

 Flourescent Antibody test with Enhanced Sensitivity
 Western blotting (WB)

Table. 13.2 Examples of Serologic Tests for Diagnosis of Infectious Diseases

Infections Test
Bordetella pertussis EIA
CMV EIA, IFA
Helicobacter pylori EIA
Hepatitis A,B,C EIA
HIV 1,2 EIA, IFA, WB
Mycoplasma CF, EIA, IFA
pneumonia EIA, IFA, LA
Toxoplasma gondi IHA, IFA, PA
Trepenoma palladum
IFA- Indirect fluorescent antibody test, WB- western blotting, HA- Indirect haemmaglutination test, PA-
particle agglutination.

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13.10 Treponema pallidum Haemagglutination Test

Serum specimen containing antibodies to Treponema pallidum are mixed in micro wells
containing red cells sensitized with treponema antigens. The antigen –antibody reaction
results in agglutination seen as a mat pattern in the bottom of the well. A negative result
is seen as a smooth ring at the bottom of the well.

13.11 Febrile Agglutinin Test for Salmonella

Also known as Widal test is used to test for the presence of salmonella O and H
antibodies in a patient’s serum. Two methods;

 Slide method (qualitative)

 Tube method (quantitative)

Both methods involve reacting antibodies in the sera with salmonella antigens.

13.12 Test of Cell Mediated Immunity

This includes Mantoux test/ tuberculin test, heaf test, etc.

Mantoux Test to test for delayed hypersensitivity reaction to Mycobacteria tuberculosis. A

tiny amount (0.1ml) of tuberculin protein is injected into the skin. After 48- 72 hrs, a
hypersensitive re action develops in a positive result seen as a swelling and redness
around the area.

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Chapter 14
Laboratory Techniques in Parasitology
14.1 Specimen Collection and Preservation

14.2 Blood Specimen

14.2.1 Timing

Whenever possible, specimens should be collected before treatment is initiated. When

malaria and babesiosis are suspected, blood smears should be obtained and examined
without delay. Since the parasitaemia may fluctuate, multiple smears might be needed.
These can be taken at 8 to 12 hour intervals for 2 to 3 days.

Microfilariae exhibit a marked periodicity depending on the species involved; therefore

the time of specimen collection is critical. If a filarial infection is suspected, the optimal
collection time for demonstrating microfilariae is:

Loa loa—midday (10 AM to 2 PM)

Brugia or Wuchereria—at night, after 8 PM
Mansonella—any time
Onchocerca—any time

14.2.2 Type of Sample

Venous blood samples provide sufficient material for performing a variety of diagnostic
tests, including concentration procedures (filariasis, trypanosomiasis). However, in
some parasitic diseases (e.g., for diagnosis of malaria in particular), anticoagulants in
the venous blood specimen can interfere with parasite morphology and staining
characteristics; this problem can be further compounded by excessive delays prior to
making the smears. In such cases, capillary blood samples are preferable.

14.2.3 Capillary blood obtained by finger prick

1. Label pre-cleaned slides (preferably frosted-end) with the patient’s name (or other
identifier) and date and time of collection.
2. Clean the site well with alcohol; allow to dry.
3. Prick the side of the pulp of the 3rd or 4th finger (alternate sites include ear lobe, or in
infants large toe or heel).
4. Wipe away the first drop of blood with clean gauze.
Prepare at least 2 thick films and 2 thin films.

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14.2.4 Venous blood obtained by venipuncture

1. Label collection tubes and pre-cleaned slides (preferably frosted-end) with the
patient’s name (or other identifier) and date and time of collection.
2. Clean the site well with alcohol; allow to dry.
3. Collect the venous blood in a vacuum tube containing anticoagulant (preferably
EDTA); alternatively, collect the blood in a syringe and transfer it to a tube with
anticoagulant; mix well.

Prepare at least 2 thick films and 2 thin films as soon as possible after collection.

14.3 Stool Specimen

1. Collect about 50-100g of the stool in a wide mouthed, dry, clean, grease-free, leak
proof container. Make sure no urine, water, soil or other material gets in the
container.
2. This table demonstrates the distribution of protozoa in relation to stool
consistency and should be taken into consideration when specimens are
received.

Figure. 14.1

3. Fresh stool should be examined, processed, or preserved immediately. An

exception is specimens kept under refrigeration when preservatives are not
available; these specimens are suitable for antigen testing only. Helminth ova
may continue to dev at room temperature but both ova and protozoan cysts can
be satisfactorily recovered up to 24hours after voiding.
4. Preserve the specimen as soon as possible. If using a commercial collection kit,
follow the kit’s instructions. If kits are not available, the specimen should be
divided and stored in two different preservatives, the most commonly used are
10% aqueous formalin and polyvinyl-alcohol (PVA), using suitable containers.
Add one volume of the stool specimen to three volumes of the preservative. 10%
formalin and PVA are suitable because they do not interfere with staining and
concentration techniques.
5. Insure that the specimen is mixed well with the preservative. Formed stool
needs to be well broken up.
6. Insure that the specimen containers are sealed well. Reinforce with parafilm or
other suitable material. Insert the container in a plastic bag.

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7. Certain drugs and compounds will render the stool specimens unsatisfactory for
examination. The specimens should be collected before these substances are
administered, or collection must be delayed until after the effects have passed.
Such substances include: antacids, kaolin, mineral oil and other oily materials,
non-absorbable anti-diarrhoeal preparations, barium or bismuth (7-10 days
needed for clearance of effects), antimicrobial agents (2-3 weeks), and gallbladder
dyes (3 weeks).
8. Specimen collection may need to be repeated if the first examination is negative.
If possible, three specimens passed at intervals of 2-3 days should be examined.
The distribution of ova and cysts in a single motion is uneven. Random sampling
of single specimen will retrieve less than 50% of potential parasites in light
infection. Increasing the sampling to 3 on consecutive days will increase the
recovery potential by 75 to 100%
9. When repeated faecal examination fails to reveal presence of suspected intestinal
parasites additional techniques may be used. These include; sigmoidoscopy for
Entamoeba histolytica and duodenal contents for Giardia lamblia and Strongyloides
stercoralis infections (see below).

Figure. 14.2

14.4 Serum/Plasma Specimens

14.4.1 Specimen Requirements

Serum/plasma is required for all parasitic disease immunodiagnostic tests. A single

sample is usually sufficient; acute and convalescent specimens are not necessary. CSF
and eye fluids (vitreous or aqueous) are acceptable for selected diseases and MUST be
accompanied by a serum specimen.

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14.4.2 Serum for all tests

 Adults – 3 ml serum/plasma separated from RBC’s.

 Children – 0.5 ml serum/plasma separated from RBC’s.
 CSF: 1.0 ml. This is acceptable only for; cysticercosis, paragonimiasis, and
schistosomiasis testing, when CNS disease is suspected.
 Eye fluids: 0.1 ml neat fluid (no washings). This is acceptable only for;
toxocariasis and toxoplasmosis.

14.5 Other Specimens

14.6 Tissue Specimens

Tissue samples, including biopsies, surgical or necropsy specimens, collected for

possible detection of parasites, should be submitted to the hospital pathologist. In most
instances, routinely stained hematoxylin and eosin sections are suitable for examination
for parasitic agents, and many times, an accurate identification of the parasite can be
established in tissue sections. Occasionally, additional sections are required to further
the diagnosis.

Other specimens include Sputum, aspirates, vaginal swab, cellulose tape/swube tube
procedure and urine specimens (see below).

