WOLLEGA UNIVERSITY

COLLAGE OF MEDICAL AND HEALTH SCIENCE

SCHOOL OF VETERINARY MEDICINE

VETERINARY MICROBIOLOGY LABORATORY MANUAL

By

Sultan Abda Neja (DVM, MSc, MvSc, Assistant Professor)

Nekemte, Ethiopia
May, 2014

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TABLE OF CONTENTS

Pages

CHAPTER 1: INTRODUCTION ........................................................................................3

CHAPTER 2: GENERAL SAFETY RULES IN MICROBIOLOGY LABORATORY ....4
2.1. Microbiology Laboratory Safety Rules and Procedures .......................................... 6
2.2. Microbiological Safety Cabinets.............................................................................. 8
2.3. Demonstration of major laboratory equipment and reagents ................................. 11
2.4. Collection and submission of clinical specimens for diagnosis of microbial
infections ............................................................................................................... 17
CHAPTER 3: METHODS OF CLEANING AND STERILIZATION .............................19
3.1. Cleaning methods................................................................................................... 19
3.2. Antiseptics and Disinfectants ................................................................................. 20
3.3. Sterilization Methods ............................................................................................. 21
CHAPTR 4: BACTERIOLOGICAL STAINING METHODS .........................................24
4.1 Smear Preparation for bacteriological Staining ...................................................... 24
4.2. Types of Bacterial Staining Technique .................................................................. 25
iii. Staining of Flagella ......................................................................................................34
CHAPTER 5: BACTERIAL CULTURE METHODS ......................................................36
5.1. Types of culture media........................................................................................... 36
5.2. Preparation of culture media .................................................................................. 37
5.3. Inoculation and Asceptic Procedures ..................................................................... 38
5.4. Types of Inoculation Methods ............................................................................... 42
5.5. Atmospheric Oxygen Requirement of an Organisms ............................................ 47
5.5.1 Determine the oxygen requirements of bacteria .............................................. 48
5.5.2 Cultivation of Anaerobic Organisms ............................................................... 49
5.6 Bacterial Count ....................................................................................................... 51
CHAPTER 6: BIOCHEMICAL TESTS ...........................................................................55
CHAPTER 7: ANTIMICROBIAL SUSCEPTIBILITY TESTS .......................................64
7.1 Factors Influencing Antimicrobial Susceptibility Testing ...................................... 64
7.2 Methods of Antimicrobial Susceptibility Testing ................................................... 66
7.2.1 Disk Diffusion .................................................................................................. 66
7.2.2 Dilution Methods ............................................................................................. 73
7.2.3 Dilution and Diffusion ..................................................................................... 80
CHAPTER 8: COMMON SEROLOGICAL TESTS ........................................................82
8.1 Principle of Antigen Antibody Interactions ............................................................ 82
8.2 Collection, Preparation and Preservation of Specimen for Serologic Tests ........... 84
8.3 Materials Necessary for Basic Serologic Tests ....................................................... 84
8.4 Common Types of serologic tests. .......................................................................... 86

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CHAPTER 9: FUNGAL CULTURE AND TESTS ..........................................................92
9.1. Characteristic Features of Fungi ............................................................................ 93
9.2. General Considerations for the Lab Diagnosis of Fungal Infections ..................... 94
9.3. Direct Examination of Clinical Specimen ............................................................. 95
9.4 Fungal Smear and Staining Technique ................................................................... 95
9.5 Culture Medias and Culturing technique ................................................................ 99
CHAPTER 10: CULTIVATION OF VIRUSES .............................................................104
10.1. Cultivation of Viruses in Cell Cultures.............................................................. 105
10.1.1 Methods Used in Diagnostic Virology: Viral Culture ................................. 105
10.1.2 Specimen Processing and Inoculation for Isolation of Virus ...................... 107
10.2 Cultivation of Viruses in Natural Hosts and Laboratory Animals ...................... 110
CHAPTER 11: REFERENCES .......................................................................................111

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CHAPTER 1: INTRODUCTION

The competency of microbiology professionals is obtained through the theoretical

knowledge gained in the class and the practical skills and applications that lead to the
better understanding and interpretations the subject matter. This laboratory manual is
designed to guide the student through basic microbiology laboratory techniques,
procedures and experiments. It provides students good techniques in practical
investigations to ensure and proceed safely and to successfully achieve the required
educational and research aims. The main portion of this manual involves the
identification of unknown microbial agents. The manual can also be used as a reference
material and guideline protocol for basic microbiological practices and research
experiments.

This laboratory manual encompasses the basic laboratory techniques which start with
description about basic laboratory safety rule, cleaning and sterilization methods,
bacterial staining techniques, bacterial culturing methods, primary identification and
secondary biochemical tests and antibiotic sensitivity tests. It also includes commonly
used serological tests, mycological and viral culture and identification technique. Each of
these technique are written detail accordingly and the steps in each tests includes
principles of the test, objectives of the test, materials and reagents used, methods
and/or procedures and their possible results and subsequent interpretations.

High standards of laboratory safety and containment that will ensure healthy working
conditions for student, laboratory staff and protection of the environment must be of the
greatest priority. They can only be achieved by careful study of the principles involved
followed by practical application to premises, facilities, operating procedures and
hygiene. Orientations or short term training must be given to all students and laboratory
personnel before they are entirely allowed to use the laboratory.

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CHAPTER 2: GENERAL SAFETY RULES IN MICROBIOLOGY
LABORATORY
Objective
 To give overview on the laboratory safety rules
 To aware the students about the risks and precautions for the laboratory associated
hazards.

Laboratory work in veterinary microbiology lab should be carried out with a minimum of
risk to the health of the staff (biosafety) and the environment (biocontainment). This
requires careful consideration of the risks involved in a particular procedure, followed by
appropriate measures to minimize the disease risk at the laboratory and of possible
release into the environment and causing disease in animals or humans. This chapter of
the manual is concerned mainly with risks from infectious agents, and also with physical
and chemical injuries in microbiology laboratories. Risks from infection are reduced by
good laboratory techniques and secure facilities, which aid in the containment of
pathogens.

The backbone of the practice of biosafety is microbiological risk assessment. While there
are many tools available to assist in the assessment of risk for a given procedure or
experiment, the most important component is professional judgment. Risk assessments
should be performed by the individuals most familiar with the specific characteristics of
the organisms being considered for use, the equipment and procedures to be employed,
animal models that may be used, and the containment equipment and facilities available.
One of the most helpful tools available for performing a microbiological risk assessment
is the listing of risk groups for microbiological agents. Infective microorganisms can be
classified in to four risk groups.
Risk Group 1 (no or low individual and community risk): A microorganism that is
unlikely to cause human or animal disease.
Risk Group 2 (moderate individual risk, low community risk): A pathogen that can
cause human or animal disease but is unlikely to be a serious hazard to laboratory
workers, the community, livestock or the environment. Laboratory exposures may cause

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serious infection, but effective treatment and preventive measures are available and the
risk of spread of infection is limited.
Risk Group 3 (high individual risk, low community risk): A pathogen that usually causes
serious human or animal disease but does not ordinarily spread from one infected
individual to another. Effective treatment and preventive measures are available.
Risk Group 4 (high individual and community risk): A pathogen that usually causes
serious human or animal disease and that can be readily transmitted from one individual
to another, directly or indirectly. Effective treatment and preventive measures are not
usually available.
Microorganisms are divided into 4 Biosafety Levels (BSL) by the Centers for Disease
Control and Prevention. The microbes used in our microbiology laboratory fall into the
BSL-1 and BSL-2 categories. The assignment of an agent to a biosafety level for
laboratory work must be based on a risk assessment. Such an assessment will take the
risk group as well as other containment factors into consideration in establishing the
appropriate biosafety level. For example, an agent that is assigned to Risk Group 2 may
generally require BSL-2 facilities, equipment, practices and procedures for safe conduct
of work. However, if particular experiments require the generation of high-concentration
aerosols, then BSL-3 may be more appropriate to provide the necessary degree of safety,
since it ensures superior containment of aerosols in the laboratory workplace. The
biosafety level assigned for the specific work to be done is therefore driven by
professional judgment based on a risk assessment, rather than by automatic assignment of
a laboratory biosafety levels. Thus, the assignment of a biosafety level takes into
consideration the organism (pathogenic agent) used, the facilities available, and the
equipment practices and procedures required conducting work safely in the laboratory.

