Performing Microbiological tests

Learning Module

Module code: HLT MLS4 MO6 1017

December, 2017

Bishoftu, Ethiopia
 Performing Microbiological Tests ORHB

Preface

This learning module was prepared based on the outcome based curriculum for level – IV
Medical Laboratory Technicians to help the trainees gain the knowledge of basic concepts of
microbiology and practical skills of microbiological tests for the purpose of providing the
trainees with the competency of performing microbiological tests.
This module was prepared by expertise from universities and colleges found in Oromia regional
state by the initiation and facilitation of Oromia Regional Health Bureau (ORHB) so as to solve
the scarcity of learning and teaching materials which focuses on occupational standards of
medical laboratory technicians’ level – IV.
Overall, this module has been divided into ten chapters being intended to provide the trainees
with the knowledge of basic concepts of general microbiology, bacteriology, virology and
mycology.
This module also helps the trainees to develop skills and attitude to identify microorganisms such
as bacteria and fungi using staining techniques and direct examination procedures, and to prepare
culture media for culture, isolation and identification of microorganisms in order to investigate
human diseases.
Furthermore, the module helps the trainees to comply with safety rules and standard operational
procedures in order to ensure quality in microbiological laboratory as well as helps the trainees
maintain records of microbiological laboratory work.

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Acknowledgments
This performing microbiological tests module and skill lab check list was developed by Experts
from Universities and Colleges in Oromia Regional State. Oromia Regional Health bureau and
Oromia TVET would like to acknowledge the following individuals and their organization for
their dedication, kind participation, expert opinions, and contributions in the development of this
learning Module.

S. no Name Organization
1. Abdi Legesse Oromia Regional Health Bureau
2. Abdisa Bukso Jimma University
3. Amsalu Mekonnin Salale University
4. Asefa Mekonnin Metu Blood bank
5. Bedado Dulo Adama Hospital Medical College
6. Belay Tafa Ambo University
7. Biruk Zerfu Addis Ababa University
8. Dejene Dessalegn East wollega zonal Health Office
9. Limeneh Habte MWU Shashmene Regional Hospital
10. Mamo Abdi OTVET
11. Mekuria Kebede OOCAA
12. Meseret Mitiku Medda Welabu University
13. Olifan Zewdie Wellega University
14. Shibiru Tufa Begna Oromia Regional Health Bureau
15. Shimelis Adugna Arsi University
16. Teferi Guji Jhpiego
17. Teferi Lemu Nekemte Health Science College
18. Teklu Shiferaw Adama Hospital Medical College
19. Waqtola Cheneke Jimma University
20. Zerihun Ataro Haramaya University
21. Zerihun Ayano Oromia Regional Health Bureau
This outcome based Module development was held on October, 2017 at Ethiopian Management
institute

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Acronyms and abbreviations

AFB Acid Fast Bacilli
Ag-Ab Antigen-Antibody
AIDS Acquired Immunodeficiency Virus
AST Antimicrobial Sensitivity Test
ATCC American Type Culture Collection
BCG Bacille Calmette - Guerin
BSC Biosafety Cabinet
cAMP Cyclic Adenosine Monophosphate
CDC Center for Disease Control and Prevention
CLSI Clinical Laboratories Standards Institute
CSF Cerebrospinal Fluid
DNA Deoxyribonucleic Acid
EF-2 Elongation Factor - 2
EHEC Enterohemorrhagic Escherichia coli
EIEC Enteroinvasive Escherichia coli
ELISA Enzyme Linked Immunosorbant Assay
EO Ethylene Oxide
EPEC Enteropathogenic Escherichia coli
EQA External Quality Assessment
ETEC Enterotoxigenic Escherichia coli
FTA Fluorescent Treponemal Antibody
H2O2 Hydrogen Peroxide
HAV Hepatitis A Virus
HBsAg Hepatitis B Surface Antigen
HCW Healthcare Worker
HIV Human Immunodeficiency Virus
HSV Herpes Simplex Virus
IgA Immunoglobulin A
KOH Potassium Hydro-Oxide
LED Light emitting diode

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LGV Lymphogranuloma Venerum

LPS Lipopolysaccharide
LTA Lipotechoic Acid
MCQ Multiple Choice Questions
NaCl Sodium Chloride
NGU Nongonococcal Urethritis
OPD Outpatient Department
PBP Penicillin Binding Protein
PCR Polymerase Chain Reaction
PFGE Pulsed Field Gel Electrophoresis
pH Power of Hydrogen
QA Quality Assurance
QC Quality Control
RNA Ribonucleic Acid
SOPs Standard Operating Procedures
TB Tuberculosis
TPI Treponema pallidum Immobilization

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Table of Contents Page

Preface........................................................................................................................................................... i
Acknowledgments ....................................................................................................................................... ii
Acronyms and abbreviations ..................................................................................................................... ii
Table of Contents ....................................................................................................................................... iv
Module Syllabus and Schedule for Performing Microbiological Tests ............................................... viii
Chapter One: Introduction to Microbiology ............................................................................................ 1
1.1. Basic concepts and branches of microbiology.....................................................................1
1.2. Historical background of Microbiology..............................................................................1
1.3. The Distribution and features of Microorganisms ..............................................................6
1.4. The Importance of studying Microbiology .........................................................................6
1.5. Definitions of common terminologies in Microbiology .......................................................6
1.6. Host and Pathogen Relationship ........................................................................................6
1.7. Normal microbial flora ......................................................................................................9
Chapter Two: Medical Bacteriology ....................................................................................................... 13
2.1. Introduction to Bacteriology ............................................................................................ 13
2.2. Nature of bacteria ............................................................................................................ 13
2.3. Classification of bacteria .................................................................................................. 14
2.3.1. Based on phenotyping ............................................................................................... 14
2.3.2. Genotypic Classification ........................................................................................... 20
2.4. Bacterial structures and functions.................................................................................... 20
2.5. Common bacteria causing Diseases in humans................................................................. 24
2.5.1. Gram positive cocci................................................................................................... 24
2.5.2. Pathogenic gram-positive rods .................................................................................. 27
2.5.3. Pathogenic Gram-Negative Cocci & Coccobacilli...................................................... 33
2.5.4. Pathogenic Gram-negative rods ................................................................................ 37
2.5.5. Genus Spirochetes ..................................................................................................... 45
Chapter Three: Medical Mycology ......................................................................................................... 58
3.1. Introduction to Mycology ................................................................................................. 58
3.2. Basic terms in mycology ................................................................................................... 59
3.3. General characteristics of fungi ....................................................................................... 59
3.4. Classification of fungi....................................................................................................... 60
3.5. Morphology of fungi ........................................................................................................ 61
3.6. Overview of fungal diseases.............................................................................................. 62
3.7. Laboratory diagnosis of fungal infections ........................................................................ 64
1.7.1 Specimens used for the diagnosis of fungal infection include .................................... 64
1.7.2 Laboratory examination methods of fungal infections .............................................. 64
Chapter Four: Medical Virology ............................................................................................................. 69
4.1 Introduction to virology ................................................................................................... 69
4.2 General Features of viruses .............................................................................................. 69

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4.3 Structure of virus ........................................................................................................... 70

4.4 Classification of viruses .................................................................................................... 71
4.5 Viral replication ............................................................................................................... 73
4.6 Transmission routes of viruses ......................................................................................... 76
4.7 Laboratory diagnosis of viral diseases .............................................................................. 78
4.7.1 Direct Examination of Specimen ............................................................................... 78
4.7.2 Indirect Examination ................................................................................................ 78
4.8 Common pathogenic DNA and RNA viruses .................................................................... 78
4.8.1 DNA VIRUSES ......................................................................................................... 78
4.8.2 RNA VIRUSES ......................................................................................................... 82
Chapter Five: Collection, Handling, Processing, Transportation and Storage of Microbiological
Samples ................................................................................................................................................... 91
5.1 Different types of specimens for microbiological tests ...................................................... 92
5.1.1 Sputum specimen ...................................................................................................... 92
5.1.2 Wound specimen....................................................................................................... 97
5.1.3 Urogenital (Urethral and Cervical) specimen: ........................................................ 102
5.1.4 Ear swabs (Discharge) ............................................................................................ 107
5.1.5 Urine specimen ....................................................................................................... 111
5.1.6 Stool specimen ........................................................................................................ 112
5.1.7 Blood specimen ....................................................................................................... 114
5.2 Microbiological Specimen Rejection Criteria ................................................................. 116
5.3 The right time for the collection of different specimens for microbiological tests ........... 118
5.4 Specimen Handling and Transportation ........................................................................ 118
5.5 Storage and Transportation of Microbiology Samples ................................................... 118
5.6 Appropriate preservation methods for the storage of microbiological............................ 119
Chapter Six: Safety and Safety Rules in Microbiology Laboratory................................................... 121
6.1. Safety in Microbiology Laboratory ................................................................................ 121
6.2. Methods of sterilization and Disinfection ....................................................................... 125
6.3. Standard precautions when handling biological materials ............................................. 135
Chapter Seven: Microscopic Examination of Wet and Stained Preparations and Identification of
Common Microbial Pathogens ........................................................................................................... 148
7.1 KOH preparation........................................................................................................... 148
7.2 Indian ink preparation ................................................................................................... 152
7.3 Preparation of Wet Mount of Bacteria ........................................................................... 156
7.4 Staining in medical microbiology ................................................................................... 162
7.4.1 Simple Staining ....................................................................................................... 163
7.4.2 Differential Stains ................................................................................................... 164
Chapter Eight: Preparation of Culture Media, Cultivation of Microorganism and Antimicrobial
Sensitivity Testing ................................................................................................................................ 186
8.1 Overview of culture media ............................................................................................. 186
8.2 Inoculation and Cultivation of Microorganisms ...................................................................... 198
8.3 Overview of antimicrobial sensitivity testing .................................................................. 202

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8.4 Antibiotic Susceptibility Testing..................................................................................... 204

Chapter Nine: Quality Assurance and SOPs in Microbiological Laboratories ................................ 210
Chapter Ten: Recording and Reporting of Microbiological Test Results ......................................... 215
10.1 Reporting and recording test results .............................................................................. 215
10.2 Standardization in reporting test results ........................................................................ 215
10.3 Recording results in the laboratory ................................................................................ 215
10.4 Confidentiality Of Data And Records Related To Microbiology Laboratory ................. 216

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Module Syllabus
Module title/Name: Performing Microbiological tests
Module code: HLT MLS4 MO6 1017
Nominal duration: 240Hours (Theory: 72, Practice: 168); (6 weeks)
Module Description:
This module aims to provide students with the knowledge of basic concepts of bacteriology, virology and
mycology. This module also helps students to develop skills and attitude to identify micro-organisms such
as bacteria and fungi using staining techniques and direct examination procedures, and to prepare culture
media for culture, isolation and identification of micro-organisms in order to investigate human diseases.
Unit of Competencies: HLT MLS 4 06 0611-Perform microbiological tests
Learning outcomes:
By the end of this module, the students will be able to:
 Classify and describe medically-significant microorganisms according to their characteristics
 Perform collection, handling, processing, transportation and storage of microbiological samples.
 Follow safety rules and standard operational procedures in order to ensure quality in
microbiological laboratory
 Apply aseptic techniques in Microbiology laboratory
 Perform microscopic examination of wet and stained preparations and identify common microbial
pathogens
 Prepare culture media and cultivate microorganism
 Assist in antibiotic sensitivity testing
 Maintain records of microbiological laboratory work
Teaching/learning methods:
Interactive Presentation
Small and large group discussion
Role play simulation (client communication during sample collection)
Demonstration and practical with coaching
Presentation
Co-operative training
Teaching/learning materials:
 TTLM (Training, Teaching and Learning Materials)
o Learning module, laboratory manual and SOPs

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 Reference books
 LCD Projector and flip chart
 White board/white board marker, chalk/black board
 Consumables (laboratory reagents, clinical samples and others)
 Microbiology laboratory equipments and materials
Methods of assessment: formative and summative assessments
 Written exam/test on underpinning knowledge
 Questioning or interview on underpinning knowledge
 Practical assessment by direct observation of tasks through simulation/Role-plays

Module Syllabus and Schedule for performing Microbiological tests

Week Day Learning Activity and allotted Time Required
Reading /
Assignment
Week - 1 Day - 1 Interactive presentation and group discussions (4hrs)
 Introduction to Microbiology
Interactive presentation (4hrs)
 Introduction to Medical Bacteriology
Day - 2 Interactive lecture (4hrs)
 Common bacteria causing diseases in humans: Gram
positive cocci and Pathogenic Gram-positive rods
Interactive presentation(4hrs)
 Common bacteria causing diseases in humans:
 Pathogenic gram-negative cocci & coccobacilli
 Pathogenic gram-negative rods (Family
Enterobacteriaceae)
Day - 3 Assessment (1hr)
 Test - 1
Interactive presentation(3hrs)
 Common bacteria causing diseases in humans:
 Pseudomonas, Genus of Vibrios and Genus of
Campylobacter
 Genus of Mycobacteria

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Week Day Learning Activity and allotted Time Required

Reading /
Assignment
Interactive presentation(4hrs)
Common bacteria causing diseases in humans:
 Genus Spirochetes: Genus Treponema, Genus of
Borellia, Genus of Lepetospira
Day - 4 Interactive presentation(4hrs)
 Common bacteria causing diseases in humans:
 Genus Chlamydia, Genus Mycoplasma and
Genus Rickettsia
Interactive presentation, Group discussion and summary
(4hrs)
 Common bacteria causing diseases in humans
Day - 5 Interactive presentation(4hrs)
 Medical Mycology
Interactive presentation(3hrs)
 Introduction to virology
Assessment (1hr)
 Quiz /test
Day - 6 Review of the previous sessions and Independent study
Week - 2 Day - 1 Assessment (1hr)
 Test -2
Interactive presentation(3hrs)
 Common pathogenic DNA viruses
Interactive presentation and group discussions (4hrs)
 Common pathogenic RNA viruses
Day - 2 Group activities and presentations (4hrs)
 Common pathogenic viruses
Interactive presentation(4hrs)
 Types of specimens for microbiological tests
Day - 3 Skill lab and group discussions (4hrs)
 Practice on collection and labeling of routine specimens
for microbiological tests
Skill lab and group discussions (4hrs)

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Week Day Learning Activity and allotted Time Required

Reading /
Assignment
 Practice on collection and labeling of routine specimens
for microbiological tests
Day - 4 Skill lab and group discussions (4hrs)
 Identify appropriate specimens for microbiological tests
Interactive presentation(2hrs)
 Specimen collection time, handling and transportation
Skill lab (2hrs)
 Demonstration of triple package system for high risky
specimens
Day - 5 Skill lab and group discussions (4hrs)
 Select appropriate preservation methods for the storage
of microbiological specimens
Interactive presentation(1hr)
 concepts of biohazard materials
Skill lab and group discussions (3hrs)
 Apply safety measures in microbiology Laboratory

Day - 6 Review of the previous sessions and Independent study

Week - 3 Day - 1 Skill lab and group discussions (4hrs)

 Safe handling of equipment and materials in
microbiology lab
Interactive presentation(4hrs)
 Methods of sterilization and Disinfection
Day - 2 Interactive presentation(2hrs)
 Methods of sterilization and Disinfection
Skill lab (2hrs)
 Apply standard precautions when handling biological
materials
Skill lab and group discussions (4hrs)
 Apply sterilization and Disinfection techniques for
microbial growth control
Day - 3 Interactive presentation(4hrs)
 Wet mount and staining techniques in microbiology

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Week Day Learning Activity and allotted Time Required

Reading /
Assignment
(4hrs)
Interactive presentation(4hrs)
 Gram staining and acid fast staining
Day - 4 Skill lab and group discussions (4hrs)
 wet mount preparation and examination
Skill lab and group discussions (4hrs)
 Wet mount preparation and examination
Day - 5 Skill lab and group discussions (4hrs)
 Thin smear preparation and staining
Skill lab and group discussions (4hrs)
 Thin smear preparation and staining
Day - 6 Review of the previous sessions and Independent study
Week - 4 Day - 1 Skill lab and group discussions (4hrs)
 Thin smear preparation and staining
Skill lab and group discussions (4hrs)
 Microscopic examination of Stained smear
Day - 2 Assessment and feedback (4hrs)
 Practical exam on wet mount preparation and
examination
Assessment and feedback (4hrs)
 Practical exam on wet mount preparation and
examination
Day - 3 Skill lab and group discussions (4hrs)
 Microscopic examination of Stained smear
Skill lab and group discussions (4hrs)
 Microscopic examination of Stained smear
Day - 4 Skill lab and group discussions (4hrs)
 Microscopic examination of Stained smear
Skill lab and group discussions (4hrs)
 Microscopic examination of Stained smear

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Week Day Learning Activity and allotted Time Required

Reading /
Assignment
Day - 5 Assessment and feedback (4hrs)
 Practical exam on smear preparation and staining
Assessment and feedback (4hrs)
 Practical exam on smear preparation and staining
Day - 6 Review of the previous sessions and Independent study
Week - 5 Day - 1 Assessment and feedback (4hrs)
 Practical exam on microscopic examination of stained
smears
Assessment and feedback (4hrs)
 Practical exam on microscopic examination of stained
smears
Day - 2 Interactive presentation(4hrs)
 Overview culture media (2hrs)
 Overview of antimicrobial sensitivity testing (1hr)
 Quality assurance methods in Microbiological
laboratories (1hr)
Skill lab and group discussions (4hrs)
 Recording or transcribing data
Day - 3 Skill lab and group discussions (4hrs)
 Prepare and sterilize culture media
Skill lab and group discussions (4hrs)
 Prepare and sterilize culture media
Day - 4 Skill lab and group discussions (4hrs)
 Perform inoculation and cultivation of culture media
Skill lab and group discussions (4hrs)
 Perform inoculation and cultivation of culture media
Day - 5 Skill lab and group discussions (4hrs)
 Perform antimicrobial sensitivity testing
Skill lab and group discussions (4hrs)
 Perform antimicrobial sensitivity testing
Day - 6 Review of the previous sessions and Independent study

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Week Day Learning Activity and allotted Time Required

Reading /
Assignment
Day - 1 Cooperative training/attachment at health facility (8hrs)
Week - 6
Day - 2 Cooperative training/attachment at health facility (8hrs)
Day - 3 Cooperative training/attachment at health facility (8hrs)
Day - 4 Cooperative training/attachment at health facility (8hrs)
Day - 5 Reflective portfolio and oral examination (8hrs)
References Books  Linne JJ, RingsrudKM (1979) Basic techniques for the
medical laboratory 2nd ed. McGraw-Hill,inc New York.
 Cheesbrough, M. (1998) District Laboratory Practice in
Tropical Countries. Part 2 Cambridge University Press
 Cheesbrough, Laboratory Manual for Tropical
Countries. Vol.II Cambridge: Butterworth-Heinemann.
Ltd
 Patrick Murray, Ken S. Rosenthal, Michael A. Pfaller.
(2005) Medical Microbiology 5th edition.
 Baron S (1996) Medical Microbiology 4th edition.
 Kanai L Mukherjee. Medical Laboratory Technology:
A Procedure Manual for Routine Diagnostic Tests, 2001
TATA McGraw Hill.

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Chapter 1: Introduction to Microbiology

Dear trainee,
In this chapter, you will learn the basic concepts and historical background of microbiology as
well as the applications of and terms used in microbiology. In addition, you will learn the
features, concepts of host pathogen relationship, and about normal flora colonizing human body.

 Objectives
At the end of this chapter, you will be able to:
 State the historical background of microbiology
 Mention the features of microorganisms
 Describe the applications of microbiology
 Define common terms in microbiology
 Describe concepts of host pathogen relationship
 Define normal microbial flora
 Describe human body parts colonized by normal flora
1.1. Basic concepts and branches of microbiology
Microbiology is a subject which deals with living organisms that are individually too small to be
seen with the naked eye. There are sub-branches of microbiology such as Medical microbiology,
Pharmaceutical microbiology, Industrial microbiology and others. As it is our area of interest,
Medical Microbiology is the study of microbes that infect humans, the diseases they cause, their
diagnosis, prevention and treatment. Further, medical microbiology is divided into the following
sub-fields:
 Medical bacteriology deals with bacteria causing diseases.
 Medical mycology deals with fungi causing diseases.
 Medical virology deals with viruses causing diseases.
1.2. Historical background of Microbiology
Man kind has always been affected by diseases which were originally believed to be
visitations by the Gods and meant to punish evil doers.
Hippocrates:Known as “Father ofMedicine”

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He observed that ill health resulted due to changes in air, winds, water, climate, food, nature of
soil and habits of people.
Varro (117-26 BC):He said disease was caused by animate particles invisible to naked eye but
which were carried in the air through the mouth and nose into the body.
Fracastorius (1500 G.C.):Proposed that agents of communicable disease were living germs,
which transmitted by
 direct contact with humans and animals
 indirectly by objects
 but this proposed idea has no proof because of lacking experimental evidence.
Antony Van Leeuwenhoek(1632-1723):Known as “Father of Microbiology”
He was describe “animalcules” (single celled organism) using simple microscope with one
lens.He was the first scientist who properly described the different shapes of bacteria.He was not
concerned about the origin of micro-organism;but, many other scientists were searching for an
explanation for spontaneous appearance of living things from,
 decaying meat
 stagnating ponds
 fermenting grains and
 infected wounds
On the bases of these observations, two major theories were formulated.
 Abiogenesis
 Biogenesis : States that life comes from pre existing life
1. Theory of Abiogenesis (non-biological origin):deals with the theory of spontaneous
generation; stating that living things originated from non-living things. Generated by Greek
philosopher Aristotle (384-322BC).
Example: -
 mice spontaneously appear in stored grain
 maggots spontaneously appear in meat.
Francesco Redi (1626-1697) :The first scientist who tried to set an experiment to disprove
spontaneous generation.He Proved that no maggots appeared in meat when flies were prevented
from laying eggs. He used three flasks and placed meat in each of the three flasks

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1. One of the flasks open

2. Another one tightly sealed and,
3. The last one covered with gauze
 He put the meat in a flask and covered it with a gauze.
 He observed that the flies laid eggs from which the maggots developed.
 He said maggots did not developed from meat but from flies egg.
 Conclusion: abiogenesis is not acceptable theory
2. Theory of Biogenesis: states that life comes from pre-existing life.Louis Pasteur (1822-
1895 GC)- was the French microbiologist and chemist who disproved the theory of
abiogenesis once and for allby his experiment.
Experiment of Louis Pasteur:
He designed a large curved flask (Pasteur goose neck flask) and placed a sterile
growth broth medium. Air freely moved through the tube; but dust particles were trapped
in the curved portion of flask. Microbial growth in the broth was not seen. Therefore
Pasteur proved that micro-organisms entered tosubstrates through the air and
micro-organisms did not evolve spontaneously. In ‘A’ air freely moved through the tube,
but dust particles were trapped in the curved portion of the flask. And no microbial
growth was observed.

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Fig-1.1 Louis Pasteur experiment

Observation
 the broth in open vessel became turbid
 no turbidity was observed in broth in a sealed vessel
Conclusion
 Organisms that grew on broths came from outside, as spores on dust, rather than
being generated within the broth
 Microorganisms are present in air but not created by air
 Refuses the concept of spontaneous generation
 Strong justification for development of germ theory of disease
Other contributions of Louis Pasteur
 Concept of pasteurization (Milk)
 The concept of sterilization
 Development of broth for growing microorganisms
 Developed the first vaccine for anthrax and rabies
 Discovery of streptococci
The germ theory of disease
The complete establishment of the germ theory of disease depended on the work of a German
scientist, Robert Koch (1843-1910).
Major achievements of Robert Koch

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1. Discovery and use of solid medium in bacteriology

2. Discovery of causative agents of tuberculosis, anthrax and cholera.
3. Koch’s phenomenon
4. Koch’s postulates
Koch’s postulates: proof of germ theory of disease
 A micro-organism can be accepted as a causative agent of an infectious disease only if
the following conditions are satisfied.
 The microorganism should be found in every case of the disease and under conditions,
which explain the pathological changes and chemical features.
 It should be possible to isolate the causative agent in pure culture from the lesion.
 When such pure culture is inoculated in to appropriate lab animal, the lesion of the
disease should be reproduced.
 It should be possible to re-isolate the bacterium in pure culture from the lesion produced
in the experimental animal
 Now a day’s additional postulate is mentioned i.e. Specific antibody to the bacterium
should be detectable in the serum during the course of the disease
Exceptions to Koch’s postulate
1. Many healthy people carry pathogens but do not exhibit symptoms of the disease.
2. Some microbes are very difficult or impossible to grow in vitro (in the laboratory) in
artificial media. Eg. T. pallidum.
3. Many pathogens are species specific. Eg. Brucella abortus cause abortion in animals
but not in humans.
4. Certain diseases develop only when an opportunistic pathogen invades immuno-
compromised host.
Major achievements of Robert Koch
 Discovery and use of solid medium in bacteriology
 Discovery of causative agents of tuberculosis, anthrax and cholera.
 Koch’s postulates
Conclusion: Generally, germ theory of disease proposes that microorganisms are the cause
of many diseases.

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1.3.The Distribution and features of Microorganisms

Microorganisms are found everywhere: in the air we breathe, water we drink, on or in our
body, soil and others.
Characteristics which make them found everywhere are:
– They exist in different forms (spore, cysts)
– They are small in size
– Easy adaptation to physical and chemical factors
– They have rapid reproduction
– Easy transmission
– They have diverse metabolism (have different enzymes)
1.4.The Importance of studying Microbiology
The popular perception among the general public why we study microbiology as a subject is that
invisible ‘bugs’ so called microbes cause diseases. In reality, only a couple of hundred out of the
half million or so known bacterial species give rise to infections in humans; these are termed
pathogens, and have tended to dominate our view of the microbial world.
It may come as something of a surprise therefore to learn that the vast majority of
microorganisms coexist alongside us without causing any harm. Indeed, many perform vital
tasks such as the recycling of essential elements, without which life on our planet could not
continue. Other microorganisms have been exploited by humans for our own benefit, for instance
in the manufacture of antibiotics and foodstuffs.
1.5.Definitions of common terminologies in Microbiology
 Micro-organism or microbe: is a microscopic organism that comprises either single cell
(unicellular), cell clusters (multicellular) or no cell at all (a cellular)
 Pathogen: is an organism with the potential to cause disease
 Pathogenicity: the ability of a pathogen to damage host cell or tissue
 Virulence factor: a factor which contributes for the pathogenicity of the microbes.
1.6.Host and Pathogen Relationship
A relation is either harms or lives at the expense of another organism (the host) and the purpose
of interaction is competition for superiority. In this case two ideas were observed
 When the host is successful healthy (pathogen removed) no more disease

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 When the pathogen gets upper hand  disease will appear

In this cases disease occurs due to imbalance between host resistance and virulence factors.
Under normal condition the host and the parasite exist as balanced in environment, but the case
of imbalance will happen it the following two ways,
 Any decrease in host resistance or increase in parasite virulence result in disease
 When host resistance impaired, organisms that are normally present may cause disease
 To understand the above two ideas, the following general Concepts are important
Host is resistance to disease when
 Host is enhanced by phagocytic cells and an intact immune system
 Initial resistance is due to nonspecific mechanisms
 Specific immunity develops over time
Host exposed to the disease (susceptible) when
 Susceptibility to some infections is higher in the very young and the very old and in
immunosuppressed patients
 Pathogen Infectivity
 Pathogen infectivity results from a disturbance in the balance between pathogen virulence
and host resistance
 The "objective" of any pathogen is to multiply rather than to cause disease; it is in the
best interest of the pathogen not to kill the host.
 Host Resistance
Numerous physical and chemical attributes of the host protect against bacterial infection. These
defenses include the antibacterial factors in secretions covering mucosal surfaces and rapid rate
of replacement of skin and mucosal epithelial cells. Once the surface of the body is penetrated,
bacteria encounter an environment virtually devoid of free iron needed for growth, which
requires many of them to scavenge for this essential element. Bacteria invading tissues encounter
phagocytic cells that recognize them as foreign through a complex signaling mechanism
involving interleukins, eicosanoids, and complement, mediate an inflammatory response in
which many lymphoid cells participate
 Host-mediated Pathogenesis
In certain infections (e.g. tuberculosis) tissue damage results from the toxic mediators released
by lymphoid cells rather than from bacterial toxins.The pathogenesis of many bacterial infections

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cannot be separated from the host immune response, for much of the tissue damage is caused by
the host response rather than by bacterial factors. Classic examples of host response-mediated
pathogenesis are seen in diseases such as Gram-negative bacterial sepsis, tuberculosis, and
tuberculoid leprosy. The tissue damage in these infections is caused by toxic factors released
from the lymphocytes, macrophages, and polymorphonuclear neutrophils infiltrating the site of
infection (Fig below.). Often the host response is so intense that host tissues are destroyed,
allowing resistant bacteria to proliferate.

Fig- 1.2Generalized mechanisms of bacterial pathogenesis: bacteria-induced toxicity or host-

mediated damage.
 Intracellular Growth
Some bacteria (e.g., Rickettsia species) can grow only within eukaryotic cells, whereas others
(e.g., Salmonella species) invade cells but do not require them for growth. Most pathogenic
bacteria multiply in tissue fluids and not in host cells. In general, bacteria that can enter and
survive within eukaryotic cells are shielded from humoral antibodies and can be eliminated only
by a cellular immune response. However, these bacteria must possess specialized mechanisms to
protect them from the harsh effects of the lysosomal enzymes encountered within the cell.
 Virulence Factors
Virulence factors help bacteria to:
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1. invade the host

2. evade host defenses
3. cause disease
The following are types of virulence factors:
 Adherence Factors: Many pathogenic bacteria colonize mucosal sites by using pili
(fimbriae) to adhere to cells.
 Invasion Factors: Surface components that allow the bacterium to invade host cells can
be encoded on plasmids, but more often are on the chromosome.
o Capsules: Many bacteria are surrounded by capsules that protect them from
opsonization and phagocytosis.
o Endotoxins: The lipopolysaccharide endotoxins on Gram-negative bacteria cause
fever, changes in blood pressure, inflammation, lethal shock, and many other toxic
events.
1.7.Normal microbial flora
Why is it called the normal flora?
The term flora is used because the majority of the organisms concerned are bacteria. It has been
estimated that humans have approximately 1013 cells in the body and something like 1014
bacteria associated with them, the majority in the large bowel. Members of groups such as
viruses, fungi and protozoa are also regularly found in healthy individuals, but form only a minor
component of the total population of resident organisms. The organisms occur in those parts of
the body that are exposed to, or communicate with, the external environment, namely the skin,
nose and mouth, and intestinal and urogenital tracts. Internal organs and tissues are normally
sterile and free from any microorganism.
 Advantages and disadvantages of the normal flora
Some of the species of the normal flora are positively beneficial to the host. The importance of
these species for health is sometimes revealed quite dramatically under stringent antibiotic
therapy. This can drastically reduce their numbers to a minimum, and the host may then be
overrun by introduced pathogens or by overgrowth of organisms normally present in small
numbers. In short, the advantage of normal flora is as follow:
 The advantages of normal flora in preventing colonization of potential pathogens can be seen
in the following ways.

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 Skin bacteria produce fatty acids, which discourage other species from invading.
 Gut bacteria release a number of factors with antibacterial activity (bacteriocins,
colicins) as well as metabolic waste products that help prevent the establishment of
other species.
 Vaginal lactobacilli maintain an acid environment, which suppresses growth of other
organisms.
 The sheer number of bacteria present in the normal flora of the intestine means that
almost all of the available ecologic niches become occupied; these species therefore
compete with others for living space.
 The disadvantages of the normal flora lie in the potential for spread into previously sterile
parts of the body. This may happen in the following ways:
 When the intestine is perforated or the skin is broken;
 during extraction of teeth (when viridans streptococci may enter the bloodstream)
 When organisms from the perianal skin ascend the urethra, and cause urinary tract
infection.
 Members of the normal flora are important causes of hospital-acquired infection when
patients are exposed to invasive treatments.
 Patients suffering burns are also at risk
 Overgrowth by potentially pathogenic members of the normal flora can occur when the
composition of the flora changes (e.g. after antibiotics) or when:
 The local environment changes (e.g. increases in stomach or vaginal pH);
 The immune system becomes ineffective (e.g.AIDS, clinical immunosuppression).
 Under these conditions, the potential pathogens take the opportunity to increase their
population size or invade tissues, so becoming harmful to the host.

Summary
 Microbiology is a subject which deals with living organisms that are individually too
small to be seen with the naked eye

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 Medical microbiology is divided into the following sub-fields:

 Medical bacteriology deals with bacteria causing diseases.
 Medical mycology deals with fungi causing diseases.
 Medical virology deals with viruses causing diseases.
 Antony Van Leeuwenhoek the scientist who describe “animalcules” (single celled
organism) using simple microscope with one lens
 Louis Pasteur was the French microbiologist and chemist who disproved the theory of
abiogenesis once and for all by his experiment.
 The complete establishment of the germ theory of disease depended on the work of a
German scientist, Robert Koch.
 Microorganisms are found everywhere: in the air we breathe, water we drink, on or in
our body, soil and others
 A relation is either harms or lives at the expense of another organism (the host) and the
purpose of interaction is competition for superiority. In this case, two ideas were
observed
 When the host is successful- healthy (pathogen removed) no more disease
 When the pathogen gets upper hand - disease will appear
 Normal flora is a member of microorganisms regularly found in or on the body of
individuals.

Self-check questions:
Instruction: Please! attempt all the questions listed below.
Part – I. Essay questions
1. What kinds of organisms are studied in Microbiology?
2. Who first discovered the simple microscope?
3. Who disproved the theory of abiogenesis?
4. Explain the germ theory of disease.
5. Define pathogen.
6. Define normal flora, and state the advantages of normal flora.
Part – II Multiple choice
1. Which one of the following statement is not trueabout the origin of microorganisms?

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A. The followers of Spontaneous generation theory believed in the creation of life from
organic matter.
B. Louis Pasteur was the most powerful opponent of Spontaneous generation.
C. The followers of biogenesis theory believed that life originated from preexisting life.
D. The critics raised on Lazzaro Spallanzani were answered by Francesco Redi.

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Chapter 2 : Medical Bacteriology

Dear trainees,
In this chapter, you will learn the features, classification and structure of bacteria. In
addition, you will learn medically important common bacterial pathogens with their distinctive
features, their laboratory diagnosis and their pathogenesis.
Objectives:
At the end of this chapter, you will be able to:
 Describe nature/features of bacteria.
 Classify bacteria based on morphology, staining characteristics as well as growth
requirements.
 List different structures of bacteria and describe their functions.
 List and describe medically important bacteria.
2. Medical Bacteriology
2.1. Introduction to Bacteriology
Bacteriology is the study of bacteria. Bacterial cytology is the study of chemistry, structure and
function of a bacterial cell.
2.2. Nature of bacteria
Bacteria are among the most widely distributed forms of life which are found in air, water, food,
and soil and etc. They are also found in the external and internalbodies of humans, animals and
plants. They are small microorganisms with a simple form of cellular organization described as
prokaryotic cells.
Life in all its diversity is composed of only two types of cells: Eukaryotic and Prokaryotic.
Prokaryotic cells are simple, one-celled organisms; such as bacteria. All other life is composed of
Eukaryotic cells. Not surprisingly, Prokaryotic cells are far simpler in structure and they are also
much smaller than Eukaryotic cells. Eukaryotic, derived from the Greek, means “true nucleus,”
whereas Prokaryotic means “before the nucleus.” In addition to having a nucleus, Eukaryotic cells
have a nucleolus containing the cell’s DNA, as well as specific membrane-bound compartments,
organelles, where specific metabolic activities occur. Prokaryotic cells do not have a nucleus or
contain these more complex inner structures. The following table depicts the comparison features
of prokaryotic and eukaryotic organisms.

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Table: Comparison features of prokaryotic and eukaryotic organisms

Characteristic features Prokaryotes Eukaryotes
Typical organisms Example: bacteria Example: protozoan, fungi, plants, animals
Type of nucleus no real nucleus real nucleus with double membrane
DNA circular (usually) linear molecules (chromosomes) with
histone proteins
RNA/protein-synthesis coupled in cytoplasm RNA-synthesis inside the nucleus and
protein synthesis in the cytoplasm
Ribosome 50s+30s=70s 60s+40s=80s
Cytoplasm structures Very few structures highly structured by endomembrane and a
cytoskeleton
Mitochondria None one to several thousand (though some lack
mitochondria)
Chloroplasts None in algae and plants
Organization usually single cells single cells, higher multicellular
organisms with specialized cells
Cell division Binary fission (simple Mitosis and Meiosis
division)

2.3. Classification of bacteria

The classification of bacteria serves a variety of different functions. Because of this variety,
bacteria may be grouped using many different typing schemes. The critical feature for all these
classification systems is an organism identified by one individual (scientist) is recognized as the
same organism by another individual.
The classification schemes most commonly used by clinicians and clinical microbiologists are
discussed below.
2.3.1. Based on phenotyping
A. Gram stain
It allows a large proportion of clinically important bacteria to be classified as either Gram
positive or negative based on their morphology and differential staining properties. Slides are

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sequentially stained with crystal violet, iodine, then destained with alcohol and counter-stained
with safranin.
Gram positive bacteria stain blue-purple and Gram negative bacteria stain red. The difference
between the two groups is believed to be due to a much larger peptidoglycan (cell wall) in Gram
positives. As a result, the iodine and crystal violet precipitate in the thickened cell wall and are
not eluted by alcohol in contrast with the Gram negatives where the crystal violet is readily
eluted from the bacteria. As a result, bacteria can be distinguished based on their morphology
and staining properties.

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Chart 2.1. Features between Gram Positive and Negative Bacteria

Gram positive bacteria have a large peptidoglycan structure. As noted above, this accounts for
the differential staining with Gram stain. Some Gram positive bacteria are also capable of
forming spores under stressful environmental conditions such as when there is limited
availability of carbon and nitrogen. Spores therefore allow bacteria to survive exposure to
extreme conditions and can lead to re-infection (e.g.,pseudomembranous colitis from
Clostridium difficle).

Cell wall of Gram positive bacteria

Fig-2.1 Cell wall gram positive bacteria

Gram negative bacteria have a small peptidoglycan layer but have an additional membrane, the
outer cytoplasmic membrane. This creates an additional permeability barrier and results in the
need for transport mechanisms across this membrane.

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Gram negative cell wall

Lipopolysaccharide

Porin proteins

Outer membrane
Periplasmic space
Peptidoglycan
Cytoplasmic membrane

Cytoplasm

Fig- 2.2 Cell wall gram negative bacteria

Some bacteria such as mycobacteria (the cause of tuberculosis) are not reliably stained due to the
large lipid content of the peptidoglycan. Alternative staining techniques (Kinyoun or acid fast
stain) are therefore used that take advantage of the resistance to destaining after lengthier initial
staining.