14.7 Stool Parasitology

14.8 Stool Specimen processing

Stool specimens can be examined fresh or preserved.

14.9 Macroscopic examination

1. Pale, frothy, suggestive of excess fat, often seen in giardiasis and tropical sprue.
2. External mucus and blood, seen in amoebic dysentery, bacillary dysentery and
inflammatory bowel disease
3. Unformed or watery faeces which may contain fresh cellular exudates or which may
contain erythrocytes. These often contain active protozoan trophozoites or cysts of
cryptosporidia.
4. Small ‘rice grain’ egg sacs of Inermicapsifer species (a rare tapeworm) may be visible.

Examination of fresh specimens permits the observation of motile trophozoites, but this
must be carried out without delay. Liquid (diarrhoeic) specimens (which are more
likely to contain trophozoites) should be examined within 30 minutes of passage (not
within 30 minutes of arrival in the laboratory!), and soft specimens (which may contain
both trophozoites and cysts) should be examined within one hour of passage.

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If delays cannot be avoided, the specimen should be preserved to avoid disintegration

of the trophozoites. Formed specimens (less likely to contain trophozoites) can be kept
for up to one day, with overnight refrigeration if needed, prior to examination.

Specimens preserved in formalin can be tested directly (wet mount, immunoassay,

chromotrope stain, UV fluorescence) or can be concentrated prior to further testing.

14.10 Specimen Collection

Fresh stool uncontaminated with urine is collected in a wide mouthed dry, leak-proof
container. About 10ml or a large teaspoon is adequate. Stool can be stored in formalin
or polyvinyl alcohol if not processed immediately.

14.11 Appearance

Report the colour and consistency of stool, the presence of blood, mucus or pus.

14.12 Microscopic Examination

Specimen containing pus and mucus should be examined immediately. For unformed
stool, a wire loop or piece of stick is used to transfer a small amount of stool on one side
of a slide and covered with a cover slip.

For formed stool, emulsify in 1 drop of normal saline. On the other side of the slide,
emulsify stool in a drop of iodine or eosin (for unformed stool) and cover with a cover
slip. View under 10x and 40x objective. Report the number of eggs or larvae or motile
trophozoites (in unformed stool) as few, moderate or numerous.

14.13 Possible Pathogens

Cysts of E histolytica, Gardia lamblia, egg of Ascaris lumbricoides, hook worm, Trichuris
trichiura, larva of Strongyloides stercoralis etc.

14.14 Stool Concentration Techniques

Concentration procedure separate parasites from faecal debris and increase the chances
of detecting parasitic organisms when these are in small numbers.

They are divided into flotation techniques and sedimentation techniques.

14.15 Flotation Technique

Flotation techniques (most frequently used: zinc sulphate or Sheather's sugar) use
solutions which have higher specific gravity than the organisms to be floated so that the
organisms rise to the top and the debris sink to the bottom. The main advantage of this

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technique is to produce a cleaner material than the sedimentation technique. The

disadvantages of most flotation techniques are that the walls of eggs and cysts will
often collapse, thus hindering identification. Also, some parasite eggs do not float.

14.16 Sedimentation Technique

Sedimentation techniques use solutions of lower specific gravity than the parasitic
organisms, thus concentrating the latter in the sediment. Sedimentation techniques are
recommended for general diagnostic laboratories because they are easier to perform
and less prone to technical errors. The sedimentation technique used by most
laboratories is the formalin-ethyl acetate technique, a diphasic sedimentation technique
that avoids the problems of flammability of ether, and which can be used with
specimens preserved in formalin, MIF (merthiolate iodine formaldehyde) or SAF
(sodium acetate, acetic acid, formaldehyde).

Formalin-Ethyl Acetate Sedimentation Concentration

1. Mix the specimen well.

2. Strain 5ml of the faecal suspension (more or less depending on its consistency)
through wetted cheesecloth-type gauze placed over a disposable paper funnel into a
15 ml conical centrifuge tube. (Conical paper cups with the tips cut off are
sufficient).
3. Add 0.85% saline or 10% formalin through the debris on the gauze to bring the
volume in the centrifuge tube to 15 ml. Distilled water may be used; however,
Blastocystis hominis may be deformed or destroyed.
4. Centrifuge at 500 × g for 10 minutes.
5. Decant supernatant. Add 10 ml of 10% formalin to the sediment and mix thoroughly
with wooden applicator sticks.
6. Add 4 ml of ethyl acetate, stopper the tube, and shake vigorously in an inverted
position for 30 seconds. Carefully remove the stopper.
7. Centrifuge at 500 × g for 10 minutes.
8. Free the plug of debris from the top of the tube by ringing the sides with an
applicator stick. Decant the top layers of supernatant.
9. Use a cotton-tipped applicator to remove debris from sides of the centrifuge tube.
10. Add several drops of 10% formalin to re-suspend the concentrated specimen.
Proceed with applicable testing.

Specimens preserved in PVA are mostly used for permanent staining with trichrome.
Prior to staining, they are processed as follows:

1. Insure that the specimen is well mixed.

2. Prepare a smear using 2 to 3 drops of the specimen depending on density.
3. Heat fix on slide warmer set at 60°C for 5 minutes or air dry completely at room
temperature.

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Slides may be trichrome stained or kept for several months in a protective slide tray or
box for future staining.

14.17 Specific staining reactions

1. Eosin 1% in saline at 370C: In saline preparation, motile parasites such as amoeba,

flagellates, larvae and ciliates can be detected and identified. Helminth eggs can be
identified. Cyst can also be detected. Cysts and amoebae (motile) are much easier to
detect in an eosin preparation. The eosin is useful as a negative background i.e. it
does not stain the living amoeba or cysts.

2. Iodine (equal volumes of Lugol’s iodine and 25% acetic acid). When allowed to flow
under the cover slip of a concentrated preparation of faeces it provides a graded
concentration of dye. It stains glycogen brown and emphasises nuclear chromatin in
amoebic cysts.

3. Burrow’s stain. When added to stool concentrations in equal volume it stains

chromatoid bodies of Entamoeba histolytica cysts deep blue after 24 hours.

4. Modified Field’s stain (Field stain A and B from Merk Ltd) Stain methanol fixed
smears for flagellates, Dientamoeba fragilis and Blastocysts hominis trophozoites.

5. Modified Ziehl-Neelsen stain for coccidia oocysts (Cryptosporidium and Isospora)

species.

6. Trichrome stain. Is recommended staining Entamoeba histolytica amoeba in tissue

(e.g. biopsies) and whenever a permanent stained faecal preparation is required to
study the nuclear detail of Entamoeba histolytica or other faecal protozoa.

7. Specific monoclonal antibody fluorescence reagents for the detection of antigens of

cryptosporidia and Giardia lamblia.

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E vermicularis Tinea saginata Trichuris trichura

A lumbricoides E histolytica Hook worm

G lamblia S mansoni T solium

S haematobium
Figure. 14.3

14.18 Urine

For the detection of Schistosoma haematobium.

14.19 Sample Collection

Collect 10-15mls of urine in a clean dry container and keep in the dark if unable to
examine immediately.

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14.20 Microscopic Examination

14.21 Report the Appearance of Urine

Whether clear, or bloody or cloudy etc. Centrifuge 10mls of urine at 1000g or allow
standing for one hour. Discard the supernatant fluid and transfer all the sediment to a
slide, cover with cover slip and view with 10x and then 40x objective. Count the number
of eggs present and report the number/10ml of urine.