Biosafety Level 1 organisms are defined as well-characterized strains of microorganisms

not known to cause disease in healthy human adults. Precautions in BSL-1 labs include
general lab safety rules such as no eating or drinking, prohibition of mouth pipetting,
practicing aseptic technique, and proper disposal of sharps and microbiological waste.
Examples of BSL-1 organisms include non-pathogenic laboratory strains of Escherichia
coli, Staphylococcus xylosus, and Bacillus megaterium.

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Biosafety Level 2 organisms are defined as moderate-risk microorganisms that are
associated with less serious human diseases whose potential for transmission is limited
and a proven treatment for the disease exists. Many BSL-2 pathogens are opportunistic,
meaning they don’t ordinarily cause disease in healthy human adults, but may cause
disease in children and immunocompromised adults. Additional precautions in BSL-2
labs include using personal protective equipment (PPE) such as disposable gloves and lab
coats and limiting lab access to trained individuals. Examples of BSL-2 organisms
include Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella.

Biosafety Level 3 organisms are defined as high-risk microorganisms with a true

potential for infection by aerosols and in which the resulting disease may have serious or
lethal consequences. Researchers in BSL-3 labs generally wear double gloves,
respirators, and disposable surgical scrubs and gowns, and work in biological safety
cabinets in isolated, negative-pressure containment rooms. Examples of BSL-3 organisms
include Mycobacterium tuberculosis and Bacillus anthracis.

Biosafety Level 4 organisms are defined as easily transmitted, very-high risk

microorganisms which cause life-threatening diseases for which there is no vaccine or
therapy. Workers in BSL-4 labs work in impermeable positive pressure “space suits”
with an external oxygen supply, and precautions such as chemical showers must be taken
before exiting the lab. Examples of BSL-4 organisms include Ebola virus, Marburg virus,
and Lassa fever virus.

2.1. Microbiology Laboratory Safety Rules and Procedures

The essential requirements for any work with infectious agents and biohazardous
chemicals are as follows:
1. The laboratory should be easy to clean, with surfaces that are impervious to water
and resistant to chemicals. There shall be a wash-hand basin and emergency
shower, including an eye bath, in each laboratory suite as appropriate for the

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 chemicals and other hazards present. Procedures shall be established for frequent
cleaning and disinfection during and at the end of the work period. Hands must be
washed before leaving the laboratory;
2. Personnel access to the work area should be restricted; appropriate security
measures such as controlled electronic access may be necessary with higher risk
agents;
3. Personal protective equipment such as long-sleeved lab coats or gowns, closed-
toe footwear, disposable gloves, masks, safety glasses, face shields, and oro-nasal
respirators, as appropriate, shall be worn in the laboratory and removed when
leaving the laboratory;
4. The laboratory door should be closed when work is in progress and ventilation
should be provided by extracting air from the room. (Where biosafety cabinets are
used, care shall be taken to balance ventilation systems.);
5. Food (including chewing gum, candy, throat lozenges and cough drops) and/or
drink shall not be stored or consumed in laboratories;
6. Smoking and/or application of cosmetics shall not take place in the laboratory;
7. Pipetting shall not be done by mouth. Always use pipetting devices;
8. Care shall be taken to minimize the production of aerosols. Be careful around
Bunsen burners. Flames cannot always been seen. Flame wires loops, or needles
before and immediately after use to transfer biological material;
9. Emergency response plans should be developed to deal with the biohazard of
spills. Some of the items addressed in the plans should include having effective
disinfectant available for cleaning spills, removal of and decontamination of
contaminated protective clothing, washing of hands, and cleaning and disinfection
of bench tops;
10. Used laboratory glassware and other contaminated material shall be stored safely.
Long hair should be secured behind your head. Materials for disposal shall be
transported without spillage in strong containers. Waste material should be
autoclaved, incinerated or otherwise decontaminated before disposal. Reusable
material shall be decontaminated by appropriate means;

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 11. No infectious materials and biohazardous fluids shall be discarded down
laboratory sinks or any other drain;
12. Label all materials with your name, date, and any other applicable information
(e.g., media, organism, etc.).
13. Any accidents or incidents shall be recorded and reported to the Safety Officer.

2.2. Microbiological Safety Cabinets

These are used at the different containment levels, as described in Section C above. They
are of three types:
Class I: An open-fronted cabinet designed specifically to provide operator and
environmental protection and not to give protection to the work being handled.
Class II: An open-fronted safety cabinet, sometimes referred to as a laminar flow
recirculating cabinet. They are designed to give operator, product and environment
protection.
Class III: These cabinets are closed, with glove ports at the front, and provide the highest
degree of containment by complete separation of work and worker. Some cabinets have a
removable glove port and are known as Class III/I cabinets, i.e. they can be used in either
mode.
Requirements for work with infectious agents
1. Known pathogens: Having decided the risk level of certain work it is then possible
to decide the appropriate ‘containment level’ that is needed to minimize the risk of
human disease, and the risk of spread of disease to animals and the environment.
2. Diagnostic specimens: Veterinary diagnostic centers readily receive specimens that
are submitted because they are suspect for a variety of diseases. The infectious nature
of the specimens is usually unknown, but they have the potential to contain biological
agents that may cause disease in animals and humans. Practices and procedures need
to be in place that will minimize the risk of occupational exposure of employees to
such pathogens.

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3. Storage of Pathogens: Storage of live pathogens requires appropriate containment
and security to avoid risks due to breakage or unauthorised use of material. Storage
facilities should be appropriately labelled to indicate the nature of the pathogens (e.g.
their Group) and the contact information for the person(s) responsible for them.
Special care must be taken when opening glass vials of freeze-dried pathogens, as
these can sometimes shatter. Care must be taken when working with liquid nitrogen
or rooms where asphyxiating gasses may be produced. In a veterinary laboratory an
important responsibility is to minimize any risk of escape of pathogens to animals,
either wild or domestic, in the outside community.
4. Transport of Infectious Material: Great care must be taken when preparing and
packing diagnostic specimens, infectious materials and pathogens for transport, to
ensure that there is no breakage of containers or leakage of contents that could put at
risk personnel in the transport system or animals that may come in contact with
contamination. Applicable local, national and international regulations for the
transportation of dangerous goods (diagnostic or clinical sample and infectious
materials) and importation of animal pathogens must be followed.
5. Laboratory Animal Facilities: Work with pathogens in laboratory animals poses
special risks. Animal rooms have to be constructed to appropriate standards and
containment levels, just as laboratories. Containment in animal houses is very
important because of the large amount of infectious agents that they may generate.
Special care must be taken to avoid injury to staff, e.g. through animals biting and
kicking or self-inoculation accidents. The animal rooms should not only provide a
suitable environment for the animals themselves but should be constructed and
ventilated in such a way as to ensure comfort for the attending personnel.

Physical and chemical hazards

Laboratory work involves many manipulations that are potentially dangerous, such as
handling glassware and work with needles or other sharp instruments. There shall be
appropriate procedures and equipment for the safe and proper disposal of needles and
other ‘sharps’. Laboratory staff should be protected from the risk of receiving a burn

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from hot solids or liquids. Autoclaves shall be fitted with safety devices to prevent
accidental opening of doors when under pressure, and be regularly serviced and tested.
Heat-protective gloves, apron and face shields with brow and chin guards shall be
provided. Extreme cold can also be a risk, for example when working with liquid
nitrogen; splashes on exposed skin can be very damaging. Gloves should be worn that
provide insulation from cold and that are also waterproof, to prevent penetration of the
liquid nitrogen. Face shields with brow and chin guards and boots should also be worn
when working with liquid nitrogen. Nitrogen evaporating from liquid nitrogen storage in
poorly ventilated rooms can lead to depletion of oxygen with fatal consequences.

Irradiation is a serious health risk that may be present due to the use of X-ray machines,
or use of gamma emitters or other sources. Equipment shall be regularly serviced and
tested. All use of radioactive material must be meticulously recorded. All staff must wear
a personal radiation-monitoring device and have annual health checks. Local and national
regulations must be followed.

A wide range of chemicals are used in veterinary microbiology laboratories, many of

which may be toxic or mutagenic, and some may be carcinogenic. It should be
remembered that it is the dose that makes the poison. Vapours are especially hazardous,
and some chemicals can be absorbed by penetration of intact skin. Steam sterilisation
may make toxic chemicals volatile and endanger personnel who unload the
autoclave/pressure steam steriliser.