Complex cell wall structure of Mycobacteria

Fig-2.3 Cell wall of Mycobacteria

B. Morphology

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Depending on morphology, Bacteria can be grouped as

 Cocci (Singular: Coccus)
 Bacilli (Singular: Bacillus)
 Vibrios (Singular: Vibrio)
 Spirilla (Singular: Spirillum)
 Spirochaetes (Singular: spirochaete)

Morphological features of bacteria

Fig 2.4 Morphological features of bacteria

C. Growth Requirements: Oxygen, Temperature, pH
Microorganisms can be grouped on the basis of their need for oxygen, temperature, pH and
others to grow.
Based on Oxygen requirements, bacteria classified as:
a. Obligate aerobes – undergo aerobic respiration
b. Obligate anaerobes – do not use aerobic metabolism
c. Facultative anaerobes – can maintain life via fermentation or anaerobic respiration or by
aerobic respiration

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d. Aerotolerant anaerobes – do not use aerobic metabolism but have some enzymes that
detoxify oxygen’s poisonous forms
e. Microaerophiles – aerobes that require oxygen levels from 5-10% and have a limited
ability to detoxify hydrogen peroxide and superoxide radicals

Figure -2.5 Oxygen requirement classification of bacteria

 Classification of bacteria based on temperature:
– Psychrophiles: Below 20°C
– Mesophiles: 25-40°C
– Thermophiles: 55-80°C
– Hyperthermophiles: upto 100°C or greater

[INSERT FIGURE 6.5]

Fig -2. 6Temperature requirement classifications of bacteria

 Classification of bacteria based on pH requirement
Organisms are sensitive to changes in acidity because H+ and OH- interfere with H bonding in
proteins and nucleic acids.
 Neutrophiles: are bacteria and protozoa that grow best in a narrow range around neutral
pH (6.5-7.5)
 Acidophiles: are bacteria and fungi that grow best in acidic habitats
 Alkalophiles: grow best in alkali habitats

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D. Biochemical reactions
Clinical microbiology laboratories typically will identify a pathogen in a clinical sample, purify
the microorganism by plating a single colony of the microorganism on a separate plate, and then
perform a series of biochemical studies that will identify the bacterial species.
2.3.2. Genotypic Classification
i. Ribosomal RNA (rRNA) sequence analysis: This has emerged as a major method for
classification. It has been used (as described above) to establish a phylogenetic tree. In
addition, it is now also used to rapidly diagnose the pathogen responsible for an infection, to
help select appropriate therapy and to identify non-cultivatable microorganisms.
ii. Molecular subtyping: Sometimes it is necessary to determine whether strains from the same
species are the same or different. For example, if there is an outbreak of infections that
appear due to the same bacterial species, the microbiologists will want to know if all of the
infections are due to the same strain. Clues can be obtained by examining the biochemical
studies or the antibiotic susceptibility profile, but a more reliable method is by molecular
analysis. Pulsed Field Gel Electrophoresis (PFGE) is the most frequently used molecular
technique. Chromosomal DNA is digested with a restriction enzyme that makes relatively
infrequent cuts in the DNA and as a result creates large DNA fragments. The DNA fragments
from the different strains are then run on a gel and compared.
2.4. Bacterial structures and functions
In general, prokaryotic cells have three architectural regions (see the diagram below). These are:
1. Appendages(proteins attached to the cell surface) in the form of flagella and pili
2. A cell envelope consisting of a capsule, cell wall and plasma membrane; and
3. A cytoplasmic region that contains the cell genome (DNA) and ribosomes and various
sorts of inclusions.

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Fig -2.7 Basic bacterial cell structure

 Functions of bacterial structures
 Pilus (plu. Pili): an elongate, hallow appendage used to transfer DNA into other cells and
in cell adhesion.
 Flagellum (plu. Flagella): it is a specialized appendage attached to the cell by a basal
body that holds a long rotating filament. The movement pushes the cell forward and
provides motility.
 Capsule: a coating or layer of molecules external to cell wall. It serves protective,
adhesive, and receptor functions.
 Cell wall: a semi rigid covering that provides structural support and shape for the cell.
 Cell membrane: a thin sheet of lipid and protein that surrounds the cytoplasm and
controls the flow of materials into and out of the cell pool.
 Bacterial chromosome or nucleoid: the site where the large DNA molecule is condensed
into a packet. DNA is the code that directs all genetics and hereditary of the cell.
 Ribosomes: are tiny particles composed of protein and RNA that are the sites of protein
synthesis.
Small group discussions:
Dear trainees, the cell wall of gram negative and gram positive bacteria is different. Thus,
you are expected to compare and contrast the cell wall of gram negative and gram positive
bacteria. In order to perform your activity, make small group and discuss on it. Let the
instructor facilitate the activity and each group reflects accordingly.

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Summary
 Bacteriology is the study of bacteria.
 Bacteria are grouped under prokaryotic cells, simple one-celled organisms.
 Bacteria can be classified based on phenotypical and genotypical schemes.
 There are different bacterial structures with different functions.
Self-check questions:
Instruction: Please! attempt all the questions listed below.
Part I: Multiple Choices
1. One describes phenotic classification of microorganisms
A. It is a genetic classification of microorganisms
B. It is based on observable morphological characteristics
C. Evolutionary relatedness is a criterion for grouping
D. Groups necessarily reflect genetic similarity
2. A typical prokaryotic organism is
A. Virus C. Fungus
B. Bacteria D. Protozoa
3. Prokaryotic organisms are characterized by
A. Lack of deoxyribonucleic acid
B. Circular and multiple chromosomes
C. Membrane bounded organelles such as ribosomes
D. Lack of membrane bounded nucleus
4. The correct order of bacterial structures from the outer surface to the most inner
compartment
A. Cell Wall---> Cell Membrane ----> Capsule ----> Cytoplasm
B. Capsule ---> Cell Membrane ----> Cell Wall ----> Cytoplasm
C. Capsule ---> Cell Wall ----> Cell Membrane ----> Ribosome
D. Capsule ---> Cell Wall ----> Ribosome ----> Cell Membrane
5. Bacteria of human pathogen are mostly found in the group _____________
A. Psychrophiles
B. Thermophiles
C. Mesophiles
D. Hyperthermophiles

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6. Cell wall of Mycobacterium is difficult to stain because

A. Acid fast cell wall resembles gram positive cell wall
B. It has a thick peptidoglycan component
C. The presence of waxy lipid in the cell wall
D. The presence of lipopolysaccharide in the cell wall
Part II: Essay Questions
Instruction: Attempt the following questions listed below
1. Bacteria are classified as gram positive and gram negative. What is a base of this
classification?
2. List different morphology and arrangement of bacteria.
3. Why Mycobactrium is distinct from the gram positive and gram negative bacteria?
4. Write the three architectural regions of prokaryotic cells and mention their examples.

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2.5. Common bacteria causing Diseases in humans

2.5.1. Gram positive cocci
These two most important genera of gram-positive cocci are medically important and cause wide
range of diseases.
 Staphylococcus
 Streptococcus
Staphylococcus
Organism:
 Genus:Staphylococcus
 Species:Staphylococcus aureus, Staphylococcus epidermidis
General Concepts:
 The staphylococci are divided in two groups based on the presence or absence of the
enzyme coagulase.
o S. aureus is coagulase-positive
o S. epidermidis (and other "non-pathogens") is coagulase-negative.
 Typically, staphylococci are opportunistic pathogens or saprophytes.
Distinctive Properties:
 Staphylococci are Gram-positive cocci usually arranged in clusters like a bunch of grapes.
 The morphologically similar streptococci can be differentiated from staphylococci by testing
for the enzyme catalase; staphylococci possess this enzyme while streptococci do not.
 Staphylococci possess both group specific and type specific antigens:
 Toxins produced by S. aureus include: hemolysins, leukocidins, enterotoxin, exfoliative toxin
and toxic shock syndrome (TSS) toxin.
 Extracellular enzymes produced by S. aureus include: coagulase, fibrinolysin, DNAse,
lipases and hyaluronidase.
Pathogenesis:
 Coagulase negative strains of Staphylococcus are generally non-invasive. Under
certain conditions, however, they may cause severe disease (e.g. S. epidermidis and
subacute endocarditis).

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 S. aureus is a common cause of boils, sties and skin infections. Serious (life-
threatening) infections (pneumonia, deep abscesses, meningitis) may occur in
debilitated persons.
 S. aureus is the most common cause of Gram-positive bacteremia, most commonly
involving hospital strains of the organism.
 S. aureus is also responsible for scalded skin syndrome and toxic shock syndrome. It
is the most common cause of food poisoning. Symptoms occur only a few hours
following ingestion of preformed enterotoxin but large amounts of toxin are required.
Laboratory diagnosis:
 Generally, a Gram stain of exudate from a lesion can demonstrate the characteristic
Gram-positive cocci arranged in clusters.
 Isolation techniques employ blood agar, mannitol salt agar or potassium-tellurite agar.
Bacteriophage testing or serotyping may be utilized.

Streptococcus
Organism:
 Genus:Streptococcus, Enterococcus
 Species:S. pyogenes (Group A), S. agalactiae (Group B), S. mutans (viridans), S.
pneumoniae, E. faecalis (Group D)
General Concepts:
 The streptococci are a very heterogeneous group of bacteria. Some members are a
part of our normal flora while others are potent pathogens.
 The primary pathogens are S. pyogenes and S. pneumoniae but other species can be
opportunistic.
 For example, S. agalactiae can produce severe neonatal disease including meningitis,
pneumonia and bacteremia in infants aged 7 days up to 3 months. E. faecalis may be
implicated in endocarditis and urinary tract infections. S. mutans is an important
contributor to dental caries.

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Distinctive Properties:
 Streptococci are Gram-positive, non-motile cocci that divide in one plane, producing
chains of cells. S. pneumoniae is a lancet-shaped diplococcus (formerly genus
Diplococcus).
 The streptococci are catalase negative (unlike Staphylococcus) and may be either
facultative or obligate anaerobes.
 Hemolysis (alpha, beta) on blood agar is an important differential characteristic.
 Lancefield groupings are based on the serology of cell wall polysaccharides (18
groups were originally established by Rebecca Lancefield).
 The M proteins of group A serve as important virulence factors that help the organism
resist phagocytosis.
 Lipoteichoic acids (LTA) mediate attachment to epithelial cells.
 Many antigenic moieties on the streptococcal cell surface resemble human muscle
and connective tissue and these similarities may be responsible for the late sequelae.
For example, S. pyogenes membrane Ags resemble cardiac, skeletal, smooth muscle,
heart valve fibroblasts and neuronal tissue.
 The capsule of S. pyogenes is composed of hyaluronic acid (like host connective
tissue) so it is non-antigenic while the capsule of S. pneumoniae is very antigenic and
is its sole virulence factor.
 Toxins produced by streptococci include: streptolysins (S & O), NADase,
hyaluronidase, streptokinase, DNAses, erythrogenic toxin (causes scarlet fever rash
by producing damage to blood vessels; requires cell to be lysogenized by phage that
encodes toxin).
Pathogenesis:
 S. pyogenes: is the leading cause of bacterial pharyngitis and tonsillitis. It may also
produce sinusitis, otitis, arthritis and bone infections. Some strains prefer skin,
producing either superficial (impetigo) or deep (cellulitis) infections.
 S. pneumoniae: is the major cause of bacterial pneumonia in adults. Its virulence is
dictated by its capsule.

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 Post-infection sequelae of S. pyogenes occur 1-3 weeks after acute disease. These
sequelae include i) acute rheumatic fever (following pharyngeal infections) and ii)
glomerulonephritis (following either pharyngeal or skin infections). These sequelae
may be due to an altered immune response (autoantibodies). Glomerulonephritis
results from deposition of Ag:Ab complexes on basement membrane of kidney
glomeruli.
 Other species/groups include:
o Group B strep (e.g. S. agalactiae): most often produce disease in animals but
are also the leading cause of neonatal septicemia and meningitis.
o Group D strep (e.g. E. faecalis): produce urinary tract infections and
endocarditis.
o Viridans species (e.g. S. mutans): are responsible for oral caries and subacute
bacterial endocarditis following dental surgery.
o Anaerobic streptococci may cause genital, brain or abdominal infections.
Diagnosis:
 Clinical:
o Diagnosis based solely upon symptomology is often not possible.
 Laboratory:
o To confirm the presence of S. pyogenes, throat swabs are used.
o For S. pneumoniae, sputum or blood samples are taken.
o The specimens may then be plated on blood agar for isolation of Gram-
positive, catalase-negative cocci.
o Useful characteristics for differentiation include the pattern of hemolysis,
bacitracin resistance or sensitivity and optochin resistance or sensitivity.
o Immunologically-based rapid test kits are often employed.
2.5.2. Pathogenic gram-positive rods
These three most important genera of gram-positive rods are medically important and cause wide
range of diseases.
 Bacillus
 Clostridium
 Clostridium

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Bacillus
Organism:
 Genus:Bacillus
 Species:anthracis, cereus
General Concepts:
 At least 48 species are known but only B. anthracis and B. cereus cause disease in
humans.
 B. anthracis is responsible for the disease anthrax. This is a disease primarily of
animals but humans can acquire via
o Handling
o inhaling or ingesting contaminated animal products.
 B. cereus is predominantly responsible for food poisoning in humans.
Distinctive Properties:
 Members of the genus Bacillusare Gram-positive, rod-shaped, spore-formers that
require oxygen. However, this is a very diverse group of organisms and some species
are actually Gram-negative or facultative.
 B. anthracisproduces a single antigenic type of capsule and several exotoxins.
 B. cereus produces enterotoxins that cause food poisoning.
Pathogenesis:
 Anthrax infections result only if the bacteria produce
o capsule, the capsule allows the bacteria to survive phagocytosis
o three exotoxins (all of which are required for virulence) include:
 edema factor (adenylate cyclase)
 protective antigen factor
 lethal factor.
 Anthrax infections are classified by route of entry:
o Cutaneous anthrax: Bacillus spores enter the skin through a cut or animal
bite and germinate. A small red lesion develops after 1-7 days, eventually
producing local necrosis (the "black eschar"). Spread of the bacteria causes
regional lymph tenderness which may be followed by a toxic septicemia and
death. Only about 5% of cutaneous infections become septic.

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o Inhalation anthrax: Bacillus spores are inhaled and ingested by alveolar

macrophages. These cells carry the bacteria to the regional lymph nodes,
causing necrotic hemorrhaging which leads to death.
o Gastrointestinal anthrax: Ingestion of contaminated meat produces
systemic symptoms which can lead to death. Mortality by gastrointestinal
anthrax may be 50%.
 B. cereus: food poisoning results from the ingestion of preformed enterotoxins,
producing predominantly vomiting and diarrhea. The vomiting form is most often
associated with ingestion of a heat stable toxin from contaminated rice, while the
diarrheal form is most often associated with ingestion of a heat labile toxin from
contaminated meat or vegetables.
Diagnosis:
 Clinical:
o Cutaneous anthrax may be suspected upon observing the characteristic "black
eschar" lesions.
o Inhalation and gastrointestinal anthrax are very difficult to diagnose based solely
on clinical presentation.
o B. cereus food poisoning may present in two different forms: the vomiting form
occurs within 1-6 hours (average 2 hours) following ingestion while the diarrheal
form occurs from 8-12 hours (average 9 hours) following ingestion.
 Laboratory:
o A Gram stain of lesion material or feces can indicate the presence of these Gram-
positive bacteria.
o Immunofluorescence techniques are also available.
Clostridium
Organism:
 Genus:Clostridium
 Species:C.perfringens, C.tetani, C.botulinum, C.difficile
General Concepts:

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 The clostridia are opportunistic pathogens. Nonetheless, they are responsible for some
of the deadliest diseases including gas gangrene, tetanus and botulism. Less life-
threatening diseases include pseudomembranous colitis (PC) and food poisoning.
 Clostridia cause disease primarily through the production of numerous exotoxins.
Distinctive Properties:
 Clostridium species are Gram-positive, rod-shaped, spore-formers. These generally
obligate anaerobes are ubiquitous saprophytes or part of our normal flora.
 Clostridia employ butyric fermentation pathways to generate energy and, as a result,
often produce a foul odor.
 C. perfringens: produces large rectangular spores and is non-motile. This species is
most often associated with wound infections but these are generally polymicrobic.
 C. tetani: produces terminal spores, giving it the appearance of a squash racket. This
species is motile and produces a single antigenic type of exotoxin.
 C. botulinum: produces oval subterminal spores and is motile. Different strains within
this species produce one of 8 exotoxin types (A,B,C1,C2,D,E,F,G). Types C and D
are encoded by bacteriophage that infect the bacteria.
 C. difficile: produces large oval subterminal spores and two different toxins; toxin A
(an enterotoxin causing fluid accumulation in the intestine) and toxin B (a cytopathic
agent). Ordinarily, this species can't compete with normal intestinal flora but, when
antibiotics eliminate this normal flora, C. difficile can flourish, producing disease.
Pathogenesis:
C. perfringens:
o Gas gangrene results from an anaerobic tissue environment caused by poor blood
supply due to trauma, surgery, etc. This acute disease is often fatal.
o One to six days following trauma, a generalized fever and pain is observed in the
affected area. This leads to rapid muscle necrosis because of the release of
bacterial exotoxins (lecithinases, hemolysins, collagenases, proteases, lipases).
o Gas gangrene generally involves muscle extremities where anaerobiosis can
occur.
C. tetani:
o Tetanus results from trauma or a puncture wound leading to tissue contamination.
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o Tetanus is a non-invasive disease occurring because of the release of exotoxins. It

produces a spasmogenic toxin that fixes to gangliosides thereby blocking the
release of the neurotransmitter glycine.
o Glycine normally prevents contraction of antagonistic muscles; therefore, muscle
spasms and convulsions (lockjaw) may occur.
o Cardiac failure can lead to death in approximately 55-65% of affected persons.
C. botulinum:
o Botulism results from the ingestion of bacterially produced neurotoxins.
o These toxins block the release of the neurotransmitter acetylcholine resulting in
double vision, slurred speech, decreased saliva, difficult swallowing and general
weakness.
o Paralysis with accompanying respiratory failure can be fatal in about 20% of
those affected.
o Botulism food poisoning can be observed about 18-36 hours following ingestion
of preformed toxin, which is heat labile.
o Infant botulism may occur via germination of spores in the intestinal tract with
subsequent toxin production, possibly accounting for some cases of Sudden Infant
Death Syndrome (SIDS).
C. difficile:
o Pseudomembranous colitis (PC) results predominantly as a consequence of the
elimination of normal intestinal flora through antibiotic therapy.
o Symptoms include abdominal pain with a watery diarrhea and leukocytosis.
o "Pseudomembranes" consisting of fibrin, mucus and leukocytes can be observed
by colonoscopy.
Diagnosis:
 Clinical:
o Gas gangrene: Symptomology and the presence of bacilli in the wound.
o Tetanus: Cramping and twitching around a wound, auditory hyperacuity and pain
in neck and jaw.
o Tetanus is similar to strychnine ingestion so must exclude the latter.

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o Botulism: Difficult to diagnose. Must demonstrate a normal cerebrospinal fluid

(CSF) to exclude other possibilities. The toxin is rarely found.
o Pseudomembranous colitis: Demonstration of pseudomembranous by
colonoscopy is diagnostic.
 Laboratory:
o Members of the genus Clostridium can be differentiated from other bacteria by
laboratory techniques including
 enzymatic digestion on egg-yolk agar plates and by using mice treated
with or without antitoxin.

Corynebacterium
Organism:
 Genus:Corynebacterium
 Species:diphtheriae
General Concepts:
 Corynebacteria belong in the family Mycobacteriaceae and are part of the CMN group
(Corynebacteria, Mycobacteria and Nocardia).
 The family Mycobacteriaceae are Gram-positive, nonmotile, catalase-positive and have a
rodlike to filamentous morphology (Corynebacteria are often pleomorphic).
 As a group, they produce characteristic long chain fatty acids termed mycolic acids. In
the image to the right, the R-groups represent these chains. For Corynebacteria, chains of
28-40 carbons are common; for Nocardia, chains of 40-56 carbons are produced; for
Mycobacteria, the chains are 60-90 carbons in length.
Distinctive Properties:
 Corynebacterial cell walls contain thin spots which lead to some Gram variability and
"ballooning" that produces a "club-shaped" cell. Old cells store inorganic phosphate,
which can appear as metachromatic granules when stained.
Pathogenesis:
 C. diphtheriae is the etiologic agent of diphtheria.

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 These organisms colonize the mucus membranes of the respiratory tract and produce the
enzyme neuraminidase which splits N-acetylneuraminic acid (NAN) from cell surfaces to
produce pyruvate which acts as a growth stimulant.
 C. diphtheriae also produces diphthin, which is a protease that inactivates IgA.
 Toxigenic strains carry the gene tox, which resides on certain bacteriophages;
lysogenization leads to toxigenicity.
Diagnosis:
 Clinical: Muscle weakness, edema and a pseudomembranous material in the upper
respiratory tract characterizes diphtheria.
 Laboratory: Tellurite media is the agar of choice for isolation of Corynebacteria, which
produce jet black colonies.
2.5.3. Pathogenic Gram-Negative Cocci & Coccobacilli
These following genera are the most medically importantgram-negative cocci &
Coccobacilliwhich cause wide range of diseases.

 Neisseria
 Haemophilus
 Bordetella
Neisseria
Organism:
 Genus:Neisseria
 Species:gonorrhoeae, meningitidis
General Concepts:
 Neisseria inhabit mucosal surfaces. There are 2 species that are pathogenic for humans:
1. N. gonorrhoeae. Also referred to as the gonococcus, it is responsible for the
disease gonorrhea.
2. N. meningitidis. Also referred to as the meningococcus, it is responsible for
meningitis.
Distinctive Properties:

 Neisseria are Gram-negative diplococci with their adjacent sides flattened.

 These organisms are aerobic, strongly oxidase-positive, have an oxidative metabolism,
are susceptible to drying and are fastidious (growth is inhibited by free fatty acids).
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 N. meningitidis is serotyped by the antigenic character of its capsular polysaccharide

Pathogenesis:
 Gonorrhea is a sexually transmitted disease. The sites of infection include the urethra (in
men) and the cervix (in women).
 Fimbriae (pili) are very important for the gonococcus to attach to host cells. N.
gonorrhoeae lacking fimbriae are avirulent.
 N. gonorrhoeae produce cytotoxic substances that damage ciliated epithelial cells in
fallopian tubes; the LPS endotoxin may be partly responsible.
 The virulence of N. meningitidis is associated with its antiphagocytic capsule.
 N. meningitidis produces proline-threonine and proline-serine proteases, but any
particular isolate will only produce one or the other.
Diagnosis:
 Clinical
o The symptoms of gonorrhea differ between the sexes. In men, a copious urethral
exudate containing Gram- negative diplococci is common. In women, disease is often
asymptomatic.
o The symptoms of meningitis usuallybegin abruptly with headache and fever.
However, confirmation of meningococcal infection requires bacteriologic culture.
 Laboratory:Neisseria may be cultured on Thayer-Martin agar or other suitable media with
incubation in 10% CO2. Neisseria are strongly oxidase-positive, Gram-negative diplococci.
N. gonorrhoeae oxidizes glucose only; N. meningitidis oxidizes both glucose and maltose.

Haemophilus
Organism:
 Genus:Haemophilus
 Species:Haemophilus influenzae
General Concepts:
 Haemophilus influenzae is responsible for producing a variety of infections including
meningitis and respiratory infections.
 Six serological types (a,b,c,d,e,f) based on the antigenic structure of the capsular
polysaccharides are recognized. Nonencapsulated strains are (by definition) nontypable.

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 Other species of Haemophilus include:

o H. parainfluenzae (pneumonia, endocarditis)
o H. ducreyi (venereal chancre)
o H. aegypticus (conjunctivitis).
Distinctive Properties:
 The genus Haemophilus is composed of Gram-negative coccobacilli.
 These organisms are fastidious and require factors X (hemin) and/or V (NAD).
 Haemophilus possess LPS in the cell wall but produce no apparent extracellular toxins.
Pathogenesis:
 The organisms colonize the nasopharynx and are spread by direct contact. Haemophilus
are capable of penetrating the epithelium to produce a bacteremia that may lead to
localization of the organisms in many organs.
 H. influenzae type b is the most common cause of bacterial meningitis in children aged 6
months-2 years. It is uncommon in adults because of protective antibody.
 Cellulitis, conjunctivitis, epiglottitis and arthritis may also result from Haemophilus
infection.
Laboratory diagnosis:
 A Gram stain of cerebrospinal fluid may reveal the organisms. One can also detect
capsular material directly.
 The organisms are cultured on chocolate agar because it contains both factors X and V.
Incubation in 10% CO2 is required.
Bordetella
Organism:
 Genus:Bordetella
 Species:pertussis, parapertussis, bronchiseptica
General Concepts:
 The genus Bordetella is responsible for respiratory disease in humans and animals.
 B. pertussis, in particular, is the etiologic agent of pertussis, more commonly known as
whooping cough.
 B. parapertussis causes a milder form of pertussis, while B. bronchiseptica mostly affects
animals but occasionally humans.
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 Bordetella causes disease by producing toxins that impair ciliary function in the
respiratory tract.
Distinctive Properties:
 Bordetella is Gram negative coccobacilli. They produce a capsule and are strict aerobes.
Only B. bronchiseptica is motile.
 Bordetella possess the heat stable endotoxin LPS and produce several exotoxins. These
include:

1. Pertussigen: A 120 kD protein exhibiting the A-B model for toxin activity.
Pertussigen is an ADP-ribosyl-transferase that interferes with the transfer of
signals from cell surface receptors. Pertussigen is also involved in mediating
attachment to respiratory epithelia.
2. Adenylate cyclase toxin: this toxin increases cAMP levels, inhibiting immune
effector cell functions.
3. Tracheal cytotoxin: This toxin causes ciliostasis and extrusion of ciliated
epithelia.
4. Dermonecrotic toxin: This heat labile substance causes tissue destruction.
5. Filamentous hemagglutinin: This is involved in attachment to host cells.
Pathogenesis:
 Whooping cough results from colonization and multiplication of Bordetella pertussis on
the mucus membranes of the respiratory tract, in particular, the ciliated epithelial cells.
 Production of toxins irritates cells causing ciliostasis and leukocytosis.
 The hallmark of pertussis is the spasmatic cough that may last 6 weeks. Occasional
secondary complications include encephalopathy, seizures and pneumonia.
Diagnosis:
 Clinical:
o Whooping cough requires a 7-14-day asymptomatic incubation period.
o This is followed by the catarrhal stage, which lasts 1-2 weeks. Symptoms include
fever, rhinorrhea and a highly infectious cough.

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o The next 2-4 weeks define the paroxysmal phase, during which the spasmatic
("whooping") cough is observed. Vomiting and leukocytosis (> 100,000
lymphocytes/mm3) are also evident.
o Finally, the convalescent phase marks the end of disease and may last 3-4 weeks or
longer. During this period, secondary complications may occur.
 Laboratory: The organisms can be grown on Bordet-Gengou agar media after 3-4 days
incubation. Immunological techniques may also be employed.
2.5.4. Pathogenic Gram-negative rods
This a group Enterobacteriaceae which are Gram negative rods, found primarily in colon of
humans and animals. The members of this groups are:Escherichia,Enterobacter,Klebsiella,
Citrobacter,Salmonella,Providencia,Shigella,Serratia,Proteus,Yersinia,Morgella,Edwardsi
ella and Hafni.Of these the following very essential to deal with.
 Salmonella
 Shigella
 Escherichia

Salmonella
Organism:
 Genus:Salmonella
 Species:typhi, enteritidis, cholerae-suis
The diseases produced by different species of Salmonella are collectively known as
"salmonelloses". These diseases occur worldwide and are most generally manifested as a self-
limiting gastroenteritis.
 Salmonellae are pathogenic because of their capacity to
o invade intestinal mucosa and
o Produce toxins.
 The salmonellae infect a variety of animals, resulting in a large animal reservoir.
o S. typhi is more specific to humans, however.
 Approximately 2000 serotypes of Salmonella are known.

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Distinctive Properties:
 The genus Salmonella is a member of the family Enterobacteriaceae. The genus is
composed of Gram-negative bacilli that are facultative and flagellated (motile).
 Salmonellae possess 3 major antigens;
o the "H" or flagellar antigen (phase 1 & 2),
o the "O" or somatic antigen (part of the LPS moiety)
o the "Vi" or capsular antigen (referred to as "K" in other Enterobacteriaceae).
 Salmonellae also possess the LPS endotoxin characteristic of Gram-negative bacteria.
Pathogenesis:
 Salmonellosis may present as one of several syndromes including gastroenteritis, enteric
(typhoid) fever or septicemia.
 Disease is initiated by oral ingestion of the bacteria followed by colonization of the lower
intestine. The bacteria are capable of mucosal invasion, which results in an acute
inflammation of the mucosal cells. This then leads to the activation of adenylate cyclase,
increased fluid production and release of fluid into the intestinal lumen, resulting in
diarrhea.
 Salmonella gastroenteritis is the most common form of salmonellosis and generally
requires an 8-48-hour incubation period and may last from 2-5 days.
 Symptoms include
o nausea,
o vomiting
o diarrhea
 Enteric or typhoid fever occurs when the bacteria leave the intestine and multiply within
cells of the reticuloendothelial system. The bacteria then re-enter the intestine, causing
gastrointestinal symptoms. Typhoid fever has a 10-14-day incubation period and may last
for several weeks. Salmonella typhi is the most common species isolated from this
salmonellosis.

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Diagnosis:
 Clinical: Clinical diagnosis of the salmonellosis is often difficult because the symptoms
closely resemble other diarrheal diseases. Isolation of the organism is required for
positive identification.
 Laboratory: Salmonella can be readily isolated and characterized using standard
bacteriologic media or rapid identification systems. Salmonellae are motile, incapable of
fermenting lactose and produce H2S. Serological techniques may be used for
epidemiological characterization.

Shigella
Organism:
 Genus:Shigella
 Species:dysenteriae
General Concepts:
 Shigella dysenteriae is responsible for bacillary dysentery, a disease most often
associated with crowded, unsanitary conditions.
 Other species of Shigella may produce milder forms of diarrheal disease.
Distinctive Properties:
 Shigellae are facultative, non-motile, Gram-negative bacilli. They possess the heat stable
endotoxin (LPS) characteristic of Gram-negative bacteria.
 Shigellae are pathogenic primarily due to their ability to invade intestinal epithelial cells.
 S. dysenteriae also produces a heat labile exotoxin that is a neurotoxin acting upon the
gray matter of the central nervous system.
Pathogenesis:
 Dysentery is an oral infection transmitted via fecal contamination of water or food.
During the 1-4-day incubation period, penetration of bacteria into the mucosal epithelial
cells of the intestine causes an intense irritation of the intestinal wall, producing cramps
and a watery, bloody diarrhea.

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Diagnosis:
 Clinical: As with other diarrheal diseases, clinical diagnosis alone is equivocal. Diarrhea,
fever and a watery bright red blood tinged stool are classical symptoms, but isolation
of the organisms is required for confirmation.
 Laboratory:
o Shigella can be readily isolated and characterized using standard bacteriologic media
or rapid identification systems.
o Shigellae are non-motile, incapable of fermenting lactose and do not produce H2S.
Serological techniques may be used for epidemiological characterization.

Escherichia
Organism:
 Genus:Escherichia
 Species: Escherichia coli
General Concepts:
 Members of the genus Escherichia are common bacteria that colonize the human large
intestine. Most are opportunistic normal flora but some are potent pathogens.
 Transmission of diarrheal disease is generally person to person, usually related to
hygiene, food processing and sanitation.
 Four general categories of pathogenic E. coli are recognized:
1. Enterotoxigenic (ETEC)
2. Enteroinvasive or "Shigella-like" (EIEC)
3. Enteropathogenic (EPEC)
4. Enterohemorrhagic (EHEC)
 Different groups are most often delineated by serology, in particular, by the immunogenic
character of the O (somatic, LPS) and H (flagellar) antigens.
Distinctive Properties:
 Escherichia are Gram-negative bacilli that ferment lactose. Most are motile by
peritrichious flagella.
 Escherichia possess a typical Gram-negative cell wall containing LPS.

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 Approximately 170 different O antigens have been delineated and some of these are
cross-reactive with Shigella, Salmonella and Klebsiella.
 Motile strains possess H (flagellar) antigens that can be used for epidemiologic purposes.
 Escherichia also possess K (capsular) antigens similar to the Vi antigen of Salmonella.
 Enterotoxigenic strains may also display colonization factor antigens (CFA/I, CFA/II).
Pathogenesis:
 E. coli diarrhea is generally acquired via ingestion of water or food that has been
contaminated by an infected person. The following table outlines the four major
classifications:
Classification(Strain) Site of Infection Disease(s) Pathogenisis
Enterotoxigenic Small intestine Traveler's diarrhea Enterotoxins ST and LT
(ETEC) Watery stool, cramps,
nausea, low fever
Enteroinvasive Large intestine Shigella-like diarrhea Tissue invasion and
(EIEC) Fever, cramps, watery destruction of epithelial
diarrhea followed by cells (plasmid-mediated)
scant, bloody stool
Enteropathogenic Small intestine Infantile diarrhea Adherence and
(EPEC) Salmonella-like with destruction of epithelial
fever, nausea, vomiting cells (plasmid-mediated)
Enterohemorrhagic Large intestine Hemorrhagic colitis SLT-I, SLT-II cytotoxins
(EHEC, O157:H7) Severe abdominal pain, ("verotoxins")
watery diarrhea
followed by grossly
bloody stool

Diagnosis:
 Clinical: Generally, clinical diagnosis of E.coli infection is equivocal. The
enterotoxigenic and enteropathogenic forms because a watery diarrhea and nausea while

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the enteroinvasive and enterohemorrhagic forms subsequently produce bloody stools.

These presentations alone, however, are not sufficient for confirmation.
 Laboratory:
o E. coli infection might be generally suspected in absence of isolation of Salmonella or
Shigella or intestinal parasites.
o The organisms themselves are easily isolated and identified by routine procedures.
o Specialized serotyping may be necessary for epidemiologic studies.

Pseudomonas
Organism:
 Genus:Pseudomonas
 Species:Pseudomonas aeruginosa, others
General Concepts:
 Greater than 140 species of Pseudomonas have been described and most are saprophytic.
 In terms of human disease, Pseudomonas is generally an opportunistic pathogen.
However, the genus is responsible for several specific diseases including glanders (P.
mallei) and melioidosis (P. pseudomallei).
 Pseudomonas infections may be serious in hospitalized patients or those with cancer or
cystic fibrosis.
Distinctive Properties:
 Pseudomonads are Gram negative rods. They are motile, nonfermentative aerobes that
can utilize acetate for carbon and ammonium sulphate for nitrogen. Many species are
resistant to high salt, dyes, weak antiseptics and most antibiotics. P. aeruginosa can grow
at 42°.
 Pseudomonas possesses the LPS endotoxin characteristic of other Gram-negative bacteria
and displays O and H antigens.
 Also, a protein called exoenzyme S is another ADP-ribosyltransferase, transferring the
ADP-ribose from NAD to other proteins (not EF-2).
 Pseudomonas has an antiphagocytic polysaccharide slime layer and many strains produce
pigments, some which are fluorescent.

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Pathogenesis:
 Pseudomonas produces localized infections following surgery or burns. Localized
infections can lead to generalized, and occasionally fatal, bacteremia.
 Pseudomonas is also responsible for a number of nosocomial infections including urinary
tract infections following catheterization, pneumonia resulting from contaminated
respirators, and eye and ear infections.
Diagnosis:
 Clinical: Diagnosis is very difficult.
 Laboratory: Pseudomonas can be easily isolated on blood agar. These organisms are
non-fermentative (oxidative), oxidase-positive, Gram-negative bacilli. They often give
off a fruity odor and some may produce fluorescent pigments. P. aeruginosa grows well
at 42°.
Vibrio
Organism:
 Genus:Vibrio
 Species:cholerae
General Concepts:
 The name Vibrio derives from the Latin because these curved rods possess a single polar
flagellum and appear "to vibrate".
 V. cholerae was first isolated in pure culture in 1883 by Robert Koch.
 V. cholerae produces the disease cholera, defined as "a metabolic disturbance of the
epithelial cells of the small bowel". Cholera is caused, in part, by a potent enterotoxin
(choleragen) and is usually a disease of poor sanitation.
 Humans are the only natural host for this organism and there have been 6 great
pandemics of cholera.
Distinctive Properties:
 The genus Vibrio is composed of Gram negative, curved rods that are motile by means of
a single polar flagellum.
 These organisms are sensitive to acid pH but tolerate alkaline pH (9.0-9.6) very well.

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Pathogenesis:
 The acid sensitivity of V. cholerae means that a large dose is required to produced
disease. Indeed, 1011 vibrios given orally fail to produce illness but if bicarbonate (e.g.
Alka-Selzer®) precedes the inoculation, then only 104 are required.
 Cholera is a disease of the small intestine, unlike most other enteric illnesses. The
bacteria penetrate the mucus layer and adhere to the mucosal cells where they
subsequently produce toxin. The potent enterotoxin choleragen is well defined. A
cholera patient may secrete 20 liters of fluid per day with 108 vibrios per ml!
Campylobacter
Organism:
 Genus:Campylobacter
 Species:Campylobacterjejuni
General Concepts:
 The genus Campylobacter is a relatively recently discovered important human pathogen.
The reason for this is that the organisms are microaerophilic, requiring low
concentrations of oxygen only.
 Indeed, Campylobacter infections occur more often than Salmonella and Shigella
diarrheas combined.
Distinctive Properties:
 Campylobacters are Gram-negative, curved rods (the name derives from the Greek
"campylo", meaning curved). These organisms are microaerophilic and motile.
 Campylobacters possess a typical Gram-negative cell wall containing LPS endotoxin.
 There are approximately 50 heat-labile "K" (capsular) and "H" (flagellar) antigens and 60
different heat-stable "O" (somatic) antigens associated with different species of
Campylobacter.
 These organisms are able to use amino acids and citric acid cycle intermediates for
growth. C. jejuni grows best at 42°.
Pathogenesis:
 A relatively small inoculum is required to cause illness; as few as 800 bacteria can
produce disease in healthy persons.

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 Illness generally occurs following a 2-4-day incubation period when the bacteria multiply
in the intestine, reaching numbers similar to Salmonella and Shigella infections (106-109
per gram of feces).
 Symptoms resemble an acute enteritis with
o Fever
o Diarrhea
o Nausea
o Abdominal pain.
 The illness is generally self-limiting but may last a week.
 C. jejuni appears to produce an enterotoxin similar to both the cholera and Escherichia
coli toxins.
Diagnosis:
 Clinical: Clinical diagnosis is difficult since the symptomology is non-specific.
 Laboratory:
o Special methods are required for isolation.
o Growth occurs in 5% O2, 10% CO2, 85% N2 at 42°.
o A Gram stain of fecal material may reveal curved ("seagull" or "comma") shaped
organisms.
2.5.5. Genus Spirochetes
These are medically important organisms under genus spirochetes which could bethe causative
agents for many diseases.
 Treponema
 Borelli
 Lepetospira
Treponema
Organism:
 Genus:Treponema
 Species:pallidum, pertenue, carateum
General Concepts:
 Three "treponematoses" are discussed: syphilis, yaws and pinta.

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 Each of these diseases is characterized by distinct clinical stages. These stages are known
as primary, secondary and tertiary.
o The primary stage involves multiplication of the bacteria at the site of entry to
produce a localized infection.
o The secondary stage occurs following an asymptomatic period and involves
dissemination of the bacteria to other tissues.
o The tertiary stage may occur after 20-30 years.
 The Treponema are highly invasive organisms; T. pallidum is the most invasive of the
species, T. carateum the least invasive.
Distinctive Properties:
 The Treponema are motile, helically coiled organisms having a corkscrew-like shape.
They stain very poorly because their thickness approaches the resolution of the light
microscope.
 Treponema is delicate organisms requiring pH in the range 7.2 to 7.4, temperatures in the
range 30°C to 37°C and a microaerophilic environment.
 The structure of these organisms is somewhat different: the cells have a coating of
glycosamino-glycans, which may be host-derived, and the outer membrane covers the
three flagella that provide motility.
 In addition, the cells have a high lipid content (cardiolipin, cholesterol), which is unusual
for most bacteria. Cardiolipin elicits "Wassermann" antibodies that are diagnostic for
syphilis.
 Treponema possess a complex antigenic makeup that is difficult to determine because the
organisms cannot be grown in vitro.
Pathogenesis:
 Treponema pallidum is capable of infecting all body tissues.
 The disease caused by T. pallidum is syphilis. This is a relatively painless, slowly
evolving disease. The host-parasite relationship leads to short symptomatic periods when
the organism multiplies, followed by prolonged asymptomatic periods when host
responses produce healing. Syphilis is strictly a person-person disease.