Exercise 1: Observe from the sample given if you can identify any of the following
ova/cysts:

14.22 Blood

Blood borne parasites of medical importance belong to 2 major phyla:

i. Protozoan - Blood protozoa of major clinical significance include members of genera

Trypanosoma (T. brucei and T. cruzi); Leishmania (L. donovani, L. tropica and L.
braziliensis); Plasmodium (P. falciparum, P. ovale, P. malariae and P. vivax); Toxoplasma
gondii; and Babesia (B. microti).
ii. Helminths – Blood helminthes of major clinical significance include members of the
genera Schistosoma (S. haematobium, S. mansoni, S. japonicum, S. intercalatum) Filaria
(W. bancrofti, Loa loa, Mansonella spp)

14.23 Sample Collection

Capillary or anticoagulated venous blood can be used. Capillary blood is collected from
the lobe of the finger which is swabbed with 70% alcohol, pricked squeezed gently to
obtain a drop of blood. For microfilaria, it is important to collect blood at the right time
of the day considering periodicity.

14.24 Wet Blood Preparation

Collect a small drop of capillary or venous blood on a slide and cover with a cover
glass. Examine the entire slide with 40x objective for the presence of motile
trypanosomes.

14.25 Making Films

Three types of films can be made in the diagnosis of haemoparasites.

14.25.1 Fresh film preparation and examination

i. Collect a drop of blood in the centre of a cover slip

ii. Lower the cover slip face down on the centre of a clean slide. The resulting film
should be one cell thick and should extend to the edges of the cover slip.

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iii. Examine at once under the low power followed by the high dry objective if
necessary.

Note: Fresh preparations are very important especially in the diagnosis of moving
parasites like Microfilaria which may be seen moving actively amongst the red cell. It is
better seen under the high power.

14.25.2 Thick Film

i. Using a dry slide, collect a large drop of blood and with a circular movement
make a film of about 1cm in diameter evenly with needle or cover slip.
ii. Make the film quickly and do not stir; stirring may lead to fibrin formation and
promote auto-agglutination of the red cells in anaemic cases.
iii. Keep the film horizontal and protect from dust and flies while it dries. Do not fix.

NB: Thick films are very valuable in concentration of parasites such that even in light
infections blood parasites such as plasmodium can still be seen.

14.25.3 Thin Blood Film

i. Place a drop of blood near the end of a dry slide. Bring the edge of a clean
spreading slide back into the drop at an angle of about 300 and then slowly push
forward to give straight edges to the film.
ii. The angle of the slide, the rate of spreading and the size of the drop can so be
combined as to produce a film of more or less uniform thickness except for the
tails at the end.
iii. Keep the film horizontal and protect from dust and flies while it dries.

Figure. 14.4 Thin and thick blood films

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14.26 Staining

Fresh films preparations are, as a rule, NOT stained. The techniques in the use of stains
are described below and should be followed:

14.26.1 Leishmans (Thin films)

i. Run 0.5ml stain on a horizontal slide, film upwards, to fix the film. Leave for
about 60 seconds.
ii. Add 1.5ml of buffered (sterile) water; leave staining for about 9 minutes. For
deeper differentiation, stain for 15-20 minutes.
iii. Rinse off the staining mixture with water for a few seconds and clean the back of
the slide and place in a slanting position to dry.

14.26.2 Giemsa

14.26.2.1 Thin films

i. Fix in methyl alcohol for about 30 seconds.

ii. Place the slide film downwards over a staining plate or inserted into a staining jar,
and add a 10%dilution of the freshly prepared working solution using buffered
water.
iii. Allow to stain for 10-20 minutes, rinse in water and air dry.

14.26.2.2 Thick Film

i. Do not fix sample prior to staining

ii. Make a 1 in 10 dilution of stalk Giemsa stain,
iii. Position thick film slide uprightly in a staining jar. Stain for about 10minutes
iv. Gently rinse in water and allow to air-dry.

NB: A slightly alkaline stain (pH 7.2) is recommended as an acidic stain may fail to
show the parasites.

14.26.3 Fields Stain

Field stain consists of 2 staining solution usually referred to as STAIN A and STAIN B.
Stain A contains a polychrome methylene blue while stain B contains eosin.

14.26.3.1 Thin Film

1. Place the slide on a staining rack. Make a 1 in 5 dilution of field stain B and cover
the film with 0.5mls of the diluted stain.
2. Add equal volume of field’s stain A immediately, mix and leave to stain for 1
minute.

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3. Rinse the stained slide with clean water. Wipe the back of slide and place in a
draining rack for the film to air-dry.

14.26.3.2 Thick Film

i. Gently dip the dried but unfixed thick film in a jar containing Field stain A for 1 or 2
seconds.
ii. Remove from solution A and immediately rinse in clean water.
iii. Dip in Stain B for 3 seconds.
iv Rinse in clean water for a few seconds.
v Place in a vertical position to dry.

When a large number of thick films require staining, Field's stain is preferred because it
is very fast. The solutions are kept in covered staining jars.

14.27 Blood Specimen

14.28 Preparing Blood films

If you are using venous blood, blood films should be prepared as soon as possible after
collection (delay can result in changes in parasite morphology and staining
characteristics).

14.28.1 Thick films

Thick films consist of a thick layer of dehaemoglobinized (lysed) red blood cells (RBCs).
The blood elements (including parasites, if any) are more concentrated (app. 30×) than
in an equal area of a thin film. Thus, thick films allow a more efficient detection of
parasites (increased sensitivity). However, they do not permit an optimal review of
parasite morphology. For example, they are often not adequate for species
identification of malaria parasites: if the thick film is positive for malaria parasites, the
thin film should be used for species identification.

Prepare at least 2 films per patient!

1. Place a small drop of blood in the centre of the pre-cleaned, labelled slide.
2. Using the corner of another slide or an applicator stick spread the drop in a circular
pattern until it is the size of a dime (1.5 cm2).
3. A thick film of proper density is one which, if placed (wet) over newsprint, allows
you to barely read the words.
4. Lay the slides flat and allow the films to dry thoroughly (protect from dust and
insects!). Insufficiently dried films (and/or films that are too thick) can detach from
the slides during staining. The risk is increased in films made with anticoagulated
blood. At room temperature, drying can take several hours; 30 minutes is the

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minimum; in the latter case, handle the film very delicately during staining. You
can accelerate the drying by using a fan or hair dryer. Protect thick films from hot
environments to prevent heat-fixing the film.
5. Do not fix thick film with methanol or heat. If there will be a delay in staining
smears, dip the thick smear briefly in water to haemolyse the RBCs.

14.28.2 Thin films

Thin films consist of blood spread in a layer such that the thickness decreases
progressively toward the feathered edge. In the feathered edge, the cells should be in a
monolayer, not touching one another.

Prepare at least 2 films per patient!

1. Place a small drop of blood on the pre-cleaned, labelled slide, near its frosted end.
2. Bring another slide at a 30-45° angle up to the drop, allowing the drop to spread
along the contact line of the 2 slides.
3. Quickly push the upper (spreader) slide toward the unfrosted end of the lower slide.
4. Make sure that the films have a good feathered edge. This is achieved by using the
correct amount of blood and spreading technique.
5. Allow the thin smears to dry. (They dry much faster than the thick films, and are less
subject to detachment because they will be fixed.)
6. Fix the films by dipping them in absolute methanol.

Note: Under field conditions, where slides are scarce, both a thick and thin films can be
prepared on the same slide. This works adequately if one makes sure that of the two
films, only the thin film is fixed.

14.29 Staining of Blood films with Giemsa

Stock Giemsa liquid solution is diluted 1: 10 with buffered distilled water 7.2 for thin
films. For thick films on the other hand, a dilution of 1:20 is used but a dilution of upto
1:50 may be used.