Procedures sufficient to protect pregnant laboratory workers should be followed at all

times. A list of hazardous chemicals shall be maintained, and a file record kept of
chemicals to which individual staff members could be exposed. Chemicals shall be
correctly stored in appropriate containers and at the correct temperature. Those that are
flammable shall be kept in a fireproof chemical store. A record must be maintained of the
purchase and use of hazardous chemicals: how much, when used, by whom and for what
purpose. Disposal of some chemicals is subject to official regulation.

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2.3. Demonstration of major laboratory equipment and reagents

Microscopes

Microscopes are one of the most important tools available to a microbiologist. In a

clinical setting, accurate diagnosis of a disease may require the determination of cell size
and shape, arrangement of cells, and whether or not the cells are motile. In addition, there
are many staining procedures that used to distinguish between different bacterial species.
All of these determinations require the use of a microscope.

There are many kinds of microscopes, including bright-field, dark-field, phase contrast,
fluorescence, and electron microscopes. Many microscopes have only one ocular, these
are termed monocular. Those with two oculars are termed binocular. In this manual,
bright-field compound microscopes, which can magnify objects up to about 1000X is
described (Figure 1). For observing bacteria, it is generally necessary to use the highest
magnification possible in order to see them clearly. This also means that the microscope
must be adjusted properly so things won’t appear fuzzy, and operated correctly so the
lenses don’t get out of alignment.

The usefulness of a light microscope is determined primarily by two factors:

magnification and resolving power. Magnification is simply the enlargement of the
specimen by using the lens systems of a given microscope. Modern light microscopes
have two lens systems, an objective lens system and the ocular lens system. The light
microscopes are equipped with a 10X ocular lens, and three objective lenses of 10X,
40X, and 100X. Some microscopes may have a fourth objective lens of 4X.

When you look at an object under a light microscope, the objective lens system magnifies
the object to produce a real image, which is projected up into the focal plane of the
ocular lens system. This lens system then produces a virtual image that you see when
you look through the microscope. The total magnification of an object is determined by

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multiplying the magnification of the ocular lens and the magnification of the objective
lens. For example, using an ocular lens of 10X together with an objective lens of 10X
gives a total magnification of 100X.

The resolving power of a lens system is defined as the smallest distance at which two
points can be seen separately, and is a function of the wavelength of light, the design and
positioning of the condenser, and the numerical aperture of the objective lens. The
condenser collects and focuses light onto the slide containing the organism being
studied. The numerical aperture is a function of the wavelength of light coming from
the condenser, the half angle of the lens aperture, and the refractive index of the medium
between the slide and the front of the lens. In most light microscope systems, the
wavelength of light and the half angle of the lens aperture are not readily changed.
However, using immersion oil for the highest-powered objective lens (100X) will
improve the resolving power. This works because immersion oil has the same refractive
index as glass, and so the amount of light reaching the objective lens is maximized (less
light is lost). Using the highest magnification and immersion oil lenses, the best light
microscopes can distinguish between objects that are about 0.2 μm apart.

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Figure 1. Parts of the microscope

Care and handling of microscopes: Microscopes are very expensive pieces of scientific
equipment and must be treated with care. Students assigned to a microscope are required
to take responsibility or sign an agreement for taking care of the microscope. Some basic
rules of microscope care include the following:
1. Always carry a microscope with two hands: one hand under the base of the
microscope and the other hand holding onto the stand or arm.
2. Keep the microscope at least 6 inches from the edge of the lab bench, and keep all
excess electrical cord up on the bench and out of the way of bunsen burners.
3. Do not touch the surfaces of the glass lenses with your fingers. They scratch easily.
4. Always remove the oil from the surface of the oil immersion lens with a Q-tip or
lens paper BEFORE putting the microscope away. This will ensure that the lens

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 is clean and not encrusted with immersion oil the next time you hope to look
through it.
5. Use only Q-tips or lens paper to clean the lenses. Never use Kimwipes, Kleenex, or
paper towels—they will scratch the lenses.
6. Do not tamper with any parts of the microscope. If it doesn’t work properly, ask
your T.A. for assistance.
7. Always start your observations using the lowest-powered objective (e.g. 4X or
10X), and always put the lowest-powered objective back into place when putting
the microscope away.
8. Never use the coarse adjustment knob to focus while the 40X and 100X objective
lenses are in place: use the fine adjustment knob.
9. Always unplug the microscope by pulling on the plug, not the cord.
10. Never look through the microscope while rapidly reducing the distance between
the objective lens and the slide. Watch from the side so that you avoid damaging
the lens by running it into the slide.

Table 1. List of equipment commonly used in Microbiology Laboratory and their

functions.
Equipment Functions
Microscopes To examine microorganisms which can’t be seen by naked eyes
Bunsen Burner To heat or boil solution in laboratory
Autoclave To sterilize the equipment medias and other solutions
Hot air Oven To sterilize metallic equipment and non-volatile solution
Refrigerator To preserve the samples, media, reagents and other specimen
Incubator used for bacterial or fungal cultures
Safety Cabinet To rescue the exposure of the operator and the lab contamination
Wire loop or used to inoculate test samples into culture media for bacterial or fungal
inoculation loop cultures, antibiograms, etc.
Petri dish to act as a supporting container to hold the culture medium
Flask To measure and prepare media
Beaker To measure and transfer the solution
Cylinders To supply adjusted amount of C2O
Staining racks To place slides to be stained
Coverslip To cover the specimen under the microscope
Balance To measure the weight of an specimen
Pipette To transfer quantified volume of specimen or solution
Microtitre plates For ELISA

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Haemagglutination Haemagglutination plate
plate
for viral culture For viral culture detection
detection
Wire mesh To place the beaker or flask on top of the Bunsen rack
Filter paper To filter the solutions
Tissue culture To grow or keep alive cells or tissue from a living organism,
bottles e.g. stem cells
Historically used for anaerobiosis; a lit candle was placed in as
Candle jar air-tight jar such that when it went out it would be because it
used up all the available oxygen
Anaerobic Jar Production of anaerobic conditions for organisms that die in the
presence of even little oxygen (anaerobiosis), e.g. tetanus bacteria
Gas-pack Releases gases to remove oxygen from a closed container, usually for
anaerobiosis
Gas cylinder To carry gas used for supply to Bunsen burner
Swab cotton To prepare swab
Water Bath Used to heat medium gently (to around 45-55 degrees Celsius) during
media preparation
Thermal cycler Used to amplify segments of DNA via the polymerase chain reaction
(PCR) process.
To draw out the air from any closed chamber before pumping
Vacuum pump
back CO2, O2 or N2, usually for anaerobiosis
Used to detect gas production in sugar fermentation media; the
Durham's tube tube is placed in an inverted fashion so that gases produced get
trapped in it and do not float away to the surface
Bijou bottle A cylindrical small glass bottle with a screw cap used as a culture
medium holder
Blood collection To collect blood by venipuncture
bottle
Castaneda's bottle Used for simultaneous solid and liquid cultures in one bottle
Universal container A cylindrical small glass bottle with a screw cap used as a culture
medium holder
McCartney's bottle For simultaneous solid and liquid cultures
Pre-sterilized
disposable Specimen collection
container
Pre-sterilized
disposable
Specimen collection
syringe / auto-
destruct syringes
Pre-sterilized
Specimen collection
disposable swabs

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/ NIH swab /
postnasal swab

Table 2. List of reagents and media commonly used in Microbiology Laboratory and their
functions.
Reagents Functions
Carbolfuchsine To stain bacteria
Crystalline violet To stain bacteria
Ethyl alcohol Decolorize the stain
Gram’s Iodine Act as mordant
Methyline blue Used in simple stain and counter stain in Acid Fast Stain
Safranine Used in gram stain
Sulpheric acid Used in spore stain
Emulsion oil Used to see specimen under high magnification
Hydrogen peroxide Used in Catalase test
Potassium hydroxide Used in KOH test
Covacks reagent Used in Indol test
Indian Inc Used to stain fungus
Geimsa Used in simple stain

Sabourad dextrose agar To culture fungus

Muller hinten agar For antibiotic sensitivity test
Brelliant green agar Used in identification of Enterobacteriacae family
Modified Edward media Selective media for Streptococcus
Mc-Conkey agar Selective and differential media for isolation of gram negative
bacteria
Mannitol salt agar Selective media for Staphylococcus
Nutrient agar General and commonly used solid media for primary culture
Nutrient broth General and commonly used liquid media for primary culture
Blood agar Enriched media for culturing of many organisms
Choccolate agar For culturing of family Pasteurellacae particularly Haemophilus
SIM media To identify Citrate utilizers
Urease agar To identify urease utilizers
CAMP media Used for identification of Staphylococcus and Listeria species
TSI agar Used in identification of Enterobacteriacae family
MRS agar Used in identification of Lactic Acid Bacteria