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 An incubation period of from 10 to 90 days precedes the clinical presentation, despite the
fact that the organisms disseminate immediately. The prominent feature of the disease is
vascular involvement, particularly arterioles and capillaries.
 Treponemal antigen-host antibody complexes may cause some immunosuppressant of the
host and production of the distinct clinical stages:
o The primary stage occurs weeks to months following infection. The principal sign of
infection is
 the hard chancre, generally found on the genitals.
 this lesion is essentially painless but filled with treponemes
 highly contagious.
o The secondary stage occurs following an asymptomatic period of 2-24 weeks. In this
stage,
 a skin rash spreads from the palms and soles towards the trunk. This rash may
last 2-6 weeks and is followed by recovery.
 on average, about 25% of patients experience relapses of the secondary stage.
o Following the secondary stage is a period of latency which may last many years and
during which there are essentially no clinical symptoms.
o The tertiary stage may erupt following the period of latency and can affect all areas of
the body and be fatal.
 Cardiovascular and neurological involvement are the most frequent causes of
death.
 Typically, however, the appearance of lesions called "gummas" mark the
tertiary stage.
 These lesions are, in fact, large granulomas resulting from hypersensitivity
reactions and they can be extremely disfiguring.
 Syphilis that occurs in utero is termed congenital syphilis. About 50% of such fetuses
abort or are stillborn.
Diagnosis:
 Clinical: The clinical manifestations of the treponematoses are generally characteristic
and readily identified.

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 Laboratory: Darkfield examination of material from a chancre can show the presence of
spirochetes. Immunological techniques including fluorescent treponemal antibody (FTA)
or T. pallidum immobilization (TPI) can be of great assistance in observing the
organisms. The Wassermann test looks for the presence of antibody against cardiolipin.
Many other tests are also available.
Leptospira
Organism:
 Genus:Leptospira
 Species:interrogans
General Concepts:
 As the name implies, Leptospira are spiral shape organisms.
 The diseases produced by Leptospira are termed "leptospirosis" and can vary from
subclinical to fatal.
 Leptospirosis is a zoonosis; man is an accidental host via contaminated animal urine.
 The leptospirosis known as "Weil's disease" was first described in 1886.
Distinctive Properties:
 Leptospira are thin, tightly coiled obligate aerobes that are highly motile.
 Their structure is similar to other spirochetes; a multilayered outer membrane, helical
shaped peptidoglycan and flagella located in the periplasmic space.
 Their nutritional requirements include long-chain fatty acids and vitamins B1 and B12.
 There are more than 180 serotypes of Leptospira described.
Pathogenesis:
 Mucosa and broken skin provide the entry for leptospires.
 The organisms produce a generalized infection with bacteremia (leptospiremic phase).
Antibody is produced and the organisms then become localized primarily in the kidneys.
Multiplication in the kidneys leads to shedding in the urine (leptospiruric phase). This
may persist for weeks, months or years.
 Leptospira produce no known exo- or endotoxins.
 Damage to the endothelial lining of capillaries and renal failure are the most common
reasons for death. Occasionally the central nervous system may become involved.

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 The host immune response is probably responsible for lesions associated with late phase
of disease. This is suggested because antibiotics are ineffective after symptoms have
persisted for more than 4 days.
 Antibody plus complement is leptospiricidal. Immunity against Leptospira is primarily
humoral.
 The cell-mediated response may be responsible for late manifestations.
Diagnosis:
 Clinical: Leptosirosis is a general febrile disease that is often misdiagnosed as meningitis
or hepatitis. Following a 7-14-day incubation period, patients experience fever, severe
headache, pain and occasional jaundice. Symptoms last about 7 days then subside. The
leptospiruric phase then lasts for several days before complete recovery.
 Laboratory: Darkfield microscopic examination of the blood or urine combined with
serologic tests is confirmatory.
Borrelia
Organism:
 Genus:Borrelia
 Species:recurrentis, hermsii, burgdorferi
General Concepts:
 Borreliae produce febrile diseases characterized by remittent fever.
 The organisms are transmitted to humans by lice or ticks.
 B. recurrentis produces epidemic relapsing fever (lice); B. hermsii causes endemic
relapsing fever (ticks); B. burgdorferi is the agent responsible for Lyme disease (ticks).
Distinctive Properties:
 The Borreliae are similar to Leptospira but somewhat fatter and have more complex
nutritional requirements.
 The cell wall contains various lipids including cholesterol.
Pathogenesis:
 Borreliae produce a generalized infection following an incubation period of about 1
week. Symptoms include fever, headache and muscle pain that lasts 4-10 days and
subsides. An afebrile period lasting 5-6 days follows and then there is a recurrence of
acute symptoms.
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 Epidemic relapsing fever (transmitted by lice) is generally more severe than endemic
relapsing fever (transmitted by ticks) and has an approximately 40% mortality if
untreated. Also, the epidemic form is generally characterized by having a single relapse,
while the endemic form may have several relapses due to cyclic antigenic variation of the
Borrelia.
 Lyme disease (transmitted by ticks) involves the production of ulcerative lesions on the
skin and may lead to arthritis or neurologic involvement.
Diagnosis:
 Clinical:
o The symptomology of the recurrent fevers is not specific enough for accurate clinical
diagnosis. With Lyme disease, however, the occurrence of a "bulls-eye" lesion on the
skin (erythema chronicum migrans, ECM) is almost always (85%) associated with
infection.
o Among cases that show ECM, about 20% progress to include arthralgia, about 50%
involve intermittent episodes of arthritis and 10% progress to chronic arthritis.
 Laboratory: Darkfield smears can be used to observe the relapsing fever Borrelia but
serologic tests (ELISA) are a better determinant for Lyme disease.

Mycobacterium

Organism:
 Genus:Mycobacterium
 Species:tuberculosis, leprae
General Concepts:
 Mycobacteria belong in the family Mycobacteriaceae and are part of the CMN group
(Corynebacteria, Mycobacteria and Nocardia).
 The family Mycobacteriaceae are Gram-positive, nonmotile, catalase-positive and have a
rodlike to filamentous morphology (Corynebacteria are often pleomorphic).
 As a group, they produce characteristic long chain fatty acids termed mycolic acids.
Distinctive Properties:
 Mycobacteria are considered "acid-fast", which means that they retain dyes following an
acid-alcohol decolorization step.
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 These organisms are very slow growers; a 5-hour division time is not uncommon.
 Different species can be differentiated based on growth rate, niacin secretion, reduction
of nitrate, caratogenesis, etc.
 Mycobacteria produce "cord factors", which are dimycolates of trehalose. This gives rise
to a pattern of growth in serpentine cords.
 Mycobacteria are called Acid-Fast Bacilli (AFB) due to their microscopic appearance
after decolorizing.

Fig- 2.8 Mycobacterium tuberculosis, rod shape

 Organisms appear red on a blue background
 M. leprae has never been cultured in vitro but can be grown on the footpads of
armadillos.
Pathogenesis:
 M. tuberculosis is the agent responsible for the disease tuberculosis.
 Initial symptoms are similar to those seen in other respiratory infections – it is important
to look for:
o Fever
o Fatigue
o Weight loss
o Chest pain
o Shortness of breath
o Congestion with coughing

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 Virulent strains of Mycobacteria have the capacity to disrupt phagosomal membranes of

alveolar macrophages while the cord factors inactivate mitochondrial membranes of
phagocytes. These properties enable the organisms to survive and multiply in phagocytes.
 Leprosy is caused by M. leprae and may appear in one of two forms; tuberculoid or
lepromatous.
Diagnosis:
 Clinical: Tuberculosis is characterized by a prolonged and productive cough.
 Laboratory:
o Microscopic demonstration of AFB in sample (acid-fast stain on sputum can reveal
the bacteria using microscope)
o Growth of TB bacilli in culture
o Molecular diagnosis
o Skin Test- A skin test can ascertain exposure, but only where the Bacille Calmette-
Guerin (BCG) vaccine is not used regularly.
o X-Ray
Rickettsia
Organism:
 Genus:Rickettsia, Rochalimaea, Coxiella
 Species:Rickettsia prowazekii (epidemic typhus), Rickettsia typhi (endemic typhus),
Rickettsia rickettsii (spotted fever), Rochalimaea quintana (trench fever), Coxiella
burnetii (Q fever)
General Concepts:
 The Rickettsia are Gram-negative, obligate intracellular bacteria that infect mammals and
arthropods.
 R. prowazekii is the agent of epidemic typhus.
 Rocky Mountain spotted fever and Q fever remain relatively common.
Distinctive Properties:
 These organisms are small, pleomorphic coccobacilli about 2 µm in length. Their
structure is typical of Gram-negative bacteria.
 Rickettsia replicate in the cytoplasm and nucleus of their host cell; Coxiella replicate only
in the phagolysosome.

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Pathogenesis:
Disease caused by Rickettsiae
 Epidemic typhus:
o Occurs in epidemics & is transmitted by body louse.
o Initial symptoms of the disease are headache and fever 6-15 days after being exposed
to R. prowazekii.
o Macular rash start after 4-6 days of illness on the trunk and axillary folds & then
spread to the extremities
o The disease is fatal especially in old age.
o The Weil Felix reaction is positive with proteus OX-19
 Brill-Zinsser disease
o Relapse of louse-borne typhus that commonly occurs many years after the primary
infection.
o R. prowazekii remains sequestrated in cells of reticuloendothelial system.
o Antibody titre to proteus OX-19 are absent.Such individuals are immune to a 2nd
infection.
 Murine typhus (endemic typhus):
o The causative organism is R. typhi.
o The vector for human infection is the rat flea. Human is accidental hosts.
o The disease rambles louse-borne typhus in pathogenesis, symptomatology &
serology.
 Replication of the bacteria causes lysis of the host cell and consequent spread to other
cells.
 Initial replication occurs at the site of entry producing a local lesion. This is followed by
dissemination via the vascular system producing vasculitis and a skin rash. These lesions
may become necrotic.
 Virulence is probably due to many factors including release of endotoxin, the production
of immune complexes and hypersensitivity reactions.
 A characteristic triad of symptoms includes fever, headache and rash (no rash with Q
fever).

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Diagnosis:
 Clinical: These diseases present as febrile illnesses after exposure to arthropods or animal
hosts or aerosols in endemic areas and are easily misdiagnosed. A delay in diagnosis may
be partly responsible for the high mortality from Spotted fever. The spread of the rash is
often characteristic: spread from the trunk to the extremities (centrifugal) is typical for
typhus; spread from the extremities to the trunk (centripetal) is typical for spotted fever.
 Laboratory:
o The use of immunofluorescent antibodies.
o The Weil-Felix test looks for the production of serum antibody that is reactive against
Proteus OX19, OX2 or OXK antigens but it is not always reliable.

Chlamydia
Organism:
 Genus:Chlamydia
 Species:trachomatis, psittaci
General Concepts:
 The Chlamydia are obligate intracellular parasites.
 C.trachomatis is responsible for the diseases trachoma, inclusion conjunctivitis,
lymphogranuloma venereum (LGV) and nongonococcal urethritis (NGU). In other
words, oculourogenital infections.
 C. psittaci produces systemic diseases including psittacosis, ornithosis and pneumonitis.
Distinctive Properties:
 The Chlamydiahas an unusual developmental cycle that involves two distinct forms:
infectious elementary bodies and intracellular reticulate bodies. Elementary bodies attach
and are internalized by susceptible host cells. Once inside, they reorganize into a
replicative form (the reticulate body). Over a 24-hour period, these reticulate bodies
divide and begin to reorganize back into elementary bodies. About 48-72 hours after
infection, the cell is lysed and numerous infectious elementary bodies are released.
 The genome of Chlamydia is only 25% the size of E. coli, making it one of the smallest
prokaryotes.
 The pathogenic mechanisms employed by Chlamydia are not well understood.

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Pathogenesis:
 C. trachomatis is spread via the fingers to the urogenital area and vis versa.
 In contrast, C. psittaci is acquired from infected birds, usually via the respiratory route.
 Trachoma is an infection of the epithelial cells of the conjunctiva, producing inclusion
bodies. Vascularization and clouding of cornea along with trichiasis (inward growth of
eyelashes) can produce scarring that may lead to blindness.
 Inclusion
o conjunctivitis is a milder form that occurs in both children and adults. This form
generally heals without scarring or blindness.
o Sexually transmitted nongonococcal urethritis (NGU) is similar to gonorrhea and
occurs with greater frequency.
o In men, a condition termed lymphogranuloma venereum (LGV) involving inguinal
lymphadenopathy ("buboes") can occur.
o Psittacosis is a respiratory disease ranging from influenza-like to pneumonia-like and
is generally acquired from infected birds.
Diagnosis:
 Clinical: Diagnosis of trachoma is usually good. Likewise, the genital vesicles associated
with LGV are characteristic. NGU can only be suspected in the absence of laboratory
findings.
 Laboratory:
o Iodine stained specimens usually show inclusion bodies that represent the replicating
bacteria.
o The Chlamydia can be cultured in tissue culture and appropriate serological tests
performed.

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Summary
In general, the following groups of bacteriaare the common disease causing pathogens in
humans.
 Gram positive cocci (Staphylococcus and Streptococcus)
 Pathogenic Gram-positive rods (Bacillus, Clostridium and Clostridium)
 Pathogenic gram-negative cocci &coccobacilli (Neisseria, Haemophilus and Bordetella
 Pathogenic gram negative rods (Salmonella, Shigella and Escherichia
 Genus Pseudomonas
 Genus Vibrios
 Genus Campylobacter, Genus Mycobacteria, Genus Spirochetes (Treponema, Borellia
and Lepetospira), Genus Chlamydia, Genus Corynebacterium and Genus Rickettsia were
dealt with general concepts, their distinctive features, pathogenesis and their
laboratory and clinical diagnosis.
Self-check questions:
Instruction: please! attempt all the questions listed below
Part – I: Multiple Choices
1. Cocci bacteria arranged in irregular, grape like, groups are known as ___________
A. Streptococci C. Coccobacilli
B. Diplococcic D. Staphylococci
2. One of the following wrongly describes Clostridium tetani
A. It produces exotoxin which is associated with muscle spasm
B. Its growth requires high concentration of oxygen in the tissue
C. Its diagnosis is mainly based on clinical presentations
D. It is acquired through puncture wounds and trauma from the soil
3. Infection of certain bacteria caused urethritis with yellow, creamy pus and painful
urination among a man having multiple sexual partners. A gram stained smear of
urethral discharge revealed many Gram negative diplococci within pus cells. This
presumptive diagnosis showed that the pathogenic organism
was______________________________
A. Neisseria gonnorheae C. Streptococcus pneumoniae
B. Neisseria meningitis D. Staphylococcus aureus

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4. From a suspected case of meningitis, an organism with the feature of “Gram negative
intracellular diplococci” was reported from the laboratory after a gram staining
procedure was applied on the CSF sample collected from the case. What would be the
etiology of meningitis?
A. Neisseria gonnorheae C. Streptococcus pneumoniae
B. Neisseria meningitis D. Streptococcus pyogenes
5. Delousing and improvement in hygienic conditions are tools for the prevention of one
of the following infectious disease
A. Syphilis C. Tuberculosis
B. Relapsing fever D. Lyme disease
6. Louse borne typhus is a febrile illness caused by
A. Salmonella paratyphi C. Salmonella typhi
B. Rickettsia prowazekii D. Borreliaspecies
7. A clinical sample for the diagnosis of leprosy is
A. Sputum C. Urine
B. Slit skin snip D. Stool
8. In the case of Borrelia infection, fever relapses because
A. Borrelia recurrentis causes epidemic relapsing fever
B. Epidemic and endemic relapsing fever follow the same clinical patterns
C. Borrelia changes its antigenic structures and antibodies are no longer effective
D. Human body lice transmit epidemic relapsing fever
9. It is a gram positive non-spore forming bacilli associated with a pseudomembrane
formation on the oropharnyx which may extend to trachea causing respiratory tract
obstruction. The organism with the above mentioned feature is
A. Corynebacterium diphtheria C. Bacillus anthracis
B. Clostridium perfringens D. Bacillus cereus
10. The most prevalent disease condition due to the infection of Bacillus anthracis is
A. Cutaneous anthrax B. Pulmonary anthrax
C. Gastrointestinal anthrax D. Wool sorter’s disease
11. Gas gangrene is mostly associated with
A. Clostridium tetani B. Clostridium botulinum

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C. Bacillus anthracis D. Clostridium perfringens

12. The most potential pathogenic strain of Haemophilus influenziae, to which vaccine has
been developed, is
A. Haemophilus influenziae type ‘a’ C. Haemophilus influenziae type ‘c’
B. Haemophilus influenziae type ‘b’ D. Haemophilus influenziae type ‘f’
13. T.pallidum is difficult to see by light microscope because
A. It is a gram negative bacteria C. It is too thin
B. It has endoflagella D. It has outer membrane
14. The stage of syphilis where there are no clinical manifestations but continuing infection
is evidenced by serologic tests
A. Primary syphilis C. Tertiary syphilis
B. Secondary syphilis D. Latent syphilis

Chapter 3: Medical Mycology

Dear trainees,
In this chapter, you will learn the features, classification and morphology of fungi. In
addition, you will learn the overview of fungal diseases as well as the general approaches
of laboratory diagnosisof fungal diseases.
 Objectives:
At the end of this chapter, you will be able to:
 Define terms (such as mycology, medical mycology, mycotoxicology, and mycosis).
 Describe features of fungi.
 Discuss general approaches in classifying fungi.
 Describe morphology of fungal elements.
 Describe the overview of fungal diseases.
 Describe laboratory methods in diagnosing fungal diseases
3.1.Introduction to Mycology
The term mycology was derived from Greek word Mykos, meaning Mushroom. Mycology
(Myco = fungus; -logy = study) is the study of fungi. Thus, as a subject, mycology deals with the
following features of fungi.
– their taxonomic classification

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– their use to humans as source for medicine

– their use in food production (e.g., beer, wine)
– their use as source of food (edible mushrooms)
– their dangers such as poisoning or infection
– their biochemical properties
– their genetic properties
3.2.Basic terms in mycology
 Medical Mycology: Study of these fungi pathogenic to humans.
 Mycologists: scientists who study fungi.
 Mycotoxicology: study of fungal toxins and their effects.
 Mycoses: diseases caused by fungi

Importance of fungi:
 Fungi inhabit almost every niche in the environment
 Humans are exposed to these organisms in various fields of life
 Fungi are beneficial as well as harmful to human
3.3.General characteristics of fungi
 Fungi are eukaryotic organisms. They are more evolutionarily advanced forms of
microorganisms as compared to the prokaryotes.
 Vegetative body of fungi may be unicellular (e.g. yeasts) or multicellular (e.g. moulds)
 Fungi reproduce by means of spores, usually wind-disseminated
 Both sexual and asexual spores may be produced in fungi depending on the species and
conditions
 Typically, fungi are not motile, although a few (e.g. Chytrids) have a motile phase
 Like plants, fungi may have a stable haploid & diploid states
 Fungal cell walls are composed of mostly of chitin, a distinctive feature of fungi, and
glucan
 Fungi have complex cytoplasm with internal organelles, microfilaments and microtubules
 Fungi are heterotrophic(“other feeding,” must feed on preformed organic material)
 Most fungi store their food as glycogen (like animals) while plants store food as starch

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 Fungal cell membranes have a unique sterol, ergosterol, which replaces cholesterol
found in mammalian cell membranes
 Fungi are insensitive to antibacterial antibiotics
3.4.Classification of fungi
 Fungi were initially classified with plants. In 1969 R.H Whittaker classified all living
organisms into five kingdoms (Monera, Protista, Fungi, Plantae and Animalia).
Traditionally the classification proceeds in this fashion:
 Kingdom-Subkingdom - Phyla/phylum - Subphyla - Class - Order - Family - Genus-
Species.However, this traditional means of fungal classification is too complicated to be
dealt, and there are alternate and more practical approaches. These are:
 Based on sexual reproduction of fungi
 Based on morphology of fungi (vegetative structure)
A. Based on Sexual reproduction, fungi can be classified as:
1. Zygomycetes: which produce through production of zygospores
2. Ascomycetes: which produce endogenous spores called ascospores in cells called
asci
3. Basidiomycetes: which produce exogenous spores called basidiospores in cells called
basidia
4. Deuteromycetes (Fungi imperfecti): fungi that are not known to produce any sexual
spores
B. Morphological classification of fungi
Based on morphology, fungi exist in two fundamental forms: hyphae, the filamentous fungi, and
yeast, the single celled budding forms. But for the sake of classification, they are studied as
moulds, yeasts, yeast like and dimorphic fungi.
1. Moulds (Molds): Filamentous fungi Eg: Aspergillus spps, Trichophyton rubrum
2. Yeasts: Single celled cells that buds Eg: Cryptococcus neoformans, Saccharomyces
cerviciae
3. Yeast like: Similar to yeasts but produce pseudohyphae Eg: Candida albicans
4. Dimorphic: Fungi existing in two different morphological forms at two different
environmental conditions. They exist as yeasts in tissue and in vitro at 37°C; and as
moulds in their natural habitat and in vitro at room temperature.

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Eg: Histoplasma capsulatum, Blastomyces dermatidis, Paracoccidiodes brasiliensis,

Coccidioides immitis
3.5.Morphology of fungi
Fungi: Are generally larger than bacteria, with individual cell diameters ranging from 1 to 30
µm. They can be:
 Monomorphic, existing as single celled yeast
o are usually large
o single cells (5 to 8 µm in diameter
o rarely form filaments.
o colonies are usually characterized by a smooth surface

Fig 3.1 Yeast cell

 Multi-cellular filamentous mold: or dimorphic (di= two, morph = form) existing as yeast
or mold.
o Is a microscopic fungus that forms large, multi-cellular aggregates of long tube like
o
o branching filaments called hyphae and mold spores.

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(a) septate hyphae (b) aseptate hyphae

Fig-3.2 Septate and aseptate of mold
3.6.Overview of fungal diseases
Fungal Allergies
 Molds grow on any damp organic surface, and spores are constantly in the air.
 Spores and volatile fungal toxins may play a role in “sick building syndrome”.
 Fungal allergies are common; and generally occur in individuals with other allergies.
Mycotoxcosis
 Some fungi produce toxic substances that poison a person who ingest them.
 These poisonous substances are collectively called mycotoxicosis (myco= fungus, toxin=
poison).
 Mycotoxcosis may result from ingestion of fungal contaminated foods. Example –
Poisonous mushroom
 Most fungal toxins are produced when the fungus grows in moist environment at
relatively high temperature.
Fungal infection:
 Fungi cause some of the most persistent and disfiguring disease still prevalent throughout
the world.
 Diseases caused by fungi are collectively called mycoses (singular, mycosis).
 The incidence of the disease (infection) is related to the degree of exposure to fungi in
living conditions, occupation, and leisure activities and to immune status.

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 There are four general categories on the basis of the primary tissue affinity of the
pathogen.
Superficial mycoses:
 Superficial mycoses are fungal infections confined to the outer deadest layers of skin,
hair and nails.
 Agents of superficial mycoses are fungi that colonize the keratinized outer layers of the
skin, hair, and nails. Example is Malassezia furfur. Infections due to these organisms
elicit little or no host immune response and are nondestructive and thus asymptomatic
and infections are generally painless.
 Symptoms superficial mycoses may include discoloration, scaling, or de-pigmentation of
the skin. They are usually of cosmetic concern only and are easy to diagnose and treat.

Cutaneous Mycoses:
 Some fungi have particular affinity for the keratin of the skin, nails, and hair. These fungi
are known collectively as the dermatophytes, and all possess the ability to cause disease
in humans and/or animals. All have in common the ability to invade the skin, hair, or
nails.
 The disease caused by these organisms is called dermatophytosis or "dermatomycosis.”
The term dermatophytosis refers to a complex of diseases caused by any of several
species of taxonomically related filamentous fungi in the genera Trichophyton,
Epidermophyton, and Microsporum.
 In each case, these fungi are keratinophilic and keratinolytic and so are able to break
down the keratin surfaces of the skin, hair, or nails. The various forms of
dermatophytosis are referred to as "tineas" or ringworm. Symptoms the diseases include:
 Itching, Scaling or ring like patches of the skin;
 Brittle or broken hairs;
 Thick discolored nails.
Subcutaneous mycoses:
 Are infections confined to the subcutaneous tissue (dermis or fatty tissues), the deeper
layer of the skin often muscle tissue
 Man is an accidental host following inoculation of fungal spores via some form of trauma

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 Tentatively identified by the presence of a characteristic tissue reaction or granule

Systemic mycoses:
Systemic mycoses affect the deep tissues and organ systems which are caused by pathogenic
and/or opportunistic fungi often creating symptoms.Two categories of fungi cause systemic
diseases.
A) Truly pathogenic fungi: those causing diseases in normal human host when the inoculums
are of sufficient size.
 Examples of truly pathogenic fungi are: Histoplasma capsulatum, Blastomyces
dermatitidis, Coccidioides immitis and Paracoccidioides brasiliensis.
B) Opportunistic fungi: are fungi which cause infections in patients who are
immunocompromised; the organisms require the patient's defenses to be lowered to cause
diseases.
o Examples of opportunistic fungi are Aspergillus species, Candida albicans and
Cryptococcus neoformans.
3.7.Laboratory diagnosis of fungal infections
Introduction:
The diagnosis of fungal infection depends entirely on the selection and collection of an
appropriate clinical specimen. The proper collection of specimen and their transport to the
clinical laboratory are of major importance for the recovery of fungi. In many instances,
specimens not only contain the etiological agent but also contaminating bacteria or fungi that
will rapidly overgrow some of the slower growing pathogenic fungi. Since overgrowth with
contaminating organism is common, it is important to transport the specimen to the clinical
laboratory as soon as possible.
1.7.1 Specimens used for the diagnosis of fungal infection include
 Respiratory tract secretions
 Cerebrospinal fluid (CSF)
 Blood and bone marrow aspirates
 Tissue biopsies from visceral organs
 Urine and urogenital specimen
 Skin, nail, hair scrapings and swabs
1.7.2 Laboratory examination methods of fungal infections
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Microscopy and culture are the main methods in which fungal infections are diagnosed in
the laboratory. In addition to these methods, there are others like immunological methods
that help in the diagnosis of fungal infections.
 Direct Microscopic methods
o Direct examination of clinical specimens could be stained or unstained.
o Direct unstained method includes saline mount and Potassium hydroxidepreparation
(10-20% KOH mount).
o The saline wet mount
 It is useful to observe the presence of fungal elements including budding
yeast, hyphae, and pseudohyphae.
 Advantage: it is a quick and simple method.
 Disadvantage: Lack of contrast which makes identification of fungal elements
somewhat difficult.
o KOH preparation
 It helps in the initial examination of keratinized tissue suspected of fungal
infection. It is useful to observe the presence of characteristic fungal elements
including hyphae, budding yeast and spherules. Do not use cotton swabs to
collect specimens since cotton fibers may resemble hyphae.
 Fungal elements may be obscured by skin, hair, or nail tissue. Thus KOH (10-
20%w/v) dissolves keratin in skin, hair or nail specimens facilitating the
observation of the organism’s morphology.
 Advantage: Rapid and simple to perform.
 Disadvantage: Has poor contrast, and clearing of some specimens may
require an extended time.
o Staining methods include
o Calcofluor:
 Calcofluor white is used as counter stain to detect fungal elements in exudates
and small skin scales.
 It can also be added to KOH solution to enhance the visibility of fungus.
o Lacto phenol cotton blue (LPCB):
 It is used to visualize microscopic fungal morphology by imparting a blue

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color to the cell walls.

o Indian ink preparation:
 It is used to identify the capsule of Cryptococcus neoformans,theyeast
cell.Capsules appear as clear halos against a dark back ground.
 Advantage: It is rapid method.
 Disadvantage: it may be difficult to interpret as White blood cells and
artifacts can be mistaken for yeast or capsules.
o Gram stain:
 Fungi stain gram positive but may often stain poorly. For example, C.
neoformans may appear pale lavender with blue inclusions because its capsule
prevents adequate staining. Candida is best demonstrated in clinical specimen
by Gram stain.
 Fungi are two to three times the size of gram- positive cocci, with the hyphae
often two to three times wider than gram- positive bacilli.
o Giemsa or wright’sstain:
 It technique is commonly used for the detection of intracellular Histoplasma
capsulatum in, thick blood film or bone marrow smear.
 The organism appears as a small, oval yeast cell staining light to dark blue in
color. C.neoformans also stains quite well with this method.
o Gomori Methanamine Sliver Nitrate stain:
 The stain provides good contrast and staining for the fungal elements. Fungi
are stained dark gray to black against a pale background.
o Periodic Acid-Schiff (PAS) stain:
 It stains the hyphae of molds and also some yeast. Periodic acid oxidizes the
hydroxyl in the carbohydrates of the cell walls of the organisms to form
aldehydes. The aldehydes react with basic fuschin dye to form a pink- purple
complex.
 Culturing methods
o Microscopic methods should be confirmed with cultures or antigen testing when
available.
o Culture media for the cultivation of fungi must include source of nitrogen, source of

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carbon and other supplements.

– Source of nitrogen such as nitrate, nitrite, amino acids or urea.
– Carbon source which is usually glucose, vitamins and minerals .
– Other supplements such as blood and anti-micorobial may also be added.
o Sabouraud's Dextrose agar is a general isolation medium and allows the growth of
most fungi. It contains peptone and glucose.
o Other specialized media used for different fungi include:
 Brain Heart Infusion
 Inhibitory Mould Agar
 Caffeic Acid Agar and Birdseed
 Corn Meal Agar
 Trichophyton Agars
 Dermatophyte Test Medium
 Sabhi Medium
 CHROM agar Candida
 Serological methods
o Antibody detection: is the detection of anti-fungal antibody which is helpful in the
diagnosis of subcutaneous and systemic mycoses.
o Antigen detection
 It is particularly useful in the diagnosis of cryptococcal meningitis from
CSF specimens.
 It is also helpful in the detection of Aspergillus and Candida antigens in
systemic infections.
 Skin tests: Delayed hypersensitivity reactions to fungal antigens can be demonstrated by
skin tests.
 Molecular techniques: Newer techniques such as DNA hybridization and PCR can be used
for the diagnosis of fungal infection.
Self-check questions:
Instruction: Dear trainees, please, attempt all the questions listed below.
1. Define medical Mycology.
2. Define fungi.

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3. List and explain the general characteristics of fungi.

4. Discuss mold and yeast form of the fungi.
5. Discuss fungal disease
6. Dimorphic fungi exhibit different morphological appearances at certain temperature
ranges. Fill the blank spaces for the questions (a – d) below saying either Mould form
or Yeast form at a specified temperature ranges.
a) At room temperature, it exhibits _____________________
b) Inside human body, it exhibits ______________________
c) At 37oC in the laboratory, it exhibits _________________
d) At 25oC in the laboratory, it exhibits __________________
7. Write the four morphological groups of fungi and explain them.

Summary
 Fungi are found almost everywhere in the environment and humans are exposed to these
organisms in various fields of life.
 Fungi are eukaryotic organisms; they are more evolutionarily advanced forms of
microorganisms as compared to the prokaryotes. Fungal elements are either unicellular (e.g.
yeasts), or multicellular (e.g. moulds) organisms.
 The traditional taxonomic classification of fungi is difficult to understand; hence, the more
practical and simpler approaches are used. These are based on sexual reproduction and
morphology of fungi.
 In general, fungal infections are classified based on the site of the body affected: superficial
mycoses, cutaneous mycoses, subcutaneous mycoses and systemic mycosis.
 The diagnosis of fungal infection depends entirely on the selection and collection of an
appropriate clinical specimen. Different laboratory methods such as direct microscopy,
culturing and serological tests are used for the diagnosis of fungal infections.

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Chapter 4: Medical Virology

Dear trainees,
In this chapter, you will learn the features and classification viruses. In addition, you will
learn the transmission routes of viruses as well as the phases of viral replication. Laboratory
diagnostic methods of viral diseases and details of common pathogenic viruses will also be
covered.
Objectives:
At the end of this chapter, you will be able to:
 Describe features of viruses.
 Discuss general approaches in classifying viruses.
 Explain transmission routes of viruses.
 Explain phases of viral replication.
 Describe laboratory methods in diagnosing viral diseases.
 List and describe common pathogenic DNA and RNA viruses.
4.1 Introduction to virology
Virology is the study of viruses. Viruses are very tiny, simple particles. In fact, they are so tiny
that they can only be seen with a special microscope called an electron microscope, and they are
so simple that they are technically not even considered ‘alive’. The structure of a virus is
extremely simple and is not sufficient for an independent life.
4.2 General Features of viruses

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 Viruses do not have cellular organization, i.e. they are acellular.

 Smallest living units measuring from 20nm –300nm.
o Largest virus is Poxvirus (300nm)
o Smallest virus is Polio virus, and foot & mouth disease virus (20nm)
 Viruses possess only one type of nucleic acid, either DNA or RNA.
 Viruses are obligate intra-cellular parasites, i.e. viruses are inert outside the host cell.
 Most viruses lack enzymes needed for protein or nucleic acid synthesis; they depend on
host living cells (use host machinery).
 Viruses do not multiply by binary fission, but multiply by budding
 Viruses can infect all forms of life (bacteria, plants, protozoa, fungi, insects, fish, reptiles,
birds, and mammals)
 Viruses cause a large number of human diseases
 Viruses are not affected by antibiotics
4.3 Structure of virus
 Viralcore:
o Found in the centre of the virus
o It is composed of viral nucleic acid, either DNA or RNA, may be double or single
stranded.
o It is used to control the viral heredity and variation, and also responsible for the
infectivity.
 ViralCapsid
o The capsid accounts for most of the virion mass. It is the protein coat of the virus. It is
a complex and highly organized entity which gives form to the virus. Subunits
called protomers which aggregate to form capsomers which in turn aggregate to form
the capsid.
o It surrounds the viral genome, outer of the viral core. It is protein in nature.
o It is symmetric in arrangement: helical symmetry; cubic or icosahedra symmetry;
complex symmetry.
o Functions of viral capsid:
 Protects the viral nucleic acid

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 Participates in the viral infection

 Shares the antigenicity
 Viral envelope
o This is an amorphous structure composed of lipid, protein and carbohydrate which
lies to the outside of the capsid. It contains a mosaic of antigens from the host and the
virus.
o Not all viruses have the envelope. Viruses can be divided into two kinds based on the
presence or absence of envelope: Enveloped virus and naked virus. A naked virus is
one without an envelope
o The functions of viral envelope are: antigenicity, infectivity and resistance; some
viruses possess neuraminidase.
 Viral spikes
o These are glycoprotein projections which have enzymatic and/or adsorption and/or
hemagglutinating activity. They arise from the envelope and are highly antigenic.
Diagram: Structure of a typical virus:

Fig 4.1 Viral main structure

4.4 Classification of viruses
A number of criteria or approaches are used for the classification of viruses.
 Based On Nucleic Acid

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o RNA viruses: Examples: Measles, Mumps, Respiratory syncytial virus (RSV),

Parainfluenza viruses (PIV), Rabies, Ebola, Influenza, HIVviruses etc.
o DNA viruses: Examples: smallpox (variola), vaccinia, varicella-zoster, herpes
viruses, Adenoviruses; etc.
 Based On Morphology (Symmetry)
o Icosahedral- the protomeres aggregate in groups of five or six to form
the capsomere. In electron micrographs, capsomeres are recognized as regularly
spaced rings with a central hole. The shape and dimensions of the icosahedron
depends on characteristics of its protomeres. All icosahedral capsids have 12 corners
each occupied by a penton capsomere and 20 triangular faces, each containing the
same number of hexon capsomeres. Icosahedral symmetry is identical to cubic
symmetry.

Fig 4.2 Virus Morphology

o Helical: the protomeres are not grouped in capsomeres, but are bound to each other
so as to form a ribbon-like structure. This structure folds into a helix because the
protomeres are thicker at one end than at the other. The diameter of the helical capsid
is determined by characteristics of its protomeres while its length is determined by the
length of the nucleic acid it encloses.
o Complex: this group comprises all those viruses which do not fit into either of the
above two groups. e.g. that exhibited by poxvirus and rhabdovirus.

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Fig 4.3 Virus complex structure

4.5 Viral replication
Exposure of the host to the virus:
Usually viral infection occurs when a virus enters the host through a physical breach (a cut in the
skin), direct inoculation (e.g. mosquito bite), or direct infection of the surface itself (inhalation of
the virus into trachea). It is usually only after a virus enters a host that it can gain access to
possible susceptible cells.
 Viral Entry: For the virus to reproduce and thereby establish infection, it must enter cells
of the host organism and use those cells' materials. To enter the cells, proteins on the
surface of the virus interact with proteins of the cell. Attachment or adsorption occurs
between the viral particle and the host cell membrane. A hole forms in the cell
membrane, then the virus particle or its genetic contents are released into the host cell,
where viral reproduction may commence.
 Viral replication: Next, a virus must take control of the host cell's replication
mechanisms. It is at this stage a distinction between susceptibility and permissibility of a
host cell is made. Permissibility determines the outcome of the infection. After control is
established and the environment is set for the virus to begin making copies of it,
replication occurs quickly.
 Viral shedding: After a virus has made many copies of itself, it usually has exhausted
the cell of its resources. The host cell is now no longer useful to the virus, therefore the
cell often dies and the newly produced viruses must find a new host. The process, by
which virus progeny are released to find new hosts, is called shedding. This is the final
stage in the viral life cycle.