14.29.1 Thin films

1. Fix thin film in methanol for 2 minutes

2. Allow to dry
3. Flood the slide with 1: 10 dilution of Giemsa (i.e. 1 part of Giemsa to 9 parts of
buffered distilled water 7.2)
4. Allow to stain for 30 – 45 minutes
5. Flood off (don’t tip off) with buffered distilled water 7.2
6. Drain and allow to dry
7. Examine microscopically using x10 and x100 objective.

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14.29.2 Thick film

1. Flood the slide with 1: 20 dilution of Giemsa (i.e. 1 part of Giemsa to 19 parts of
buffered distilled water 7.2)
2. Allow to stain for 30 – 45 minutes
3. Flood off with buffered distilled water 7.2
4. Drain and allow to dry
5. Examine microscopically using x10 and x100 objective

NB: For thick and thin films on same slide staining may be carried out with 1: 10
dilution of the Giemsa stock.

14.30 Special Procedures for Detecting Microfilariae

14.31 Blood microfilariae

i. Capillary (finger prick) blood: Since microfilariae concentrate in the peripheral

capillaries, thick and thin films prepared from finger prick blood are recommended.
ii. Anticoagulated (EDTA) venous blood (1 ml) should be concentrated by one of the
following methods:

14.31.1 Centrifugation (Knott’s technique)

o Prepare 2% formaldehyde (2 ml of 37% formaldehyde + 98 ml H2O).

o Mix 9 ml of this 2% formaldehyde with 1 ml of patient’s venous blood.
Centrifuge at 500 × g for 10 minutes; discard supernatant. Sediment is composed of
WBCs and microfilariae (if present).
o Examine as temporary wet mounts.
o Prepare thick and thin smears; allow to dry; dip in absolute methanol before

Giemsa staining to enhance staining of microfilariae.

14.31.2 Filtration

a. Place Millipore® or Nucleopore® membrane filter (5 µm pore) in filter holder with

syringe attachment.
b. Mix 1 ml of venous blood (in EDTA) with 10 ml of 10% Teepol® 610 (Shell Co.);
allow to stand for several minutes to allow lysis; transfer to a 10 ml Luer-Loc®
syringe; attach the filter apparatus.
c. Force the solution through the 5 µm pore filter, followed by several syringes of
water to wash out the remaining blood, then 1 or 2 syringes full of air to clear excess
fluid.

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d. Prepare a temporary wet mount by removing the filter and placing it on a glass
slide, adding a drop of stain or dye and a coverslip.
e. For permanent preparations, pass 2 to 3 ml of methanol through the filter while it is
still in the holder; remove filter and dry it on a glass slide; then stain it with Giemsa
stain, horizontally (so that the filter does not wash off the slide); coverslip filter
before examining.

14.32 Microscopic Examination

Examine your slide preparations under the microscope. Examine the thick film first,
using an oil immersion or high dry lens to determine if parasites are present. Be aware
of the patient's platelet and leucocyte counts. Malaria is usually associated with a
normal or reduced leucocyte numbers.

A leucocytosis is only found in terminal cases. Platelet numbers are moderately or

markedly reduced in some 80% of patients with malaria. Parasites may appear distorted
if the patient has been treated or has had inadequate or ineffective prophylaxis.

Study demonstrations and the picture below showing examples of haemoparasites. Pay
attention to the Morphology of each parasite. Mixed infections can be seen.

View with 40x and oil immersion objective and report the presence of plasmodium
trophozoites or gametocytes or chromatin bodies or trypanosomes and microfilaria etc.

Schizoint and merozoites in thin films Ring form & Microfilaria in

gametocytes blood

Ring forms seen in stained thick film

Figure. 14.5

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Figure. 14.6

Figure. 14.7

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Figure. 14.8

Adapted from: © RPH Laboratory Medicine 1998-2002. Webbed by Bill McConnell

Figure. 14.9

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14.33 Assessment of Malaria Parasite density

14.33.1 Thick Blood Film

14.33.1.1 Double counter technique

2 tally counters are used. All parasite species and forms including gametocytes are
counted as they are encountered along with leucocytes up to 200 leucocytes. If less than
10 parasites are encountered at the time 200 leucocytes are counted, the search for
parasites is continued until 500 leucocytes are encountered.

Parasite count is calculated using the formula (No. of parasites/ตl) :

No. of Parasites
-----------------------------x WBC count (8000/µl)
No. of WBCs counted

Or if 200 leukocytes are counted, then:

No. of parasites counted x 40 = No. of parasites/µl

If 500 WBCs were counted then:

No. of parasites counted x 16 = No. of parasites/µl

Average of 8000/µl has been taken as standard WBC count.

NB: WBC: White blood count

14.33.1.2 Earle and Perez method

The number of asexual parasites in a known volume of blood (usually 5µl) spread as a
thick film are counted in toto; this is quite cumbersome and used only in research.

The "plus system”

This is the most commonly used techniques but least accurate as variation in the
thickness of the film results in false variation in parasite count.

+ = 1–10 per 100 thick fields.

++ = 11-100 per 100 thick fields.
+++ = 1–10 per thick field.
++++ = >10 per thick field.

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14.33.2 Thin Blood Film

14.33.2.1 Percentage of parasitaemia

The number of infected red cells (and not number of parasites) in 1000 RBCs is
converted to percentage. Following careful scan the total number of red cells and the
number of parasitised red cells are tabulated separately.

Calculate percentage parasitaemia as follows:

No. of parasitised red cells
-------------------------------------- x 100
No. of red cells counted,

A value of 2% and above are of clinical concern.

NB: RBC: Red blood cells

14.34 Skin Snip

For Onchocerca volvulus, streptocerca etc. it is collected using a sterile needle and scalpel
blade or razor blade. Immerse skin snip in a tube containing 1ml of physiological saline
for 4 hours. Then remove snip and place on a slide and cover with a cover slip. Also
centrifuge the tube and transfer sediment to a slide and view with 10x and 40x objective
for microfilaria.

14.35 Other Clinical Specimens

Other specimens collected for parasitological diagnosis include CSF, spleen aspirates,
liver aspirates, bone marrow aspirates, muscle tissue, sputum.

Other parasitological tests include serology, immunoassay techniques etc.

Xenodiagnosis used to diagnose Chaga’s disease (T. cruzi). Triatomid bugs are allowed to
feed on patient or on a sample of the patients blood, then kept (30-60 days) and
examined for parasites. Mosquitoes have similarly been used to diagnose filarial
worms.

14.36 Isolation of Leishmania Organisms

The diagnosis of leishmaniasis is made by microscopic identification of the non-motile,

intracellular form (amastigote) in stained sections from lesions, and by culture of the
motile, extracellular form (promastigote) on suitable media.

14.37 Sputum Specimens

Microscopic examination of sputum is used in identifying Paragonimus westermani eggs,

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Strongyloides stercoralis larvae, Ascaris lumbricoides larvae, hookworm larvae, and rarely
Entamoeba histolytica. Sputum should be obtained from the lower respiratory passages
rather than a sample consisting mainly of saliva. Sputum specimens should be collected
first thing in the morning.

A sputum sample can be examined in several ways:

1. The unfixed specimen may be centrifuged and then the sediment examined as a
direct wet mount.
2. If the sputum is too viscous, an equal volume of 3% sodium hydroxide may be
added, then centrifuge, and examine the sediment.
3. The specimen may be preserved in 10% formalin and a formalin-ethyl acetate
concentration procedure may be completed and the sediment examined using either
a wet mount or a stained preparation.
4. The specimen may also be preserved in PVA if protozoa are suspected and stained
with trichrome stain.