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2.4. Collection and submission of clinical specimens for diagnosis of microbial
infections

The collection of infectious agent from the host or from other origin should be under the
safety guidelines. The protective measures are quite important. The principles of
laboratory safety are needed to be considered at field during collection and submission of
laboratory specimen. Based on the specimen type the methods and precaution needed
varies as described below.
General Specimen
1. Specimen should be refrigerated as soon as possible. Otherwise, contaminants might
overgrow and the agent might die.
2. Collect samples before antibiotic therapy and sites where the agent might be
concentrated.
3. Select animals in the acute stage.
4. Submit a full history and tentative diagnosis
5. Take appropriate precaution not to contaminate yourself with possible zoonotic
diseases.
6. Collect specimen aseptically and pack
Specimen for bacterial Isolation
a. Abortion cases: whole faetus, if not faetal abomasal content, lung, liver and lesion,
placenta, serum from the cow/sheep, uterine discharge and placenta cotyledons. For
suspected leptospiral abortion: 20 ml of mid-stream urine.
b. Bovine mastitis: Milk samples immediately after the case has been detected and
before antibiotic treatment.
c. Abscesses: If possible 3ml of abscess and scrapping from the wall of the abscess
d. Specimen for anaerobic culture: Should be collected immediately after death or
maintain anaerobic conditions. Bone marrow is suitable for Black leg or Malignant
oedema; for suspected entertoxaemia, collect about 20 ml of intestinal content.
e. Urine samples:
f. Skin: Skin scrapping
g. Blood: For suspected bacteraemia cases.

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Specimen for viral diseases
For routine diagnosis of viral diseases, serum samples are appropriate. If viral isolation is
possible, preserve the sample in 50% buffered glycerol. For serum collection, about 10
ml of blood is collected and left to coagulate for one hour. After centrifugation, the serum
is collected into another test tube. The serum should be transported frozen and avoid
freezing and thawing.
Specimen for mycotic infection
Cutaneous infection: Disinfect the area with 70% alcohol. Digest in 10% NaOH or KOH
in order to digest the keratinized tissue.

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CHAPTER 3: METHODS OF CLEANING AND STERILIZATION

Objectives:
 Students will practice on cleaning and wrapping of glassware and apparatus
 Introduce them on techniques of sterilization
 Storage of sterile glassware

3.1. Cleaning methods

Cleaning is used for equipment: first by tap water, soap, savlone and then other
detergents (for equipments) and also for wound (antiseptics like phenols)
Clean up
 Working surfaces must be cleared after use. If cultures have been used the
benches must be swabbed with disinfectant.
 Discarded cultures, empty media tubes and all contaminated material must be
placed in the appropriate labeled receptacles.
 Discard containers must be carefully and securely packed and never overloaded.
Plastic Petri dishes
 Must never be stacked above the lip of the discard container.
 Cultures and contaminated paper towels, gloves etc. must be autoclaved at 121ºC
for 15 minutes before disposal.
 Slides, pipettes and Pasteur pipettes must be discarded in the appropriate
containers of Hypochlorite (sodium chlorate 1). They must be soaked for at least
24 hours before disposal.
 Never discard sharp or broken items in a way which would endanger anyone (see
“Spillage management” page 3).
 After sterilization, all materials can be disposed of with normal waste. Care must
be taken that glass is adequately packaged to prevent injury.
 Before leaving the laboratory, laboratory coats must be removed and hands
washed with hot water and soap.

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3.2. Antiseptics and Disinfectants

The emergence of anti-microbial soaps, lotions and other products has seen a huge
increase over the last few years. The number of choices is excessive and the consumer is
often unaware that many of the anti-microbial agents are no more effective than basic
soap and water. The effectiveness of an anti-microbial is dependent upon many factors
such as the concentration of the antimicrobial agent, the amount of contamination, the
sensitivity of the contaminating organisms, temperature, and length of exposure.

To understand antimicrobial agents one must first understand the difference between
antiseptics and disinfectants. Antiseptics are normally safe for application to living
tissue such as the human skin and throat. Antiseptics normally are more bacteriostatic in
that they prevent bacterial multiplication, but do not kill the organism. Disinfectants,
however, are considered germicidal or bacteriocidal. Germicides are chemicals that are
usually lethal to bacteria and are meant to be used on non-living areas such as floors,
work benches, etc.

There are specific characteristics that identify an ideal antimicrobial. A food antiseptic
often is chosen for its ability to work on specific organisms effectively. Several
antiseptics as well as disinfectants are described on Table 3. An ideal disinfectant should
be highly effective even when diluted, nontoxic, colorless, odorless, stable in any
concentration, harmless to all surfaces, biodegradable, inexpensive and, if it is a phenolic,
a good phenol coefficient.

Disinfection is the destruction, inhibition or removal of microbes that may cause disease
or other problems e.g. spoilage. It is usually achieved by the use of chemicals.

Choice, preparation and use of antiseptics and disinfectants

Specific disinfectants at specified working strengths are used for specific purposes. The
choice is now much more straightforward as the range available from suppliers has

20
decreased. The most commonly used disinfectants are 0.25% chlorine (as Sodium
hypochlorate I) and 70% ethanol, aldehides or methylated spirit. Ethylene oxide is
gaseous disinfectant. Other antiseptics like phenols are also effective against growing
bacteria.

Table 3. Classes of antimicrobials and their use category.

3.3. Sterilization Methods

Sterilization means the complete destruction of all the micro-organisms including

spores, from an object or environment. It is usually achieved by heat or filtration but
chemicals or radiation can be used. There are two types of sterilization commonly used in
our laboratory. These are Dry heat sterilization and moist heat sterilization. But generally

21
sterilization methods can be physical, chemical or Mechanical method.

Physical methods include Heat (Dry and moist heat), Filtration in which very small
size are utilized to sterilize substances such as sera, toxins, vitamin, antibiotic
preparations, and other labile substances such as sugars. Irradiation in which rays such
as ultraviolet, gamma rays and x-rays damage microorganisms affecting the cell
membranes, essential enzymes or the nucleic acids. In our laboratory we were practiced
on some of the chemical methods and mainly on physical methods of sterilization; as I
described them below.
Moist Sterilization:
Use of the autoclave/pressure cooker
Principle
Steam under pressure is used to produce a temperature of 121ºC which if held for 15
minutes will kill all micro-organisms including bacterial endospores. Materials that do
not resist dry heat such as rubber equipment, plastic materials, tips of micropipate, most
media, round bottom flask etc. are sterilized using this method. High temperature like in
Boiling (at 100 0c) is also the simple method practiced usually against vegetative form of
bacteria. Water bath is also used to keep the autoclaved media at around 45-50 0c before
dispensing it to the Petri dish.

Dry Heat Method

Dry heat method is important in that it does not affect the glass surface; do not corrode
metals, well to sterilize powders, oil and other viscous substances in that it can be
stralized without getting moist or evaporated. Dry heat includes red hot (to stralize
inoculating loop), Flaming (used to fix the smears like in M. tuberculosis and to reduce
air contamination at mouth of broth tube during sub culture). Hot air oven uses a very
high temperature (160-180oC) for one hour. Used for sterilization of flasks, test tubes and
pipettes placed in pipette canisters. Petri dish, universal tube, conical flasks, graduated
centrifuge tube and swab applicator can be foiled by alumunium foil and sterilized under
hot air oven. However it takes longer time to sterilize, less penetration capacity and thus
less efficient.

22
Sterilization of equipment and materials
Wire loop: Heat to redness in Bunsen burner flame. Empty glassware and glass (not
plastic!) pipettes and Petri dishes: Either, hot air oven, wrapped in either greaseproof
paper or aluminium and held at 160ºC for 2 hours, allowing additional time for items to
come to temperature (and cool down!). Or, autoclave/pressure cooker. Culture media
and solutions: Autoclave/pressure cooker. Glass spreaders and metal forceps: Flaming
in alcohol (70% industrial methylated spirit).