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 Viral latency: Some viruses can "hide" within a cell, either to evade the host cell
defences or immune system, or simply because it is not in the best interest of the virus to
continually replicate. This hiding is deemed latency. During this time, the virus does not
produce any progeny; it remains inactive until external stimuli such as light or stress
prompts it to activate.
 Below is the summarized viral replication cycle:
o Adsorption-Viruses can enter cells via phagocytosis, viropexis or
adsorption. Adsorption is the most common process and the most highly specific
process. It requires the interaction of a unique protein on the surface of the virus with
a highly specific receptor site on the surface of the cell.
o Penetration-This occurs by one or more processes. Enveloped viruses fuse their
envelope with the membrane of the host cell. This involves local digestion of the viral
and cellular membranes, fusion of the membranes and concomitant release of the
nucleocapsid into the cytoplasm. Naked viruses bind to receptor sites on the cellular
membrane, digest the membrane and enter into the cytoplasm intact. Both naked and
enveloped viruses can be ingested by phagocytic cells. However, in this process they
enter the cytoplasm enclosed in a cytoplasmic membrane derived from the phagocytic
cell.
o Uncoating -During this stage cellular proteolytic enzymes digest the capsid away
from the nucleic acid. This always occurs in the cytoplasm of the host cell. The
period of the replication cycle between the end of the uncoating stage and maturation
of new viral particles is termed the eclipse. Thus during the eclipse stage, no complete
viral particles can be viewed within the cell.
o Replication of nucleic acid: Replication of viral nucleic acid is a complex and
variable process. The specific process depends on the nucleic acid type.
o Maturation and Release
 Naked viruses -Maturation consists of two main processes: the assembly of
the capsid, and its association with the nucleic acid. Maturation occurs at the
site of nucleic acid replication. After they are assembled into mature viruses,
naked virions may become concentrated in large numbers at the site of
maturation, forming inclusion bodies. Naked virions are released in different

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ways, which depend on the virus and the cell type. Generally, RNA-
containing naked viruses are released rapidly after maturation and there is
little intracellular accumulation; therefore, these viruses do not form
predominant inclusion bodies. On the other hand, DNA-containing naked
icosahedra viruses that mature in the nucleus do not reach the cell surface as
rapidly, and are released when the cells undergo autolysis or in some cases are
extruded without lysis. In either case they tend to accumulate within the
infected cells over a long period of time. Thus, they generally produce highly
visible inclusion bodies.
 Enveloped viruses -In the maturation of enveloped viruses, a capsid must first
be assembled around the nucleic acid to form the nucleocapsid, which is then
surrounded by the envelope. During the assembly of the nucleocapsid, virus-
coded envelope proteins are also synthesized. These migrate to the plasma
membrane (if assembly occurs in the cytoplasm) or to the nuclear membrane
(if assembly occurs in the nucleus) and become incorporated into that
membrane. Envelopes are formed around the nucleocapsids by budding of
cellular membranes.
 NOTE: Enveloped viruses will have an antigenic mosaicism characteristic of the virus
and the host cell. Viruses are slowly and continuously released by the budding process
with the results that: (a) the cell is not lysed; and (b) little intracellular accumulation of
virus occurs; and (c) inclusion bodies are not as evident as with naked viruses.
 Complex viruses -These viruses, of which the poxvirus is a good example, begin the
maturation process by forming multilayered membranes around the DNA. These layers
differentiate into two membranes: The inner one contains the characteristic nucleoid,
while the external one acquires the characteristic pattern of the surface of the
virion. These form very characteristic cytoplasmic inclusion bodies. The viruses are
generally released from the cell via cell lysis.

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Fig 4.4 General viral replication cycle

 Products of viral replication:

o Virion: the complete infectious unit of virus particle.
o Defective virus: deficiency in some aspects of replication, but interferes with the
replication of normal viruses.
o Abortive infection: viruses enter into cells, but cannot bio-synthesize their
components or not assemble virions.
4.6 Transmission routes of viruses
 The route of transmission depends on the source of the virus (the tissue site of viral
replication and secretion) and the ability of the environment and the body route to the
target tissue.
 Non-enveloped viruses (naked viruses) can withstand drying, the effect of detergents, and
extremes of PH and temperature. Non-enveloped viruses are generally transmitted by the
respiratory and fecal-oral routes and can often be acquired from contaminated objects.
 Unlike the non-enveloped viruses, enveloped viruses are comparatively fragile. They
require an intact envelope for infectivity. These viruses must remain wet and are spread:

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o A. In respiratory droplets, blood, mucus, saliva, or semen.

o By injection
o By organ transplantation
 Animals can also act as vectors that spread viral disease to other animals or humans.
 Animals can also be reservoirs.
 Viral diseases that are shared by animals or insects and humans are called zoonoses.
 Arthropods, including mosquitoes, ticks, and sand flies can act as vectors for toga
viruses, flavi-viruses, bunya viruses, and reo-viruses. These viruses are often referred to
as arbo-viruses because they are arthropod borne.

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4.7 Laboratory diagnosis of viral diseases

In general, diagnostic tests can be grouped into 3 categories.
1. Direct detection
2. Indirect examination (virus isolation), and
3. Serology
4.7.1 Direct Examination of Specimen
 In direct examination, the clinical specimen is examined directly for the presence of virus
particles, virus antigen or viralnucleic acids.
 Methods for direct examination are:
o Electron Microscopy morphology / immune electron microscopy
o Light microscopy histological appearance - e.g. inclusion bodies
o Antigen detection immunofluorescence, ELISA etc.
o Molecular techniques for the direct detection of viral genomes
4.7.2 Indirect Examination
 In indirect examination, the specimen is inoculated into cell culture, eggs or animals in an
attempt to grow the virus. This is called virus isolation.
4.7.3 Serology
 This involves the detection of rising titres of antibody for viruses.
 The majority of common viral infections can be diagnosed by serology.
4.8 Common pathogenic DNA and RNA viruses
4.8.1 DNA VIRUSES
General characteristics:
o double stranded except parvovirus
o are icosahedral, except pox viruses which are brick shaped or complex symmetry
o replicate their DNA in the nucleus except poxvirus
I. Adenoviruses
o Adenoviruses are double-stranded DNA viruses and belong to the family
Adenoviridae
o There are 51 different serotypes of adenoviruses (each designated by a number)
o Several disease syndromes associated with different serotypes
Route of transmission or spread

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o Respiratory adenoviruses are spread by the respiratory route.

o Enteric adenoviruses (adenovirus 40 and 41) are spread via the faecal–oral route
o Adenoviruses causing conjunctivitis are very infectious and spread by direct
contamination of the eye. Patients are infectious while they are symptomatic.
Reservoir: ubiquitous in humans and animals
Incubation period: 2–5days.
At-risk groups: Immunocompromised persons who often have prolonged carriage of
the virus especially in enteric infections
Symptoms:
o Respiratory adenoviruses cause a range of respiratory symptoms from mild coryza
to pneumonia. Clinical symptoms include fever, cough and sore throat due to
pharyngitis and tonsillitis. Some infections are asymptomatic. It is difficult to
differentiate adenovirus infection from other respiratory virus infections
symptomatically, although adenoviruses, unlike influenza viruses, do not usually
produce myalgia.
o Some adenoviruses can also cause a maculopapular rash. Rarely death occurs due
to disseminated adenovirus infection.
o Enteric adenoviruses cause diarrhea, vomiting and fever, particularly in children
less than 2 years of age. The diarrhea lasts for an average of 8 days (range 3–
11days) longer than diarrhea caused by rotaviruses.
o Ocular adenoviruses cause conjunctivitis with red, sore infected conjunctiva. It is a
very infectious condition and scrupulous infection-control procedures are
necessary to prevent spread, particularly by the direct-contact route.
Laboratory diagnosis: PCR and Viral culture
Prophylaxis: There is no prophylaxis available.
II. Herpes simplex virus (HSV1 and HSV2)
o Herpes simplex virus is a double-stranded DNA virus and a member of the
Herpesviridae family.
Route of transmission or spread
o Close personal contact (kissing, sexual contact) and has incubation period of 2–12
days (mean of 4 days).

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Reservoir: human mucosa and ganglia

At-risk groups: these include immunocompromised persons and neonates.
Symptoms:
o Primary HSV infection occurs when a person first encounters the virus.
o It first infects an area of skin then the virus travels down a sensory nerve and
establishes a latent infection in the dorsal route ganglia and becomes dormant.
o Most infected persons don't experience a reactivated infection where the virus
reactivates and causes another clinical episode at the site of the primary infection.
o Herpes simplex virus can infect various sites in the body. The most severe
infection is HSV encephalitis which has a 70% mortality rate if untreated.
Infection in immunocompromised patients can be severe or fatal.
o Neonatal herpes: Babies who acquire HSV infection from the mother’s genital
tract at the time of delivery are at risk of severe or fatal neonatal HSV infection.
Body sites infected Manifestations
Skin HSV causes fluid-filled skin blisters (vesicles) which can sometimes be
difficult to distinguish from varicella-zoster virus (VZV) infection.
Mouth and lips Primary infection in children often occurs in the mouth and on the lips.
Infection can be missed unless severe gingivostomatitis is present.
Reactivate dinfection is usually seen as a cold sore on the lip.
Genitals HSV causes vesicles and shallow ulcers on the labia, cervix, and penis
and perianal area. Herpes simplex virus type 2 is more frequently found
that HSV type 1.
CNS Encephalitis is the most severe HSV infection which is often severe or
fatal and requires prompt treatment and diagnosis. Meningitis usually
associated with HSV type 2.
Eye HSV causes keratoconjunctivitis in the eye. Repeated reactivated
infection of the cornea can cause corneal scarring and blindness.
Laboratory diagnosis
o Virus culture takes 1–3 days to get a positive result.
o Polymerase chain reaction (PCR) can provide a result in a few hours.

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o HSV antibody tests are not useful in the diagnosis of acute HSV infection but are
helpful for epidemiological studies.
III. Varicella-Zoster Virus
Transmission is through respiratory droplets. Incubation period is 10–23 days (mean
of 14 days). Infectious period is from 2 days before the onset of symptoms until 5 days
after the rash or all the skin lesions are fully crusted.
Reservoir is human mucosa and nerves.
Diseases: chicken pox and shingles
At-risk groups
o Immunocompromised persons and Pregnant women
o Unborn babies in the first 20 weeks of pregnancy, and babies one week before or
after delivery.
Prevention
o Vaccination
o Varicella-zoster immunoglobulin for post exposure prophylaxix of
immunocompromised
IV. Hepatitis B virus
o Hepatitis B virus (HBV) is a member of the Hepadnaviridae family and has a
double-stranded circular DNA.
o It has two major proteins: hepatitis B surface antigen (HBsAg) which is an outer
protein expressed in excess when the virus replicates in the liver; and hepatitis B
core antigen, an inner protein, which is expressed only within hepatocytes in the
liver.
o A third protein, hepatitis B envelope antigen (HBeAg) is also shed in the blood
when the virus replicates, and its presence is associated with high infectivity.
Route of transmission or spread
o The routes of transmission are parenteral (blood exposure), Sexual and Vertical
(from mother to baby).
o Infection can develop from 6 weeks to 6 months after exposure to the virus.
Infectivity is related to the presence of HBs Ag in the blood. Hepatitis BeAg is a

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marker of high infectivity whereas the presence of antibody to HBe Ag (anti-HBe)

denotes low or absent infectivity.
Laboratory diagnosis
o Serology: usually detection of HBsAg in serum
Prevention
o An effective recombinant vaccine made of the outer viral protein is available for
both pre- and post-exposure prophylaxis for hepatitis B virus.
4.8.2 RNA VIRUSES
General characteristics
o All are single stranded except reoviruses
o Most are enveloped except picornavirus, calcivirus, reovirus
i. Poliovirus
o Poliovirus is human enterovirus which is a causative agent of poliomyelitis. It is a
member of the family of Picornaviridae.
o Poliomyelitis (often called polio or infantile paralysis) is an acute viralinfectious
disease which spreads from person to person primarily via the fecal-oral route.
o Prevention is through oral polio vaccine or intramuscular polio vaccine.
ii. Rhinoviruses
o Are single-stranded RNA viruses and belong to the Picornaviridae family.
o The name rhinovirus derived from the fact that these viruses infect the nasal
passages, and they are one of several causes of the ‘common cold’. There are over
100 serotypes of rhinovirus.
o Route of spread is by direct contact with respiratory secretions, fomites and large
droplets through the nose and eyes.
o Incubation period is 1–3 days. Infected individuals are usually infectious for about
5 days when they are sneezing and have a nasal discharge.
Laboratory diagnosis
o Diagnosis is usually clinical and laboratory investigations are normally not
indicated.
Prophylaxis and prevention
o There are no viral vaccines available for preventing rhinovirus infections.

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o The spread of rhinoviruses can be reduced by strict hand washing after patient
contact.
iii. Hepatitis A virus (HAV)
o HAV is a single-stranded RNA virus which belongs to the genus Hepatovirus in
the family Picornaviridae.
o Spread by the faeco–oral route through eating and drinking of contaminated food
and water
o Rarely may it be transmitted through blood transfusion through a viraemic donor.
o Average incubation period is 2–6 weeks. Virus is shed in the faeces of the infected
individual from two weeks before jaundice develops to about one week after the
jaundice. Maximum amount of virus is shed before the jaundice develops;
therefore, patients are most infectious in the late incubation period.
o Infection is almost always self-limiting and chronic infection with hepatitis A does
not occur. Rarely deaths do occur, mainly in elderly patients and those with
chronic liver disease.
o Fulminant hepatitis may occur in a very small minority and is an urgent reason for
liver transplant without which the mortality rate is high.
Prophylaxis
o Pre-exposure: Killed HAV vaccine is available. Two doses given one year apart
can offer protection for up to 10 years. A booster may be required at 10 years but
recent evidence suggests that the two doses will give lifelong protection.
o Post-exposure: Normal human immunoglobulin and/or hepatitis A vaccine should
be given to household contacts within 14 days of exposure.
iv. Rotavirus
o Rotavirus is a double-stranded RNA virus belonging to the family Reoviridae. It
spreads among humans by the faeco–oral and respiratory routes. Patients are most
infectious when symptomatic with diarrhea and vomiting
o There are seven different groups (A–G). Group-A rotaviruses are the major cause
of human infection, but groups B and C also infect humans.
o The incubation period of rotavirus is 1–2 days.
Laboratory diagnosis of rotavirus infection

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o Dip stick/rapid test device, PCR, Electron microscopy

Prophylaxis: There are oral vaccines which can prevent rotavirus associated disease.
v. Influenza viruses
o Influenza A and B viruses are RNA viruses and belong to the family Myxoviridae.
The RNA genome is split into eight segments which allows the influenza ‘A’
strains to exchange genetic information with each other giving rise to new strains
all the time.
o Both influenza A and B viruses have got two important surface proteins, namely
haemagglutinin (H), which is responsible for attaching the virus to the cell surface,
and neuraminidase (N). These two proteins are used in the nomenclature of strains
(e.g. H2N3 and H5N1).
o Some influenza ‘A’ strains infects other animals such as birds and pigs. Infections
can spread from these animals to humans sometimes causing an outbreak.
o Usually these infections are associated with single cases or clusters of cases in
humans.
o There is always the fear that these avian viruses will mutate becoming much more
infectious to humans and causing a worldwide pandemic.
Route of spread
o Influenza viruses spread by the respiratory route
o Incubation period: 1–2 days. Patients are infectious while they have respiratory
symptoms (especially when coughing)
Prevention: vaccination
vi. Mumps virus
o Mumps virus is a single-stranded RNA virus and belongs to the family
Paramyxoviridae.
Route of spread
o Infection is spread by aerosol or hand and fomite contact with infected salivary or
respiratory secretions.
o Mumps is an endemic childhood infection worldwide. Cases occur all year round,
though in temperate climates they tend to peak in colder months

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Incubation period: 10–21 days, average of about 2 weeks. Virus is shed in the saliva
and respiratory secretions, maximum infectivity is at 48 hours prior to appearance of
parotitis, but patients are infectious from about a week before to after the appearance
of parotitis. The virus can be shed in the urine for much longer than this.
Laboratory diagnosis: Serology, PCR, viral culture
Prevention: vaccination
vii. Measles virus
o Measles is an RNA virus belonging to the family Paramyxoviridae.
Route of spread
 Measles is highly infectious with a high secondary infection rate in contacts,
especially household contacts. The infection is spread by the respiratory droplet
route. Measles has a worldwide prevalence with most infections occurring in
childhood.
 Incubation period: 10–15 days, an average of two weeks.
Symptoms:
 The typical measles rash is preceded by a 2–3 day prodromal illness, which consists
of cough, fever, conjunctivitis and rhinitis.
 At this stage, typical white lesions called Koplik’s spots can be seen in the inside of
cheek buccal mucosa in a proportion of cases; these are diagnostic of measles.
Patients are highly infectious in the prodromal stage and the virus is shed and spread
from respiratory secretions. The prodromal stage is followed by the appearance of a
maculopapular rash, which first appears on the face and neck and then spreads to the
trunk and limbs. The rash and fever fade by 4–5 days.
Complications:
 Secondary bacterial infection: Causing otitis media, laryngotracheitis, and
bronchopneumonia. These are common inchildren with measles in developing
countries due to poor nourishment, and the causeof high measles mortality rates
there.
 Encephalitis (Acute post-infectious measles encephalitis): This typically occurs
about a week or 10days after the rash disappears. It is accompanied by headache,
irritability, loss ofconsciousness and fever. This is due to demyelination as a result of

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auto-immunereaction to the measles virus and therefore the virus cannot be found in
the centralnervous system.
Laboratory diagnosis
 For the diagnosis of measles, blood is usually the specimen of choice. Urine, CSF
and brain biopsy can also be used. Serology and PCR are the methods of diagnosis
for measles.
Prophylaxis
o Pre-exposure: Live attenuated measles vaccine as triple vaccine with mumps and
rubella (MMR) is recommended at 13–15 months (to allow maternal antibody to
disappear as otherwise it may interfere with vaccine take) with a pre-school
booster.
o Post-exposure: The MMR vaccine can be given within 72 hours of exposure as
post-exposure prophylaxis. Normal immunoglobulin should be given as post-
exposure prophylaxis to those at risk in whom MMR is contraindicated (e.g.
pregnant and immunocompromised patients)
viii. Rabies virus
o Rabies virus belongs to the family Rhabdoviridae.
Route of spread
o Rabies is a zoonotic disease that is transmitted from animals (particularly dogs,
foxes, wolves, jackals, monkeys and bats) to man.
o Infection can be transmitted from a bite or scratch via a puncture wound through
the skin, or through a lick on an open wound or sore.
o Rabies occurs in every region of the world, but there are some countries such as
the UK, Hawaii, Panama and Australia that have eradicated the infection.
o Rabies can be reintroduced into countries that have eradicated it. Rabies is
endemic in Ethiopia.
o The incubation period in man is usually 1–3 months, but it can be as short as 10
days and as long as 2 years following exposure. The incubation period in the dog is
usually from 14 to 60 days, but it may be much longer.

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o Once infected, animals remain infectious via infected saliva. Rabies is usually a
fatal infection in animals but asymptomatic infection, especially in bats, is
recognized. The latter may provide a long-term reservoir of infection.
Symptoms
 For the first 2–4 days, patients usually develop malaise, fever, headache, sore throat
and lack of appetite.
 The virus first multiplies in the tissue around the site of inoculation. It then moves
into local nerves. Pain and tingling around the site of inoculation in the infected limb
is usually the first indication that the virus has entered the nervous system.
 These symptoms usually travel up the limb or spread around the face or neck,
depending on the site of infection. Jerky movements and increased muscle tone may
well follow. Dilation of the eye pupils and excessive secretion of tears and saliva
often occur next. The patient may next become anxious and frightened when
examined or disturbed, and the patient’s temperature rises to 38–40 0C.
 Localized paralysis may follow, resulting in difficulty in swallowing and in the
patient being terrified of drinking. The fear of drinking water (hydrophobia) is very
suggestive of rabies. Patients may be very excited or apathetic. With very few
exceptions, patients die within a week of the onset of symptoms.
Laboratory diagnosis
o Several laboratory methods and clinical specimens can be used to diagnose rabies. All the
tests should be performed in a high-security specialist laboratory.
Prevention and treatment
 Once symptoms have become established, there is no effective treatment other than
supportive care.
 Pre-exposure prophylaxis is with 3 doses of rabies vaccine.
 Post-exposure prophylaxis is either by 5 doses of rabies vaccine over a period of a
month, or if the risk of rabies exposure is likely by means of vaccine and human
anti-rabies immunoglobulin.
 It is very important to seek urgent medical advice in the case of a bite or a lick or
scratch from a suspect animal, especially if it was behaving aggressively.

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 Even in the case of previous rabies vaccine, it is strongly advised to have post-
exposure vaccine where a significant risk of exposure has occurred.
ix. Human immunodeficiency virus (HIV)
o Human immunodeficiency virus is an RNA virus which belongs to a special class of
viruses called retroviruses within the genus lentivirus (lenti – slow) within the family
Retroviridae (retro – backwards), so called because viruses (including HIV) in the family
possess a reverse transcriptase (RT) enzyme to convert the viral RNA template into DNA
which integrates in the cellular DNA to cause persistent infection.
o The other virus in the genus lentivirus is simian immunodeficiency virus (SIV), which
infects monkeys.
o Human immunodeficiency virus is closely related to SIV which causes a similar illness to
acquired immunodeficiency syndrome (AIDS) in rhesus monkeys, and there is good
evidence now that the virus was introduced into humans from monkeys in the first half of
the twentieth century through hunting and human consumption.
o Outside of a human cell, HIV exists as roughly spherical particles (sometimes called
virions)
o There are two known HIV viruses that cause human infection namely HIV-1 and HIV-2.
o Human immunodeficiency virus 1 is further divided into three groups: ‘major’ group, M;
‘outlier’ group, O; and ‘new’ group, N.
 Group M has several subtypes or clades (subtypes A to K)
 Other human viruses in the family Retroviridae are human T-cell leukemia virus
(HTLV) 1 and 2
Structure of HIV
o HIV particles surround themselves with a coat of fatty material known as the viral
envelope (or membrane). Projecting from this are around 72 little spikes which are formed
from the proteins gp120 and gp41. Just below the viral envelope is a layer called the matrix
which is made from the protein p17.

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Fig 4.5 Structure of HIV

o The viral core (or capsid) is usually bullet-shaped and is made from the protein p24.
o Inside the core are three enzymes required for HIV replication called reverse
transcriptase, integrase and protease.
o Also held within the core is HIV's genetic material which consists of two identical strands
of RNA. Almost all organisms, including most viruses, store their genetic material on long
strands of DNA. Retroviruses are the exception because their genes are composed of RNA
(Ribonucleic Acid).
o RNA has a very similar structure to DNA. However, small differences between the two
molecules mean that HIV's replication process is a bit more complicated than that of most
other viruses.
Route of spread or transmission of HIV
– Sexual contact
– Mother-to-child (vertical transmission)
– Blood transfusion
– Contaminated needle
Laboratory diagnosis
– Serology (ELISA and rapid test algorithms)
– Direct detection of virus: p24 antigen ELISA, Cell culture, PCR

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Self-check questions:
Instruction: Please! attempt all the questions listed below
Part – I: Multiple Choices
1. One is false about adenoviruses
A. Adenoviruses are RNA viruses
B. Adenoviruses causing conjunctivitis are very infectious
C. Enteric adenoviruses spread through faecal-oral route
D. Adenoviruses are ubiquitous in humans and animals
2. One is false about HSV
A. It is transmitted through kissing and sexual contact
B. Immunocompromised persons are the risk for its infection
C. It is a double stranded DNA virus
D. None of the above
3. The major outer protein of HBV which is expressed in excess while replicating in liver is
A. HBs antigen C. HBe antigen
B. HB core antigen D. HB core antigen and HBe antigen
4. HBV is not transmitted through _______________
A. Sexual contact C. Parenteral injections
B. Trans-placental D. Faeco-oral
5. Which one is false about poliovirus?
A. It is a causative agent of poliomyelitis
B. It is transmitted through faecal-oral route
C. It is not vaccine preventable
D. It leads to temporary or permanent paralysis
6. Hepatitis A virus is transmitted through _________
A. Faeco-oral route C. Parenteral injections
B. Sexual intercourse D. Aerosol droplets
7. Genital herpes transmission can be reduced or prevented by all of the following except
A. Male condom
B. Abstinence
C. Contraceptive pills

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D. Female condom
Part – II: Short Essay
1. Write the features of viruses
2. Explain the structure of virus
3. Write the general Viral replication cycle
4. Explain the general Laboratory diagnosis of viral diseases
Summary:
 Viruses are very tiny particles that they can only be seen with a special microscope called
an electron microscope.
 Viruses are obligate intra-cellular parasites lacking enzymes needed for protein or nucleic
acid synthesis; they use host machinery.
 Viruses are classified based their nucleic acid, either RNA or DNA, and their structural
symmetry. Viral nucleic acid is the major criteria for classifying viruses.
 Viruses are transmitted through respiratory droplets, blood, mucus, saliva, or semen from
infected individuals to healthy persons.
 Laboratory diagnosis of viral diseases can be direct detection, indirect detection (viral
isolation) and serological tests. Serology is the most widely used viral diagnostic methods.

Chapter 5: Collection, Handling, Processing, Transportation and Storage of

Microbiological Samples
Dear trainees,
In this chapter, you will learn different types of clinical specimens used in microbiological
tests. Furthermore, you will gain knowledge and skills of collecting, handling, processing,
transporting and storing of different types of microbiological samples.
 Objectives
At the end of this chapter you will be able to:
 describe different types of specimens for microbiological tests
 practice collection of routine specimens for microbiological tests
 identify appropriate specimens for microbiological tests
 demonstrate acceptance and rejection criteria of microbiological specimen
 describe the right time for the collection of different specimens for microbiological
tests

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 select appropriate preservation methods for the storage of microbiological specimens

for delayed tests or future references
5.1 Different types of specimens for microbiological tests
The correct type of specimen to be collected will depend on the pathogens to be isolated.
Demonstration of pathogenic organisms in the patient specimen is the most definitive test in
microbiology. However, failure to demonstrate pathogens in a single specimen is NOT definitive
and may only indicate that:
 The pathogen was absent or scant in that particular specimen
 The sample was taken at a stage of the disease when the pathogen was rare
 Viability was lost between the times of collection and arrival in the laboratory
 The pathogen cannot be detected by this method of testing.
 General principle of Specimen Collection in diagnostic microbiology
 Collect the specimen from the actual site of infection without contaminating adjacent
tissues and secretions.
 Collect the specimen at the best time possible (e.g., early morning sputum for AFB
microscopy/culture)
 Collect enough amount of sample by using appropriate collection devices such as sterile,
leak-proof specimen containers.
 Use appropriate transport media to transport sample to other places
 Collect specimens before administration of antimicrobial agents whenever possible.
 Label the specimen properly and fill out test request form completely.
 Lessen transport time and maintain an appropriate environment between collection of
specimens and delivery to the laboratory.
5.1.1 Sputum specimen
Purpose:
A sputum specimen is obtained for microscopy/culture to identify the microorganism
responsible for lung infections and management lung diseases. The sputum should be
collected in a sterile wide-mouthed container with a secure, tight-fitting cover and sent to the
laboratory without delay. If the sputum is allowed to stand after collection, overgrowth of
contaminating bacteria may take place before the examination is carried out.
Supplies and facility

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 Laboratory coat
 Gloves
 Goggles
 Laboratory request form
 Wide-necked, leak-proof containers for sputum specimens/ sputum cup
 Water and soap
 Permanent Marker
 AFB Log book
 Coughing area
Procedure:
1. Observe proper hand hygiene and gather equipment.
2. Wear the laboratory coat, don gloves and goggles
3. Read the order correctly
4. Label the sputum cup/container
5. Record the patient identification (Name, Age, address etc.)
6. Introduce yourself.
7. Uncap the container/sputum cup but avoid touching the inside to ensure that it’s sterile.
8. Provide privacy for the patient and explain the entire procedure
9. Using the sterile collection container provided, instruct the patient to bring 13-15ml of
sputum specimen.
10. If you don’t get an adequate sample on the first try, have him continue to cough until you
are able to collect a minimum amount of sputum specimen.
11. Once you have collected the specimen, securely cap the container. Remove and discard
your gloves and wash your hands thoroughly.
12. Record the amount, consistency, and color of the sputum collected, as well as the time
and date.
Important: The specimen must be sputum, not saliva. Sputum is best collected in the morning
soon after the patient wakes and before and before any mouth wash. Results of smears and
cultures will be highly misleading.
Expected pathogens:
 Bacteria – Ex - streptococcus pneumonia, Haemophilus influenzae, M. tuberculosis, etc.

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Skills Practice Session 5.1

Topic: Collection of Sputum for Acid Fast Bacilli (AFB) Detection:
Purpose:
The purpose of this activity is to enable you practice those skills necessary to collect sputum
sample, and to achieve competency in these skills.
Instructions:
This activity should be conducted in a skill laboratory/healthy facility. You should review
Learning Guide for sputum collection for AFB test before beginning the activity. The trainer
should demonstrate the steps/tasks in each learning guide one at a time. Under the guidance of
the trainer, you should then work in groups and practice the steps/tasks in the Learning Guide for
sputum collection and observe each other’s performance; while one learner/group doing the
sputum collection activity, another learner/group should use the Learning Guide to observe
performance. You should then rotate roles. You should be able to perform the steps/tasks before
skills competency is assessed using the Checklist for Sputum collection.
Conditions/ Situation for the operations:
 This activity could be done in skill laboratory or in healthy facility
Resources (Equipment and Materials)
 Laboratory coat
 Gloves
 Goggles
 Laboratory request form
 Wide-necked, leakproof containers for sputum specimens/sputum cup
 Water and soap
 Permanent Marker
 AFB Log book
 Coughing area
Precaution: When a sputum specimen is being collected, adequate safety precautions must be
taken to prevent the spread of infectious organisms and to avoid contaminating the outside of the
container. Use a phenol-containing disinfectant to wipe the outside of the container after
collecting the specimen

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Standard Precautions
 Always wash hands before and after obtaining and handling specimens.
 Cover cuts and lesions with waterproof dressing.
 Wear disposable aprons and appropriate gloves if there is likelihood of contact or
contamination with body fluids.
 Take care not to contaminate the outside of the container with sputum or body fluid.
 Do not use the specimen container for any other purpose.
 Always ensure the top is closed securely.

Activity: Role play

Objective: Practice sputum collection.
The time: 45 minutes
Mr. Y came from OPD with laboratory request for sputum examination. Now, make a group of
two to collect sputum sample; one student acting as a patient from your group and the other
trainee being a laboratory professional to collect the sputum. Let the instructor facilitate the
activity and give feedback to the role players.

Discussion questions
 Did the trainee collect the sputum sample properly?
 Did the trainee follow pre-analytical stage in collection of sputum sample?
 Did the trainee demonstrate good attitude during collection of the sputum sample?
 Did the trainee practice good communication skill during collection of the sputum
sample?

Procedure-Learning Guide/Checklist
Learning Guide 5.1
Collection of Sputum for Acid Fast Bacilli (AFB) Detection
(To be completed by Participant/students)

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Rate the performance of each step or task observed using the following rating scale:
1. Needs Improvement: Step or task not performed correctly, out of sequence (if necessary), or
is omitted
2. Competently Performed: Step or task performed correctly in proper sequence (if necessary)
but participant/student does not progress from step to step efficiently
3. Proficiently Performed: Step or task performed efficiently and precisely in the proper
sequence (if necessary)
Collection of Sputum for Acid Fast Bacilli (AFB) Detection
(Some of the following steps/tasks should be performed simultaneously)

STEP/TASK

improvement

Competently

Proficiently
performed

performed

Remark
Needs
Getting ready
1. Wearing the Laboratory coat
2. Washing your hand with soap
3. Wearing the gloves
4. Preparing the necessary equipment
5. Greeting the patient respectfully and with kindness.
6. Reading the order (request form)
7. Labeling the sputum cup/container
8. Recording the patient identification (Name, Age, address
etc.)
9. Orienting the patient to bring approximately 10-15ml sputum
at right place
10. Receive the sputum sample and put at appropriate area
11. Remove gloves and apron and perform hand hygiene

Critical aspect of the competency

During accomplishment of Collection of Sputum for Acid Fast Bacilli (AFB) Detection,

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the trainees should be:

 Able to describe the process of sputum collection
 Able to follow pre analytical stages of procedures
 Able to apply safety procedures during collection of sputum sample
 Able to develop good attitude towards patients and professionals
5.1.2 Wound/Pus specimen
Purpose :
To provide the appropriate interventions for the needs of the individual patient while reassessing
the clinical status of the patient in response to all interventions and disease processes. When you
collect the wound exudates for microscopy and culture, it must be free from contamination. The
ultimate goal is to enable identification of organism(s) causing infections.
Supplies and Equipment :
 70% Alcohol or Detergent wipe (for decontaminating)
 Sterile gauze
 Dressing pack
 Dressing trolley
 Sterile swabbing solution (sodium chloride 0.9% is normally used to clean wounds)
 Disposable gloves
 Bag to dispose of used items
 Sterile swab stick
 Tran swab (dual tube with swab stick plus charcoal transport medium)
 Eye protection/Goggles
 Laboratory coat
 Laboratory request form
 Water and soap
 Permanent Marker
 Log book
Procedures:
1. Keep proper hand hygiene and gather equipment.
2. Wear the laboratory coat, put on gloves and goggles.

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3. Read the request properly.

4. Positively identify the patient.
5. Label the sample container.
6. Record the patient identification (Name, Age, address etc.)
7. Introduce yourself.
8. Before collecting a swab, remove all excessive debris and dressing product residue
without unduly disturbing the wound surface. This can be achieved by using a gently
stream of sterile 0.9% sodium chloride. Normal saline cleanses the contaminants without
destroying the pathogen.
9. Remove excess saline with a sterile gauze. This exposes the wound to ensure a good
culture is collected
10. Wait for 1 -2 minutes to allow the organisms to rise to the surface of the wound.
11. If fresh pus or wound fluid is present, ensure this is collected on the swab.
12. Once collected, the swab should be placed in the charcoal medium.

Skills Practice Session 5.2

Topic: Collection of Wound/Pus Specimen

Purpose:
The purpose of this activity is to enable you practice those skills necessary to collect wound/pus
specimen, and to achieve competency in these skills.
Instructions
This activity should be conducted in a skill laboratory/healthy facility. You should review
Learning Guide for Wound/Pus for microbial test before beginning the activity. The trainer
should demonstrate the steps/tasks in each learning guide one at a time. Under the guidance of
the trainer, you should then work in groups and practice the steps/tasks in the Learning Guide for
Wound/Pus specimen collection and observe each other’s performance; while one learner/group
doing the Wound/Pus collection specimen activity, another learner/group should use the
Learning Guide to observe performance. You should then rotate roles. You should be able to
perform the steps/tasks before skills competency is assessed using the Checklist for Wound/Pus
specimen collection.
Conditions/Situation for the operations:

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 This activity should be done in skill laboratory or in healthy facility.

Resources (Equipment and Materials):
 70% Alcohol (for decontamination)
 Sterile gauze
 Dressing pack
 Dressing trolley
 Sterile swabbing solution (sodium chloride 0.9% is normally used to clean wounds)
 Disposable gloves
 Bag to dispose of used items
 Sterile swab stick
 Tran swab (dual tube with swab stick plus charcoal transport medium)
 Eye protection/Goggles
 Laboratory coat
 Laboratory request form
 Water and soap
 Permanent Marker
 Log book
Precaution: When wound/Pus Specimen is being collected, adequate safety precautions must be
taken to prevent the spread of infectious organisms and to avoid contaminating the outside of the
container.

Standard Precautions
Always wash hands before and after obtaining and handling specimens
 Cover cuts and lesions with waterproof dressing
 Wear disposable aprons and appropriate gloves if there is likelihood of contact or
contamination with blood or body fluids.
 Take care not to contaminate the outside of the container with blood or body fluid
 Do not use the specimen container for any other purpose
 Always ensure the top of the container is closed securely

Activity: Role play

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Objective: Practice Wound/Pus Specimen collection.

Mr.(R) came from surgical OPD with laboratory request so that you are responsible to collect
Wound/Pus Specimen. Make a group of two; one student acting as a patient from your group and
the other trainee being a laboratory professional to collect wound/pus. Let the instructor facilitate
the activity and give feedback to the role players.
Discussion questions
 Did the trainee collect the wound/pus specimen properly?
 Did the trainee follow pre-analytical stage in collection of wound/pus specimen?
 Did the trainee demonstrate good attitude during collection of wound/pus specimen?
 Did the trainee practice good communication skill during collection of the wound/pus
specimen?

Procedure-Learning Guide/Checklist
Learning Guide 5.2
Collection of Wound/Pus specimen
(To be completed by Participant/students)
Rate the performance of each step or task observed using the following rating scale:
1. Needs Improvement: Step or task not performed correctly, out of sequence (if necessary), or is
omitted
2. Competently Performed: Step or task performed correctly in proper sequence (if necessary) but
participant/student does not progress from step to step efficiently
3. Proficiently Performed: Step or task performed efficiently and precisely in the proper sequence (if
necessary)

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Collection of Wound/Pus Specimen

(Some of the following steps/tasks should be performed simultaneously)
STEP/TASK

improvement

Competently

Proficiently
performed

performed
Remark
Needs
Getting ready
1. Wear the Laboratory coat
2. Wash your hand with soap
3. Wear the gloves
4. Prepare the necessary equipment
5. Greet the patient respectfully and with kindness.
6. Read the request form
7. Label the sample container
8. Record the patient identification (Name, Age, address etc.)
9. Before collecting a swab, remove all excessive debris and
dressing product residue without unduly disturbing the wound
surface. Normal saline cleanses the contaminants without
destroying the pathogen.
10. Remove excess saline with a sterile gauze. This exposes the
wound to ensure a good culture is collected
11. Wait for 1 -2 minutes to allow the organisms to rise to the
surface of the wound
12. If fresh pus or wound fluid is present, ensure this collected on
the swab.
13. Place in the charcoal medium.
14. Remove gloves and gown and perform hand hygiene

Critical aspect of the competency

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During accomplishment of collection of wound/Pus Specimen, the trainees should be:

 Able to know the process of wound/Pus Specimen collection

 Able to follow pre analytical stages of procedures
 Able to apply safety procedures during collection wound/Pus Specimen

5.1.3 Urogenital (Urethral and Cervical) specimen:

Purpose
Accurate diagnosis of genital infections depends on the separation of pathogens from normal
flora. The female genital tract is colonized by a wide array of organisms. However, many
infections arise from this normal flora upon activation by patient condition and history, and other
organisms; and in some instances, it is important to identify the carrier state regardless of patient
symptoms.
Supplies and Equipments:
 Clean, dry container with lid
 Sterile cotton wool swab
 Disposable gloves
 Bag to dispose of used items
 Laboratory coat
 Laboratory request form
 Water and soap
 Sterile warm water
 Sterile physiological saline
 Permanent Marker
 Log book
Procedures:
1. Keep proper hand hygiene and gather equipment.
2. Wear the laboratory coat, put on gloves and goggles
3. Read the request form correctly
4. Positively identify the patient.
5. Label the sample container
6. Record the patient identification (Name, Age, address etc.)

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7. Introduce yourself.
8. Cleans the urethral opening using a swab moistened with sterile physiological saline.
/Moisten a vaginal specimen with sterile warm water/physiological saline and insert into
the vagina.
9. Gently massage the urethra from above downwards, and collect a sample of pus on a
sterile cotton wool swab/ Pass a sterile cotton wool swab into the endocervical canal and
gently rotate the swab to obtain a specimen. The patient should not have passed urine
preferably for 2hours before the specimen is collected.
10. Make a smear of the discharge on a slide for staining by the Gram technique and label
the specimen.
Expected pathogens: Candida albicans (microscopic examination), Chlamydia trachomatis,
Gardnerella vaginalis (microscopic examination), Haemophilus ducreyi, Neisseria gonorrhoeae,
Treponema pallidum (dark-field microscopy)
Activity: Demonstration on model (Manikin)
Objective: Urogenital (Urethral and Cervical) Specimen collection.
Notice: Let the students practice this skill at Nursing or Midwifery skill laboratory.
Mr. (X) has a problem of infection on urogenital organ and there is discharge on his organ. Now
you are going to practice to collect urogenital specimen on model found in nursing skill
laboratory. Collect Urogenital (Urethral and Cervical) specimen as the model used as a patient
and let another group/trainee collect the Urogenital (Urethral and Cervical) specimen using the
following instruction. Let the trainer facilitate the activity
Discussion Question
 Did the group/trainee collect the Urogenital (Urethral and Cervical) sample?
 Did the group/trainee follow pre analytical stage in collection of Urogenital (Urethral and
Cervical) sample?
 Did the trainee/group show/shows good attitude and good communication skill during
collection of the Urogenital (Urethral and Cervical) sample?