14.38 Induced Sputum and Bronchoalveolar Lavage (Bal) For Pneumocystis jirovecii

Sputum, induced sputum, and bronchoalveolar lavage (BAL) material are commonly
used for diagnosing Pneumocystis jirovecii (previously classified as Pneumocystis carinii)
infections. To examine these specimens for cysts of P. jiroveci, prepare smears of the
sediment and examine after staining with the recommended procedure (Giemsa or
methenamine silver stain).

14.39 Aspirates

Duodenal aspirates may be useful in demonstrating Giardia lamblia or Strongyloides

stercoralis larvae. Material collected following intubations through the nose and stomach
into the upper small intestine may be submitted to the laboratory. Centrifuge the
specimen at 500 × g for 2 to 3 minutes and examine the wet mount. An unfixed
specimen can be examined immediately or if the specimen cannot be examined within 1
to 2 hours after collection, it should be preserved in 10% formalin.

Jejunal fluid can be sampled using the string test (Enterotest capsule, Hedeco) for
Giardia trophozoites and Strongyloides larvae.

Sigmoidoscopy material and abscesses of the liver and lung may demonstrate amoebic
trophozoites. Material from the mucosal surface or from visible lesions should be
aspirated. This material can be examined immediately in a 0.85% saline wet mount
preparation (or part of this material could be placed in formalin) or can be fixed in PVA.
Once fixed in PVA, the material can be stained using trichrome stain and examined for
trophozoites of Entamoeba histolytica.

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Lymph node material, bone marrow, and spleen may be examined for the presence of
motile trophozoites of Trypanosoma brucei gambiense or Trypanosoma brucei rhodesiense.
For Leishmania donovani infections, material obtained by needle aspiration from bone
marrow or spleen can be used to demonstrate amastigote stages. Smears can then be
prepared by fixing in methanol and staining with Giemsa stain.

Skin ulcers may demonstrate the amastigote stages in cutaneous and mucocutaneous
leishmaniasis. Permanent stained smears made with these specimens by fixing in
methanol and staining with Giemsa stain.

14.40 Other Specimens

14.41 Vaginal Swabs for Detection and Susceptibility of Trichomonas

Demonstration of Trichomonas vaginalis trophozoites is usually done by preparing wet

mounts made from vaginal swabs or scrapings. If the specimen cannot be examined
immediately, it should be preserved in PVA and stained smears examined later.

Susceptibility Testing: T. vaginalis is usually highly susceptible to metronidazole.

However, cases of resistant T. vaginalis have been reported and may be increasing.

14.42 Cellulose Tape or Swube Tube Procedure for Demonstration of Pinworm Eggs

The most reliable and widely used technique for demonstrating pinworm eggs
(Enterobius vermicularis) is the cellulose tape or swube tube procedure. The adhesive
part of the swube tube or tape is applied to the peri-anal area first thing in the morning.
Specimens should be collected on three consecutive mornings prior to bathing. If an
infection is present, eggs and sometimes adult worms of Enterobius vermicularis will be
present on the tape and can be seen under the microscope.

14.43 Urine Specimens

The definitive diagnosis of urinary schistosomiasis (Schistosoma haematobium) is

established by demonstration of S. haematobium eggs in urine. An increased number of
eggs are shed in the urine around midday, so an optimum urine specimen for diagnosis
should be collected at noon. The specimen should be immediately centrifuged at 400 ×
g and the sediment examined by wet mount.

Trichomonas vaginalis motile trophozoites may also be found in the urine, especially in
infected male patients. To look for the presence of trophozoites, the urine specimen
should be centrifuged at 400 × g, the sediment mixed with a drop or two of saline, and
examined by wet mount. Temporary stains, such as methylene blue or malachite green,
are also helpful.

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14.44 Biochemical Diagnosis

Based on isoenzyme patterns, isoenzymes are variants of the same enzyme, they have
same biochemical function, but because of mutations in non-essential parts of the
molecule have slightly different isoelectric points and so migrate differently on gels.
This combined with enzymes specific stains allows isoenzyme patterns to be visualised.
Isoenzyme patterns are under genetic control and parasites with different banding
patterns are genetically distinct. This enables species and sub-species to be
distinguished.

14.45 Advantages of isoenzyme diagnosis

 Simple technique can run many samples at the same time.

 Large number of typing enzymes available, so diagnosis is not based on a single
pattern

14.46 Disadvantages of isoenzyme diagnosis

 Contamination by host tissue

 Relative expensive equipment
 Enzymes are labile – get false negatives.

14.47 Immunodiagnosis

14.47.1 Antibody Based Methods

Serological methods are based on identifying specific antibodies in the blood of infected
individuals. An early method was to inject a small amount of crude parasite extract
under the skin and see if there was an inflammatory response (e.g. Casone hydatid test).
The advantage of serological methods is that they can be developed into field based
tests for mass screening; the disadvantage is that they do not distinguish between
current and past infections.

Antibody diagnosis is routinely used in sleeping sickness surveillance. Anti-

trypanosomal IgM in the blood is detected by a simple, cheap, rapid card agglutination
test called CATT (card agglutination test for trypanosomiasis). The regent is freeze
dried trypanosomes which have been stained blue on a white plastic card. A drop of
blood is added and if antibodies are present the trypanosomes clump.

The Fatala kit for diagnosing T. cruzi uses indirect agglutination. Red blood cells are
coated with two recombinant parasite proteins and again a positive result is shown by
agglutination. Another test for T. cruzi uses three recombinant proteins.

Other tests also in use include

ELISA (enzyme linked immunosorbent assay) is used to diagnose Onchocerciasis,

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Amoebiasis, Schistosomiasis, Giardia, Hydatid disease, Strongyloides, Toxocariasis,

toxoplasmosis.

IFAT (Immunofluorescence): Amoeba (E. histolytica, Naegleria, Acanthamoeba),

cysticercosis, fascioliasis, filariasis, Giardiasis, Leishmaniasis, malaria, pneumocystis,
Trichiniasis.

CIEP; counter-current immunoelectrophoresis. Amoebiasis

CAP (cellulose acetate precipitation) Amoebiasis

DAT (Direct agglutination test) for Leishmaniasis.

There are two major disadvantages to antibody based methods:

 Cross reactions resulting in false positives

 High cost of development

14.47.2 Antigen based tests

Instead of looking for antibodies you can instead look for parasite antigens. The
advantage of antigens is that hey are only present when there is an active infection, the
disadvantage is low sensitivity. Western blotting is the most straightforward way of
detecting parasite antigens, the proteins in host serum are separated by gel
electrophoresis, the proteins are transferred to a membrane which is then probed with
suitably labelled specific anti-parasite antibody. Another way is to separate parasite
proteins on a gel, transfer to a membrane and probe with host sera. This approach is
often used to try and identify immunogenic proteins, but is not feasible for routine
screening. Sandwich ELISA is another method of detecting antigens, but again is not
suitable for mass screening.

Antigen capture assays have now been developed into a 'dipstick format' suitable for
self diagnosis or mass screening.

14.48 DNA Based Tests

DNA diagnosis is based either on Southern blotting or Polymerase chain reaction

technology (PCR). Basically both methods are looking for patterns which are somehow
unique to the parasite.