23
CHAPTR 4: BACTERIOLOGICAL STAINING METHODS

4.1 Smear Preparation for bacteriological Staining

Principle
A “smear” of bacteria or yeast is made on a microscope slide, fixed, stained, dried and,
without using a coverslip, examined with the aid of a microscope. A culture on agar
medium is much preferable to a liquid culture for making a smear. A smear that is thin
and even enables the shape and arrangement of cells to be clearly seen and ensures that
the staining procedure is applied uniformly. One way of achieving this detail involves
smear preparation and simple staining.

Making Smear
Procedure
1. Clean a plain microscope slide thoroughly using lens tissue.
2. Label a microscope slide with a marker pen to record the culture being used, date
and initials; this is also a useful reminder of which side of the slide is being used.
3. Flame a wire loop to ensure that no culture accidentally remains from a previous
operation.
4. Transfer one or two loopfuls of tap water on to the centre of the slide.
5. Flame loop and allow to cool.
6. Using aseptic technique, transfer a very small part of a single colony from a plate or
slope of agar medium into the tap water. (If the amount of culture on the loop is
easily visible you have taken too much!)
7. Make a suspension of the culture in the tap water on the slide and thoroughly but
gently spread it evenly over an oval area of up to 2 cm length.
8. Flame the loop.(If it is necessary to use a liquid culture or sample, the use of tap
water to prepare the smear will probably be unnecessary and may result in a smear
with too few cells).
9. Dry the suspension by warming gently over a Bunsen burner flame and then “fix” it
by quickly passing it through the flame a few times. This is called a heat-fixed
smear; it should be visible to the naked eye as a whitish area. Fixing is necessary to
ensure that cells adhere to the slide and to minimize any post mortem changes

24
 before staining.

Principles of Bacterial Staining

Since living bacteria are generally colorless and almost invisible because of their lack of
contrast with the water in which they may reside, staining is necessary in order to make
them readily visible for observation of intracellular structures as well as overall
morphology. Simply speaking, a stain is a substance that adheres to a cell, giving the cell
color. Different stains have different affinities for different organisms, or different parts
of organisms. They may be used to differentiate different types of organisms or to view
specific parts of organisms. Stains are actually salts composed of a positive and a
negative ion – one of the ions is colored and is known as the Chromophore. The color of
basic dyes is in the positive ion. The color of acidic dyes is in the negative ion. At a pH of
7.0, bacteria are slightly negatively charged. Hence, most bacteria are stained primarily
with basic stains. Acidic dyes are not attracted to the negatively charged bacterial surface,
so that acidic dyes stain the background – this technique is called negative staining.
Generally there are three types of stain used in most microbiology laboratory.

4.2. Types of Bacterial Staining Technique

A. Simple Stains: A simple stain is an aqueous or alcohol solution of a single basic

dye. The bacterium takes up stain and becomes colored, while the background
remains unstained. Simple stains are typically used on bacterial smears which
have been heat-fixed and thus contain non-living microbes. Simple stains are used
in microbiology to determine shape, cell arrangement and size. Occasionally, a
mordant is added to the solution to intensify the stain – like iodine. Examples of
simple stains include methylene blue, carbolfuchsin, crystal violet, and safranin.
Procedure
1. Put the slide with the fixed smear uppermost on a staining rack over a sink or
staining tray.
2. Thoroughly cover the smear with stain and leave for, usually, 30 seconds.

25
 3. Hold the slide with forceps (optional but avoids stained fingers), at a 45° angle
over the sink.
4. Rinse off the stain with tap water.
5. Blot dry the smear with filter/fibre free blotting paper using firm pressure but not
sideways movements that might remove the smear.
6. Examine under oil immersion.
7. When finished, dispose of slides into discard jar.
Suitable stains include basic dyes (i.e. salts with the colour-bearing ion, the chromophore,
being the Cation) such as methylene blue, crystal violet and safranin. Staining solutions
(relevant to procedures described below)
B. Negative, indirect, or background staining
Principle
Negative, indirect, or background staining is achieved by mixing bacteria with an acidic
stain such as nigrosin, India ink, or eosin, and then spreading out the mixture on a slide
to form a film. The above stains will not penetrate and stain the bacterial cells due to
repulsion between the negative charge of the stains and the negatively charged bacterial
wall. Instead, these stains either produce a deposit around the bacteria or produce a dark
background so that the bacteria appear as unstained cells with a clear area around
Objective
• To stain background for unstained micro-organisms
• To determine overall bacterial morphology without the use of harsh staining or
heat-fixing techniques that change the shape of cells
• Negative staining is also good for viewing capsules
• This might be the case when the bacterium does not stain well (e.g., some of the
spirochetes is very delicate cell that is easily distorted by heat-fixing; thus,
Negative staining is the procedure of choice in the clinical laboratory)
Materials
• 24- to 48-hour tryptic soy broth cultures of Bacillus subtilis Micrococcusluteus
and Spirillum volutans
• Dorner’s nigrosin solution, India ink, or eosin blue
• clean microscope slides

26
 • inoculating loop
• immersion oil,microscope,lens paper and lens cleaner
• wax pencil, Bunsen burner
Procedure
a) With a wax pencil, label the left-hand corner of three glass slides with the names of
the respective bacteria.
b) Use an inoculating loop to apply a small amount of bacteria to one end of a clean
microscope slide
c) Add 1 to 2 loops of nigrosin, India ink, or eosin solution to the bacteria and mix
thoroughly.
d) Spread the mixture over the slide using a second slide.
e) The second slide should be held at a 45°angle so that the bacteria-nigrosin solution
f) Allow the smear to air dry .Do not heat-fix!
g) With the low-power objective, find an area of the smear that is of the optimal
thickness for observation.
h) Use the oil immersion lens to observe
Result: The background is stained
C. Differential Stains: Differential stains differentiate between different types of
bacteria. It is a type of staining that allows you to distinguish between types of
bacteria or between specific structures in a bacterium. A differential stain
typically uses two or more reagents – a primary stain and a counter stain.
Examples of differential stains include gram stain, acid-fast stain, capsule stain,
endospore stain, and flagella stain.

i. Gram Stain
The Gram stain is the most useful and widely employed differential stain in bacteriology.
It is a differential stain which distinguishes bacteria based on cell wall properties.
Bacterial cell walls are composed primarily of peptidoglycan and bacteria can be
classified into two main groups dependent on the amount of peptidoglycan present in
their cell wall. Gram-positive organisms have a thick layer of peptidoglycan, whereas
Gram-negative organisms have a thin layer of peptidoglycan, plus an additional outer

27
membrane that is absent in Gram-positive organisms. In the gram staining procedure, the
primary stain is crystal violet, and all cells take up the purple crystal violet stain.
Following the primary stain, Gram’s Iodine is applied to the bacterial smears. The iodine
acts as a mordant, enhancing the ability of the stain to enter and bind to the bacteria.
Specifically, the iodine binds with crystal violet and locks it into peptidoglycan of
bacteria. It also intensifies the purple color. The decolorizing agent used in the gram
staining procedure is 95% ethanol, which is a lipid solvent that melts the Gram negative
outer membrane and leads to decolorization of Gram-negative cells. It also dehydrates
proteins, helping the primary stain to remain in Gram-positive cell walls. The counter
stain then used is Safranin, which stains the decolorized Gram-negative cells pink. Thus,
at the end of the staining procedure, Gram-positive cells are purple and Gram-negative
cells are pink. Note: It is preferable to use fresh cultures for the Gram stain. Old cultures
may stain “Gram-variable” (a mix of purple and pink) because they decolorize easily.

Principle
Bacteria also differ from one another chemically and physically and may react differently
to a given staining procedure. This is the principle of differential staining. Differential
staining can distinguish between types of bacteria based on the thickness of the cell wall.
It divides bacteria into two groups: Gram negative and gram positive.