Skills Practice Session 5.3

Collection of Urogenital Specimen collection
Purpose:

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The purpose of this activity is to enable you to practice those skills necessary to provide
urogenital specimen, and to achieve competency in these skills.
Instructions:
This activity should be conducted in a skill laboratory/healthy facility. You should review
Learning Guide for Urogenital specimen for microbial test before beginning the activity. The
trainer will demonstrate the steps/tasks in each learning guide one at a time. Under the guidance
of the trainer, you should then work in groups and practice the steps/tasks in the Learning Guide
for Urogenital specimen on model (Manikin) and observe each other’s performance; while one
of you/group doing the urogenital specimen activity; another learner/group should use the
Learning Guide to observe performance. You should then rotate roles. You should be able to
perform the steps/tasks before skills competency is assessed using the Checklist for Urogenital
Specimen collection.
Conditions/ Situation for the operations
 This activity could be done in skill laboratory or in healthy facility
Resources (Equipment tools and Materials)
 Clean, dry container with lid
 sterile cotton wool swab
 Laboratory request form
 Disposable gloves
 Bag to dispose of used items
 Laboratory coat
 Laboratory request form
 Water and soap
 sterile warm water
 sterile physiological saline
 Permanent Marker
 Log book
Precaution: When a collection of Urogenital Specimen is being collected, adequate safety
precautions must be taken to prevent the spread of infectious organisms and to avoid
contaminating the outside of the container.

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Standard Precautions
Always wash hands before and after obtaining and handling specimens
 Cover cuts and lesions with waterproof dressing
 Wear disposable aprons and appropriate gloves if there is likelihood of contact or
contamination with blood or body fluids.
 Take care not to contaminate the outside of the container with blood or body fluid
 Do not use the specimen container for any other purpose
 Always ensure the top is closed securely

Procedure-Learning Guide/Checklist
Learning Guide 5.3
Collection of Urogenital specimen
(To be completed by Participant/students)

Rate the performance of each step or task observed using the following rating scale:
1. Needs Improvement: Step or task not performed correctly, out of sequence (if necessary), or
is omitted
2. Competently Performed: Step or task performed correctly in proper sequence (if necessary)
but participant/student does not progress from step to step efficiently
3. Proficiently Performed: Step or task performed efficiently and precisely in the proper
sequence (if necessary)

Collection of Urogenital specimen

(Some of the following steps/tasks should be performed simultaneously)
STEP/TASK
improvement
Competently

Proficiently
performed

performed

Remark
Needs

Getting ready
1. Wear the Laboratory coat

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Collection of Urogenital specimen

(Some of the following steps/tasks should be performed simultaneously)
STEP/TASK

improvement
Competently

Proficiently
performed

performed

Remark
Needs
2. Wash your hand with soap
3. Wear the gloves
4. Prepare the necessary equipment
5. Greet the patient respectfully and with kindness (be careful this
is a sensitive issue).
6. Read the order
7. Label the sample container
8. Record the patient identification (Name, Age, address etc.)
9. Cleans the urethral opening using a swab moistened with sterile
physiological saline/Moisten a vaginal specimen with sterile
warm water/physiological saline and insert into the vagina.
10. Gently massage the urethra from above downwards, and collect
a sample of pus on a sterile cotton wool swab/ Pass a sterile
cotton wool swab into the endocervical canal and gently rotate
the swab to obtain a specimen. The patient should not have
passed urine preferably for 2hours before the specimen is
collected.
11. Make a smear of the discharge on a slide for staining by the
Gram technique and label the specimen.
12. Remove gloves and gown and perform hand hygiene

Quality criteria:

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During accomplishment of Collection of Urogenital specimen, the trainees should be:

 Able to understand the process of Urogenital specimen collection
 Able to follow pre analytical stages of procedures
 Able to apply safety procedures during collection Urogenital specimen
 Able to develop good attitude towards patients, professions and environment

5.1.4 Ear swabs (Discharge)

Purpose
No antibiotics or other therapeutic agents should have been in the aural region for about three
hours prior to sampling the area as this may inhibit the growth of microorganisms.
Supplies and Equipment
 70% Alcohol or Detergent wipe (for decontaminating)
 Sterile swab/cotton
 Disposable gloves
 Bag to dispose of used items
 Sterile swab stick
 Tran swab (dual tube with swab stick plus charcoal transport medium)
 Eye protection/Goggles
 Laboratory coat
 Laboratory request form
 Water and soap
 Permanent Marker
 Log book
Expected pathogens:
S. aureus, Other beta-haemolytic streptococci, S.pyogenes, H. influenzae, P. aeruginossa,
S. preumoniae, Klebsiella specia etc.
Precaution:While collecting ear discharge specimen, adequate safety precautions must betaken to
prevent the spread of infectiousorganisms and to avoid contaminating the outside of the
container.

Standard Precautions:

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Always wash hands before and after obtaining and handling specimens
 Cover cuts and lesions with waterproof dressing
 Wear disposable aprons and appropriate gloves if there is likelihood of contact or
contamination with blood or body fluids.
 Take care not to contaminate the outside of the container with blood or body fluid
 Do not use the specimen container for any other purpose
 Always ensure the top of the container is closed securely
Procedures:
1. Keep your hand hygiene and gather equipment.
2. Wear the laboratory coat, put on gloves and goggles
3. Read the order correctly
4. Positively identify the patient.
5. Label the sample container
6. Record the patient identification (Name, Age, address etc.)
7. Introduce yourself.
8. Place a sterile swab into the outer ear and gently rotate to collect the secretions.
9. If there is purulent discharge, this should be sampled.
10. For deeper ear swabbing a speculum may be used. Only experienced medical staff
should undertake this procedure as damage to the eardrum may occur.
11. Collect a specimen of the discharge on sterile cotton.
12. Place swab in transport medium.
13. Remove gloves and apron and perform hand hygiene.
Activity: Role play
Objective: Practice ear discharge specimen collection.
Mr.X came from surgical OPD with laboratory request so that you are responsibleto collect ear
discharge specimen. Make a group of two; one student acting as a patient from your group and
the other trainee being a laboratory professional to collect ear discharge specimen. Let the
instructor facilitate the activity and give feedback to the role players.
Discussion questions
 Did the trainee collect the ear discharge specimen properly?
 Did the trainee follow pre-analytical stage in collection of ear discharge specimen?

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 Did the trainee demonstrate good attitude during collection of ear discharge specimen?
 Did the trainee practice good communication skill during collection of ear discharge
specimen?

Skills Practice Session 5.4

Topic: Collection of Ear Discharge Specimen
Purpose:
The purpose of this activity is to enable you practice skills necessary to collect ear discharge
specimen, and to achieve competency in these skills.
Instructions:
This activity should be conducted in a skill laboratory/healthy facility. You should review
Learning Guide for discharge specimen for microbial test before beginning the activity. The
trainer should demonstrate the steps/tasks in each learning guide one at a time. Under the
guidance of the trainer, you should then work in groups and practice the steps/tasks in the
Learning Guide for discharge specimen collection and observe each other’s performance; while
one trainee/group doing the Ear discharge collection specimen activity, another trainee/group
should use the Learning Guide to observe performance. You should then rotate roles. You should
be able to perform the steps/tasks before skills competency is assessed using the Checklist for ear
discharge collection.
Conditions/ Situation for the operations:
This activity could be done in skill laboratory or in healthy facility
Resources (Equipment and Materials):
 70% Alcohol or Detergent wipe (for decontaminating)
 Sterile swab/cotton
 Disposable gloves
 Bag to dispose of used items
 Sterile swab stick
 Tran swab (dual tube with swab stick plus charcoal transport medium)
 Laboratory coat
 Laboratory request form

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 Water and soap

 Permanent Marker
 Log book

Procedure-Learning Guide/Checklist
Learning Guide 5.4
Collection of Ear discharge specimen
(To be completed by Participant/students)
Rate the performance of each step or task observed using the following rating scale:
1. Needs Improvement: Step or task not performed correctly, out of sequence (if necessary), or
is omitted
2. Competently Performed: Step or task performed correctly in proper sequence (if necessary)
but participant/student does not progress from step to step efficiently
3. Proficiently Performed: Step or task performed efficiently and precisely in the proper
sequence (if necessary)

Collection of Ear discharge

(Some of the following steps/tasks should be performed simultaneously)

STEP/TASK
improvement

Competently

Proficiently
performed

performed
Remark
Needs

Getting ready
1. Wear the Laboratory coat
2. Wash your hand with soap
3. Wear the gloves
4. Prepare the necessary equipment
5. Greet the patient respectfully and with kindness.
6. Read the order
7. Label the sample container
8. Record the patient identification (Name, Age, address etc.)
9. Place a sterile swab into the outer ear and gently rotate to collect the
secretions.
10. If there is purulent discharge this should be sampled.

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Collection of Ear discharge

(Some of the following steps/tasks should be performed simultaneously)

STEP/TASK

improvement

Competently

Proficiently
performed

performed
Remark
Needs
11. For deeper ear swabbing a speculum may be used. Only
experienced medical staff should undertake this procedure as
damage to the eardrum may occur.
12. Collect a specimen of the discharge on sterile cotton.
13. Place swab in transport medium.
14. Remove gloves and apron and perform hand hygiene
Quality criteria:
During accomplishment of Collection of Ear discharge specimen, the trainees should be:
 Able to describe the process of Ear discharge specimen collection
 Able to follow pre-analytical stage of procedures
 Able to apply safety procedures during collection Ear discharge specimen
 Able to develop good attitude towards patients and professionals

5.1.5 Urine specimen

Purpose:
Urine is the specimen most frequently submitted for culture. It also presents major problems in
terms of proper specimen collection, transport, culture Techniques, and interpretation of results.
As with any other specimen sub-mitted to the laboratory, the more comprehensive the
information provided by the submitting physician, the more the laboratory is able to provide the
best possible culture data.
Supplies and Equipment
 Clean, dry container with lid
 Cotton ball
 Laboratory request form

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 Disposable gloves
 Bag to dispose of used items
 Laboratory coat
 Laboratory request form
 Water and soap
 Permanent Marker
 Log book
Procedures
1. Keep hand hygiene and gather equipment.
2. Wear the laboratory coat, put gloves
3. Read the request correctly
4. Positively identify the patient.
5. Label the sample container
6. Record the patient identification (Name, Age, address etc.)
7. Introduce yourself.
8. Instruct the patient to use the cotton ball to clean urethral area thoroughly to prevent
external bacteria from entering the specimen.
9. Let the patient void into the container. It must be the mid-stream urine.
10. Label the specimen container with patient identifying information, and send to the lab
immediately. A delay in examining the specimen may cause a false result when bacterial
determinations are to be made.
11. Wash your hands and instruct the patient to do it as well.
12. Note that the sample was collected.
Expected pathogens:
Candida albicans, Enterococci, Escherichia coli, Mycobacterium tuberculosis, Other
Enterobacteriaceae, Staphylococci, Pseudomonas and other non-fermenters,
Staphylococcus saprophyticus.

5.1.6 Stool specimen

Purpose
Stool culture plays an important role in understanding and treating intestinal illness. It can
confirm the presence of harmful bacteria. It may also show what treatments may work to kill an

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invasive organism. If no dangerous bacteria are present in the stool culture but symptoms still
exist, other explanations like irritable bowel syndrome, a parasitic infection, or other diagnosis
can be explored.
Supplies and Equipment
 Laboratory coat
 Gloves
 Laboratory request form
 Water and soap
 Permanent Marker
 Log book
 Clean bedpan and cover (an extra bedpan or urinal if the patient must void)
 Specimen container and lid
 Wooden tongue blades
 Paper bag for used tongue blades
 Plastic bag for transport of container with specimen to laboratory
Procedure
1. Keep hand hygiene and gather equipment.
2. Wear the laboratory coat, put on gloves and goggles
3. Read the order correctly
4. Positively identify the patient.
5. Label the sample container
6. Record the patient identification (Name, Age, address etc.)
7. Introduce yourself.
8. Discuss the test and the procedure with the patient. Ask him to tell you when he feels the
urge to have a bowel movement.
9. Wear gloves when handling any bodily discharge.
10. Bedpan should be provided when the patient is ready. If the patient wants to urinate first,
provide the urinal for a male patient or provide the extra bedpan for a female patient.
Avoid mixing urine or regular toilet paper into the sample.

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11. With the use of a tongue blade, transfer a portion of the feces to the specimen container.
Don’t touch the specimen because it is contaminated. It is not necessary to keep the
specimen sterile because the gastrointestinal tract is not sterile.
12. Immediately cover the container and label it with the patient’s name and other needed
information.
13. Fill out the appropriate laboratory request form completely, noting any special
examination ordered.
14. Take the specimen to the lab immediately; examination for parasites, ova, and organisms
must be made while the stool is warm.
15. With regard to an infant patient, place the diaper in a leakproof bag, label it, and take the
diaper and request form to the lab as soon as possible. However, it can be difficult to
keep urine away from the stool sample.
Expected pathogens:
C. perfrigens type A and C, B. cereus (toxin), S. aureus (toxin), Shigella spp., Salmonella
spp., E. coli ETEC, EIEC, EPEC), V. cholerae O1, Y. enterocolitica

5.1.7 Bloodspecimen
Purpose:
Blood is cultured to detect and identify bacteria or other cultivable microorganisms (yeasts,
filamentous fungi). The presence of such organisms in the blood is called bacteremia or
fungaemia, and is usually pathological. In healthy subjects, the blood is sterile. A blood culture
is being done to determine which specific organism or bacteria is causing the problem and how
best to combat it.
Supplies and Equipment:
 Gloves
 vacutainer tube
 vacutainer tube holder and Two-way needle
 sterile syringe and needle (if the syringe method is used)
 tourniquet
 gauze pads or cotton,
 70% alcohol or suitable skin antiseptic

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 test tubes with or without anticoagulant (for syringe method)

 Sharp container
 Band Aid (to stop further bleeding) Laboratory coat
 Laboratory request form
 Water and soap
 Permanent Marker
 Log book
Procedure:
1. Keep hand hygiene and gather equipment.
2. Wear the laboratory coat, don gloves and goggles
3. Read the order correctly
4. Positively identify the patient.
5. Label the sample container
6. Record the patient identification (Name, Age, address etc.)
7. Introduce yourself.
8. Assemble the necessary materials and equipment
9. thread the short end of the double-pointed needle into the holder and push the tube
forward until the top of the stopper meets the guide mark on the holder
10. Identify the right patient and allow him/her to sit comfortably preferably in an armchair
stretching his/her arm.
11. Reassure the patient
12. Apply the tourniquet
13. Prepare the arm by swabbing the antecubital fossa with a gauze pad or cotton moistened
with 70% alcohol
14. Grasp the back of the patient’s arm at the elbow and anchor the selected vein by drawing
the skin slightly taut over the vein
15. Insert the needle properly into the vein
16. Then the point of the needle is advanced 0.5-1.0cm into the subcutaneous tissue (at an
angle of 450) and is pushed forward at a lesser angle to pierce the vein wall
17. When the needle is properly in the vein, the vacuum tube is pushed into the needle holder
all the way so that the blood flows into the tube under vacuum

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18. The tourniquet should be released the moment blood starts entering the vacuum tube
19. After drawing the required blood sample, apply a ball of cotton to the puncture site and
gently withdraw the needle
20. Instruct the patient to press on the cotton
21. Remove the tube from the vacutainer holder and if the tube is with anticoagulant, gently
invert several times
22. Label the tubes with patient’s name, hospital number and other information required by
the hospital (before the patient leaves the collection area)
23. Re-inspect the venipuncture site to ascertain that the bleeding has stopped.
24. Re-inspect the venipuncture site to ascertain that the bleeding has stopped.
25. Re-inspect the venipuncture site to ascertain that the bleeding has stopped.
Expected pathogen
S. aureus, S. pneumonia, S. pyogenes,C.perfringens,S.typhi,H.influanzae,P.aeruginosa,Klebsiclla
strains, Proteus spp., N.meningitides, Y.pestis et
5.2 Microbiological Specimen Rejection Criteria
Purpose
To ensure that samples and requisitions are accurately identified with required information. This
policy defines conditions that would render a specimen unacceptable for processing in
Microbiology. The laboratory reserves the right to refuse improperly labeled specimens. The
laboratory takes measures to maintain specimen integrity during the process of following up on
the receipt of an improperly identified specimen. The laboratory recognizes that, in certain cases
where the specimen is less common, involves an invasive procedure or could not otherwise be
easily recollected, it may be acceptable to apply an exception to specimen rejection. Exceptions
are applied using strict and explicit criteria in accordance with established procedures. The table
below outlines the basic criteria for rejection of requests for microbiology tests.

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Category Criterion For Rejection Action

Identification Container not identified  Write "container not identified” and call
ordering unit to send someone to verify the ID
if the request form is attached to the specimen.
 Otherwise, do not process unless "one of a
kind".
Container and request form  Call ordering floor or physician office to send
have different ID's correct request forms or resolve the problem

Request form Insufficient information  Call ordering floor or physician office for
marked on form additional necessary information
Specimen Specimen grossly  Call ordering floor or physician to send repeat
contaminated. specimen.
 If they are unable to re-collect, ask them to
come down to clean it in the laboratory.
Unlabeled Specimens  Call ordering floor or physician office to send
the labeled specimens and resolve the
problem

Specimen submitted in  Notify ordering floor or physician office of
improper container. problem.

Excessive delay between  If specimen was not stored properly, notify

specimen collection and ordering unit and request repeat specimen.
arrival in laboratory.  Do not process unless patient care is likely to
be compromised by delay.
Inadequate specimen for  Call physician for priority of requests and to
number of tests requested request additional material.
 Report as specimen had inadequate volume as
reason for lack of performance of certain tests.
Dry swab received for  Notify ordering floor or physician office that
culture dry swabs are not suitable for culture.
 Do not process. Sent report out with note
indicating reason for not performing test.
Improper specimen for test.  Call ordering floor or physician office to verify
test request.
 Do not process, and send documented reason
for not performing test
Unsatisfactory expectorated  See sputum screening procedure.
sputum.
Duplicate specimen (other  Send notice that only one of the received
than duplicate blood) specimens will be processed unless the
laboratory is notified and reasons are given for

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processing duplicate(s).

5.3 The right time for the collection of different specimens for microbiological tests
Time of Collection:
Specimens must be taken if at all possible before antibiotics are administered. If antibiotics have
already been started, then the requisition sheet must be so marked so that everyone is aware. The
timing of blood specimens is very important. Detection of positive blood cultures of course
depends on the pathogenic process of the organism. With some diseases the bacteremia occurs
only in the early stages of the infection, while in other cases there is continuous presence of
bacteria. In many cases the presence of bacteria in the blood is transient and can best be found
after a chill when the patient spikes a fever. During a chill the bactericidal properties of blood are
accentuated; the micro vessels constrict and become clogged with cells and bacteria during the
chill.
5.4 Specimen Handling and Transportation
The proper collection and transport of clinical specimens is critical for the isolation,
identification, and characterization of agents that cause bacterial meningitis. Optimally,
clinical specimens should be obtained before antimicrobial therapy commences in order to avoid
loss of viability of the etiological agents.
 Specimens must be collected in an appropriate specimen container to maintain the
integrity of the specimen.
 After collection, specimens must be labeled with the patient’s full name (or unique code
number in the case of anonymous testing) and one other unique identifier such as the
admission/identification or accession number.
 Most specimens should be stored between 2-8°C. Specific handling/storage information
is included in the test-specific.
5.5 Storage and Transportation of Microbiology Samples
Treatment of the patient should not be delayed while awaiting collection of specimens or results
from the laboratory and a specimen should be obtained in all suspect cases as bacterial pathogens
can still be detected even after antimicrobial therapy has begun. It is important that samples for
Microbiological testing are transported to the laboratory as quickly as possible. This is
particularly important when bacterial culture is requested as undue delays can lead to distorted

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microbiological findings. For example, significant pathogens may be overgrown by colonizing or

commensal microorganisms, and fastidious organisms may loose their viability.
The use of appropriate transport media such as
 Boric acid containers for urine samples
 Amies with charcoal” transport swabs help to reduce these problems in the short term.
 Microbiology samples held overnight should be refrigerated in order to reduce the
possibility of bacterial overgrowth.
5.6 Appropriate preservation methods for the storage of microbiological
The method of preserving tissues, fluids, and cultures will depend on their anticipated use(s).
Samples stored for periodic access such as assay reference materials should be aliquotted to
avoid potential problems associated with repeated retrieval and return to storage. They may be
stored separately from specimens or samples stored for historical, long-term preservation.
Storage conditions should be managed to maintain viability, biochemical, and immunological
properties of the samples to the maximum extent possible. Considerations for preserving the
integrity of the samples must include protection from desiccation (e.g. as can happen in certain
freezers), frequent or extreme temperature fluctuations, UV degradation, humidity,
contamination, and the potential for loss of identification and associated archive documentation.
Unique or valuable isolates and materials should be stabilized and stored using at least two
different procedures and storage locations.
 Storage at ultra-low temperatures (e.g. liquid nitrogen, cryopreservation in freezers at –
140°C or lower) is considered the optimum method for long-term storage of biological
materials.
 Storage at low-freezer temperatures of –80°C and –20°C is common for periods that may
range from months to 5–10 years.
 Ultra-low freezing may not be a practical choice as it is expensive to maintain, but cost
must be balanced with the fact that biological degradation of the sample over time is an
increasing risk at warmer freezer temperatures.
 Reference materials that are to be accessed with any regularity should be stored in
appropriately-sized aliquots to allow access while minimizing the number of times the
“master stock” is handled.

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 Repeated freezing and thawing of samples should be avoided as it can denature antigens,
result in loss of viability of fastidious agents, and can precipitate the over-growth of
contaminants or unwanted microorganisms in the sample.
Summary
 Prior to the application of different microbiological diagnostic techniques, there must be
appropriate collection, handling, processing, transportation and storage of
microbiological samples.
 Different types of clinical specimens can be used for microbiological tests to diagnose
infectious diseases.
 The correct type of specimen should be collected to isolate the pathogenic organism (s).
Hence, the general principles of specimen collection in diagnostic microbiology should
be applied.
 The right time for the collection of different specimens for microbiological tests should
be identified to properly demonstrate the suspected pathogenic organisms.
 The proper collection and transportation of clinical specimens is critical for the isolation,
identification, and characterization of pathogenic organisms.
 In the case, inappropriate specimen arrives to the microbiology laboratory, the specimen
rejection policy must be implemented.

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Chapter 6: Safety and Safety Rules in Microbiology Laboratory

Dear trainees,
In this chapter, you will learn safety measure and how to ensure self-handling of equipment
and materials in medical microbiology. You will describe methods of sterilization and
disinfection to have safe working environment. Furthermore, you willdeal about biohazard
materials and how to handle them.
Objectives:
At the end of this chapter, you will be able to:
 Apply safety measures in microbiology Laboratory
 Ensure Safe handling of equipment and materials in microbiology laboratory
 Describe methods of sterilization and Disinfection
 Apply sterilization and Disinfection techniques for microbial growth control
 Describe basic concepts of biohazard materials
 Apply standard precautions when handling biological materials
6.1. Safety in Microbiology Laboratory
Safety in a microbiology laboratory is important in the prevention of infection as Microbiology
laboratory cultures, manipulates, and uses virulent and/or potentially pathogenic
microorganisms. In addition to microorganisms, there are some chemicals used in this
laboratory that are potentially harmful. Many procedures involve glassware, open flames, and
sharp objects that can cause trauma/ damage if used improperly.
“An experiment done well is, an experiment done safely."
a. General Safety Rules in microbiology laboratory
1. The laboratory procedures must be read prior to attending that laboratory session.
2. Smoking, eating, and drinking in the laboratory are absolutely prohibited in the laboratory at
any time.
3. Only closed-toe shoes are to be worn in the laboratory. Sandals or open toed or canvas
shoes are not permitted because of the constant danger of cuts and infections from broken
glass found on the lab floors and the possibility of chemical spills.
4. Keep hands and other objects away from your face, nose, eyes, ears, and mouth. The
application of cosmetics in the laboratory is prohibited in the laboratory
5. Work areas/surfaces must be disinfected before and after use.

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6. Laboratory coats must be worn and buttoned while in the laboratory. Laboratory coats should
not be worn outside the laboratory.
7. Protective eyewear must be worn when performing any exercise or procedure in the
laboratory.
8. Long hair should be secured behind your head to minimize fire hazard or contamination of
experiments.
9. Hands must be washed before leaving the laboratory.
10. Upon entering the laboratory, coats, books, and other paraphernalia e.g. purses, briefcases etc
should be placed in specified locations and never on bench tops (except for your lab manual).
11. Never pipette anything by mouth (including water). Always use pipetting devices.
12. Label all materials with applicable information.
13. Dispose of wastes in their proper containers (see Biohazard Waste Disposal below).
14. When handling chemicals note the hazard code on the bottle and take the appropriate
precautions indicated.
15. Do not pour chemicals down the sink.
16. Return all chemicals, reagents, cultures, and glassware to their appropriate places.
17. Do not pour biohazard fluids down the sink.
18. Glassware should be washed with soap and water, and then rinsed with distilled water.
19. Flame transfer loops, wires, or needles before and immediately after use to transfer biological
material.
20. Do not walk about the laboratory with transfer loops, wires, needles, or pipettes containing
infectious material.
21. Be careful around Bunsen burners. Flames cannot always have been seen.
22. Report any broken equipment, immediately; report any broken glass, especially those
containing infectious materials.
23. If you are injured in the laboratory, immediately contact your course instructor.
24. Spills, cuts and other accidents should be reported to the instructor in case further treatment is
necessary.
25. Familiarize yourself with safety equipment in the laboratory and emergency escape routes.
26. Always wipe and clean the lenses of your microscope before putting it away. Use the
appropriate tissue paper and cleaning solution for this purpose.

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27. Use appropriate universal precautions with all biological fluids.

28. Do not remove any materials from the laboratory without the written permission of the course
instructor or technical assistant.
b. Safety of equipment and materials in microbiology laboratory
Monitoring and safe handling of equipments used in the Microbiology Laboratory is very
important. Many organisms are sensitive to specific temperatures and/or atmospheric conditions;
so incubators, freezers and refrigerators must be monitored to insure that the desired conditions
are maintained. Other equipment used in the laboratory such as centrifuges, microscopes,
autoclaves, balances, and pipettes must be checked for proper functioning.
Every effort should be made to prevent equipment from becoming contaminated. To reduce the
likelihood of equipment malfunction that could result in leakage, spill or unnecessary generation
of aerosolized pathogens:
 Review the manufacturer’s documentation. Keep for future reference.
 Use and service equipment according to the manufacturer’s instructions.
 Ensure that anyone who uses a specific instrument or piece of equipment is properly
trained in setup, use and cleaning of the item.
 Ensure that equipment leaving the laboratory for servicing or disposal is appropriately
decontaminated.
Equipment Safety measures
Incubators The temperatures of incubators and refrigerators should be recorded as soon as
and they are opened in the morning and before ambient temperature can affect them.
refrigerators To facilitate that process, tape a log sheet on the door of the instrument and
complete daily. Some thermometers are built into the door so the temperature can
be seen without exposing the inside to room temperature, but their temperatures
should still be recorded.
Incubators that are used to enhance atmospheric conditions such as carbon dioxide
(CO2) and anaerobic conditions should also be checked and results recorded.
If candle jars are used to generate CO2, a growth control plate with a CO2 sensitive
organism should be incubated along with plates from patients’ samples as it is
difficult to monitor the exact amount of CO2 in the jar.

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If any problems are noted, corrections should be made immediately. If cultures

have been incubated in incubators in which temperature or atmospheric conditions
were not maintained for a significant length of time, primary samples should be re-
plated. If an adequate sample is not still available a new one should be obtained
from the patient. As a rule, all samples should be kept until the report is finalized.
Autoclaves Staff operating the autoclave must receive training that covers detailed instructions
for use and safety precautions. Autoclaves are used to sterilize material used in the
microbiology laboratory to prevent contamination of samples being testing and to
decontaminate waste material before it is discarded.
When operating an autoclave, it is important to be sure the temperature is high
enough to kill any organisms. Chemically treated tape strips with indicators that
change color at a prescribed temperature are often used. A color change shows
that the autoclave achieved the desired temperature. Cards and discs are also
available and demonstrate similar sensitivity to temperature.
When using these indicators, they should be placed inside the batch – not just on
the outside surface.
Biological indicators use glass vials containing specific concentrations of
temperature resistant spores of Bacillus stereothermophilus. A color change in the
vials shows a pH change indicating that the spores were killed in the autoclave.
Biological indicators are superior to chemical indicators because they not only
show that the temperature has been met, but they also verify that desired time and
pressure were achieved during the sterilization process.
Remember that if the autoclave was overloaded or if the volume was too great, the
material may not be sterile even if the indicators work. Procedure manuals should
describe the maximum load that can be used in each autoclave.
A log book should be kept for each autoclave or other means of sterilization.
Maintain the following information:
· date that the material was sterilized;
· start and end time of sterilization;
· maximum temperature achieved;

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· number of trays or baskets included in the run;

· results of indicator;
· Signature of user.
Microscopes The microscope is one of the most valuable tools available to laboratory staff, but
is often poorly maintained. Dirty, contaminated, or damaged lenses make reading
and interpretation of material difficult and unreliable. Always keep non-immersion
lenses free of oil.
Establish Kohler illumination so the light source will illuminate the sample for
maximum viewing. Kohler illumination will eliminate the need to move the
condenser up and down when changing lenses, a common misuse of the
microscope.
Always focus on objects using the 10x lens first and then rotate to other lenses as
needed. This single process will prevent scratching of higher powered lenses.
All staff should be trained on proper care and use of the microscope using
accepted references.

6.2. Methods of sterilization and Disinfection

objectives:
 Define sterilization and disinfection
 Differentiate between sterilization and disinfection
 List methods of sterilization/disinfection
 State the principles of sterilization/disinfection methods
 Describe the application of sterilization/disinfection in healthcare settings
Sterilization:
 Sterilization is defined as the process where all the living microorganisms, including
bacterial spores are killed. Sterilization can be achieved by physical, chemical and
physiochemical means. Chemicals used as sterilizing agents are called chemisterilants.
To be effective, sterilization must be preceded by meticulous cleaning (mechanical or
manual) to remove all foreign materials from objects prior to undergoing sterilization

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Disinfection :
 Disinfection is the process of elimination of most pathogenic microorganisms (excluding
bacterial spores) on inanimate objects. Disinfection can be achieved by physical or
chemical methods. Chemicals used in disinfection are called disinfectants. Different
disinfectants have different target ranges, not all disinfectants can kill all
microorganisms. Some methods of disinfection such as filtration do not kill bacteria, they
separate them out.
 Sterilization is an absolute condition while disinfection is not. The two are not
synonymous.
Definition of other terms:
 Decontamination is the process of removal of contaminating pathogenic microorganisms
from the articles by a process of sterilization or disinfection.
 Asepsis is the employment of techniques (such as usage of gloves, air filters, uv rays etc)
to achieve microbe-free environment.
 Antisepsis is the use of chemicals (antiseptics) to make skin or mucus membranes devoid
of pathogenic microorganisms
 Bacteriostasis is a condition where the multiplication of the bacteria is inhibited without
killing them.
 Bactericidal is chemical that can kill or inactivate bacteria.

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i. Physical Methods
Heat
 Heat is considered to be the most reliable method of sterilization of articles that can
withstand heat.
 Heat acts by oxidative effects as well as denaturation and coagulation of proteins.
 Those articles that cannot withstand high temperatures can still be sterilized at lower
temperature by prolonging the duration of exposure.
Factors affecting sterilization by heat are:
 Nature of heat: Moist heat is more effective than dry heat
 Temperature and time: temperature and time are inversely proportional. As temperature
increases the time taken decreases
 Number of microorganisms: More the number of microorganisms, higher the temperature
or longer the duration
 Nature of microorganism: Depends on species and strain of microorganism, sensitivity to
heat may vary. Spores are highly resistant to heat

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 Type of material: Articles that are heavily contaminated require higher temperature or
prolonged exposure. Certain heat sensitive articles must be sterilized at lower temperature
 Presence of organic material: Organic materials such as protein, sugars, oils and fats
increase the time required
Action of heat
 Dry heat acts by protein denaturation, oxidative damage and toxic effects of elevated
levels of electrolytes
Dry Heat
 Red heat: Articles such as bacteriological loops, straight wires, tips of forceps and searing
spatulas are sterilized by holding them in Bunsen flame till they become red hot
 Flaming: This is a method of passing the article over a Bunsen flame, but not heating it to
redness. Articles such as scalpels, mouth of test tubes, flasks, glass slides and cover slips
are passed through the flame a few times. Even though most vegetative cells are killed,
there is no guarantee that spores too would die on such short exposure.
 Incineration: This is a method of destroying contaminated material by burning them in
incinerator. Articles such as soiled dressings; animal carcasses, pathological material and
bedding etc should be subjected to incineration. This technique results in the loss of the
article, hence is suitable only for those articles that have to be disposed.
Hot air oven:
 This method was introduced by Louis Pasteur.
 Articles to be sterilized are exposed to high temperature (160° C) for duration of one hour
in an electrically heated oven.
 The oven should be fitted with a thermostat control, temperature indicator, meshed
shelves and must have adequate insulation.
 Articles sterilized: Metallic instruments (like forceps, scalpels, scissors), glasswares (such
as petri-dishes, pipettes, flasks, all-glass syringes), swabs, oils, grease, petroleum jelly.
 Advantages
o It is an effective method of sterilization of heat stable articles
o The articles remain dry after sterilization

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 Disadvantages
o Since air is poor conductor of heat, hot air has poor penetration
o Cotton wool and paper may get slightly charred
o Glasses may become smoky
o Takes longer time compared to autoclave
 Infra-red rays
o Infrared rays bring about sterilization by generation of heat
o Articles to be sterilized are placed in a moving conveyer belt and passed through a
tunnel that is heated by infrared radiators to a temperature of 180°C
o The articles are exposed to that temperature for a period of 7.5 minutes
o Articles sterilized included metallic instruments and glassware
o It requires special equipments and mainly used in central sterile supply department
 Moist Heat
o Moist heat acts by coagulation and denaturation of proteins
 At temperature below 100°C
o Pasteurization: This process was originally employed by Louis Pasteur. Currently
employed in food and dairy industry. There are two methods of pasteurization, the
holder method (heated at 63°C for 30 minutes) and flash method (heated at 72°C for
15 seconds) followed by quickly cooling to 13°C. This method is suitable to destroy
most milk borne pathogens like Salmonella, Mycobacteria, Streptococci,
Staphylococci and Brucella, however Coxiella may survive pasteurization.
o Serum bath: The contaminating bacteria in a serum preparation can be inactivated by
heating in a water bath at 56°C for one hour on several successive days. Proteins in
the serum will coagulate at higher temperature. Only vegetative bacteria are killed
and spores survive.
 At temperature 100°C
o Boiling: Boiling water (100°C) kills most vegetative bacteria and viruses
immediately. Certain bacterial toxins such as Staphylococcal enterotoxin are also heat
resistant. Some bacterial spores are resistant to boiling and survive; hence this is not a
substitute for sterilization. When absolute sterility is not required, certain metal

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articles and glass wares can be disinfected by placing them in boiling water for 10-20
minutes. The lid of the boiler must not be opened during the period.
o Steam at 100°C: Instead of keeping the articles in boiling water, they are subjected to
free steam at 100°C. A steamer is a metal cabinet with perforated trays to hold the
articles and a conical lid. The bottom of steamer is filled with water and heated. Sugar
and gelatin in medium may get decomposed on autoclaving; hence they are exposed
to free steaming for 20 minutes for three successive days. This process is known as
tyndallisation (after John Tyndall) or fractional sterilization or intermittent
sterilization. The vegetative bacteria are killed in the first exposure and the spores that
germinate by next day are killed in subsequent days.
 At temperature above 100°C
o Autoclave: Sterilization can be effectively achieved at a temperature above 100°C
using an autoclave. Water boils at 100°C at atmospheric pressure, but if pressure is
raised, the temperature at which the water boils also increases. In an autoclave the
water is boiled in a closed chamber. As the pressure rises, the boiling point of water
also raises. At a pressure of 15 lbs inside the autoclave, the temperature is said to be
121°C. Exposure of articles to this temperature for 15 minutes sterilizes them.
Advantages of steam: It has more penetrative power than dry air, it moistens the
spores (moisture is essential for coagulation of proteins).
 Advantage: Very effective way of sterilization, quicker than hot air oven.
 Disadvantages: Drenching and wetting of articles may occur, trapped air may
reduce the efficacy, takes long time to cool.
 Radiation
o Two types of radiation are used, ionizing and non-ionizing.
o Non-ionizing rays are low energy rays with poor penetrative power while ionizing
rays are high-energy rays with good penetrative power
o Since radiation does not generate heat, it is termed "cold sterilization." Fruits and
vegetables are irradiated to increase their shelf life.
 Filtration
o Filtration does not kill microbes; it separates them out. Membrane filters with pore
sizes between 0.2-0.45 μm are commonly used to remove particles from solutions that

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can't be autoclaved. It is used to remove microbes from heat labile liquids such as
serum, antibiotic solutions, sugar solutions, urea solution.

ii. Chemical Methods of Disinfection

 Disinfectants are those chemicals that destroy pathogenic bacteria from inanimate
surfaces.
 Some chemical have very narrow spectrum of activity and some have very wide.
 Those chemicals that can sterilize are called chemisterilants.
 Those chemicals that can be safely applied over skin and mucus membranes are called
antiseptics.
Classification of disinfectants
1. Based on consistency
a. Liquid (E.g., Alcohols, Phenols)
b. Gaseous (Formaldehyde vapor, Ethylene oxide)
2. Based on spectrum of activity
a. High level
b. Intermediate level
c. Low level
3. Based on mechanism of action
a. Action on membrane (E.g. Alcohol, detergent)
b. Denaturation of cellular proteins (E.g., Alcohol, Phenol)
c. Oxidation of essential sulphydryl groups of enzymes (E.g., H2O2, Halogens)
d. Alkylation of amino-, carboxyl- and hydroxyl group (E.g., Ethylene Oxide,
Formaldehyde)
e. Damage to nucleic acids (Ethylene Oxide, Formaldehyde)

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 Alcohols
o Mode of action: Alcohols dehydrate cells, disrupt membranes and cause coagulation
of protein. Examples: Ethyl alcohol, isopropyl alcohol and methyl alcohol.
o Application: A 70% aqueous solution is more effective at killing microbes than
absolute alcohols. 70% ethyl alcohol is used as antiseptic on skin.
o Disadvantages: Skin irritant, volatile (evaporates rapidly), inflammable.
 Aldehydes
o Mode of action: Acts through alkylation of amino-, carboxyl- or hydroxyl group, and
probably damages nucleic acids. It kills all microorganisms, including spores.
Examples: Formaldehyde, Gluteraldehyde
o Application: 40% Formaldehyde (formalin) is used for surface disinfection and
fumigation of rooms, chambers, operation theatres, biological safety cabinets, wards,
sick rooms etc. It also sterilizes bedding, furniture and books. 10% formalin with
0.5% tetraborate sterilizes clean metal instruments. 2% gluteraldehyde is used to
sterilize thermometers, cystoscopes, bronchoscopes, centrifuges, anasethetic
equipments etc.
o Disadvantages: Vapors are irritating (must be neutralized by ammonia), has poor
penetration, leaves non-volatile residue, activity is reduced in the presence of protein.