In Southern blotting DNA is extracted from isolated cysts or eggs, cut by specific
restriction enzymes and separated by gel-electrophoresis. The DNA probe hybridises
with the complementary sequence on the gel. Originally radioactive phosphorus was
used to label the probe, now more often a fluorescent label is used. The key is finding a

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specific probe. The same thing can be done with RNA Northern Blot), mRNA is
extracted, run on gel (no need to digest) and hybridise with a specific RNA probe.

The main problems are first getting specific probes (to avoid cross reactions) and
secondly getting enough DNA or RNA. The polymerase chain reaction provides rapid
amplification of target DNA, so low levels of parasitaemia can be detected. It is possible
to amplify malaria sequences from whole blood; the parasite does not have to be
isolated.

Again the key is to have a DNA sequence that is unique to the parasite. Main
disadvantages are cost, the requirement for fairly sophisticated laboratory facilities and
cross reactions which give false positives. Finding a unique DNA sequence can be
difficult and primers developed in the lab, in isolation, often fail in practice because
they cross react with other things in the extracts. For example a battery of primers has
been developed to distinguish between the different strains of Cryptosporidium, in the
lab with pure cultures they work beautifully. But when they are used with sewage
samples you get cross reactions with fungi. Again in real extracts you often get
compounds which inhibit the key polymerase reaction .PCR also does not distinguish
between live and dead parasites. A modification of PCR - Reverse Transcriptase PCR
(RT-PCR) measures mRNA and this is only found in living organisms. It involves the
synthesis of complimentary DNS (cDNA) from mRNA, then PCR with specific primers.

If you have not got specific primers for your parasite you can try Random
Amplification of Polymorphic DNA (RAPD - PCR). A randomly designed primer is
taken and used in the PCR reaction. The random primer will (hopefully) attach at
several different points in the parasite DNA and so will amplify a number of different
pieces of DNA of differing lengths. So, rather like RFLP techniques you get a pattern of
different length fragments and with luck the fragment pattern will be characteristic of
the parasite species.

A real-time PCR test is available for confirmation of amoebiasis that detects E. histolytica
at the species level. If molecular diagnosis is necessary, specimens should be
unpreserved and kept refrigerated or frozen.

14.49 Culture

Most clinical laboratories do not provide culture techniques for the diagnosis of
parasitic organisms.

14.50 Malaria Culture

Plasmodium falciparum can now be maintained in continuous culture in human

erythrocytes incubated at 38 degrees C in RPMI 1640 medium with human serum under
an atmosphere with 7 percent carbon dioxide and low oxygen (1 or 5 percent). The

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original parasite material, derived from an infected Aotus trivirgatus monkey, was
diluted more than 100 million times by the addition of human erythrocytes at 3- or 4-
day intervals. The parasites continued to reproduce in their normal asexual cycle of
approximately 48 hours but were no longer highly synchronous. They have remained
infective to Aotus.

14.51 Leishmanial culture

Leishmanial culture is also available on needle aspirates or punch biopsy lesions. CDC
can provide culture medium (typically Novy-MacNeal-Nicolle (NNN) medium). Keep
this refrigerated until it is used (stable for 2-4 weeks) and bring it to room temperature
right before inoculation.

The needle aspirate should be obtained by first drawing approximately 0.1 ml of

preservative-free sterile 0.9% NaCl into a 1.0 to 3.0ml syringe. Insert the needle (23-27
gauges) through intact skin into the dermis of the active border. Move the needle back
and forth repeatedly under the skin simultaneously rotating the syringe and applying
gentle suction until pink-tinged tissue juice is noted in the hub of the syringe. After the
aspirate is obtained, discharge into the leishmanial culture medium.

For biopsy specimens, obtain 1 or 2 full thickness punch specimens at the active border
of the lesion. Use 1 specimen for culture and the other for impression smears. For
culture, place the punch biopsy into the culture medium. The culture tubes should be
kept at room temperature prior to and during shipment. The cultures will be kept 4
weeks at CDC and if the culture becomes positive and grows, isoenzyme studies will be
performed for species identification.

14.52 Trypanosomiasis culture

The NNN medium is also used for the cultivation of trypanosomiasis.

14.53 Entamoeba histolytica

Most intestinal amoeba do not culture well as E. histolytica. Robinsons medium may
provide a higher yield of amoebae as trophozoites than microscopy alone.

14.54 Strongyloides stercoralis/ hookworms

Routine culture of faeces for Strongyloides should be made on all clinically suggestive
cases when microscopy is negative. The Charcoal culture method has been used;

 Comminute 10g of faeces in tap water until it forms a thick suspension.

 Mix in an equal volume of granulated hardwood charcoal
 Place a piece of No. 1 whatman filter paper into a Petri dish and moisten with water.

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 Transfer the faecal/charcoal mixture onto the filter paper and add water until it
glistens. Seal the dish to prevent evaporation.
 Keep the sealed dish in the dark at room temperature for 5-7days. Check daily and
replenish with water, if necessary, to keep the culture moist.
 After 5 days pipette 2-3 ml water around the filter paper.

The transformation of Strongyloides and hookworm larvae from rhabditiform to

infective filariform occurs and the larvae will migrate into the water. Inspect for a
further 2 days, using an inversion or dissecting microscope, for active filariform larvae.
If none is seen, formolize the culture and pass it through a wire sieve. Concentrate the
filtrate by the formol-ether technique and the deposit may reveal larvae.

14.55 Animal Innoculation

Most laboratories have neither the time nor the facilities for animal care to provide
animal inoculation procedures for the diagnosis of parasitic infections. Host specificity
for many parasites also limits the types of laboratory animals available for these
procedures.

Hamsters are used for recovery of leishmanial organisms. After intraperitoneal or

intratesticular inoculation, infection is allowed to develop and aspirates from spleen
and testis are stained and examined for intracellular organisms.

Mice are used for the isolation of Toxoplasma gondii. A mouse is inoculated through the
peritoneum; it develops a fulminating infection that leads to death within a few days.
Organisms are easily recovered from the ascetic fluid stained and examined.

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Chapter 15
Laboratory Techniques in Mycology
Iheanacho Nwadioha
15.1 Specimens

Include skin, nail, hair, CSF, Pleural aspirates, corneal scrapings, sputum, pus, bone
marrow aspirates, blood cultures etc.

Methods of specimen collection: Scrapings of scale is best taken from the leading edge
of the rash after the skin has been cleaned with alcohol. Skin stripped off with adhesive
tape can be used; it is then stuck on a glass slide. Hair is better pulled out from the
roots. Brushings can be collected from an area of scaly scalp. Nail clippings, or skin
scraped from under a nail. Skin biopsy can be used. Moist swab can be taken from a
mucosal surface (inside the mouth or vagina). Samples are either transported in a sterile
container or a white paper envelope.

15.2 Direct Microscopic Examination

15.3 Wet Preparation

15.4 Unstained Wet Mount

e.g vagina swab sometimes using saline in preparation.

15.5 KOH Preparation

Place KOH on a slide and add small pieces of specimen to it. Cover with a cover slip
and allow to stand for 5-30 minutes in a damp petri dish to prevent it from drying out
while waiting for complete clearing. Then examine with 10x or 40x objective for
presence of hyphae and conidia.

15.6 Staining: Calcoflour White – KOH Stain

To a drop of calcoflour white and a drop of 10% KOH glycerin, add a portion of the
clinical specimen and allow for about 5minutes. Examine using 10x and 40x fluorescent
microscope at 400-500nm.

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15.7 Gram Stain

As described in previous chapters.

15.8 Indian Ink

Use in detection of yeasts of Cryptococcus neoformans in Cerebro Spinal fluid (CSF).