Objective
• To make certain that a given bacterial" strain” is truly gram positive or gram
negative
Materials
Wire loop (inoculating loop), Culture media (mother Culture), Microscope, Staining rack,
water, Bunsen burner,
Procedure
a) Fix the smear
b) Stain with the primary stain – crystal violet - for 30 seconds
c) Wash crystal violet off with water

28
 d) Add iodine for 10 seconds (mordant) (the mordant can also be added without
washing the crystal violet)
e) Wash iodine off with water
f) Decolorize with ethyl alcohol for 10 – 20 seconds (decolorizing agent)
g) Wash the alcohol off with water
h) Counter stain with safranin for 30 seconds
i) Wash the safranin off with water
j) Blot the smear dry
k) Observe under oil immersion
Result:
Gram-positive bacteria are deep purple in color and gram-negative bacteria are pinkish to
red in color

Interpretation
The Gram stain does not always yield clear results. Species will differ from one another
in regard to the ease with which the crystal violet-iodine complex is removed ethanol.
Gram-positive cultures may often turn gram negative if they get too old. Thus, it is
always best to Gram stain young, vigorous cultures rather than older ones. Furthermore,
some bacterial species are gram variable. That is, some cells in the same culture will be
gram positive and some, gram negative. Indistinct Gram-stain results can be confirmed
by simple test using KOH test which is an alternative test to gram stain.

ii. Acid-Fast Staining (Ziehl-Neelsen and Kinyoun)

The acid-fast stain is a differential stain which distinguishes bacteria based on cell wall
properties. Acid-fast bacteria have a waxy substance called mycolic acid in their cell
walls, which comprises up to 60% of the cell wall components. They also contain
polymers called arabinogalactan and lipoarabinomannan. The primary stain in the acid-
fast staining procedure is carbol fuchsin, which contains phenol to solubilize the cell
wall and allow the stain to enter the cell. The procedure requires the application of heat
in order for the stain to penetrate the waxy cell wall. All cells will take up the reddish-
pink primary stain. Once the cells have been allowed to cool after the primary stain, the

29
cells are decolorized. The decolorizer used in the acid-fast staining procedure is acid
alcohol, which decolorizes all cells except acid-fast cells. The acid alcohol is unable to
penetrate the waxy cell wall of acid-fast microorganisms. The counterstain then used is
methylene blue, which stains the decolorized non acid-fast cells blue. Thus, at the end of
the staining procedure, acid-fast cells are reddish-pink and all other cells are blue.

Principle
Cell walls of some bacterial genera (Mycobacterium and Nocardia) and the parasite
Cryptosporidium are impermeable to many dyes. It does not take dyes of gram stain.
However, these microorganisms can be stained by heating them with carbolfuchsin and
once stained, they resist decolorized by acid-alcohol, and hence are termed acid-fast.
Objective
 To differentiate Acid fast microorganism from other non-acid fast groups by
making use of the difference in retention of carbolfuchsin.
Materials
• Bunsen burner, inoculating loop hot plate, microscope, Staining rack, immersion
oil. 1-ml pipettes with pipettor, Mother Culture (Tryptic soy broth culture of
Escherichia coli, or Mycobacterium spp from LJ agar) or swab from clinical case.
Stock Reagents
Concentrated carbol fuchsin
 Basic fuchsin 1.0 g
 Ethanol 95% (vol/vol) 10.0 ml
 Phenol 5.0 g
 Distilled water 100.0 ml
The basic fuchsin dissolved in the ethanol, the phenol is then dissolved in the distilled
water and the two solutions mixed together. The solution is allowed to stand for a few
days and then filtered into a clean container.
Acid-alcohol (decolorizer)
 Concentrated hydrochloric acid 8.0 ml
 Ethanol 95% (vol/vol) 97.0 ml
The acid should be added to the alcohol!

30
Methylene blue (counter stain)
 Methylene blue 8.0 g
 Ethanol 95% (vol/vol) 300.0 ml
 Distilled water 1300.0 ml
 Potassium hydroxide 0.13 g
Procedure
a) Prepare film, dry in air and fix by passing through flame.
b) Place the slide over the two parallel rectangular rods (staining rack)
c) Flood with carbol fuchsin and heat at intervals until steam rises without
boiling the fluid. Gently. Allow for at least 5 minutes: (stain must not be
allowed to dry on the slide. Add more stain if required. To keep surface of
slide covered.
d) Wash in water.
e) Decolorize by flooding slide with acid-alcohol for 1-2 minutes and wash
with water.
f) Counter stain with methylene blue or malachite green for 30-60 seconds.
g) Wash in water, blot dry
h) Examine under oil immersion and write your observation.
Result
• Acid-fast microorganisms will retain the primary stain and appear red
• Microorganisms that are not acid-fast, termed non-acid-fast, will appear blue or
brown due to the counterstaining with methyleneblue after they have been
decolorized by the acid-Alcohol.
Interpretation
D. Special Stains: Special stains are required to demonstrate some special structures of
bacteria such as the spore, capsule, flagella, etc. Thus it includes Negative stain for
capsules – virulent organisms, Endospore stain – Clostridium and Bacillus, Flagella
stain – motile organisms and Hanging drop for motility.

i. Endospore Staining (Schaeffer-Fulton or Wirtz-Conklin)

The endospore stain is a differential stain which stains bacterial endospores or spores.

31
Some bacteria, including those belonging to the genera Clostridium and Bacillus, have
the capacity to produce metabolically inactive cells called spores. Spores are highly
resistant to hostile chemical and physical conditions and are produced by a process called
sporulation if environmental conditions become unfavorable for normal vegetative
growth. The spore structures have a tough outer covering composed primarily of keratin
which makes them resistant to heat, radiation, disinfectants, and desiccation. The bacteria
will remain in this suspended state until conditions become favorable again and they can
germinate and return to their vegetative state. The term endospore refers to the spore
structure contained within a mother cell. The term spore refers to the spore structure that
exists free of the mother cell. Endospores may be located in the middle of the cells
(central), at the end (terminal), or between the end and the middle of the cells
(subterminal). The endospores themselves may be round or oval. There are two methods
of staining endospores of which one method is listed in the procedure bellow.

The primary stain in the endospore staining

procedure is malachite green, which stains
both vegetative cells and endospores. As
with the acid-fast stain, heat is required to
penetrate the endospore coat.

Once the cells have cooled, the cells are decolorized with water, which selectively
removes the malachite green from all vegetative cells but not from endospores. The
counterstain then applied is safranin, which stains the decolorized vegetative cells pink.
Thus, at the end of the staining procedure, the endospores are dark green, and vegetative
cells are pink. Note: Sometimes the endospores don’t take up the malachite green very
well. In those cases, the endospores will appear as clear ovals or circles within a pink
vegetative cell.

Principle
Bacteria in genera such as Bacillus and Clostridium produce quite a resistant structure
called an endospore which can’t be stained with ordinary differential stain such as Gram

32
stain. But, once stained, they strongly resist decolonization. While, the rest of the cell will
be easily decolorized and counterstained.
Objective
 To determine whether the bacteria produce endospore or not.

Procedure
1. Prepare film, dry in air and fix by passing through flame
2. Flood with carbol fuchsin solution.
3. Heat at intervals until steam rises without boiling the fluid. Allow to act for 5
minutes.
4. Wash with water
5. Decolorize with 0.5% H2SO4 for one minute and wash in water.
6. Counterstain with 1% methylene blue or 0.5% malachite green.
7. Wash in water, blot and dry
8. Examine under oil immersion and write your observation
Result
Spore: pink, Vegetative Bacilli = blue/green

ii. Capsule Staining (Anthony’s procedure employs two reagents).

Principles
Many bacteria have a glycocalyx or slimy layer surrounding them, which are usually
referred to as a capsule which cannot be always determined by simple staining
procedures, such as using negative staining and India ink. An unstained area around a
bacterial cell will occur. The principle behind is that, the primary stain (crystal violet),
which gives the bacteria cell and its capsular material a dark purple color will be
decolorized by Copper sulfate which removes excess primary stain as well as color from
the capsule. At the same times, the copper sulfate acts as a counter stain by being
absorbed into the capsule and turning it a light blue or pink.
Procedure

33
1. With a wax pencil, label the left-hand corner of a clean glass slide with the name of the
bacterium that will be stained.
2. As shown in figure 14.3, aseptically transfer a loopful of culture with an inoculating
loop to the slide. Allow the slide to air dry. Do not heat-fix! Heat-fixing can cause the
bacterial cells to shrink and give a false appearance to the capsule.
3. Place the slide on a staining rack. Flood the slide with crystal violet and let stand for 4
to 7 minutes
4. Rinse the slide thoroughly with 20% copper sulfate
5. Blot dry with bibulous paper
6. Examine under oil immersion (a coverslip is not necessary) and draw the respective
bacteria in the report. Capsules appear as faint-halos around dark cells.
Result: light blue or pink capsule

iii. Staining of Flagella

Because of their extreme thinning, flagella are best demonstrated with electron
microscope; metal-shadowed film or films made with phosphotungistic acid (PTA) for
“negative staining” are employed. Flagella can also be demonstrated by the light
microscope, using special staining methods which require most careful attention to detail
technique. To make possible their resolution, the flagella must be thickened at least ten-
fold by superficial deposition of stain. In spite of this, their characteristic arrangement
and wave form are generally distinguishable.
Staining by Leifson’s method
Principle of the stain: The stain, basic fuchsin with tannic acid, is deposited on the
bacteria and flagella from an evaporating alcoholic solution. The degree of staining is
controlled by an exact determination of the duration of exposure. Good results depend to
a large extinct on preliminary thorough cleaning and flaming of the glass slide.