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Gluteraldehyde requires alkaline pH and only those articles that are wettable can be
sterilized.
 Phenol
o Mode of action: Act by disruption of membranes, precipitation of proteins and
inactivation of enzymes. Examples: 5% phenol, 1-5% Cresol, 5% Lysol (a saponified
cresol), hexachlorophene, chlorhexidine, chloroxylenol (Dettol).
o Applications: Joseph Lister used it to prevent infection of surgical wounds. Phenols
are coal-tar derivatives. They act as disinfectants at high concentration and as
antiseptics at low concentrations. They are bactericidal, fungicidal, mycobactericidal
but are inactive against spores and most viruses.
o Disadvantages: It is toxic, corrosive and skin irritant Chlorhexidine is inactivated by
anionic soaps. Chloroxylenol is inactivated by hard water.
 Halogens
o Mode of action: They are oxidizing agents and cause damage by oxidation of
essential sulfydryl groups of enzymes. Chlorine reacts with water to form
hypochlorous acid, which is microbicidal. Examples: Chlorine compounds (chlorine,
bleach, hypochlorite) and iodine compounds (tincture iodine, iodophores).
o Applications: Tincture of iodine (2% iodine in 70% alcohol) is an antiseptic. 10%
Povidone Iodine is used undiluted in pre and postoperative skin disinfection. Chlorine
gas is used to bleach water. Household bleach can be used to disinfect floors.
Household bleach used in a stock dilution of 1:10. In higher concentrations chlorine is
used to disinfect swimming pools. Used at a dilution of 1:10 in decontamination of
spillage of infectious material.
o Disadvantages: They are rapidly inactivated in the presence of organic matter. Iodine
is corrosive and staining. Bleach solution is corrosive and will corrode stainless steel
surfaces.
 Surface Active Agents
o Mode of actions: They have the property of concentrating at interfaces between lipid
containing membrane of bacterial cell and surrounding aqueous medium. These
compounds have long chain hydrocarbons that are fat soluble and charged ions that
are water-soluble. Since they contain both of these, they concentrate on the surface of

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membranes. They disrupt membrane resulting in leakage of cell constituents.

Examples: These are soaps or detergents.
o Application: They are active against vegetative cells, Mycobacteria and enveloped
viruses. They are widely used as disinfectants at dilution of 1-2% for domestic use
and in hospitals
o Disadvantages: Their activity is reduced by hard water, anionic detergents and
organic matter. Pseudomonas can metabolise cetrimide, using them as a carbon,
nitrogen and energy source.
 Hydrogen Peroxide
o Mode of action: It acts on the microorganisms through its release of nascent oxygen.
Hydrogen peroxide produces hydroxyl-free radical that damages proteins and DNA.
o Application: It is used at 6% concentration to decontaminate the instruments,
equipments such as ventilators. 3% Hydrogen Peroxide Solution is used for skin
disinfection and deodorising wounds and ulcers. Strong solutions are sporicidal.
o Disadvantages: Decomposes in light, broken down by catalase, proteinaceous
organic matter drastically reduces its activity.
 Ethylene Oxide (EO)
o Mode of action: It is an alkylating agent. It acts by alkylating sulfydryl-, amino-,
carboxyl- and hydroxyl- groups.
o Properties: It is a cyclic molecule, which is a colorless liquid at room temperature. It
has a sweet ethereal odor, readily polymerizes and is flammable.
o Application: It is a highly effective chemisterilant, capable of killing spores rapidly.
Since it is highly flammable, it is usually combined with CO2 (10%CO2+ 90%EO) or
dichlorodifluoromethane. It requires presence of humidity. It has good penetration
and is well absorbed by porous material. It is used to sterilize heat labile articles such
as bedding, textiles, rubber, plastics, syringes, disposable Petri dishes, and complex
apparatus like heart lung machine, respiratory and dental equipments.
o Disadvantages: It is highly toxic, irritating to eyes, skin, highly flammable,
mutagenic and carcinogenic.

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6.3. Standard precautions when handling biological materials

iii. Concepts of biohazardous materials
Working in a laboratory usually involves working with various chemical, physical, and
biological hazards. Because the hazards vary from laboratory to laboratory, employers must
address the hazards specific to their laboratories. Standard precautions are meant to reduce the
risk of transmission of blood borne and other pathogens from both recognized and unrecognized
sources. They are the basic level of infection control precautions which are to be used, as a
minimum, in the health care settings.
Preparing for laboratory work :
Before starting to work in a laboratory, you must familiarize with the following:
 The hazards of the materials in the lab, as well as appropriate safe handling, storage and
emergency protocols. Read labels and material safety data sheets (MSDSs) before
moving, handling or opening chemicals. Never use a product from an unlabeled
container, and report missing labels to your supervisor.
 The agents, processes and equipment in the laboratory. If you are unsure of any aspect of
a procedure, check with your supervisor before proceeding.
 The location and operation of safety and emergency equipment such as fire extinguishers,
eye wash and shower, first aid and spill response kits, fire alarm pull stations, telephone
and emergency exits
 Emergency spill response procedures for the materials you will handle
During laboratory work:
 Restrict laboratory access to authorized persons only. Children are not permitted in labs.
 Smoking; eating; drinking; storing food, beverages or tobacco; handling contact lenses
are not permitted in laboratories.
 Wear lab coats (knee length) and safety glasses in laboratories employing chemicals,
biohazards or radioisotopes. Open shoes, such as sandals, should never be worn in the
lab.
 Keep work places clean and free of unwanted chemicals, biological specimens, Avoid
leaving reagent bottles, empty or full, on the floor.
 Work only with materials once you know their safe handling and storage.

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 Never pipette by mouth; use mechanical transfer devices.

 Keep exits and passageways clear at all times.
 Ensure that accesses to personal protective equipments are not blocked.
 Report accidents and dangerous incidents (“near-misses”) promptly to your supervisor
 Wash your hands thoroughly before leaving the laboratory.
 Perform procedures that liberate infectious bio-aerosols in a biological safety cabinet
 Handle all human blood and body fluids as if potentially infectious.
Cleaning up before leaving:
Perform a safety check at the end of each experiment and before leaving the lab. Make sure to:
 Turn off gas, water, electricity, vacuum and compression lines and heating apparatus
 Return unused materials, equipment and apparatus to their proper storage locations
 Dispose of all waste material.
 Remove defective or damaged equipment immediately, and arrange to have it repaired or
replaced
 Decontaminate any equipment or work areas that may have been in contact with
hazardous materials.
 Leave behind protective clothing (lab coats, gloves, etc.) when leaving the laboratory.

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Fig-6. 1. Symbols to be identified by all laboratory professionals

Fire safety :
Laboratory fires can by caused by Bunsen burners, runaway chemical reactions, electrical
heating units, failure of unattended or defective equipment, or overloaded electrical circuits.
Familiarize yourself with the operation of the fire extinguishers and the location of pull stations,
emergency exits and evacuation routes where you work. In the event that the general alarm is
sounded use the evacuation routes established for your area and follow the instructions of the
Evacuation Monitors. Once outside of the building, move away from the doors to enable others
to exit.

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The fire triangle:

Fire cannot occur without an explosion source, fuel and an oxidizing atmosphere (usually air),
the three elements that comprise what is called the “fire triangle”:

Fig-6.2 The fire triangle

Fire will not be initiated if any one
one of these elements is absent, and will not be sustained if one of
these elements is removed. This concept is useful in understanding prevention and control of
fires. For example, the coexistence of flammable vapours and explosion sources should be
avoided,, but when flammable vapours cannot be controlled, elimination of explosion sources is
essential.
Learn how to use the extinguisher in your lab, as there will be no time to read instructions during
an emergency. Attempt to fight small fires only, and only if there is an escape route behind you.
Remember to have the extinguisher recharged after every use.
 P: Pull and twist the locking pin to break the seal.
 A: Aim low, and point the nozzle at the base of the fire.
 S: Squeeze the handle to release the extinguishing
extin agent.
 S: Sweep from side to side until the fire is out.
 Be prepared to repeat the process if the fire breaks out again.
iv. Safety precautions in microbiology laboratory
Universal/Standard Precautions:
Precautions
These guidelines refer to the precautions, consistently
consistently used for all patients regardless of their
infection status and diagnosis. The main objective is to prevent exposure of staff and patients to

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blood and body fluids.

 Don’t eat, drink, smoke or apply cosmetics (including lip balm).
 Don’t insert or remove contact lenses.
 Don’t bite nails or chew on pens.
 Don’t mouth pipette.
 Limit access to the laboratory to trained personnel only.
 Assume all patients are infectious for HIV or other blood borne pathogens.
 Use appropriate barrier precautions to prevent skin an mucous membrane exposure,
including wearing gloves at all times and masks, goggles, gowns or aprons if there is a
risk of splashes or droplet formation.
 Wash hands thoroughly and other skin surfaces after gloves are removed and
immediately after any contamination.
 Avoid injuries to sharps such as needles and scalpels.
Standard Precautions:
In 1996, CDC developed a new system of standard precaution synthesizing the features of
universal precautions and body substance isolation. Standard precautions are used in the care of
all patients and apply to blood, all body fluids, secretion and excretion except sweat regardless of
whether they contain visible blood.
Standard precautions are guidelines and procedures designed to reduce the risk of transmission
of microorganisms from both recognized and unrecognized sources of infection in healthcare
settings.
Standard Precautions Include:
 Hand washing
 Barrier protection
 Safe handling of sharp items
 Safe handling of specimen (blood etc)
 Safe handling of spillage of blood/body fluid
 Use of disposable/ sterile items

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Hand Washing:
 Single most important method to limit cross transmission of nosocomial pathogens
 Multiple opportunities exist for HCW hand contamination by Direct patient care and
Inanimate environment
 This is an ideal safety precaution and gloves should not be regarded as a substitute for
hand washing.
For General Patient Care:
 Wash hands thoroughly in running water with soap without missing any area. For
effective hand washing first wash palms
palms with fingers followed by back of hands,
knuckles, thumbs, fingertips and wrists, Rinse and dry thoroughly.
 Wash hand immediately after accidental contamination with blood/body fluid, before
eating and drinking and after removing gowns/coats and gloves.
 Leave soap bars in dry containers to prevent contamination with microorganism
microorganism.

Fig- 6.3 stages of effective hand hygiene

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v. Application of Personal protective equipment

Gloves:
 Wear while collecting/ handling blood specimens and blood soiled items.
 Wear while disposing waste.
 Remove before handling door knobs, telephone, pens performing office work.
 Discard if cracked, discolored or punctured.
 Discard if blood spills on them.
 Don’t reuse disposable gloves.
 Wash hands when gloves are removed or changed.
Masks:
 Wear masks and protective glasses if splashing or spraying of blood/body fluids is
expected.
 Mask of cotton wool, gauze, or paper masks are ineffective. Paper mask with synthetic
material for filtration are an effective barrier against microorganism.
Caps:
 Cover hair completely in aseptic units, operating rooms, or performing selected invasive
procedure.
Gown and Aprons:
 Wear clean clothes made up of a material easy to clean.
 Change after exposure to blood and body fluids.
 Wear gown or apron of plastic or water resistant paper when splashes of blood or other
body fluids are likely to occur.
Occlusive Bandage:
 Cover all skin defects e.g. cuts, scratches or other breaks with waterproof dressing before
patient care.
Safe Handling of Sharps:
 Take extreme care to avoid autoinoculation.
 Discard all chipped or cracked glassware in appropriate containers.
 Don’t manipulate disposable needles. Never bend, break, recap or remove needle from
syringe.

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 Dispose your own sharps. Don’t pass used sharps directly from one person to another.
 Discard needles in puncture proof rigid containers (Plastic or cardboard boxes) after
disinfection in 0.5-1% sodium hypochlorite solution. Use needle shredder if available for
needles or needles along with syringe nozzle.
 Send sharp disposable containers for disposal when three-fourth full.
Safe Handling of Specimen:
 Collect specimens, specially blood and body fluids in pre sterilized containers properly
sealed to prevent leakage or spillage.
 Use autoclaved/ pre-sterilized disposable syringes and needles for vene-punture and
lancets/ cutting needles for finger pricks.
 Cover cuts in hands properly with waterproof adhesive bandages.
 Wear disposable gloves while collecting blood/body fluids and maintain proper asepsis.
 Wash hands thoroughly with soap and water, particularly after handling specimens.
Safe Handling of Blood/Body Fluid Spills
 Cover spills of infected or potentially infected material on the floor with paper towel/
blotting paper/ newspaper.
 Pour 1% sodium hypochlorite solution on and around the spill area and cove with paper
for at least 30 minutes.
 After 30 minutes, remove paper with gloved hands and discard.
Use of Disposable Sterile Items
 Ensure proper handling of disposable/ sterile item before use.
 There should be no recirculation of disposable items
vi. Biosafety Cabinet (BSC)
Biosafety cabinet (BSC) - also called a biological safety cabinet or microbiological safety
cabinet - is an enclosed, ventilated laboratory workspace for safely working with materials
contaminated with (or potentially contaminated with) pathogens requiring a defined biosafety
level. Several different types of BSC exist, differentiated by the degree
of biocontainment required.

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Purposes:
The primary purpose of a BSC is to serve as a means to protect the laboratory worker and the
surrounding environment from pathogens. All exhaust air is HEPA-filtered as it exits the
biosafety cabinet, removing harmful bacteria and viruses.
Classes:
The U.S. Centers for Disease Control and Prevention (CDC) classifies BSCs into three classes.
These classes and the types of BSCs within them are distinguished in two ways: the level of
personnel and environmental protection provided and the level of product protection provided.
BSC Class I:
Class I cabinets provide personnel and environmental protection but no product protection. In
fact, the inward flow of air can contribute to contamination of samples.
BSC Class II:
Class II cabinets provide both kinds of protection (of the samples and of the environment) since
makeup air is also HEPA-filtered. Class II cabinets are the commonly used cabinets in clinical
and research laboratories.
BSC Class III:
The Class III cabinet, generally only installed in maximum containment laboratories, is
specifically designed for work with BSL-4 pathogenic agents, providing maximum protection.
The enclosure is gas-tight, and all materials enter and leave through a dunk tank or double-
door autoclave. Gloves attached to the front prevent direct contact with hazardous materials
(Class III cabinets are sometimes called glove box). These custom-built cabinets often attach into
a line, and the lab equipment installed inside is usually custom-built as well.
vii. Safe disposal of biohazardous materials
Biomedical laboratories are special and have unique work environments that may pose
identifiable infectious disease risks to persons in or near them. Correct Biohazard Waste
handling is therefore necessary to reduce or eliminate exposure to laboratory staff, other persons
and the outside environment to potentially hazardous materials: such as blood or other body
fluids, lab wastes and specimens which might be contaminated with agents known to be
infectious to humans.

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Biohazardous Waste: Segregation, Collection & Disposal Guide

Sharps :
This includes items which are sharp enough to puncture skin and contaminated with unsterilized
biological materials. Example devices include:
 needles & lancets,
 scalpels & razor blades,
 glass slides,
 glass Pasteur pipettes,
 Biologically-contaminated broken glass.
This category also includes all sharps-associated medical devices (i.e., syringes).
Collection & Storage:
These devices need to be placed into a sharps container immediately after use. Sharps containers
are biohazard-marked, solid-walled, puncture-proof containers that are leak-proof on the sides
and bottom.
NOTE: Sharps containers must be containers designed for that purpose. Cardboard boxes or
repurposed food/beverage containers are not acceptable!
Sharps container lids have a restricted access opening to prevent devices from being accessed
once inside the container. Assure that the lid is properly and completely installed before using it
for sharps collection. If the opening requires you to drop the device in vertically, it should be
closed when the container is not in use. Keep the container free of visible contamination and
store it in an upright position.
Treatment & Disposal:
Permanently close the container when it is 3/4ths full or when items do not freely fall into the
container, regardless of the fullness level. (Do not force objects into a container or shake the
container to make more space!) Place full containers in the designated biohazardous waste
pickup point.
If containers will be picked up by an outside vendor (i.e., Stericycle), place the closed container
in the vendor-supplied waste container for pickup and disposal. Sharps containers must be
autoclaved using a validated waste treatment cycle or incinerated for final treatment. At
Vanderbilt, sharps containers are collected by Environmental Services or an outside vendor and
ultimately treated and disposed of by an outside vendor.

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Liquid Biohazardous Waste

This includes bulk biological liquids such as culture media, pooled clinical specimen liquids, etc.
Collection & Storage:
Vacuum flasks and liquid “pour-off” containers should be charged with disinfectant prior to use
to help prevent growth of contamination in the flask during the holding period. Collection
vessels need to be labeled with the biohazard label and name of the disinfectant. Non-breakable
vessels should be used whenever possible.
Vacuum flasks should be stored in a non-breakable and leak-proof secondary container when not
maintained inside a biosafety cabinet (BSC). Vacuum flasks must also be equipped with an
overflow flask and/or HEPA filter on the line to protect vacuum lines in the event of a flask
malfunction. Flasks should be discharged and cleaned weekly, or when they are half-full,
whichever comes first, to prevent overflow and prevent growth of contaminants.
Treatment & Disposal:
Treated liquid waste may be disposed of via the lab sink. Use a lab coat, gloves and splash
goggles (or safety glasses with a face shield) when discharging waste to the drain. Use care to
minimize generating “splash back” and thoroughly rinse the sink following waste discharge.
Liquid waste may also be autoclaved and then disposed of via the sink. NOTE: If you will
autoclave your waste, you should not pre-treat with disinfectant, or you should instead use only a
disinfectant that is safe to autoclave based on information from the manufacturer. Bleach is not
safe to autoclave.
Solid, non-sharps biohazardous waste:
This includes lab consumables that have come in contact with viable biological materials that
contain recombinant or synthetic nucleic acids, clinical specimens in a lab setting, and any lab
materials that are regarded as potentially infectious. Examples include:
 Gloves used for biological material manipulations,
 Disposable culture flasks, serological pipettes, pipette tips and well plates,
 Waste items contaminated with blood in a manner that would present a personnel
exposure risk (i.e., more than incidentally contaminated).

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Collection & Storage:

Collect waste in a solid-walled, leak-proof container lined with an autoclaveable biohazard bag.
The container needs to have a lid and be marked with the biohazard symbol. The container
needs to be closed when not actively in use.
Bench-top biohazardous waste containers should meet the same criteria. Bags should be secured
closed and transferred to the floor container when full. If the container has no lid, the bag needs
to be securely closed and transferred to the floor container at the end of the day.
Waste generated in a BSC should be collected in a biohazard container inside the BSC whenever
procedures permit.
Collect serological pipettes separately (i.e., placed in pipettekeeper boxes, in a dedicated bag, or
in a box lined with a biohazard bag) in order to prevent bag puncture during the waste handling
process.
Treatment & Disposal:
Bags should be securely closed and transferred to a leak-proof secondary container in the
designated biohazardous waste pickup point in the lab. (Heavy bags should be double-bagged to
further prevent leakage during the handling process.)
If bagged waste will be picked up by an outside vendor (i.e., Stericycle), place the securely
closed bag in the vendor-supplied waste container for pickup and disposal. These waste bags are
autoclaved or incinerated for final treatment by the vendor.
NOTE: Bagged waste should always be stored in a secondary container designed to contain a
leak including when being transported to autoclave facilities. (Double-bagging alone is not
sufficient to contain a leak if the bags are ruptured.) Bagged waste treated on site must be treated
using an established autoclave cycle that has been validated for effective sterilization of this
waste. Waste picked up by Environmental Services is treated in this manner.
Pathological Biohazardous Waste:
Pathological waste includes removed human (or animal) organs, tissues and body parts that have
been exposed to infectious agents.
Collection and Disposal Procedures:
To prevent potential leaks, pathological waste should be double-bagged and stored similar to
liquid waste in secondary containers. It is commonly disposed of through incineration or
chemical treatment, but not autoclaving.

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Self-check Question:
PART I. Say true or false
1. 70% alcohol is more effective than 95% alcohol at killing microbes.
2. Dry heat is more effective than moist heat at killing microbes.
PART III. MCQ
1. Which one of the following disinfectant has a broader range of activity?
A. Low level disinfectant C. High level disinfectant
B. Intermediate level disinfectant D. All are equally effective
PART III. Essay
1. Differentiate between sterilization and disinfection, and then give two examples for each.
2. Why sterilization and disinfection are important in healthcare settings?
3. What are standard precautions?
4. How would you handle blood/body fluid spills?
5. Write measures to prevent fire accidents
6. Explain steps of hand washing
Summary
 Safety in a microbiology laboratory is important because:
o Microbiology laboratory cultures, manipulates, and uses virulent and/or potentially
pathogenic microorganisms.
o There are also chemicals used in this laboratory that are potentially harmful.
o Many procedures in the laboratory involve glassware, open flames, and sharp objects
that can cause trauma/ damage if used improperly.
 In order to prevent the infections caused by microorganisms and harms of laboratory
chemicals, the general safety rules in microbiology laboratory must be followed.
 As integral part of laboratory safety, monitoring and safe handling of equipments used in the
Microbiology Laboratory is very important.
 There are different sterilization and disinfection methods used in microbiology laboratory so
as to prevent the contamination of professionals as well as the surrounding environment.
 Standard precautions are meant to reduce the risk of transmission of blood borne and other
pathogens from both recognized and unrecognized sources.

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Chapter 7: Microscopic Examination of Wet and Stained Preparations and

Identification of Common Microbial Pathogens
Dear trainees,
In this chapter, you will learn the technique of wet mount and staining preparations that are
used in medical microbiology. Additionally, you will develop knowledge and skills of
performing the preparation of smears as well as examination of wet mount and stained
preparations in medical microbiology.
Objectives:
At the end of this chapter you will be able to
 Describe wet mount techniques in microbiology
 Perform microscopic examination of wet mount preparation
 Describe types of staining techniques in medical microbiology
 Describe the principle of staining in medical microbiology
 Explain terms related to staining techniques
 Perform different staining of smeared samples
 Perform microscopic examination of stained preparation
7.1 KOH preparation
 It helps in the initial examination of keratinized tissue suspected of fungal infection. It is
useful to observe the presence of characteristic fungal elements including hyphae,
budding yeast and spherules. Do not use cotton swabs to collect specimens since cotton
fibers may resemble hyphae.
 Fungal elements may be obscured by skin, hair, or nail tissue. Thus KOH (10-20%w/v)
dissolves keratin in skin, hair or nail specimens facilitating the observation of the
organism’s morphology.
o Advantage: Rapid and simple to perform.
o Disadvantage: Has poor contrast, and clearing of some specimens may require an
extended time.
Supplies and Equipment
 KOH reagent
 70% alcohol
 Laboratory coat

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 Disposable Gloves
 Laboratory request form
 Water and soap
 Pencil
 Log book
 Microscope
 Slides
 pencil
 Cover glasses/cover slip
 Surgical blade
Procedures of KOH Preparation
1. Apply proper hand hygiene and gather equipment.
2. Wear the laboratory coat and don gloves
3. Add one drop of 10-20%KOH reagent on slide
4. Place a small portion of the material (skin scrapings, hair, and nail) to be examined
5. Press cover slip down on sample
6. Warm the slide gently to dissolve keratinized cells. Do not boil.
7. Allow specimen to clear, approximately 20 minutes.
8. Examine under low and high- dry magnification.
Note: Do not use cotton swabs to collect specimens, since cotton fibers may resemble
hyphae.
Interpretation:
 Observe for the presence of characteristic fungal elements, including hyphae, budding
yeast, and spherules.
 Dermatophytes appear as translucent branching rod- shaped filaments (hyphae) of
uniform width, with lines of separation (septa) appear at intervals.
 The uniform width and characteristic bending and branching distinguish hyphae from
hair and other debris.

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Skills Practice Session 7.1

Perform microscopic examination of KOH Preparation
Purpose:
The purpose of this activity is to enable you to practice those skills necessary to provide practice
microscopic examination of KOH preparation, and to achieve competency in these skills.
Instructions:
This activity should be conducted in a skill laboratory/healthy facility. You should review
Learning Guide for microscopic examination of KOH preparation before beginning the activity.
The trainer should demonstrate the steps/tasks in each learning guide one at a time. Under the
guidance of the trainer, you should then work in groups and practice the steps/tasks in the
Learning Guide for microscopic examination of KOH preparation for bacteria and observe each
other’s performance; while one learner/group doing the microscopic examination of KOH
Preparation activity, another learner/group should use the Learning Guide to observe
performance. You should then rotate roles. You should be able to perform the steps/tasks before
skills competency is assessed using the Checklist microscopic examination of KOH preparation
Conditions/ Situation for the operations:
 This activity could be done in skill laboratory or in healthy facility
Resources (Equipment and Materials):
 Microscope
 KOH reagent
 70% alcohol
 Laboratory coat
 Disposable Gloves
 Laboratory request form
 Water and soap
 Pencil
 Log book
 Microscope
 Slides
 pencil

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 Cover glasses/cover slip

 Surgical blade
Precaution: When you tried to find the sample to be examined, adequate safety precautions
must be taken to prevent the spread of infectious organisms and to avoid contamination
generally.

Standard Precautions
 Always wash hands before and after obtaining and handling specimens
 Cover cuts and lesions with waterproof dressing
 Wear disposable aprons and appropriate gloves if there is likelihood of contact or
contamination with blood or body fluids.
 Take care not to contaminate the outside of the container with blood or body fluid
 Do not use the specimen container for any other purpose
 Always ensure the top is closed securely
Procedure-Learning Guide/Checklist
Learning Guide 7.1
Perform microscopic examination of KOH Preparation
(To be completed by Participant/students)

Rate the performance of each step or task observed using the following rating scale:
1. Needs Improvement: Step or task not performed correctly, out of sequence (if necessary), or
is omitted
2. Competently Performed: Step or task performed correctly in proper sequence (if necessary)
but participant/student does not progress from step to step efficiently
3. Proficiently Performed: Step or task performed efficiently and precisely in the proper
sequence (if necessary)
Perform microscopic examination of KOH Preparation
(Some of the following steps/tasks should be performed simultaneously)

STEP/TASK
improvement
Competently

Proficiently
performed
performed

Remark
Needs

Getting ready
1. Wearing the Laboratory coat

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2. Washing your hand with soap

3. Wearing the gloves
4. Preparing the necessary equipment
5. Greeting the patient respectfully and with kindness.
6. Reading the order (request form)
7. Labeling the slide
8. Recording the patient identification (Name, Age, address etc.)
9. Getting the sample to be performed

10. Adding one drop of 10-20%KOH reagent on slide

11. Place a small portion of the material (skin scrapings, hair, and
nail) to be examined
12. Press cover slip down on sample

13. Warm the slide gently to dissolve keratinized cells. Do not boil.

14. Allow specimen to clear, approximately 20 minutes

15. Examine under low and high- dry magnification

16. Clean the working area

17. Remove gloves and gown and perform hand hygiene

Quality criteria:
During accomplishment of performing microscopic examination of KOH Preparation, the
trainees should be:
 Able to know the principle of KOH preparation
 Able to follow pre analytical, analytical and post analytical stages of procedures
 Able to apply safety procedures during collection and processing of fungal specimen
 Able to develop good attitude towards patients and professionals
7.2 Indian ink preparation
 It is used to identify the capsule of Cryptococcus neoformans, the yeast cell. Capsules
appear as clear halos against a dark back ground.
 Advantage: It is rapid method.

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 Disadvantage: it may be difficult to interpret as White blood cells and artifacts can be
mistaken for yeast or capsules.
Supplies and Equipment:
 Indian Ink reagent
 70% alcohol
 Laboratory coat
 Disposable Gloves
 Laboratory request form
 Water and soap
 Pencil
 Log book
 Microscope
 Slides
 pencil
 Cover glasses/cover slip
 Surgical blade
Procedure of Indian ink preparation:
1. Apply proper hand hygiene and gather equipment.
2. Wear the laboratory coat and don gloves
3. Add one drop of India ink to specimen/CSF
4. Apply a cover slip.
5. Examine the preparation microscopically using 10x and 40x objective.
Interpretation:
 Capsules appear as clear halos against a dark back ground.
Skills Practice Session 7.2
Topic: Perform microscopic examination of Indian ink preparation
Purpose:
The purpose of this activity is to enable you to practice those skills necessary to provide practice
microscopic examination of Indian ink preparation, and to achieve competency in these skills.

Instructions:
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This activity should be conducted in a skill laboratory/healthy facility. You should review
Learning Guide for microscopic examination of Indian ink preparation before beginning the
activity. The trainer should demonstrate the steps/tasks in each learning guide one at a time.
Under the guidance of the trainer, learners should then work in groups and practice the
steps/tasks in the Learning Guide for microscopic examination of Indian ink preparation for the
fungi and observe each other’s performance; while one of you/group doing the microscopic
examination of Indian ink preparation activity, another of you/group should use the Learning
Guide to observe performance. You should then rotate roles. You should be able to perform the
steps/tasks before skills competency is assessed using the Checklist microscopic examination of
Indian ink preparation
Conditions or situation for the operations:
 This activity could be done in skill laboratory or in healthy facility
Resources (Equipment and Materials):
 Microscope
 Indian Ink reagent
 70% alcohol
 Laboratory coat
 Disposable Gloves
 Laboratory request form
 Water and soap
 Pencil
 Log book
 Microscope
 Slides
 pencil
 Cover glasses/cover slip
 Surgical blade

Precaution: When you tried to find the sample to be examined, adequate safety precautions

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must be taken to prevent the spread of infectious organisms and to avoid contamination
generally.

Standard Precautions:
 Always wash hands before and after obtaining and handling specimens
 Cover cuts and lesions with waterproof dressing
 Wear disposable aprons and appropriate gloves if there is likelihood of contact or
contamination with blood or body fluids.
 Take care not to contaminate the outside of the container with blood or body fluid
 Do not use the specimen container for any other purpose
 Always ensure the top is closed securely

Procedure-Learning Guide/Checklist
Learning Guide 7.2
Perform microscopic examination of Indian ink preparation
(To be completed by Participant/students)

Rate the performance of each step or task observed using the following rating scale:
1. Needs Improvement: Step or task not performed correctly, out of sequence (if necessary), or
is omitted
2. Competently Performed: Step or task performed correctly in proper sequence (if necessary)
but participant/student does not progress from step to step efficiently
3. Proficiently Performed: Step or task performed efficiently and precisely in the proper
sequence (if necessary)
Perform microscopic examination of Indian ink preparation
(Some of the following steps/tasks should be performed simultaneously)

STEP/TASK
improvement
Competently

Proficiently
performed

performed

Remark
Needs

Getting ready
1. Wearing the Laboratory coat
2. Washing your hand with soap
3. Wearing the gloves

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4. Preparing the necessary equipment

5. Greeting the patient respectfully and with kindness.
6. Reading the order (request form)
7. Labeling the slide
8. Recording the patient identification (Name, Age, address
etc.)
9. Getting the sample to be performed

10. Add one drop of India ink to specimen/CSF

11. Press cover slip down on sample

12. Examine the preparation microscopically using 10x and 40x
objective
13. Clean the working area

14. Remove gloves and gown and perform hand hygiene

Quality criteria :
During accomplishment of performing microscopic examination of Indian Ink
Preparation, the trainees should be:
 Able to know the principle of Indian Ink preparation
 Able to follow pre analytical, analytical and post analytical stages of procedures
 Able to apply safety procedures during collection and processing of fungal specimen
 Able to develop good attitude towards patients, professions and environment
Self-check questions:
Instruction: Dear trainees, please! attempt all the questions listed below
1. What is the purpose of Indian Ink preparation?
2. What is the function of potassium hydroxide(KOH) reagent in KOH preparation?

7.3 Preparation of Wet Mount of Bacteria

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A wet mount is a fast way to observe bacteria. Direct examination of living microorganisms is
very useful in determining size, shape, and movement. In a wet mount, the specimen is
suspended in a drop of liquid (usually water) located between slide and cover glass. The water
refractive index of the water improves the image quality and also supports the specimen. In
contrast to permanently mounted slides, wet mounts cannot be stored over extended time
periods, as the water evaporates. For this reason, a wet mount is sometimes also referred to as a
“temporary mount” to contrast it from the “permanent mounts”, which can be stored over longer
times. Aim to prepare wet mount of a bacteria, for observing its natural shape, size and
arrangement in living condition. Bacteria cells can be seen easily and clearly, when colored by
stains, but in most of the staining processes, the cells die and lose their natural shape and size due
to heat-fixation as well as due to exposure to chemicals (stains, acid and alcohol).
Principle:
In wet mount, a drop of the bacteria suspension is placed on a slide, covered with a cover slip
and observed under a compound microscope or preferably under a dark-field or phase-contrast
microscope using oil-immersion objective.
Materials and Supply:
 Microscope
 Slides
 pencil
 Cover glasses/cover slip
 immersion oil
 specimen
 Water/normal saline
 Droppers/pipette
 Laboratory coat
 Disposable gloves
 Laboratory request form
 Log book

Procedure:

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1. Apply proper hand hygiene and gather equipment.

2. Wear the laboratory coat and don gloves
3. A slide is cleaned properly under tap water; such that water does not remain as drops on
its surface
4. The slide is dried by wiping with bibulous paper and subsequently moving over flame or
keeping in the sun.
5. If the bacteria have been grown on a solid medium (as slant culture or plate culture), a
drop of distilled water is put at the center of the slide. A loop is sterilized by heating it
over a flame to red hot. It is cooled to room temperature. Then, aseptically a small
portion of bacteria culture is scraped by the loop and transferred to the drop of water. A
suspension of bacteria is made by gently mixing the loop of bacteria in the water drop, in
such a way that, the drop does not spread. If grown in a liquid medium as suspension,
aseptically a drop of the bacteria suspension is placed at the center of the slide directly,
using a flame-sterilized loop.
6. Petroleum jelly or similar material is applied surrounding the drop, so that, when the
cover slip covers the drop, evaporation and effect of air current is minimized.
7. A cover slip is kept in a slanting position near the drop of bacteria suspension with one
edge touching the slide.
8. Slowly the cover slip is lowered, till it touches the surface of the drop.
9. Then, the cover slip is released, so that the drop of bacteria suspension spreads in all
directions under the cover slip. Care should be taken, so that no air bubble is formed in
the suspension.
10. The slide is clipped on the stage of a compound microscope and observed using low
power and high dry objectives. Then, a drop of immersion oil is put on the cover slip and
observed using oil-immersion objective of the microscope. Preferably, a phase- contrast
or dark-field microscope should be used for clear observation.
Important: The wet mount technique is a very valuable tool for the microbiologist, because it is
a quick way to observe the size and shape of an organism

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Observations (Under Oil-immersion Objective)

1. Shape of bacteria:
 Spherical (coccus)
 Rod-shaped (bacilli)
2. Arrangement of bacteria:
 Comma-like (vibrio)
 Spiral (spirochetes)
 Pairs (diplobacillus/diplococcus)
 In fours (tetrads)
 In chains (streptococcus/streptobacillus)
 Grape-like clusters (staphylococcus)
 Cuboidal (sarcinae or octet)
3. Size of bacteria:
 By eye estimation, make drawing of the field under oil-immersion objective.

Skills Practice Session 7.3

Topic: Perform microscopic examination of wet mount preparation for bacteria
Purpose:
The purpose of this activity is to enable you to practice those skills necessary to provide practice
microscopic examination of wet mount preparation, and to achieve competency in these skills.
Instructions:
This activity should be conducted in a skill laboratory/healthy facility. You should review
Learning Guide for microscopic examination of wet mount preparation of bacteria before
beginning the activity. The trainer should demonstrate the steps/tasks in each learning guide one
at a time. Under the guidance of the trainer, you should then work in groups and practice the
steps/tasks in the Learning Guide for practice microscopic examination of wet mount preparation
of bacteria and observe each other’s performance; while one of you/group doing the microscopic
examination of wet mount preparation for bacteria activity, another learner/group should use the
Learning Guide to observe performance. You should then rotate roles. You should be able to
perform the steps/tasks before skills competency is assessed using the Checklist for microscopic
examination of wet mount preparation for bacteria.

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Conditions or situation for the operations:

 This activity could be done in skill laboratory or in healthy facility
Resources (Equipment and Materials):
 Microscope
 Slides
 pencil
 Cover glasses/cover slip
 immersion oil
 specimen
 Water/normal saline
 Droppers/pipette
 Laboratory coat
 Disposable gloves
 Laboratory request form
 Log book
Precautions:
 When you tried to find the sample to be examined, adequate safety precautions must
betaken to prevent the spread of infectiousorganisms and to avoid contamination
generally
 Visualization of microorganisms with the wet mount technique can be difficult due to the
size of the organisms.
 Because organisms are transparent when suspended in an aqueous solution you are able
to see them easier by lowering the condenser.
By lowering the condenser, you lower the resolution and make the bacteria unresolved, which in
turn makes them easier to see, but sacrifices the ability to see fine details.