Centrifuge CSF for 5-10minutes and place the sediment on a slide containing one drop
of Indian ink. Cover with a cover slip and view with 10x and 40x magnifications. Look
for oval cells surrounded by a large unstained capsule.

15.9 Germ Tube Test

Described in chapter 10.
15.10 Culture

Fungi are grown on culture media like Sabourand Dextrose Agar, for easy identification
through their colonial morphology, and examination of mycelia for presence of septate
or nonseptate hyphae and presence or absence or morphology of microconidia,
macroconidia etc. Media may or may not contain cycloheximine and antibiotics to
inhibit the growth of contaminating mold and bacteria. Other culture media include;

 Brain heart infusion agar,

 Corn meal agar,
 Potato flake agar,
 Yeast extract phosphate agar,
 Niger seed agar,
 Urea agar etc.
Incubation can be at room temperature or 35-370C

15.11 Lactophenol Cotton Blue Mount

This is has been used for examination of fungal element (mycelia) after culture. To a
drop of lactophenol cotton blue, pick a little mycelia growth and tease on a drop of LPC,
cover with a cover slip and view with 10x and 40x objective. Examine for hyphae, macro
and microconidia.

15.12 Other Identification Techniques

Include biochemical tests like urease tests, sugar fermentation tests etc., immunoassays,
molecular diagnosis like PCR, In-situ hybridization with nucleic acid probes etc.

15.13 Antifungal Susceptibility Testing

Using antifungal drugs like fluconazole, voriconazole; griseofulvin; caspofungins, micafungin;

amphotercin B; flucytosine etc.

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Chapter 16
Laboratory Techniques in Medical Virology
Odimayo Simidele
16.1 Specimen Collection, Transport and Storage

A wide range of specimens can be analysed in the virology laboratory and they include,
throat swab and aspirates, bronchial washings, sputum, stool and rectal swab, urine,
CSF, bone marrow, blood, body fluids, skin lesions and tissues. All specimens should be
placed in an ice pack and sent to the laboratory IMMEDIATELY. Swabs should be
placed in a viral transport medium which contains serum, gelatin and albumin to
stabilize the medium and antibiotics to prevent the overgrowth of bacteria. Viral
transport media include Stuart’s medium, Amies’ transport media, eagle tissue culture
media etc. Samples that cannot be processed immediately are refrigerated but should be
analysed within 24 hours. Otherwise samples can be stored frozen.

Successful laboratory diagnosis of viral infections requires:

 Obtaining the appropriate selection of specimens

 Timing of specimen collection
 Transport of the specimens
 Effective and timely processing of specimen

Regardless of chosen method of assay, maintenance of specimen quality throughout

testing is essential for diagnosing viral infections.

16.1.1 Specimen collection

For an active disease process of viral etiology, diagnosis usually requires collection of a
specimen from the affected organ since this is usually where the highest level of viral
replication occurs.

Clinical symptoms often help identify the target organ(s) involved and help determine
the appropriate specimen(s) to collect.

An understanding of the viral pathogenesis is also helpful in determining the most

appropriate specimen(s) for testing.

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Examples:

Blood for most viruses except Astro, Calici, Rota, Influenza, Rhino, Mumps, RSV,
Papilloma, Polyoma and Pox viruses

Bone marrow for CMV, HHV-6, Parvo B19, VZ Viruses

Tissue for most viruses

CSF for Adeno, Arbo, Arena, CMV, EBV, HSV, Mumps, Polyoma, Rabies, Retro, VZV,
HHV-6 and Enteroviruses.

Feces for Adeno, Calici, Rota, Astro, Corona, CMV, Entero, HAV, HDV, HEV, HSV &
Rubella (both in neonates)

Respiratory specimens (Throat swab, nasal swab, nasal washings, Nasopharyngeal

aspirate, Bronchoalveolar lavage) for Influenza, Paraifluenza, Rhino, RSV, Rubella,
Measles, Human metapneumonea, Corona, CMV, Arena, Adeno, filo, HSV,
retroviruses.

Skin specimens for CMV, HSV, HHV-8, Pox, Rabies and VZViruses.

Genital specimens for Adeno, CMV, HSV, papiloma retroviruses.

Saliva for CMV, HHV-8, Mumps, Retroviruses

Amniotic fluid for CMV, HSV, Parvo B19, Rubella viruses

Eye specimens (Conjunctiva fluids/scrapings, cornea fluid/scraping, aqueous and

vitreous fluids) for Adeno, CMV, HSV, VZV, Enteroviruses.

Urine for Rubella, Polyoma, Mumps, measles, Filo, Entero, CMV, Arena, Adenoviruses.

Pericardial fluid for Enteroviruses

16.1.2 Timing of Collection

This is very important in relation to symptoms in the detection of viral pathogens,

because specimens with high levels of virions, viral antigens or nucleic acids improve a
laboratory’s ability to make a diagnosis.

It is important to remember that viral titres decrease after the acute phase of an illness.
For many virus infections viral shedding begins before the onset of symptoms and
disappears when symptoms resolve. Duration of shedding depends on the type of virus

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and the organ or system involved. The immunocompetence of the patient also affects
the period and duration of virus shedding.

On the average, for optimal recovery of most viruses, specimens should be collected
within one to three (1-3) days of onset of symptoms.

The test method to be used will also guide on specimen collection.

Other factors to consider include:

The swab type/composition

The container used for collection

The type of anticoagulant e.g Heparin inhibits PCR and infectivity of HIV-1.

Use of Viral Transport Medium depends on the specimen source. VTM can be used for
most respiratory specimens except nasal washings and broncho alveolar lavage), most
swabs and tissues to prevent drying; but should generally not be used for sterile
samples e.g. CSF.

Other things important include information on the age, sex, patient history, immune
status, season of the year etc. all these information helps the laboratory chose the most
suitable assay to use thereby improving diagnosis or detection.

The patient’s data, specific site of collection and specific diagnositic tests required
should all be clearly filled out in the request form.

16.1.3 Transport Conditions

Transportation of non-blood specimens should be maintained at 40C with wet ice/cold

packs provided the transit time is less than 1hr.

VTM should be used if appropriate for the type of specimen collected.

Avoid freezing of specimens however if delay of more than 24hr is anticipated, use dry
ice ( - 600C). Freezing affects the yield of RSV, CMV and VZV.

Components of VTM usually consists of the following:

 Buffer to control the pH

 Substances to maintain an appropriate osmotic environment
 Proteins to stabilize the virus
 Antibiotics to prevent the growth of contaminants

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16.1.4 Processing of Specimens

Safety is of paramount importance. Standard precautions should always be

remembered and ideally processing should be done using a biosafety cabinet suitable
for the BSL of the organisms concerned, though most are BSL-2 organisms.

A BSC does 3 things

1. It protects the lab personnel from laboratory acquired infection

2. It prevents cross-contamination between samples
3. It protects specimen from environmental contamination

16.2 Specimen Processing

Specimen processing should be done with a high precautionary sense. Biological safety
cabinet are often needed. Processing may be done by microscopy, Culturing in cell
lines, antigen detection or molecular detection and quantitation.

16.3 Virus Detection Methods

16.4 Cytology and Histology

This involves examination of tissues and cells for viral inclusion bodies and cytopathic
effects which are visible under light microscopy. Smears are made and stained by
Giemsa, papanicoula or haematoxylin and eosin stains (H&E).

16.5 Electron Microscopy (EM)

Electron Microscopy (EM); used mainly for viruses that do not grow readily in cell
cultures. It is labour intensive and of low sensitivity. Immune EM involves reacting
specific antibody to sample suspension to form bound aggregates that are detected
more easily than single viral cells.