Procedure
1. Clean the slide with absolute alcohol, rubbing with a fine cotton cloth.
2. Then immerse them in a concentrated sulpheric acid saturated with potassium
dichromate for several days at room temperature or for an hour at 90 0C.
3. Using forceps transfer the slide to cleaned Coplin jars in which they will be kept for
rinsing, drying and storing it as overcrowd. Rinse thoroughly with tap water and
finally with distilled water. Allow to drain and dry in air with the jar inverted on
clean bottling-paper.

34
4. Store with the jar closed to prevent contamination by air-born dusts.
5. Just before use, flames the slide for a few seconds, passing it with each face
downwards about six times through a blue Bunsen flame.
6. Place on a clean warmed metal rack and allow it to cool. Mark or number the slide
with a diamond while holding with forceps.

35
CHAPTER 5: BACTERIAL CULTURE METHODS

Ingredients of culture media

1. Distilled water
2. Nutrient that supports the growth of the bacteria
3. Agar: a solidifying agent
4. Buffer: substances added to media used to maintain the pH of the media at a suitable
pH range. Phosphates are usually used as buffer.
5. Growth factors: materials that enhance the growth of fastidious organisms. e.g.
Vitamins, blood, serum
5.1. Types of culture media

1. Based on their consistency : solid medium, liquid medium and semi-solid medium
2. Based on Oxygen requirement: Aerobic media, Anaerobic media
3. Based on the constituents/ ingredients: simple medium, complex medium synthetic or
defined medium and Special media
4. Based on the use, media can also be classified in to special and ordinary media.
Ordinary media supports the growth of a wide variety of organisms. eg. Nutrient agar,
nutrient broth

Special media
 Enriched media: it is either solid or liquid (Broth) form. Substances like blood,
serum, egg are added to the basal medium. It is used to grow fastidious bacteria
that are exacting in their nutritional needs. Liquid media used to isolate pathogens
from a mixed culture. Such media is incorporated with inhibitory substances to
suppress the unwanted organism. Eg: Blood agar, Chocolate agar, Selenite F
Broth – for the isolation of Salmonella, Shigella, Alkaline Peptone Water – for
Vibrio cholerae
 Selective media: The inhibitory substance is added to a solid media. Eg: Mac
Conkey’s medium for gram negative bacteria, TCBS (V. cholerae), LJ medium

36
 (M. tuberculosis), Wilson and Blair medium (S. typhi), Potassium tellurite
medium (Diphtheria bacilli)
 Indicator media: These media contain an indicator which changes its colour
when a bacterium grows in them.Eg: Blood agar, Mac Conkey’s medium,
Christensen’s urease medium
 Differential media: A media which has substances incorporated in it enabling it
to distinguish between bacteria. Eg: Mac Conkey’s medium, Peptone, Lactose,
Agar, Neutral red, Taurocholate, I also distinguish between lactose fermenters &
non lactose fermenters.
 Sugar media: Media containing any fermentable substance. Media consists of 1%
of the sugar in peptone water. It may contain a small tube (Durham’s tube) for the
detection of gas by the bacteria Eg: glucose, arabinose, lactose, starch etc.
 Transport media: Media used for transporting the samples. Delicate organisms
may not survive the time taken for transporting the specimen without a transport
media. Eg: Stuart’s medium (non-nutrient soft agar gel containing a reducing
agent) and Buffered glycerol saline(enteric bacilli)
 Anaerobic media: These media are used to grow anaerobic organisms. Eg:
Robertson’s cooked meat medium, Thioglycolate medium.
 Complex media: Media other than basal media. They have added ingredients.
Provide special nutrients
 Synthetic or defined media: Media prepared from pure chemical substances and
its exact composition is known Eg: peptone water – 1% peptone + 0.5% NaCl in
water

5.2. Preparation of culture media

Important points
i) Before preparation, the media need to be kept at storage room or in the shelf at room
temperature according to manufacturer’s instructions and the expiring date need
to be cheeked before it is used.
ii) Take the required amount of the powder and rehydrate tablets or powder in distilled
water according to manufacturer’s instructions.

37
iii) Before sterilization, ensure ingredients are completely dissolved, using heat if
necessary. If Bunsen heat is used, shake the beakers regularly. Steam is the most
commonly use. The medium becomes clear if completely dissolved.
iv) The media usually autoclaved at 121 for 15 min and then the agar preparation is kept
in water bath until dispensed in Petri dishes or test tubes.
v) Avoid wastage by preparing only sufficient for either immediate use (allowing extra
for mistakes) or use in the near future. Normally allow 15-20 ml medium/ Petri dish.
vi) Dispense in volumes appropriate for sterilization in the autoclave/pressure cooker.
Agar slopes are prepared in test tubes or Universal/McCartney bottles by allowing
sterile molten cooled medium to solidify in a sloped position. Bottles of complete,
sterile media are available from suppliers but are expensive.
vii) Store stocks of prepared media at incubator temperature or at room temperature away
from direct sunlight and for sterility cheek, incubate it for 24 hours.
viii) For longer storage, refrigerate media using screw cap bottles to avoid desiccation.

5.3. Inoculation and Asceptic Procedures

Essential points
There are several essential precautions that must be taken during inoculation procedures
to control the opportunities for the contamination of cultures, people or the environment.
 Operations must not be started until all requirements are within immediate reach
and must be completed as quickly as possible.
 Vessels must be open for the minimum amount of time possible and while they
are open all work must be done close to the Bunsen burner flame.
 On being opened, hold it horizontally and warm the neck of a test tube or bottle
immediately by flaming so that any air movement is outwards
 During manipulations involving a Petri dish, avoid contamination
 The parts of sterile pipettes that will be put into cultures or sterile vessels must not
be touched or allowed to come in contact with other non-sterile surfaces, e.g.
clothing, the surface of the working area, outside of test tubes/bottles.
It is essential that agar plates should have a well-dried surface by either incubating the
plates for several hours, e.g. overnight, beforehand or put them in a hot air oven (ca 55-

38
60ºC) for 30-60 minutes with the two halves separated and the inner surfaces directed
downwards.

Flaming procedure

Wire loops are sterilized using red heat in a Bunsen flame before and after use to destroy
contaminants and spores. The flaming procedure is designed to heat the end of the loop
gradually because after use it will contain culture, which may “splutter” on rapid heating
with the possibility of releasing small particles of culture and aerosol formation. Follow
the procedures shown on Figure 3 for routine use.
1. Position the handle end of the wire in the light blue cone of the flame. This is the cool
area of the flame.
2. Draw the rest of the wire upwards slowly up into the hottest region of the flame,
(immediately above the light blue cone).
3. Hold there until it is red hot.
4. Ensure the full length of the wire receives adequate heating.
5. Allow to cool then use immediately.
6. Do not put the loop down or wave it around.
7. Re-sterilize the loop immediately after use.

Fig. 3. Inoculating wire loop flaming procedure

39
Flaming the neck of bottles and test tubes
1. Loosen the lid of the bottle so that it can be removed easily.
2. Lift the bottle/test tube with the left hand.
3. Remove the lid of the bottle/cotton wool plug with the little finger of the right
hand. (Turn the bottle, not the lid.)
4. Do not put down the lid/cotton wool plug.
5. Flame the neck of the bottle/test tube by passing the neck forwards and back
through a hot Bunsen flame.
6. Replace the lid on the bottle/cotton wool plug using the little finger & turn the
bottle, not the lid.
Inoculation of media in screw cap bottles and test tubes is usually done by using a wire
loop.