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Standard Precautions
 Always wash hands before and after obtaining and handling specimens
 Cover cuts and lesions with waterproof dressing
 Wear disposable aprons and appropriate gloves if there is likelihood of contact or
contamination with blood or body fluids.
 Take care not to contaminate the outside of the container with blood or body fluid
 Do not use the specimen container for any other purpose
 Always ensure the top is closed securely

Procedure-Learning Guide/Checklist
Learning Guide 7.3
Perform microscopic examination of wet mount preparation for bacteria
(To be completed by Participant/students)

Rate the performance of each step or task observed using the following rating scale:
1. Needs Improvement: Step or task not performed correctly, out of sequence (if necessary), or
is omitted
2. Competently Performed: Step or task performed correctly in proper sequence (if necessary)
but participant/student does not progress from step to step efficiently
3. Proficiently Performed: Step or task performed efficiently and precisely in the proper
sequence (if necessary)
Perform microscopic examination of wet mount preparation for bacteria
(Some of the following steps/tasks should be performed simultaneously)

STEP/TASK
Improvement
Competently

Proficiently
performed

performed

Remark
Needs

Getting ready
1. Wearing the Laboratory coat
2. Washing your hand with soap
3. Wearing the gloves
4. Preparing the necessary equipment
5. Greeting the patient respectfully and with kindness.
6. Reading the order (request form)

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7. A slide is cleaned properly under tap water, such that

water does not remain as drops on its surface
8. Make sure the is slide is dried by wiping with bibulous
paper and subsequently moving over flame or keeping in
the sun
9. Getting the sample to be performed
11. Apply a petroleum jelly or similar material is applied
surrounding the drop of sample
12. Apply a cover slip in a slanting position near the drop of
bacteria suspension
13. Make move Slowly the cover slip is lowered and released
14. Make observation using low power and high dry
objectives
Quality criteria :
During accomplishment of Perform microscopic examination of wet mount preparation
for bacteria, the trainees should be:
 Able to follow pre analytical, analytical and post analytical stages of procedures
 Able to apply safety procedures during collection and processing of microbial sample
for wet mount
 Able to develop good attitude towards patients, professions and environment
7.4 Staining in medical microbiology
Staining is technique used in microscopy to enhance contrast in the microscopic image. Stains
and dyes are frequently used in biological tissues for viewing, often with the aid of different
microscopes. Stains may be used to define and examine bulk tissues (highlighting, for example,
muscle fibers or connective tissue), cell populations (classifying different blood cells, for
instance), or organelles within individual cells. Bacteria have nearly the same refractive index as
water, therefore, when they are observed under a microscope they are opaque or nearly invisible
to the naked eye. Different types of staining methods are used to make the cells and their internal
structures more visible under the light microscope. Microscopes are of little use unless the
specimens for viewing are prepared properly. Microorganisms must be fixed & stained to
increase visibility, accentuate specific morphological features, and preserve them for future use

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Terms related to staining:

Stain: A stain is a substance that adheres to a cell, giving the cell color. The presence of color
gives the cells significant contrast so they are much more visible. Different stains have different
affinities for different organisms, or different parts of organisms. They are used to differentiate
different types of organisms or to view specific parts of organisms
Staining: is an auxiliary technique used in microscopy to enhance contrast in the microscopic
image. Stains and dyes are frequently used in biology and medicine to highlight structures in
biological tissues for viewing, often with the aid of different microscopes.
Fixation: Fixation by itself consists of several steps–aims to preserve the shape of the cells
tissue involved as much as possible. Sometimes heat fixation is used to kill, adhere, and makes
them permeable so it will accept stains
Staining techniques
 Direct staining- The organism is stained and background is left unstained
 Negative staining-The background is stained and the organism is left unaltered
Kinds of stains: Stains are classified as

 Simple stain
 Differential stain
 Structural or special stains
7.4.1 Simple Staining
The staining process involves immersing the sample (before or after fixation and mounting) in
dye solution, followed by rinsing and observation. Many dyes, however, require the use of a
mordant, a chemical compound that reacts with the stain to form an insoluble, coloured
precipitate. When excess dye solution is washed away, the mordant stain remains. Simple
staining is one step method using only one dye. Basic dyes are used in direct stain and acidic dye
is used in negative stain. A simple staining technique is used to study the morphology better, to
show the nature of the cellular contents of the exudates and also to study the intracellular
location of the bacteria.
Common dyes used in simple stains are
 Methylene blue
 Dilute carbol fuchsin

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 Polychrome methylene blue

7.4.2 Differential Stains
Differential Stains use two or more stains and allow the cells to be categorized into various
groups or types. Both the techniques allow the observation of cell morphology, or shape, but
differential staining usually provides more information about the characteristics of the cell wall
(Thickness). Gram staining (or Gram’s Method) is an empirical method of differentiating
bacterial species into two large groups
 Gram-positive and
 Gram-negative based on the chemical and physical properties of their cell wall.
The Gram stain is almost always the first step in the identification of a bacterial organism, while
Gram staining is a valuable diagnostic tool in both clinical and research settings, not all bacteria
can be definitively classified by this technique, thus forming Gram variable and Gram in
determinate groups as well. The word Gram is always spelled with a capital, referring to Hans
Christian Gram, the inventor of Gram staining
Principle of Gram Staining:
When the bacteria are stained with primary stain Crystal Violet (CV) and fixed by the mordant,
some of the bacteria are able to retain the primary stain and some are decolorized by alcohol. The
cell walls of gram positive bacteria have a thick layer of protein-sugar complexes called
peptidoglycan and lipid content is low. Decolorizing the cell causes this thick cell wall to
dehydrate and shrink which closes the pores in the cell wall and prevents the stain from exiting
the cell. So the ethanol cannot remove the Crystal Violet-Iodine complex that is bound to the
thick layer of peptidoglycan of gram positive bacteria and appears blue or purple in colour. In
case of gram negative bacteria, cell wall also takes up the CV-Iodine complex but due to the thin
layer of peptidoglycan and thick outer layer which is formed of lipids, CV-Iodine complex gets
washed off. When they are exposed to alcohol, decolorizer dissolves the lipids in the cell walls,
which allows the crystal violet-iodine complex to leach out of the cells. Then when again stained
with safranin, they take the stain and appear red in color.
Gram Reaction:
Gram-positive bacteria are those that are stained dark blue or violet by Gram staining. This is in
contrast to Gram-negative bacteria, which cannot retain the crystal violet stain, instead taking up
the counter stain (safranin or fuchsine) and appearing red or pink. Gram-positive organisms are

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able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall.
Gram- positive cell walls typically lack the outer membrane found in Gram-negative bacteria.
Gram-negative bacteria are those bacteria that do not retain crystal violet dye in the Gram
staining protocol. In a Gram stain test, a counter stain (commonly safranin) is added after the
crystal violet, coloring all Gram-negative bacteria with a red or pink color. The test itself is
useful in classifying two distinct types bacteria based on the structural differences of their cell
walls. On the other hand, Gram-positive bacteria will retain the crystal violet dye when washed
in a decolorizing solution.
Supplies and Equipment
 Crystal Violet, the primary stain
 Iodine, the mordant
 A decolorizer made of acetone and alcohol (95%)
 Safranin, the counterstain
 Laboratory coat
 Disposable Gloves
 Laboratory request form
 Water and soap
 Pencil
 Log book
 Microscope
 Slides
 pencil
 Cover glasses/cover slip
 immersion oil
 Bunsen burner
Gram’s stain reagents preparation
1. Crystal violet stain
 Crystal violet ……………. 20g
 Ammonium oxalate ……… 9g
 Ethanol or methanol absolute ………. 95ml
 Distilled water ………………………. to 1 litre
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2. Gram’s iodine
 Potassium iodide …………………. 20 g
 Iodine ……………………………… 10 g
 Distilled water ……………………… to 1 litre
NB: Should be stored in a brown bottle
3. Acetone -alcohol
To make 1 litre
 Acetone ………………………… 500ml
 Ethanol or methanol absolute …. 475 ml
 Distilled water …………………. 25ml
4. Safranin
a) Prepare a stock solution
 Safranine O ------------------------------------------------ 2.5g
 Ethanol (95%) --------------------------------------------100ml
Mix until all the safranin is dissolved. Transfer the solution to a glass stoppered bottle.
Label the bottle (Safranin stock solution) and write the date.
b) Prepare a working solution in a glass stoppered bottle
 Stock solution ----------------------------------------------10ml
 Distilled water ----------------------------------------------90ml
Label the bottle (Safranine working solution) and write the date
5. Neutral red; 1g/l (w/v)
To make 1 liter
 Neutral red ……………. 1g
 Distilled water ………… 1 litre
Gram Staining Procedure/Protocol:
1. Apply proper hand hygiene and gather equipment.
2. Wear the laboratory coat and don gloves
3. Smear from specimen or culture.
4. Allow the smear to air-dry.
5. Rapidly pass slide three times through flame.
6. Cover fixed smear with crystal violet for one minute and wash with tap water.

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7. Tip off the water and cover the smear with Gram’s iodine for one minute.
8. Wash off iodine solution with tap water.
9. Decolorize with acetone-alcohol for 30 seconds.
10. Wash off the acetone-alcohol with clean water.
11. Cover the smear with safranin for one minute.
12. Wash off the stain and wipe the back of the slide. Let the smear air-dry.
13. Examine the stained smear with oil immersion objective to look for bacteria.
Results:
 Gram positive bacteria ……………………. purple
 Yeast cells …………………………………. Dark purple
 Gram negative bacteria ……………………. Pale to red
 Nuclei of pus cell …………………………… Red
 Epithelia cells ………………………………. Pale red

Reporting of Gram’s stained smear

The report should include the following:

 Number of bacteria present whether many, moderate, few or scanty
 Gram reaction of the bacteria whether Gram positive or Gram negative
 Morphology and arrangement of the bacteria whether cocci, diplococci, streptococci,
rods, or Coccobacilli, also whether the organisms are intracellular.
 Presence and number of pus cells.
 Presence of yeast cells and epithelia cells

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Factors which contribute to false Gram’s staining result:

1. False Gram positive reaction
 Preparation of too tick smear – the stain will not be fully washed
 Application of crystal violet -presence of sediment in crystal violet -this can be avoided
by filtering the stain before use.
 Fixing before drying brings alteration of cell wall and morphology.
 Decolourizing time - when insufficient decolourization time is used or (when not fully
decolorized).
2. False Gram Negative reactions
 Making smear from old culture.
 Cell wall damage due to antibiotic.
 Excessive heat fixation of smear.
 Over decolourization of smear (decolourization for longer time).
 Use of an iodine solution which is too old (i.e. yellow instead of brown in colour) (its
mordant effect will be decreased.
Quality Control: Always check new batches of stain and reagents for correct staining reactions
using a smear containing known Gram positive and Gram negative organisms.
Skills Practice Session 7.4
Topic: Perform microscopic examination of Gram staining for bacteria
Purpose
The purpose of this activity is to enable you to practice those skills necessary to provide practice
microscopic examination of Gram staining for bacteria, and to achieve competency in these
skills.
Instructions:
This activity should be conducted in a skill laboratory/healthy facility. You should review
Learning Guide for microscopic examination of Gram staining for bacteria before beginning the
activity. The trainer should demonstrate the steps/tasks in each learning guide one at a time.
Under the guidance of the trainer, you should then work in groups and practice the steps/tasks in
the Learning Guide for practice microscopic examination of Gram staining for bacteria and
observe each other’s performance; while one learner/group doing the microscopic examination
of Gram staining for bacteria activity, another learner/group should use the Learning Guide to

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observe performance. You should then rotate roles. You should be able to perform the steps/tasks
before skills competency is assessed using the Checklist microscopic examination of Gram
staining for bacteria
Conditions or situation for the operations:
This activity could be done in skill laboratory or in healthy facility
Resources (Equipment and Materials):
 Crystal Violet, the primary stain
 Iodine, the mordant
 A decolorizer made of acetone and alcohol (95%)
 Safranin, the counterstain
 Laboratory coat
 Disposable Gloves
 Laboratory request form
 Water and soap
 Pencil
 Log book
 Microscope
 Slides
 Cover glasses/cover slip
 immersion oil
 Bunsen burner
Precaution: When you tried to find the sample to be examined, adequate safety precautions
must be taken to prevent the spread of infectious organisms and to avoid contamination
generally.

Standard Precautions:
Always wash hands before and after obtaining and handling specimens
 Cover cuts and lesions with waterproof dressing
 Wear disposable aprons and appropriate gloves if there is likelihood of contact or
contamination with blood or body fluids.
 Take care not to contaminate the outside of the container with blood or body fluid

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 Do not use the specimen container for any other purpose

 Always ensure the top is closed securely
Procedure-Learning Guide/Checklist
Learning Guide 7.4
Perform microscopic examination of Gram staining for bacteria
(To be completed by Participant/students)
Rate the performance of each step or task observed using the following rating scale:
1. Needs Improvement: Step or task not performed correctly, out of sequence (if necessary),
or is omitted
2. Competently Performed: Step or task performed correctly in proper sequence (if
necessary) but participant/student does not progress from step to step efficiently
3. Proficiently Performed: Step or task performed efficiently and precisely in the proper
sequence (if necessary)
Perform microscopic examination of Gram staining for bacteria
(Some of the following steps/tasks should be performed simultaneously)

improvement
Competently

Proficiently
performed

performed
Remark
Needs
STEP/TASK

Getting ready
1. Wearing the Laboratory coat
2. Washing your hand with soap
3. Wearing the gloves
4. Preparing the necessary equipment
5. Greeting the patient respectfully and with kindness.
6. Reading the order (request form)
7. Labeling the sample slide
8. Recording the patient identification (Name, Age, address
etc.)
9. Smearing from specimen or culture
10. Allow the smear to air-dry
11. Rapidly pass slide three times through flame.
12. Cover fixed smear with crystal violet for one minute and
wash with tap water.
13. Tip off the water and cover the smear with Gram’s iodine
for one minute

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14. Wash off iodine solution with tap water.

15. Decolorize with acetone-alcohol for 30 seconds.
16. Wash off the acetone-alcohol with clean water.
17. Cover the smear with safranin for one minute
18. Wash off the stain and wipe the back of the slide. Let the
smear air-dry.
19. Examine the stained smear with oil immersion objective
to look for bacteria
Quality criteria:
During accomplishment of Perform microscopic examination of Gram staining for bacteria, the
trainees should be:
 Able to understand the principle of gram staining
 Able to follow pre analytical, analytical and post analytical stages of procedures
 Able to apply safety procedures during collection and processing of microbial sample
for gram stain
 Able to develop good attitude towards patients, professions and environment
Acid-Fast Staining (Ziehl–Neelsen stain):
The Ziehl–Neelsen stain, also known as the acid-fast stain, widely used differential staining
procedure. In this type some bacteria resist decolourization by both acid and alcohol and hence
they are referred as acid-fast organisms. This staining technique divides bacteria into two groups
namely acid-fast and non-acid-fast. This procedure is extensively used in the diagnosis of
tuberculosis and leprosy. Mycobacterium tuberculosis is the most important of this group, as it is
responsible for the disease called tuberculosis (TB) along with some others of this genus.
Principle:
Mycobacterial cell walls contain a waxy substance composed of mycolic acids. The property of
acid fastness is related to the carbon chain length of the mycolic acid found in any particular
species. Sputum smear is heat –fixed, flooded with a solution of carbilfusin (a mixture of basic
fuschin and phenol) and heated until steam rises. The heating allows melting of cell wall of
mycobacteria which facilitate entrance of the primary stain into the bacterium. After washing
with water, the slide is covered with 3% HCL (decolourizer). Then washed with water and
flooded with methylene blue (Mycobacterium tuberculosis) and malachite green (Mycobacterium
leprae).

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Supply and Materials

 Carbol-fuchsin
 Acid-Alcohol
 Methylene blue/Malachite green
 Laboratory coat
 Disposable Gloves
 Laboratory request form
 Water and soap
 Pencil
 Log book
 Microscope
 Slides
 Cover glasses/cover slip
 immersion oil
 Bunsen burner
Procedure:

1. Apply proper hand hygiene and gather equipment.

2. Wear the laboratory coat and don gloves
3. Prepare the smear from the primary specimen and fix it by passing through the flame and
label clearly
4. Place fixed slide on a staining rack and cover each slide with concentrated carbol fuchsin
solution.
5. Heat the slide from underneath with sprit lamp until vapor rises (do not boil it) and wait
for 3-5 minutes.
6. Wash off the stain with clean water.
7. Cover the smear with 3% acid-alcohol solution until all color is removed (two minutes).
8. Wash off the stain and cover the slide with 1% methylene Blue for one minute.
9. Wash off the stain with clean water and let it air-dry.
10. Examine the smear using the 100x oil immersion objective to look for acid fast bacilli.
Results:

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 AFB............. Red, straight or slightly curved rods, occurring single or in a small group
 Cells......................................... Blue
 Background Material ………. Blue
Reporting system
 0 AFB/100 field …………………………………No AFB seen
 1-2 AFB/ 300 field………………………………. (±) or scanty
 1-9 AFB/100 field………………………………………….1+
 1-9AFB/10 field.……………………………………………2+
 1- 9AFB/field…… ………………………………………....3+
 >9 AFB/field……………… ……………………………….4+
N.B: AFB means number of acid fast bacilli seen.

Fig-7.1 AFB under the microscope

Quality Control: Always check new batches of stain and reagents for correct staining reactions
using a smear containing known positive and negative (control) organisms.
Skills Practice Session 7.5
Topic: Perform microscopic examination of Acid Fast Bacilli Detection (AFB) -M. tuberculosis

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Purpose:
The purpose of this activity is to enable you to practice those skills necessary to provide practice
microscopic examination of Acid Fast Bacilli Detection, and to achieve competency in these
skills.
Instructions:
This activity should be conducted in a skill laboratory/healthy facility. You should review
Learning Guide for microscopic examination of Acid Fast Bacilli Detection before beginning the
activity. The trainer should demonstrate the steps/tasks in each learning guide one at a time.
Under the guidance of the trainer, you should then work in groups and practice the steps/tasks in
the Learning Guide for practice microscopic examination of Acid Fast Bacilli Detection for
bacteria and observe each other’s performance; while one learner/group doing the microscopic
examination of Acid Fast Bacilli Detection activity, another learner/group should use the Learning
Guide to observe performance. You should then rotate roles. Learners should be able to perform
the steps/tasks before skills competency is assessed using the Checklist microscopic examination
of Acid Fast Bacilli Detection for M. tuberculosis
Conditions or situation for the operations:
This activity could be done in skill laboratory or in healthy facility
Resources (Equipment and Materials):
 Crystal Violet, the primary stain
 Iodine, the mordant
 A decolorizer made of acetone and alcohol (95%)
 Safranin, the counterstain
 Laboratory coat
 Disposable Gloves
 Laboratory request form
 Water and soap
 Pencil
 Log book
 Microscope
 Slides

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 Cover glasses/cover slip

 immersion oil
 Bunsen burner
Precaution: When you tried to find the sample to be examined, adequate safety precautions
must be taken to prevent the spread of infectious organisms and to avoid contamination
generally.

Standard Precautions
Always wash hands before and after obtaining and handling specimens
 Cover cuts and lesions with waterproof dressing
 Wear disposable aprons and appropriate gloves if there is likelihood of contact or
contamination with blood or body fluids.
 Take care not to contaminate the outside of the container with blood or body fluid
 Do not use the specimen container for any other purpose
 Always ensure the top is closed securely

Procedure-Learning Guide/Checklist
Learning Guide 5
Perform microscopic examination of Acid Fast Bacilli Detection (AFB) -M. tuberculosis
(To be completed by Participant/students)
Rate the performance of each step or task observed using the following rating scale:
1. Needs Improvement: Step or task not performed correctly, out of sequence (if necessary), or
is omitted
2. Competently Performed: Step or task performed correctly in proper sequence (if necessary)
but participant/student does not progress from step to step efficiently
3. Proficiently Performed: Step or task performed efficiently and precisely in the proper
sequence (if necessary)

Perform microscopic examination of Acid Fast Bacilli Detection (AFB) -M. tuberculosis
(Some of the following steps/tasks should be performed simultaneously)

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improvement

Competently

Proficiently
performed

performed
Remark
Needs
STEP/TASK

Getting ready
1. Wearing the Laboratory coat
2. Washing your hand with soap
3. Wearing the gloves
4. Preparing the necessary equipment
5. Greeting the patient respectfully and with kindness.
6. Reading the order (request form)
7. Labeling the sample slide
8. Recording the patient identification (Name, Age, address
etc.)
9. Prepare the smear from the primary specimen and fix it by
passing through the flame and label clearly
10. Place fixed slide on a staining rack and cover each slide
with concentrated carbol fuchsin solution.
11. Heat the slide from underneath with sprit lamp until vapor
rises (do not boil it) and wait for 3-5 minutes.
12. Wash off the stain with clean water.
13. Cover the smear with 3% acid-alcohol solution until all
color is removed (two minutes)
14. Wash off the stain and cover the slide with 1% methylene
Blue for one minute.
15. Wash off the stain with clean water and let it air-dry.
16. Examine the smear using the 100x oil immersion
objective to look for acid fast bacilli.
Quality criteria :
During accomplishment of Perform microscopic examination of AFB staining for M.
tuberculosis, the trainees should be:
 Able to understand the principle of AFB staining

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 Able to follow pre analytical, analytical and post analytical stages of procedures
 Able to apply safety procedures during collection and processing of AFB sample for
AFB stain
 Able to develop good attitude towards patients, professions and environment

Auramine-Rhodamine Staining for Acid fast bacilli (AFB): M. tuberculosis

One of the earliest methods devised for the detection of tubercle bacilli is the microscopic
staining technique. Mycobacteria possess cell walls that contain mycolic acid which complex
with dyes resulting in the characteristic known as “acid-fastness.” Acid-fast microscopy is the
most rapid, initial step in diagnosis and provides information about the number of acid-fast
bacilli present. The use of fluorescent dyes for the detection of acid-fast bacilli in clinical
specimens was described by Hagemann in 1937.
Principle:
Acid-fast mycobacteria resist decolourization by acid-alcohol after primary staining owing to the
high lipid (mycolic acid) content in their cell walls. The identification of mycobacteria with
auramine O is due to the affinity of the mycolic acid in the cell walls for the fluorochrome. The
dye will bind to the mycobacteria, which appear as bright yellow, luminous rods against a dark
background. The potassium permanganate helps prevent non-specific fluorescence. All acid-
fast organisms will be stained by auramine O, including some parasites. Slides stained with
auramine O may be restained with Ziehl-Neelsen or Kinyoun stain directly, as long as the oil has
been removed. This provides a convenient method of confirming and differentiating morphology
of positive slides with the traditional stains. The fluorochrome stains are recommended for
specimen examination because of their increased sensitivity and speed.
Supply and Materials:
 Primary stain (Auramine Rhoda mine Stain): Dissolve 1.5 g of Auramine O and 0.75 g
Rhoda mine B in a solution of 75 ml glycerol (glycerine), 10 ml heated phenol crystals,
and 50 ml of distilled water. This solution is usually cleared by filtering through glass
wool.
 Decolorizer (Acid alcohol)-Carefully add 0.5 ml of concentrated hydrochloric acid to
100 ml of 70% ethanol.

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 Counter stain (Potassium Permanganate)-Dissolve 0.5 g of potassium permanganate

(KMn04) in 100 ml of distilled water.
 Quaternary ammonium compounds jar (only if a loop is used, not needed with
disposable sticks).
 Diamond pencil or lead pencil (if frosted-end slides are available)
 Filter paper, small, appropriate for funnel size (when necessary)
 Funnels, small, for filtering solutions in use (when necessary)
 Forceps
 Lens paper or soft tissue paper
 Plastic containers for waste disposal
 Disposable loops or wooden sticks
 Fluorescence microscope or LED Microscope with objectives of 20x or 25x, 40x
(ideally specific for fluorescence microscopy), 100x and eyepieces of 10x
 Immersion oil for microscopy
 Slide staining rack
 Slide boxes
 Slides supports
 New, clean slides
 Timer
Procedure :
1. Apply proper hand hygiene and gather equipment.
2. Wear the laboratory coat and don gloves
3. Make a thin smear of the material for study and heat fix by passing the slide through the
4. flame of a Bunsen burner or use a slide warmer at 65-75 C. Do not overheat.
5. Flood smears with Auramine O-Rhodamine B solution and allow to stain for 15 minutes,
making certain that the staining solution remains on the smear. Do not apply heat to
smear.
6. Rinse smears with chlorine free water until no color appears in the effluent. Chlorine may
interfere with fluorescence; therefore, rinse with distilled or deionized water.

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7. Flood the smear with the decolorizing agent for 2 to 3 minutes, and then wash with
distilled water.
8. Flood smears with acid alcohol and allow de-staining for 2 minutes.
9. Rinse the slide thoroughly with distilled water and shake off excess fluid.
10. Flood smears with potassium permanganate and counter stain for 2 minutes. Time is
critical with potassium permanganate because counter staining for a longer time may
quench fluorescence of acid-fast bacilli.
11. Rinse thoroughly with distilled water and allow to air dry. Do not blot.
12. Examine microscopically using a fluorescent microscope as soon as possible. Use a 20x
or 40x objective for screening, and a 100x oil immersion objective to observe the
morphology of fluorescing organisms.
Results:
 Positive Test: Acid-fast organisms fluoresce yellow or bright orange against a dark
background (color may vary with the filter system used)
 Negative Test: Non-acid-fast organisms will not fluoresce or may appear a pale yellow,
quite distinct from the bright acid-fast organisms
Reporting of Auramine-Rhodamine Stained Smear:
 The minimum number of fields to examine before reporting a smear as negative for
acid-fast organisms at specific magnification are as follows:
Magnification Number of Fields
200x 30
250x 30
400x 55
450x 70
Reporting of smears:
 If no fluorescent rods are seen, report the smear as AFB NOT SEEN.
 If Fluorescent AFB are seen, report the smear as AFB positive, and give an indication of the
number of bacilli present in plus signs (1+ to 4+) as follows:
Number of AFB seen (450X Number of AFB seen (250X Reported As
Magnification) Magnification)
0 AFB per 70 Field 0AFB per 30 Fields AFB Not Seen
1-2 AFB per 70 Fields 1-2 AFB per 30 Fields Doubtful; repeat with
another specimen
2-18 AFB per 50 Fields 1-9 AFB per 10 Fields 1+
4-36 AFB per 10 Fields 1-9 AFB per Field 2+
4-36 AFB per Field 10-90 AFB per Field 3+

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>36 AFB per Field >90 AFB per Field 4+

Fig-7.2 Auramine-Rhodamine Stain

Quality Control:
 Include positive and negative controls with each batch’s reading. Read control slides before
other smears.
 If results are unacceptable, re-stain smears of that day together with new controls,
payingattention to correct technique; if these controls are also unacceptable.
Limitations of Auramine-Rhodamine Stain:
1. A positive staining reaction provides presumptive evidence of the presence of
mycobacteria. A negative staining reaction does not indicate that the specimen will be
culturally negative. Therefore, cultural methods must be employed.
2. Most strains of rapid growers may not appear fluorescent.
3. It is recommended that all negative fluorescent smears be confirmed with Ziehl-Neelsen
stain; at least 100 fields should be examined before being reported as negative.
4. Reagents like Auramine-Rhodamine are possible carcinogen, Acid alcohol and Potassium
permanganate is also strong irritant to skin, eyes and respiratory system.
5. Caution is required while handling and staining using such reagents.
6. Excessive exposure to the counterstain may result in a loss of brilliance of the fluorescing
organism.
7. Turbidity may develop in the stain, but it will not interfere with the effectiveness of the
stain. Shake the bottle before using.

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8. Stained smears should be observed within 24 hours of staining because of the possibility
of the fluorescence fading.
Quality Control: Always check new batches of stain and reagents for correct staining reactions
using a smear containing known positive and negative (control) organisms.
Skills Practice Session 7. 6
Topic: Perform microscopic examination of Auramine-Rhodamine Staining for Acid fast bacilli
(AFB): M. tuberculosis
Purpose:
The purpose of this activity is to enable you to practice those skills necessary to provide practice
microscopic examination of Auramine-Rhodamine Staining, and to achieve competency in these
skills.
Instructions
This activity should be conducted in a skill laboratory/healthy facility. Learners should review
Learning Guide for microscopic examination of Auramine-Rhodamine Staining before beginning
the activity. The teacher should demonstrate the steps/tasks in each learning guide one at a time.
Under the guidance of the teacher, learners should then work in groups and practice the
steps/tasks in the Learning Guide for practice microscopic examination of Auramine-Rhodamine
Staining Acid Fast Bacilli for bacteria and observe each other’s performance; while one
learner/group doing the microscopic examination of Auramine-Rhodamine, Acid Fast Bacilli
Detection activity, another learner/group should use the Learning Guide to observe performance.
Learners should then rotate roles. Learners should be able to perform the steps/tasks before skills
competency is assessed using the Checklist microscopic examination of Auramine-Rhodamine
Staining for Detection of M. tuberculosis
Conditions or situation for the operations
This activity could be done in skill laboratory or in healthy facility
Resources (Equipment and Materials)
 Primary stain (Auramine Rhodamine Stain): Dissolve 1.5 g of Auramine O and 0.75 g
Rhodamine B in a solution of 75 ml glycerol (glycerine), 10 ml heated phenol crystals,
and 50 ml of distilled water. This solution is usually cleared by filtering through glass
wool.

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 Decolorizer (Acid alcohol)-Carefully add 0.5 ml of concentrated hydrochloric acid to 100

ml of 70% ethanol.
 Counter stain (Potassium Permanganate)-Dissolve 0.5 g of potassium permanganate
(KMn04) in 100 ml of distilled water.
 Quaternary ammonium compounds jar (only if a loop is used, not needed with disposable
sticks).
 Diamond pencil or lead pencil (if frosted-end slides are available)
 Filter paper, small, appropriate for funnel size (when necessary)
 Funnels, small, for filtering solutions in use (when necessary)
 Forceps
 Lens paper or soft tissue paper
 Plastic containers for waste disposal
 Disposable loops or wooden sticks
 Fluorescence microscope or LED Microscope with objectives of 20x or 25x, 40x (ideally
specific for fluorescence microscopy), 100x and eyepieces of 10x
 Immersion oil for microscopy
 Slide staining rack
 Slide boxes
 Slides supports
 New, clean slides
 Timer
Precaution: When you tried to find the sample to be examined, adequate safety precautions
must be taken to prevent the spread of infectious organisms and to avoid contamination
generally.

Standard Precautions
 Always wash hands before and after obtaining and handling specimens
 Cover cuts and lesions with waterproof dressing
 Wear disposable aprons and appropriate gloves if there is likelihood of contact or
contamination with blood or body fluids.
 Take care not to contaminate the outside of the container with blood or body fluid

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 Do not use the specimen container for any other purpose

 Always ensure the top is closed securely
Procedure-Learning Guide/Checklist
Learning Guide 7.6
Perform microscopic examination of Auramine-Rhodamine Staining for Acid fast bacilli (AFB):
M. tuberculosis
(To be completed by Participant/students)
Rate the performance of each step or task observed using the following rating scale:
1. Needs Improvement: Step or task not performed correctly, out of sequence (if necessary), or
is omitted
2. Competently Performed: Step or task performed correctly in proper sequence (if necessary)
but participant/student does not progress from step to step efficiently
3. Proficiently Performed: Step or task performed efficiently and precisely in the proper
sequence (if necessary)
Perform microscopic examination of Auramine-Rhodamine Staining for Acid fast bacilli
(AFB): M. tuberculosis
(Some of the following steps/tasks should be performed simultaneously)

improvement
Competently

Proficiently
performed

performed
Remark
STEP/TASK Needs

Getting ready
1. Wearing the Laboratory coat
2. Washing your hand with soap
3. Wearing the gloves
4. Preparing the necessary equipment
5. Greeting the patient respectfully and with kindness.
6. Reading the order (request form)
7. Labeling the sample slide
8. Recording the patient identification (Name, Age, address etc.)
9. Make a thin smear of the material for study and heat fix by
passing the slide through the flame of a Bunsen burner or use a
slide warmer at 65-75 oC. Do not overheat.

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10. Flood smears with Auramine O-Rhodamine B solution and

allow to stain for 15 minutes, making certain that the staining
solution remains on the smear. Do not apply heat to smear.

11. Rinse smears with chlorine free water until no color appears in
the effluent. Chlorine may interfere with fluorescence;
therefore, rinse with distilled or deionized water.
12. Flood the smear with the decolorizing agent for 2 to 3
minutes, and then wash with distilled water
13. Flood smears with acid alcohol and allow de-staining for 2
minutes
14. Rinse the slide thoroughly with distilled water and shake off
excess fluid.
15. Flood smears with potassium permanganate and counter stain
for 2 minutes. Time is critical with potassium permanganate
because counter staining for a longer time may quench
fluorescence of acid-fast bacilli.
16. Rinse thoroughly with distilled water and allow to air dry. Do
not blot.
17. Examine microscopically using a fluorescent microscope as
soon as possible. Use a 20x or 40x objective for screening, and
a 100x oil immersion objective to observe the morphology of
fluorescing organisms.
Quality criteria :
Perform microscopic examination of Auramine-Rhodamine Staining for Acid fast bacilli (AFB):
M. tuberculosis, the trainees should be:
 Examination of Auramine-Rhodamine florescence Staining
 Able to follow pre analytical, analytical and post analytical stages of procedures
 Able to Apply safety procedures during collection and processing of AFB sample for
AFB fluorescence stain
 Able develop good attitude towards patients, professions and environment

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Summary
 Different microbiological preparations can be used for microscopic examinations in
microbiology laboratory.
 The routinely used preparations for microscopic examinations in microbiology laboratory
are wet mount and stained preparations.
 The wet mount preparation in microbiology laboratory is Saline, KOH and Indian ink
preparations, and the stained preparations include gram staining and Acid fast staining
but not limited to what are mentioned above.
Self-check questions:
Instruction: Dear trainees, please! attempt all the questions listed below
1. What are a shape and arrangement of bacteria can be observed in wet mount preparation
of bacteria?
2. Write the common dyes used in simple stains.
3. Describe the function of each of the following in the Gram stain
a. Mordant: ____________________________________________________________
b. Primary stain: _________________________________________________________
c. Decolorizerand Counterstain_____________________________________________
4. Gram positive bacteria stains ___________________________________________color
5. Gram negative bacteria stains ___________________________________________color
6. Organisms that resist decolourization by acid and alcohol are called as_______________
7. What is the primary stain used in the acid-fast staining procedure? __________________
8. What is the purpose of the heat/steam during the acid-fast staining procedure?
________________________________________________________________________
9. The color of acid fast bacteria in acid fast staining is______________________________
10. Explain the reporting system of Auramine-Rhodamine Staining
11. Write the reagent used Auramine-Rhodamine Staining.

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Chapter 8: Preparation of Culture Media, Cultivation of Microorganism and

Antimicrobial Sensitivity Testing
Dear trainees,
In this chapter, you will learn the types of culture media and their preparations. Besides, you
will develop knowledge and skills in assisting inoculation and cultivation of microorganisms,
and also practice antibiotic sensitivity testing.
Objectives :
At the end of this chapter, you will be able to:
 Describe concepts and types of culture media
 Describe concepts of antimicrobial agents
 Prepare and sterilize culture media
 Perform inoculation and cultivation of culture media
 Perform antimicrobial sensitivity testing
8.1 Overview of culture media
A culture medium is an artificial environment that provides sources of carbon, energy and
nitrogen in the form of available carbohydrates and amino acids for the growth of bacteria.
Bacterial culture media can be classified in different ways; based on consistency, based on
nutritional component and based on its functional use.
A. Classification based on consistency
Culture media are liquid, semi-solid or solid and biphasic.
i. Liquid media
Liquid media are sometimes referred as “broths” (e.g.nutrient broth). These are available for use
in test-tubes, bottles or flasks. In liquid medium, bacteria grow uniformly producing turbidity.
Certain aerobic bacteria and those containing fimbriae (Vibrio and Bacillus) are known to grow
as a thin film called ‘surface pellicle’ on the surface of undisturbed broth. Bacillus anthracis is
known to produce a stalactite growth (spikes hanging from the surface) on ghee containing
broths. Sometimes the initial turbidity is cleared due to autolysis, which is seen in penumococci.
Long chains of Streptococci when grown in liquid media tend to entangle and settle to the
bottom forming granular deposits. Liquid media tend to be used when a large number of bacteria
have to be grown. These are suitable to grow bacteria when the numbers in the inoculum is
suspected to be low. Inoculating a specimen in the liquid medium also helps to dilute any

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inhibitors of bacterial growth. This is the practical approach in blood cultures. Culturing in liquid
medium can be used to obtain viable counts (dilution methods). Colony morphology of bacteria
is not visible in liquid media and presence of more than one type of bacteria cannot be
differentiated.
ii. Solid media
Any liquid medium can be converted to a solid medium by addition of solidifying agents such as
agar-agar, egg yolk or serum. While serum and egg yolk are normally liquid, they can be
rendered solid by coagulation using heat. Serum containing medium such as Loeffler’s serum
slope and egg containing media such as Lowenstein Jensen medium and Dorset egg medium are
solidified as well as disinfected by a process called inspissations.
Agar-agar (simply called agar) is the most commonly used solidifying agent. It is an unbranched
polysaccharide obtained from the cell membranes of some species of red algae. It melts at 95oC
and solidifies at 42oC. Agar incorporated in to media can withstand sterilization by autoclaving.
Agar doesn’t contribute any nutritive property. It is not hydrolyzed by most bacteria and is
usually free from growth promoting or growth retarding substances. However, it may be a source
of calcium and organic ions. Most commonly, it is used at concentration of 1-3% to make a solid
agar medium. Agar is available as fibers (shreds) or as powders.
iii. Semi-solid media
Reducing the concentration of agar to 0.2 - 0.5% renders a medium semi-solid. Such media are
fairly soft and are useful in demonstrating bacterial motility and separating motile from non-
motile strains (U-tube and Cragie’s tube). Certain transport media such as Stuart’s and Amies
media are semi-solid in consistency. Hugh and Leifson’s oxidation fermentation test medium as
well as mannitol motility medium are also semi-solid.
iv. Biphasic media
A culture system may comprise of both liquid and solid medium in the same bottle. This is
known as biphasic medium (Castaneda system for blood culture). The inoculum is added to the
liquid medium and when subcultures are to be made, the bottle is simply tilted to allow the liquid
to flow over the solid medium. This obviates the need for frequent opening of the culture bottle
to subculture and minimizes the risk of contaminating the medium. This is mainly used when the
culture need to be kept for a long period with sub culturing: i.e. – Fungal blood cultures, Brucella
blood cultures.

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B. Classification based on nutritional component

Media can be classified as simple, complex and synthetic (or defined). While most of the
nutritional components are constant across various media, some bacteria need extra nutrients.
Those bacteria that are able to grow with minimal requirements are called non-fastidious and
those that require extra nutrients are said to be fastidious.
Simple media such as peptone water, nutrient agar can support most non-fastidious bacteria.
Complex media such as blood agar have ingredients in which the exact components are difficult
to estimate. Synthetic or defined media are specially prepared media for research purposes where
the composition of every component is well known.
C. Classification based on functional use or application
These include simple/basal media, enriched media, enrichment media, selective media,
indicator/differential media, transport media and storage media.
i. Basal media are basically simple media that supports most non-fastidious bacteria. Peptone
water, nutrient broth and nutrient agar are considered as basal media.
ii. Enriched media
Addition of extra nutrients in the form of blood, serum, egg yolk or sugars to basal media makes
them enriched media. Enriched media are used to grow nutritionally exacting (fastidious)
bacteria. Blood agar, chocolate agar, Loeffler’s serum slope and Dorset’s egg medium are some
of the enriched media. In most instances, enriched media are non-selective.
iii. Enrichment media
Enrichment media contain a substance that's specially formulated to enhance the growth of the
wanted bacteria. Colony number will be more when compared to the number in the original
specimen. It controls the growth of unwanted bacteria. Enrichment media are usually broth
media. Examples of enrichment media are Selenite F Broth, Tetrathionate Broth and Buffered
Listeria Enrichment Broth.
iv. Selective media
These are designed to inhibit unwanted commensal or contaminating bacteria and help to recover
pathogen from a mixture of bacteria. It does not enhance the growth of any bacteria. The colony
number is same as that is in the specimen. Selective media are generally agar based. Any agar
media can be made selective by addition of certain inhibitory agents that do not affect the
pathogen. Various approaches to make a medium selective include addition of antibiotics, dyes,

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chemicals, alteration of pH or a combination of these. Examples of selective media are

MacConkey agar, Blood Tellurite agar and Thiosulphate Citrate Bile salt Sucrose agar.
v. Differential media or indicator media
Certain media are designed in such a way that different bacteria can be recognized on the basis
of their colony colour. Various approaches include incorporation of dyes, metabolic substrates
etc, so that those bacteria that utilize them appear as differently coloured colonies. Such media
are called differential media or indicator media. Examples: MacConkey agar, Cystine Lactose
Electrolyte Deficient (CLED) agar, Thiosulphate Citrate Bile salt Sucrose (TCBS) agar, Xylose
Lysine Deoxycholate (XLD) agar etc.
Blood agar is also considered differential because it is used to distinguish pathogenic bacteria
based on the effect of bacterial enzymes known as haemolysins which lyse red blood cells. But it
is a non-selective differential medium.
vi. Transport media
Clinical specimens must be transported to the laboratory immediately after collection to prevent
overgrowth of contaminating organisms or commensals. When the patient stays away from the
laboratory and there is a risk that the pathogen in the clinical specimen may not survive or may
be overgrown by the commensals during the time the specimen is transported to the laboratory, a
transport medium is used. Such media prevent drying (desiccation) of specimen, maintain the
pathogen to commensal ratio and inhibit overgrowth of unwanted bacteria. Some of these media
(Stuart’s and Amie’s) are semi-solid in consistency. Addition of charcoal serves to neutralize
inhibitory factors. Cary Blair medium, 1% alkaline peptone water and Venkatraman
Ramakrishnan medium are used to transport feces from suspected cholera patients. Sach’s
buffered glycerol saline is used to transport feces from patients suspected to be suffering from
bacillary dysentery.
vii. Anaerobic media
Anaerobic bacteria need special media for growth because they need low oxygen content,
reduced oxidation –reduction potential and extra nutrients. Media for anaerobes may have to be
supplemented with nutrients like haemin and vitamin K. Boiling the medium serves to expel any
dissolved oxygen. Addition of 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05%
cysteine or red hot iron filings can render a medium reduced. Robertson Cooked Meat Medium
(RCMM) that is commonly used to grow Clostridium spps contains a 2.5 cm column of bullock

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heart meat and 15 ml of nutrient broth. Before use, the medium must be boiled in water bath to
expel any dissolved oxygen and then sealed with sterile liquid paraffin. Methylene blue or
resazurin is an oxidation-reduction potential indicator that is incorporated in the thioglycollate
medium. Under reduced condition, methylene blue is colourless.
viii. Storage media
Prepared sterilized media in individual screw capped bottles as broths or nutrient agar are used to
store organisms for further studies or future reference. Poured plates of agar media deteriorate
quickly when held at room temperature on bench and are often contaminated. They can only be
held for short periods not exceeding 7 -10 days. For longer durations of storage, bijou bottles
with agar slants or glycerol in small screw capped tubes are used. Screw caps prevent the
evaporation of water and keep the agar slants wet.
What are the common ingredients of culture media?
Culture media may be prepared in the laboratory from basic ingredients or it may be purchased
ready for use. It is important to have a balance of nutrients. Excess of certain nutrients may
actually inhibit the growth of certain organisms. Hence a culture medium should have the
following properties.
1. Water
2. Gelling agents
3. Essential nutrients- proteins, peptides, amino acids
4. Energy- carbohydrates
5. Essential metals and minerals
6. Buffering agents
7. Indicator substances
8. Selective agents
9. Other components
Water:
This is essential for the growth of all micro-organisms. Tap water is often suitable for culture
media. It must be free from any chemicals that inhibit bacterial growth. If the local water supply
found unsuitable, glass distilled or demineralized water should be used. Small amounts of copper
are highly inhibitory to bacterial growth, so that copper distilled water should not be used for
preparation of media.