Using electron microscope, stool specimen can be prepared and stained negatively for
electron microscopy using phosphotungstic acid. Electron Microscopes can be used to
examine objects on a very fine scale. Such examination can yield information about the
topography, morphology, composition and crystallography of the virus.

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Figure. 16.1

16.6 Immunodiagnosis (Antigen-antibody detection)

These include complement fixation test, neutralization test, precipitation tests, ELISA,
immunofluorescent techniques, e.t.c.

16.6.1 Neutralization Test

Loss of infections properties of a virus due to a reaction with specific antibody, e .g the
CPE of virus can be neutralized with specific immune sera; haemagglutination can be
inhibited (haemagglutination inhibition); neutralization of haemabsorption;
neutralization of development of CPE, neutralization of disease in animals.

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16.6.2 Haemagglutination

May be done using red blood cells of different sources, e. g human, chick, guinea-pig at
various temperatures. This technique is especially useful with myxoviruses, arboviruses
and poxviruses.

16.6.3 Complement Fixation

May be specific as with the myxoviruses, or there may be group specificity as with the
adenoviruses.

16.6.4 Precipitation Test

Is rarely used since a high concentration of virus particles is required.

16.6.5 Immunofluorescent antibody tests

Fluorescent dyes (Fluorochromes) illuminated by ultra-violet (UV) light are used to

show the specific combination of an antigen with its antibody. The antigen antibody
complexes are seen fluorescing against a dark background.

16.6.6 Gel diffusion

Antigen and antibody diffuse towards each other and where they meet in optimal
proportion a visible line of precipitation forms. The thickness of the line of precipitation
is a semi quantitative measure of the amounts of antigen and antibody that combine.

16.6.7 Radial haemolysis

Specific antibody is incorporated into the agar gel and wells are cut to contain the
antigen, which diffuses radially. A ring of precipitation forms round a well that
contains the corresponding antigen. The techniques is used mainly to detect and
measure immunoglobulins in serum and other specimens for example the detection of
viral diagnosis complement are added to the agar gel and incubated at 37C.

16.6.8 ELISA

As its name suggest, the enzyme linked immunosorbent assay uses an enzyme system
to show the specific combination or an antigen with its antibody. The enzyme system
consist of an enzyme, which is labelled or linked, to a specific antibody or antigen, a
substrate which is added after the antigen-antibody reaction. The substrate is acted on
(usually hydrolysed) by the enzyme attached to the antigen-antibody complexes, to
give a colour change. The intensity of the colour gives an indication of the amount of
bound antigen or antibody.

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16.6.9 Complement fixation Test

The CFT is a technique that has been used over many years to detect and quantify
antibody that does not agglutinate or precipitate when reacted with its antigen but can
be demonstrated by its use, or fixation of complement.

16.6.10 Haemagglutination

Many viruses possess ability to adhere on to the red blood cells of appropriate animal
species. Attachment is the result of interaction between the virus particles and the red
cells, and takes place at specific receptor sites on the cell surface. In a suspension
containing a large excess of virus particles, the cell agglutinate and when they are
allowed to settle, a rounded characteristic agglutination pattern is produced.

Antigen Detection; immunofluorescence, ELISA e.t.c.

16.7 Molecular Biology Techniques

Viral nucleic acids are detected using specific nucleic acid probes labelled with
chromogenic, fluorescent or radiologic tags. They can be amplified with the PCR for
easy identification and quantification. Identification can be done on amplicons or
directly on specimen by electropherotyping.

16.7.1 Probing

There are quite a number of oligonucleotide probes for some viruses including CMV,
measles, rotaviruses, yellow fever, Rabies, etc. These probes detect presence of
homologous viral genome in specimens collected. The results can be made available as
soon as possible.

16.7.2 Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) can be used to amplify the viral genome in any
specimen suspected of viral particles. The technique is rapid as results are made
available immediately (real time PCR) or using electropherotyping or blotting after few
hours of sample processing.

16.7.3 Electropherotyping

This is molecular biology technique used to analyse viruses based on the molecular
weight and charge on the nucleic acid of the viral genome.

16.8 Viral Culture

This can be done by eggs, or animal inoculation, orby cell/tissue cultures. In viral
culture, living cells/tissues serve as media for the growth of viruses. Tissue cultures can

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be primary, secondary or continuous cell lines. Single layer cultures are derived from
disaggregation of cells in animal tissue using enzymes like trypsin or chelating agents
like versene. Most laboratories depend on cell culture only. There are 3 types of cell
cultures:

Primary cells - e.g. Monkey Kidney. These are essentially normal cells obtained from
freshly killed adult animals. You can only passage the cells once or twice. You can
passage

Semi-continuous cells like Human embryonic kidney and skin fibroblasts over limited
number of times. However, ccontinuous cell lines passage can be done indefinitely.

Cell cultures require two types of media; growth and maintenance media. Inoculated
cell cultures are incubated at 350C for 5-28 days. The inoculums and growth medium is
removed after 12-24 hours and replaced with maintenance media which is changed
periodically. Cells are investigated periodically for presence of virus indicated by
presence of dead or dying cells (cytopathic effect).

16.9 Anti Viral Susceptibility Testing

Anti Viral Susceptibility Testing include phenotypic and genotypic methods. Genotypic
methods are used to detect determinants of resistance. Response to viral agents can also
be measured by monitoring the viral load.

16.10 Phenotypic assays

Cell cultures infected with virus is used to which various dilutions of antivirus are
added. Measures the ability of the virus to grow in the presence of various
concentrations of antiviral agent. Any decrease in viral activity is then measured. In
determining this, the quality control of cell culture media and cell lines is very
important and uninoculated cells act as negative controls.

Examples:

 PRA – Plaque reduction assays

 DU – Dye uptake method
 DNA hybridization
 EIA
 Neuraminidase inhibition
 Yield Reduction Assays
 PBMC cocultivation & recombinant assays for HIV-1

16.11 Genotypic assay

Genotypic assay determines the sequence of nucleotides in the viral genome and
compare the sequence to that of the wild-type virus.

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Examples:

 Analysis of viral NA to detect specific mutations

 DNA sequencing by automated sequencers
 PCR amplification and restriction enzyme digestion
 Hybridization to microarrays of oligonucleotide probes

Simultaneous testing of control strains is crucial.

16.12 Further Reading

Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. 8 TH

Edition, 2015.
Monica Chesbrough. District Laboratory Practice in tropical countries. Cambridge
University Press. Second Edition. 2006. The Edinburgh Building, Cambridge CB2
8RU, UK
James H. Jorgensen, and Mary Jane Ferraro. Antimicrobial Susceptibility Testing: A
Review of General Principles and Contemporary Practices. Clin Infect Dis. (2009)
49 (11): 1749-1755. doi: 10.1086/647952
RPH Laboratory Medicine 1998-2002. Webbed by Bill McConnell [email protected]:
https://en.wikipedia.org
Nagwa Mohamed Amin Aref. Virological tests, An Overview of Microbial Diagnosis.
www.freelivedoctor.com
Jawetz, Melnick and Adelberg’s Medical Microbiology 22nd Edition.
James H. Jorgensen, and Mary Jane Ferraro. Antimicrobial Susceptibility Testing: A
Review of General Principles and Contemporary Practices. Clin Infect Dis. (2009)
49 (11): 1749-1755. doi: 10.1086/647952
Eddy D M. A manual for assessing health parasites and designing practice policies. The
explicit approach. American College of Physician. Philadelphia. 1992

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