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Gelling agents:
Agar-agar (simply called agar) is the most commonly used solidifying agent. It is an unbranched
polysaccharide obtained from the cell membranes of some species of red algae. It melts at 95oC
and solidifies at 42oC. So it remains un-melted at all incubation temperatures. Agar doesn’t
contribute any nutritive property. It is being decomposed or liquefied by only few varieties of
bacteria and is usually free from growth promoting or growth retarding substances. However, it
may be a source of calcium and organic ions. Most commonly, it is used at concentration of 1-
3% to make a solid agar medium. New Zealand agar has more gelling capacity than the Japanese
agar. Agar is available as fibers (shreds) or as powder.
Peptones:
This is a general term for the water-soluble products obtained from the breakdown of animal or
plant proteins such as heart muscle, fibrin, soya flour or casein. Peptones provide nitrogen for
growing organisms. Apart from the standard grade bacteriological peptone, some manufacturers
supply special grades of peptone recommended for particular purposes (Mycological peptone).
Meat extract:
These provide organisms with a further supply of amino acids, and also with essential growth
vitamins and minerals including phosphates and sulphates.
Yeast extract:
This gives a wide range of amino acids, growth factors, carbohydrates and inorganic salts. This
may be substituted from meat extract in culture media.
Carbohydrates :
Simple or complex sugars are added to many culture media, to provide bacteria with sources of
carbon and energy. Carbohydrates are also added to media to assist in the differentiation of
bacteria. e.g. Lactose is added with an indicator to MacConkey agar and Deoxycholate Citrate
agar to differentiate lactose fermenting and lactose non fermenting species of Enterobacteriaceae.
Essential metals and minerals :
For cell growth, sulphates are required as sources of sulphur and phosphates are sources of
phosphorous. Culture media should also contain traces of magnesium, potassium, iron, calcium
and other elements which are required for bacterial enzyme activity. NaCl is also an essential
ingredient of most culture media.

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Preparation and sterilization of culture media:

The preparation of media from commercial dehydrated products is simple and straightforward.
Each bottle of dehydrated medium has instructions for preparation on its label.
For example, to prepare a liter of tryptic soy broth:
1. Suspend 30 g of the dehydrated medium in 1,000 ml of distilled water.
2. Mix thoroughly in a 2-liter Erlenmeyer flask (always use a flask that holds twice the
volume of media you are preparing).
3. Dispense and sterilize for 15 to 20 minutes at 121°C (15 lbs pressure).
As noted, the amount of powder for 1,000 ml of water will be indicated. If the medium lacks
agar, the powder will usually dissolve without heating. If it contains agar, you must heat the
medium until it starts to simmer or boil in order to completely dissolve the agar. Specific heating
instructions are given for each type of medium. For example, to prepare a liter of Vogel-Johnson
agar:
1. Suspend 61g of the dehydrated medium in a liter of distilled water.
2. Mix until a uniform suspension is obtained.
3. Heat with constant agitation and simmer for 1 minute.
4. Dispense 100-ml amounts into 250-ml flasks and sterilize by autoclaving at 121°C for 20
minutes.
Sterilizing the petri dishes, glass tubes & other containers
Heating by exposure to hot air is an accepted method for sterilizing loads that cannot be reliably
penetrated by steam and can tolerate the high temperatures required. e.g. 160 °C for 2hrs
Method:
1. Ensure articles are thoroughly clean and dry.
2. Stainless steel canisters, aluminium foils, wrappings of craft papers and stoppers of cotton
wool may be used to retain sterility after processing the items.
3. Plug glass test tubes with cotton wool stoppers and place them vertically in metal baskets.
Cover the top of the metal basket with an aluminium foil or a piece of craft paper. Do not put
screw capped bottles in a hot air oven unless their caps and liners are made of a material that
will resist distortion at the sterilizing temperatures in a hot air oven.
4. Flasks are also plugged with cotton wool stoppers and the top is covered with a piece of craft
paper or aluminium foil.

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5. Position the individual articles to allow free circulation of hot air between and around the
items.
Sterilizing cycle:
 Set the controls to ensure that the sterilization hold time does not start until the sterilizing
temperature, detected by thermocouples placed on the load, has been reached.
 Set the sterilization hold time to160°C for 2 hours, or 170°C for 1 hour or 180°C for 30
minutes.
Cooling:
1. Do not attempt to open the chamber door until the chamber and load have been cooled to
below 80°C as glassware is liable to crack if cold air is admitted suddenly while it is still
very hot.
2. Use protective gloves to remove items from the oven.
Glassware for media such as Koser’s citrate, in which there is a single source of element as
carbon or nitrogen must be chemically clean. A recommended method of ensuring this is to boil
all tubes/bottles in 20% nitric acid for 5 – 10 minutes and then wash and rinse well with glass
distilled water. Tubes/bottles are dried in an oven in the inverted position in baskets lined with
filter or blotting paper to prevent the mouths of tubes/ bottles touching the metal.
Why are culture media sterilized before use?
Describe the method you use to sterilize.
Culture media used in the isolation of pathogenic microorganisms or for the study of
microorganisms should be free from any microorganisms that could affect the interpretation of
the results. So they should be sterilized before use.
The choice of method to be used to sterilize a medium depends on whether the ingredients are
decomposed by heat or not. If autoclaving will not damage the medium, it is the best method of
sterilization. Follow manufacturer’s instructions before you sterilize each culture medium.
Autoclaving:
In this method of sterilization, pressure is used to produce high temperature steam. It is based on
the principle that, when water is boiled at an increased pressure, the temperature at which it boils
and of the steam that it forms, rises. Hot saturated steam rapidly penetrates and gives up its latent
heat when it condenses on cooler objects.

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Autoclaving is used to sterilize most agar and fluid culture media. It ensures the destruction of
vegetative cells as well as bacterial endospores by coagulating and denaturing microbial proteins
and enzymes.
Method
1. Add the correct volume of water to the autoclave.
2. Place the wire baskets containing bottles or tubes of culture media, with caps
loosened, in the inner chamber of the autoclave. Do not overload.
3. Secure the lid of the autoclave, open the air-cock, and close the draw-off knob.
4. Adjust the safety valve to the required pressure i.e. 10psi to give a temperature of
1150C or 15psi to give 1210C. Larger volumes of media require longer sterilization
times. Sometimes lower temperatures such as 1150C for times ranging 10- 20 minutes
are recommended for sterilization of media containing ingredients that are not very
stable to heat.
5. Apply heat electrically or use gas or a primus stove.
6. When all the water droplets have been expelled and only steam is emerging, wait for
1 minute, and closes the air-cock. This will cause the pressure to rise and with it, the
temperature of the steam.
7. When the required temperature has been reached, and the excess steam begins to be
released from the safety valve, reduce the heat and begin the timing. Most culture
media are sterilized at a holding time of 15 minutes.
8. At the end of the sterilizing time, turn off the heat, and allow the autoclave to cool
naturally.
9. Check that the pressure gauge is showing zero. When at zero, open the air-cock and
then wait for a few minutes before opening the lid to allow time for the autoclave to
become fully vented.
10. After removing the culture media, tighten the caps of bottles.
A bacteriological method of monitoring the performance of an autoclave is to use a strip of filter
paper impregnated with spores of Bacillus stearothermophilus as an indicator with the sterilizing
load and to see whether the organisms grow or not when plated. If the organisms grow, the
process of sterilization is unsatisfactory.

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Other methods used to sterilize culture media, are steaming at 1000C on several occasions
(Tynderlization) and filtration.
Tynderlization: Steaming at 1000C on one occasion or several occasions
This is used to sterilize media containing ingredients that would be broken down or inactivated at
temperatures above 1000C. This is not a definite way of sterilizing media.
Steaming can be performed in a steam sterilizer, such as an Arnold or Koch steamer. The bottles
of media with loosened caps are placed on perforated trays above the boiling water. During
boiling all the vegetative cells will be destroyed but not necessarily all the spores. Upon keeping
the spores will germinate to form vegetative cells and on subsequent boiling they will also be
destroyed. After sterilization, when the medium has cooled, the caps of the bottles are tightened.
Filtration:
This provides a means of removing bacteria from fluids. It is used mainly to sterilize additives
that are heat sensitive and cannot be autoclaved, or less stable substances that need to be added to
a sterilized medium immediately before it is used. Several different types of filters can be used,
including those made from sintered glass, asbestos or inert cellulose esters.
Decontamination of used culture media plates
All cultures to be discarded must render noninfectious by sterilization, even if they are
apparently ‘negative’. Sterilization is equally important for cultures in reusable containers or in
disposable containers such as plastic petri dishes that may be incinerated, as it is of paramount
importance that no living infective material leaves the laboratory.
There are 3 practical methods by which cultures may be sterilized.
a. Incineration
b. Autoclaving
c. Chemical disinfection
Autoclaving :
Discarded culture plates are usually first collected in stainless steel pails or in autoclavable
plastic bags held in pails. They are best autoclaved open, in multipurpose autoclaves with high
pre-vacuum or vacuum pulsing to remove all air from the plates, before exposure to pure stream
under pressure. Care must be taken to ensure that molten agar does not escape into the chamber
drain, which would block the drain when the agar cools.

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Cultures are non-infectious after autoclaving at 1210C at 15lb for 15 minutes and can be
discarded into a bin containing clinical waste.
Chemical disinfection
Chemical disinfectants are used to disinfect instruments used in the microbiology laboratory.
Five percent phenol or 1% hypochlorite is used to disinfect instruments contaminated with
organisms (Pasteur pipettes, glass slides, cover slips). When there is a spillage of blood, 10%
hypochlorite solution is used.
Describe the way you wash the used plates and bottles step by step
1. Autoclave the used plates and bottles
2. Wear protective heavy duty gloves
3. Remove the agar from the plates manually
4. Wash Petri dishes and bottles with soap and tap water
5. Rinse with distilled water
6. Dry in moderate heat
Petri dishes:
1. Put the Petri dishes into a canister or wrap them in foil paper
2. Put them into the hot air oven at 160°C for 2 hours or 180°C for 1 hour
Bottles:
1. Loosen the caps of the bottles
2. Put the bottles in a wire basket
3. Autoclave the bottles at 1210C at 15 lb for 15 minutes
4. Tighten the caps when taken out of the autoclave
What is the method you use to store the prepared culture media?
Prepared sterilized media in individual screw capped bottles can be stored at room temperature
for weeks, but some deterioration is likely to occur. It is essential to have some form of cold
storage in the laboratory for the preservation of blood, serum and culture media.
For a smaller laboratory, one of the domestic refrigerators of 1-2m3 capacity is suitable. Larger
laboratories require a corresponding larger cabinet, insulated cold room with the refrigerating
plant outside. The temperature should be maintained between 4 -5oC. It should never be low as
freezing since this may be detrimental.

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Plates of agar media can be kept for several days not exceeding 7- 10 days at room temperature
or in a refrigerator. However, when kept at room temperature, changes in hydration with
concentration of ingredients may occur, the pH may alter and bacterial or fungal contamination
may occur.
When stored in the refrigerator, care should be taken to prevent evaporation of water from the
medium. This can be achieved by packing the plates in plastic re-sealable bags. When storing in
containers, it is advisable to store them in a screw capped container than a cotton plugged
container to minimize evaporation.
Strong light is detrimental to most media and storage in a dark place is preferable especially for
those containing dyes.
Egg media should be kept stored for long periods (about 2 weeks) before use to provide the
opportunity to the contaminants that grow only at room temperature to develop visible colonies.
Aseptic Technique:
Aseptic technique is a method that prevents the introduction of unwanted organisms into an
environment. When changing wound dressings, aseptic technique is used to prevent possible
infection. When working with microbial cultures aseptic technique is used to prevent introducing
additional organisms into the culture.
Microorganisms are everywhere in the environment. When dealing with microbial cultures it is
necessary to handle them in such a way that environmental organisms do not get introduced into
the culture. Microorganisms may be found on surfaces and floating in air currents. They may fall
from objects suspended over a culture or swim in fluids. Aseptic technique prevents
environmental organisms from entering a culture.
Doors and windows are kept closed in the laboratory to prevent air currents which may cause
microorganisms from surfaces to become airborne and more likely to get into cultures. Transfer
loops and needles are sterilized before and after use in a Bunsen burner to prevent introduction of
unwanted organisms. Agar plates are held in a manner that minimizes the exposure of the surface
to the environment. When removing lids from tubes they are held in the hand and not placed on
the countertop during the transfer of materials from one tube to another. All of these techniques
comprise laboratory aseptic technique.

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8.2 Inoculation and Cultivation of Microorganisms

The purpose of using cultural techniques in microbiology is to demonstrate the presence of
organisms which may be causing disease and when indicated, to test the susceptibility of
pathogens to antimicrobial agents.
Before inoculating a culture medium check the medium for visual contamination, or any change
in its appearance which may indicate deterioration of the medium; e.g. darkening in colour.
When inoculating, or seeding, culture media an aseptic (sterile) technique must be used. This
will:
– prevent contamination of cultures and specimens,
– prevent infection of the laboratory worker and the environment.
Instruments used to seed culture media
 Wire loops – Original type of inoculating wire was platinum. But nichrome wires are widely
used now. They are re-usable. The wire is sterilized by holding it almost vertically in a
Bunsen flame until the entire wire become red-hot and is used to pick colonies once it is
cooled. Plastic disposable wire loops of different diameters are available for use now. Wire
loops are used to pick and spread colonies on agar media, to transfer and spread clinical
specimens on agar surfaces and to transfer colonies into broth media.
 Straight wires – Used for stab cultures or picking off single colonies (Sterilize using a
Bunsen flame before use).
 Sterilized wooden sticks – Used for spread inoculums on agar surface.
 Pipettes – Used to inoculate agar media and broth media.
 Bunsen flame – Used to sterilize wire loops, straight wires and tips of the pipettes and
mouths of the containers used in the procedure.
 Agar media/broth media – Pre-sterilized and use to inoculate specimens and colonies of
organisms.
Aseptic techniques
 Flame sterilizes wire loops, straight wires, and metal forceps before and after use.
 Flame the necks of specimen bottles, culture bottles, and tubes after removing and before
replacing caps, bungs, or plugs.
 When inoculating, do not let the tops or caps of bottles and tubes touch an unsterile surface.
This can be avoided by holding the top or cap in the hand.

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Inoculation of media in petri dishes:

The technique used to inoculate media in petri dishes (plates) must provide single colonies for
identification. It must also show whether a culture is pure or mixed, i.e. consisting of a single
type of organism or several different organisms. A pathogen must be isolated in pure culture
before it can be identified and tested for antimicrobial sensitivity.
The inoculation of media in petri dishes is referred to as ‘plating out’ or ‘looping out’. It is not
necessary to use whole plates of media for every specimen. Considerable savings can be made by
using a half or even a third of a plate (especially when the medium is a selective one). The area
of medium used must be sufficient to give separate colonies. Before inoculating a plate of culture
medium, the surface of the medium must be dried; otherwise single colonies will not be formed.
To do this, remove the lid of the plate and place this face upwards on an incubator shelf. Invert
the base containing the medium and let it rest at an angle on the lid. Usually 30–40 minutes
incubation at 35–370C is sufficient time to dry the surface of an agar plate.
Inoculating technique:
 Using a sterile loop or swab of the specimen, apply the inoculum to a small area of the plate
(the ‘well’) as shown in Fig. 7.6.
 Flame sterilizes the loop. When cool, or using a second sterile loop, spread the inoculum as
shown in Fig. 7.6 (follow the steps 2 through to 5). This will ensure single colony growth.
Note: A simplified technique of inoculating plates is shown in Fig. 7.7. This can be used by
medical and nursing staff when culturing specimens directly from patients, e.g. urogenital
specimens for the isolation of Neisseria gonorrhoeae. The techniques of inoculating half a plate
and a third of a plate of medium are shown in Fig. 7.8 and Fig. 7.9.

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Inoculation of slopes:
To inoculate slopes such as Dorset egg medium or Loeffler serum, use a sterile straight wire to
streak the inoculum down the centre of the slope and then spread the inoculum in a zig-zag
pattern as shown in Fig. 7.10. To inoculate a slope and butt medium, such as Kligler iron agar,
use a sterile straight wire to stab into the butt first and then use the same wire to streak the slope
in a zig-zag pattern (see Fig. 7.11).
Inoculation of stab media (deeps):
Use a sterile straight wire to inoculate a stab medium. Stab through the centre of the medium as
shown in Fig. 7.12, taking care to withdraw the wire along the line of inoculum without making
further stab lines.

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Inoculation of fluid media:

Broths and other fluid media are inoculated using a sterile wire loop, straight wire, or Pasteur
pipette depending on whether the inoculum is a colony, a fluid culture, or a specimen. When
using a wire loop to subculture colonies, hold the bottle or tube at an angle and rub the loop
against the side of the container below the level of the fluid.
Labelling of inoculated media:
Using a grease pencil or marker pen, label inoculated media with the date and the patient’s
number. Always label the base of a culture plate, not the lid (lids can be accidentally switched).
Label a slope on the underside of the medium so that the wording does not obscure the culture. A
stab culture should be labelled above the level of the agar. When a plate is to be incubated
anaerobically it should be marked ‘An O2’ or when in a carbon dioxide atmosphere, it should be
marked ‘CO2’.
Incubation of Inoculated Media:
Inoculated media should be incubated as soon as possible. A delay in incubation can affect the
viability of pathogens especially anaerobes, pneumococci, meningococci, gonococci, and
Haemophilus influenzae. It can also increase the risk of plates becoming contaminated from
small insects and dust. Uninoculated and inoculated media must be protected from sunlight.
Microorganisms require incubation at the temperature and in the humidity and gaseous
atmosphere most suited to their metabolism. The length of time of incubation depends on how
long an organism takes to develop the cultural characteristics by which it is recognized.
Temperature of incubation:
The temperature at which a microorganism grows best is referred to as its optimum temperature.
The temperature below which growth stops (not necessarily resulting in death) is called the
minimum temperature, and that above which growth stops and death occurs is called the
maximum temperature. The temperature selected for routine culturing is 35–370C with most
microbiologists recommending 350C in preference to 360C or 37 0C. In general, the growth of
microorganisms is more affected by slight rises above their optimum temperature than by
reductions below it.

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Colony morphology
Colony morphology will vary with the medium on which the organism is grown. But it will give
an idea the organism in question. Colony morphology gives important clues as to the identity of
their constituent microorganisms. Important classes of colony characteristics include:
1. Size
2. Shape
3. Surface
4. Margin
5. Elevation
6. Texture
7. Translucency (Clear, translucent or opaque)
8. Colour
9. Pigmentation
10. Changes brought about in the medium (Haemolysis)
8.3 Overview of antimicrobial sensitivity testing
Concepts of antimicrobial agents :
The word antimicrobial was derived from the Greek words anti (against), mikros (little) and bios
(life) and refers to all agents that act against microbial organisms. This is not synonymous with
antibiotics, a similar term derived from the Greek word anti (against) and biotikos (concerning
life). By strict definition, the word “antibiotic” refers to substances produced by microorganisms
that act against another microorganism. Thus, antibiotics do not include antimicrobial substances
that are synthetic (sulfonamides and quinolones), or semisynthetic (methicillin and amoxicillin),
or those which come from plants (alkaloids) or animals (lysozyme).
In contrast, the term “antimicrobials” include all agents that act against all types of
microorganisms – bacteria (antibacterial), viruses (antiviral), fungi (antifungal) and protozoa
(antiprotozoal). Notice that the term “antibacterials”, being the largest and most widely known
and studied class of antimicrobials, is often used interchangeably with the term “antimicrobials’’.
Antibiotics versus Antimicrobials:
 An ANTIBIOTIC is a low molecular substance produced by a microorganism that at a
low concentration inhibits or kills other microorganisms.
 An ANTIMICROBIAL is any substance of natural, semisynthetic or synthetic origin

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that kills or inhibits the growth of microorganisms but causes little or no damage to the
host.
 All antibiotics are antimicrobials, but not all antimicrobials are antibiotics.
Modes of action (mechanism of action) of antimicrobial agents
 Five modes of antimicrobial actions:
– Inhibition of cell wall synthesis
– Disruption of cell membrane function
– Inhibition of protein synthesis
– Inhibition of nucleic acid synthesis (i.e., inhibition of replication of genetic
material or transcription)
– Action as antimetabolites
What is antimicrobial resistance?
 Antimicrobial resistance (AMR) is resistance of a microorganism to an antimicrobial
medicine to which it was previously sensitive
 Resistant organisms are able to withstand attack by antimicrobial medicines, such as
antibiotics, antivirals, and antimalarials
 So that standard treatments become ineffective and infections persist and may
spread to others
 AMR is a consequence of:
 Misuse of antimicrobial medicines
 Mutation of the microbes
 Acquiring of a resistance gene
Mechanisms of bacterial drug resistance
 Four main mechanisms
 Drug inactivation or modification: for example, enzymatic deactivation of
penicillin G in some penicillin-resistant bacteria through the production of β-
lactamases
 Alteration of target site: for example, alteration of PBP—the binding target site of
penicillins
 Alteration of metabolic pathway: for example, some sulfonamide-resistant bacteria
do not require para-aminobenzoic acid (PABA), an important precursor for the

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synthesis of folic acid and nucleic acids in bacteria inhibited by sulfonamides,

instead, like mammalian cells, they turn to using preformed folic acid
 Reduced drug accumulation: by decreasing drug permeability and/or increasing
active efflux (pumping out) of the drugs across the cell surface
8.4 Antibiotic Susceptibility Testing
Main methods available for antibiotic susceptibility testing in the laboratory
1. Disk diffusion techniques
2. Dilution techniques (to get a minimum inhibitory concentration - MIC)
3. Automated methods (VITEK 1, VITEK 2)
4.Epsilometer testing (E-test)
Disc diffusion method
Discs of blotting papers impregnated with a known volume and appropriate concentration of an
antimicrobial is placed on a plate of sensitivity testing agar, inoculated with a standard inoculum
of a test organism. If the bacteria are susceptible to a particular antibiotic, an area of clearing
surrounds the discs where bacteria are not capable of growing (called a zone of inhibition). The
size of the zone and the rate of antibiotic diffusion are used to estimate the bacteria's sensitivity
to that particular antibiotic.
Kirby-Bauer CLSI modified disc diffusion technique
The validity of this carefully standardized technique depends on, for each defined species
 Using discs of correct antimicrobial content
 An inoculum which gives confluent growth
 A reliable Mueller Hinton agar
The test method must be followed exactly in every detail. After incubation at 350C for 16–18
hours, zone sizes are measured and interpreted using standards. These are derived from the
correlation which exists between zone sizes and MICs.
The test should only be used for well-evaluated bacterial species.
Not suitable for bacteria that are slow-growing, need special nutrients, or require CO2 or
anaerobic incubation.
CLSI technique .
Preparation of Mueller Hinton agar:
 Prepare and sterilize the medium as instructed by the manufacturer.

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 The pH of the medium should be 7.2–7.4.

 Pour the medium into 90 mm diameter sterile petri dishes to a depth of 4 mm (about 25ml
per plate).
 Care must be taken to pour the plates on a level surface so that the depth of the medium is
uniform.
Note: If the medium is too thick the zones will be falsely small and if too thin the inhibition
zones will be falsely large.
 Unmodified Mueller Hinton agar is not suitable for susceptibility testing H. influenzae,
S. pneumoniae, N. gonorrhoeae. The addition of lyzed blood will enable such organisms
to be tested.
Selection of antimicrobial discs:
 The choice of antimicrobials to be included in susceptibility tests will depend on the
pathogen, the specimen, range of locally available antimicrobials, and local prescribing
policies.
 Consultation between laboratory, medical, and pharmacy staff is required.
 The range of first choice drugs should be limited and reviewed at regular intervals.
Additional drugs should be included only by special request. Where there is cross-
resistance, only one member from each group of related antimicrobials should be
selected.
Note: Paper antimicrobial discs are commercially available from most manufacturers of culture
media. Most paper discs can be used for 1 year or longer from the date of manufacture providing
they are stored correctly (–200C or working stock at 2–80C) in an airtight container with an
indicating desiccant.
 Discs that have expired should not be used.
 Quality control of discs is essential.
 About 1 hour before use, the working stock of discs should be allowed to warm to room
temperature, protected from direct sunlight.
Important: Decreasing control zone size with a particular antimicrobial disc is often an indication
of deterioration of the antimicrobial due to moisture or heat.

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3. Control strains
 Control strains are used to test the performance of the method. The following strains of
bacterial species are recommended.
o Staphylococcus aureus ATCC 25923.
o Escherichia coli ATCC 25922.
o Pseudomonas aeruginosa ATCC 27853
o Haemophilus influenzae ATCC 49766
o Enetrococcus faecalis ATCC 29212
Sources of control strains:
o Reference Laboratories should supply local laboratories.
Storage of control strains:
For prolonged storage, maintain stock cultures at -200C or below or in liquid nitrogen in a
suitable stabilizer (50% foetal calf serum, 10-15% glycerol in tryptic soy broth, defibrinated
sheep blood or skim milk) or in the freeze- dried form.
Subculture frozen or freeze-dried stock cultures on appropriate media and incubate under
appropriate conditions for the organisms. Subculture the frozen or freeze-dried stock cultures
twice before use in the testing. The 2nd sub culture is referred to as Day 1 working culture.
Prepare a new subculture each week to create a working culture. Prepare a new working culture
each day.
Prepare new primary cultures from frozen or freeze-dried stock cultures at least once a month.
Procedure of AST:
Direct colony suspension method is the most convenient method for inoculum preparation.
1. Transfer 3–5 well-isolated colonies of similar appearance of the test organism and emulsify
in 3–4 ml of sterile physiological saline or nutrient broth and vortex to mix.
2. In a good light match the turbidity of the suspension to the 0.5 McFarland turbidity standards
(vortex the standard immediately before use) by viewing against a printed card or sheet of
paper.
3. Use a sterile swab to get the organism in suspension. Remove excess fluid by pressing and
rotating the swab against the side of the tube above the level of the suspension.
4. Streak the swab evenly over the surface of the Muller Hinton agar in three directions, rotating
the plate approximately 600 to ensure even distribution and finally take the swab round the

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edge of the agar surface. With the petri dish lid in place, allow 3–5 minutes (no longer than
15 minutes) for the surface of the agar to dry.

5. Using sterile forceps, needle mounted in a holder, or a multidisc dispenser, place the
appropriate antimicrobial discs, evenly distributed on the inoculated plate (no more than 5
discs on a 90 mm plate and no more than 12 discs on a 150 mm plate). Can use a template as
to ensure the discs are correctly placed.
Note: The discs should be about 15 mm from the edge of the plate and no closer than about 25
mm from disc to disc.
6. Each disc should be lightly pressed down to ensure its contact with the agar. It should not be
moved once it is placed. Within 15 minutes of applying the discs, invert the plate and
incubate it aerobically at 350C for 16–18 hours (temperatures over 350C invalidate results
for oxacillin and cefoxitin).

7. fter overnight incubation, examine the test plates to ensure the growth is confluent or near
confluent. Using a ruler on the underside of the plate or using a vernier calliper, measure the
diameter of each zone of inhibition in milimeters. The endpoint of inhibition is where growth
starts.

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8. Using the interpretative chart, interpret the zones sizes of each antimicrobial, reporting the
organism as ‘Resistant’, ‘Intermediate/Moderately susceptible’, or ‘Susceptible’.
 Quality control should be done to monitor disc potency and culture media.

Discs place on a 90 mm plate Discs placed on a 150 mm plate

Self-check questions:
Instruction: Attempt the following questions listed below
1. Define a culture medium
2. How can you classify culture media?

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Summary
 A culture medium is an artificial environment that provides sources of carbon, energy and
nitrogen in the form of available carbohydrates and amino acids for the growth of bacteria.
 Bacterial culture media can be classified in different ways; based on consistency, based on
nutritional component and based on its functional use.
 Culture media may be prepared in the laboratory from basic ingredients or it may be
purchased ready for use.
 Culture media used in the isolation of pathogenic microorganisms should be free from any
microorganisms that could affect the interpretation of the results. So they should be
sterilized before use.
 The purpose of using cultural techniques in microbiology is to demonstrate the presence of
organisms which may be causing disease and when indicated, to test the susceptibility of
pathogens to antimicrobial agents.
 Different methods are available for antibiotic susceptibility testing of bacteria in the
laboratory.

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Chapter 9: Quality Assurance and SOPs in Microbiological Laboratories

Dear trainees,
In this chapter, you will learn the internal and external quality control in medical
microbiology to assure the test results.
Objectives:
At the end of this chapter, you will be able to:
 Explain internal quality control in microbiology
 Explain external quality control in microbiology
Need for quality assurance and SOPs in microbiology:
Microbiological investigations are important in the diagnosis, treatment, and surveillance of
infectious diseases and policies regarding the selection and use of antimicrobial drugs. It is
therefore essential that test reports:
 are reliable,
 standardized,
 provide the information that is required at the time it is needed,
 in a form that can be understood.
Quality assurance is also required to minimize waste and ensure investigations are relevant and
used appropriately.
WHO in its publication Basic laboratory procedures in clinical bacteriology states that quality
assurance in microbiology must be:
 Comprehensive: to cover every step in the cycle from collecting the specimen to sending
the final report to the doctor as shown opposite;
 Rational: to concentrate on the most critical steps in the cycle;
 Regular: to provide continuous monitoring of test procedures;
 Frequent: to detect and correct errors as they occur.
The following apply to the QA of the pre-analytical, analytical, and post-analytical stages of
microbiological procedures and should be incorporated in microbiological SOPs.
Pre-analytical stage: SOPs need to describe:
o Selection and appropriate use of microbiological investigations.
o Collection and transport of specimens.
o How to fill in a request form correctly.

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o Checks to be made when the specimen and request form reach the laboratory.
Appropriate use of microbiological investigations
o This aspect of QA requires collaboration between laboratory personnel, clinicians,
and public health officers as discussed at the beginning of this subunit.
o The fewer the resources the more important it is to establish priorities based on
clinical and public health needs.
o Clear guidelines should be provided on the use and value of specific microbiological
investigations.
Collection and transport of microbiological specimens
o Specimens for microbiological investigation must be collected correctly if pathogens
are to be successfully isolated and identified, reports are not to be misleading, and
resources are not to be wasted.
o Written instructions for the collection of specimens must be issued by the laboratory
to all those responsible for collecting microbiological specimens.
Request form:
Each specimen must be accompanied by a request form which details:
o The patient’s name, age (whether an infant, child, adult), gender, outpatient or inpatient
number, ward or health centre, and home area/village.
o Type and source of specimen, and the date and time of its collection.
o Investigation required.
o Clinical note summarizing the patient’s illness, suspected diagnosis and information on
any antimicrobial treatment that may have been started at home or in the hospital.
Note: The clinical note will help to report usefully the results of laboratory investigations.
o Name of the medical officer requesting the investigation.
Checking a specimen and request form:
o SOPs should include the procedures to be followed when specimens reach the laboratory,
particularly checks to ensure that the correct specimen has been sent and the name on the
specimen is the same as that on the request form.
o Also included should be how to handle and store specimens that require immediate
attention, e.g. C.S.F., blood cultures, unpreserved urine, swabs not in transport media,
faecal specimens containing blood and mucus, and wet slide preparations.

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o Examples of specimens which should not be accepted for microbiological investigations

include:
 dry faecal swabs,
 saliva instead of sputum,
 eye swabs that have not been freshly collected,
 any specimen not collected into a correct container,
 a leaking specimen (sample may be contaminated).
Analytical stage: The following should be included in microbiologicalSOPs, covering the
analytical stage:
o Detailed procedures for examining different specimens
o Staining techniques and quality control (QC) of stains
o Aseptic techniques and safe handling of infectious material
o Preparation and QC of culture media and preservation of stock strains used in performance
testing
o Inoculation of broth and agar culture media and plating out techniques as per the standards
o Reading and interpretation of cultures as per the standards
o Techniques used to identify pathogens and the QC of diagnostic reagents, strips, and discs
o Antimicrobial susceptibility testing and QC of procedure and discs
o Cleaning and QC of equipment used in the microbiology laboratory, e.g. microscope,
incubator, anaerobic jar, centrifuge, water bath/heat block, autoclave, hot-air oven, and
refrigerator
o Immunological techniques and QC of antigen and antibody reagents.
o Safe working practices
o Disposal of specimens and cultures
o Cleaning of glassware, plastic ware, etc
o Sterilization procedures and their control
o The sterilization of glassware by dry heat
Important: As part of QC, the performance of staff must be monitored, all techniques must be
demonstrated to new members of staff, the results of QC tests must be recorded and signed, and
the work of newly qualified staff supervised.
Post-analytical stage: SOPs need to include:

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o Reporting and verifying of microbiological test results.

o Taking appropriate action(s) when a result has serious patient or public health
implications.
o Interpreting test reports correctly.
Reporting results:
o The terminology and format used in reporting microscopic preparations, cultures, and
antimicrobial susceptibility tests should be standardized and agreed between laboratory
personnel, clinicians, and public health officers.
o Any preliminary report of microscopic findings or isolation of a pathogen from a primary
culture must be followed by a full written report.
o All reports must be concise and clearly presented.
o The use of rubber stamps can be helpful in standardizing the report and making it easy to
understand, e.g. stamps that list the presence or absence of recognized pathogens or that list
the antibiotics against which an isolate has been tested.
o When using a stamp, care must be taken to position it correctly and sufficient ink must be
used to reproduce clearly the entire stamp.
Verifying and interpreting reports:
o Before leaving the microbiology laboratory, all reports must be checked for correctness and
clarity and signed by the person in charge of the department.
o Reports which are urgently needed for patient care or the management of an epidemic must
reach the clinician or public health officer/epidemiologist as soon as possible. Those
receiving the reports should consult the laboratory when any part of the report is unclear.
o Improvement in the quality and usefulness of microbiological reports will only be achieved
by effective communication between those requesting tests and laboratory staff.
o A record of the results of all investigations must be kept by the laboratory, e.g. as carbon
copies, work sheets, or in record books. Copies of work sheets should be dated and filed
systematically each day.
External quality assessment:
o Whenever possible the regional public health laboratory should organize an external quality
assessment (EQA) scheme to help district microbiology laboratories.

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o An EQA scheme should include testing for major pathogens. It should not be too
complicated, costly, or time-consuming for district laboratories.
o The main objective of an EQA scheme is to confirm that a laboratory’s SOPs and internal
QC procedures are working satisfactorily.
o EQA schemes help to identify errors, improve the quality of work, stimulate staff motivation.
o Instructions and a report form (to be returned with results after 1 week) should be sent with
the specimens to each participating laboratory.
o Each specimen should be examined in the same way as routine clinical samples (not
recognized as a QC specimen).
o Refresher courses should be held periodically to maintain competence and motivation and to
introduce new tests.
Self Check:
Instruction: Attempt the following questions listed below
1. Discuss pre- analytical stage in laboratory testing procedures
2. Discuss analytical stage in laboratory testing procedures
3. Discuss post analytical stage in laboratory testing procedures
Summary
 Microbiological investigations play a pivotal role in the diagnosis, treatment and
surveillance of infectious diseases. It is also helpful for the selection and use of
antimicrobial drugs. Hence, microbiological test results should be reliable and
standardized.
 It is the quality assurance that ensures the investigation results are relevant.
 Quality assurance needs to be applied during the pre-analytical, analytical and post-
analytical phases of microbiological procedures.
 To strengthen the district microbiology laboratories, regional public health laboratory
should organize an external quality assessment (EQA) scheme at regular intervals.

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Chapter 10: Recording and Reporting of Microbiological Test Results

Dear trainees,
In this chapter, you will how to report record, interpreted and maintain confidentiality of
microbiological data.
Objectives:
At the end of this chapter, you will be able to:
 Describe how to report and record microbiological test results.
 Interpret and record data related to microbiology laboratory activities.
 Maintain confidentiality of data and records related to microbiology laboratory.
10.1 . Reporting and recording test results
 Laboratory staff should provide as much relevant information as possible to assist those
requesting tests to interpret the results of tests correctly and use the information in the
best possible way to benefit patients and the community.

 Reports should be clearly and neatly written.

10.2 .Standardization in reporting test results
 Standardization in the presentation of reports and use of measurement units.
 It helps in the interpretation and comparison of results,
 It contributes to the efficiency of a laboratory service, and is of value when patients are
referred from one health unit or hospital to another.
 Use SI units in the reporting of tests results. So, that everybody can understood and
interpret the results.
10.3 Recording results in the laboratory
In district laboratories, records of test results can be kept by retaining carbon copies of reports,
using
work sheets, or recording test results in registers (exercise books).

 Records of test results must be reliable and enable patients’ results to be found quickly.

 Test records are also required when preparing work reports and estimating the workload
of the laboratory.

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 If carbon copies or work sheets are used these must be dated and filed systematically
each day.

 If registers are used, backing cards which are headed and ruled can be placed behind
pages to avoid having to rule and head each page separately.

 The cards must be heavily ruled with a marker pen so that the lines can be seen clearly.

 Separate registers, each with its own cards, can be prepared to record the results of
hematological, microbiological, clinical chemistry, urine and faecal tests.

Daily checks on the performance of equipment, e.g. temperature readings should be recorded in a
quality control (QC) book or on separate sheets as part of equipment control procedures.
10.4 Confidentiality of data and records related to microbiology laboratory
 The medical laboratory professional’s duty to keep patient information confidential has been
a cornerstone of medical laboratory ethics.
 Medical laboratory professionals shall preserve absolute confidentiality on all he knows
about his patient even after the patient has died.

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