Fig. 4. Flaming the neck of bottle

CULTURE TRANSFER TECHNIQUES

The purpose of this lab exercise is to learn how to do primary culture or subculture
microorganisms in various types of microbiological media. Simply stated, primary

40
culturing is if the sample obtained from the host is to be cultured for the first time on
culture media.For primary culturing of unknown organism, ordinary enriched media like
nutrient or blood agar is used. The subculturing is transferring microorganisms from one
media type to another. Various media types used in microbiology labs include agar slants,
agar deeps, agar plates, and broths. An agar slant is a solid media in a test tube with a
slanted surface on which to culture the microorganism. These are typically inoculated by
streaking the surface of the slant with a sterile loop. An agar deep is a solid media in a
test tube which does not have a slanted surface. These are typically inoculated by
stabbing the media with a sterile needle. An agar plate is a solid media which is
contained in a Petri plate, providing an optimal surface on which to culture
microorganisms. Like the agar slants, these are inoculated by streaking the surface with a
sterile loop. Broth tubes are a liquid medium which can be inoculated by a sterile loop,
needle, or pipette. Remember that when inoculating any cultures, it is essential to practice
sterile technique at all times! It is important to know what sterile media looks like. Take
a minute to observe your uninoculated cultures. You will be responsible for making sure
there are no contaminates present in the media we provide for you.
Media Needed:
1 Nutrient agar slant
1 Nutrient agar deep
1 Nutrient broth tube
Cultures Needed:
Serratia marcescens broth cultures and slants
Procedure:
1. Label each tube appropriately.
2. Using a sterile needle, obtain a small amount of culture from the broth tube
containing Serratia marcescens. Use the needle to inoculate a nutrient agar deep tube
by stabbing the needle into the agar deep.
3. Using a sterile loop, obtain a loop full of culture from the broth tube containing
Serratia marcescens. Use the loop to inoculate a nutrient agar slant tube by streaking
the agar slant with a zig-zag motion.

41
4. Using a sterile loop, obtain a loop full of culture from the slant of Serratia
marcescens. Use the loop to inoculate a nutrient broth tube by gently swishing the
loop around in the liquid broth.
5. Make sure all caps are loose, but secure.
6. Incubate at 30 °C for 48 hours.
Result
The presence of colony grown on the cultured media is considered as culture positive.
The absence of colony growth on the cultured media is considered as culture negative.

5.4. Types of Inoculation Methods

A). Streak culture: It is used for the isolation of bacteria in pure culture from clinical
specimens. Platinum wire or Nichrome wire loop is used for preparing a streak plate.
When working with microorganisms, it is desirable to start with single, isolated colonies
to ensure you are working with a pure culture. Cultures that are visible on the surface of
solid media are called colonies. A colony forms on a plate when a single microbe is
inoculated onto the surface of the plate and reproduces until there are enough cells to
form a visible colony. Since a colony theoretically forms from a single cell, a colony
should then represent a pure culture. One way to obtain single, isolated colonies is using
the quadrant streak method. The quadrant streak plate method allows sequential
dilution of the original microbial material over the entire surface of a fresh plate. As the
original sample is diluted by streaking it over successive quadrants, the number of
organisms decreases. Usually by the third or fourth quadrant only a few organisms are
transferred, and these produce single, discrete colonies.
Objective: To obtain single isolated pure colonies.
Media:
 Nutrient agar plate
Cultures:
 Mixed broth culture or primary culture positive plate.
Procedure:
1. Divide the agar plate into 4 quadrants.

42
2. Take loopful of the specimen or inoculum from the mixed broth culture or primary
culture
3. Transfer it onto the surface of the plate in Quadrant 1 with a sterile loop and streak
the loop very gently using a back and forth motion.
4. Sterilize the loop, allow to cool and go back to the edge of Quadrant 1 and extend the
streaks into Quadrant 2, going back into Quadrant 1 twice.
5. Sterilize the loop, allow to cool and go back to the edge of Quadrant 2 and extend the
streaks into Quadrant 3, going back into Quadrant 2 twice.
6. Sterilize the loop, allow to cool and go back to the edge of Quadrant 3 and extend the
streaks into Quadrant 4, going back into Quadrant 3 twice. Be careful NOT to go
back into Quadrant 1!
7. Tape plate closed on both sides. Make sure the plate is labeled with your name, date,
and the organism(s), and incubate upside down (to prevent condensation from getting
on to agar) at 30 °C.
8. Examine the isolate on the streak plate.
Results:
Single, isolated colonies will be seen particularly at 3rd and 4th quadrant.

43
Figure 10. Streak Plate (Quadrant Inoculation Method)
B). Pour Plate: A pour plate is one in which a small amount of inoculum from broth
culture is added by pipette to a molten, cooled agar medium in a test tube or bottle, mix
thoroughly and pour into a Petri dish to solidify. Pour plates allow micro-organisms to
grow both on the surface and within the medium. Most of the colonies grow within the
medium and are small in size; the few that grow on the surface are of the same size and
appearance as those on a streak plate. If the dilution and volume of the inoculum, usually
1 cm³, are known, the viable count of the sample i.e. the number of bacteria or clumps of
bacteria, per cm³ can be determined.
Procedure

44
 a) First 1 ml of the inoculum per plate is added to the molten agar cooled to
45oC and mix well.
b) Flame the neck of the bottle.
c) Lift the lid of the Petri dish slightly with the right hand and pour the mixture
into the Petri dish (15 agar medium for one plate).
d) Flame the neck of the bottle and replace the lid.
e) Gently rotate the dish to ensure that the medium covers the plate evenly.
f) Allow the plate to solidify.
g) Seal and incubate the plate in an inverted position.
h) Incubate at 37 oC, colonies will be distributed throughout the depth of the
medium.

C). Using a spreader: Spread plates, also known as lawn plates, should result in a
culture spread evenly over the surface of the growth medium. This means that they can be
used to test the sensitivity of bacteria to many antimicrobial substances, for example
mouthwashes, garlic, disinfectants and antibiotics. Sterile spreaders are used to distribute
inoculum over the surface of agar plates. Wrapped glass spreaders may be sterilized in a
hot air oven. They can also be sterilized by flaming with alcohol as shown on Figure 9 as
follows.
1. Dip the lower end of the spreader into a small volume of 70% alcohol contained
in a vessel with a lid (either a screw cap or aluminium foil).
2. Ignite the alcohol and the alcohol will burn and sterilize the glass.
3. Remove the spreader from the flame and allow the alcohol to burn off.
4. Do not put the spreader down on the bench.

45
Fig. 9. Flaming a glass spreader

The spread plate can be used for quantitative work (colony counts) if the inoculum is a
measured volume, usually 0.1 cm3, of each of a dilution series, delivered by pipette.
Procedure:
1. With the pipette, remove a small amount of broth.
2. With the left hand, partially lift the lid of the Petri dish containing the solid
nutrient medium.
3. Place a few drops of culture onto the surface about 0.1 cm3 (ca 5 drops, enough to
cover a 5 pence piece).
4. Replace the lid of the Petri dish and discard the pipette in a discard jar.
5. Dip a glass spreader into alcohol, flame and allow the alcohol to burn off.
6. Lift the lid of the Petri dish to allow entry of spreader.
7. Place the spreader on the surface of the inoculated agar and, rotating the dish with
the left hand move the spreader in a top-to-bottom or a side-to-side motion to
spread the inoculum over the surface of the agar. Make sure the entire agar
surface is covered. (This operation must be carried out quickly to minimize the
risk of contamination).
8. Replace the lid of the Petri dish and let it dry.
9. Seal and incubate the plate in the inverted position.

Incubation
The lid and base of an agar plate should be taped together with 2-4 short strips of

46
adhesive tape as a protection from accidental (or unauthorized!) opening during
incubation. Agar plates must be incubated with the medium-containing half (base) of the
Petri dish uppermost (Inverted) otherwise condensation will occur on the lid and drip
onto the culture. This might cause colonies to spread into each other and risk the spillage
of the contaminated liquid.

5.5. Atmospheric Oxygen Requirement of an Organisms

Bacterial growth is also dependent on the presence of oxygen in the environment, as

different bacteria have different oxygen requirements depending on the types of enzymes
they possess. The major bacterial oxygen classes are aerobes, microaerophiles, obligate
anaerobes, aerotolerant anaerobes, and facultative anaerobes. Aerobes require
atmospheric O2 (20%), and use O2 as the final electron acceptor in the electron transport
system. Microaerophiles require O2 at below atmospheric concentrations, typically 2-
10%. Microaerophiles have a limited ability to neutralize toxic oxygen, so excess O2 will
kill the bacteria. However, microaerophiles do use O2 as final electron acceptor in the
electron transport system. Obligate Anaerobes cannot survive in the presence of any
oxygen. Obligate anaerobes lack the enzymes necessary to break down the toxic by-
products of oxygen. In these bacteria, the final electron acceptor in the electron transport
system is a molecule other than O2. Aerotolerant Anaerobes grow equally well in the
presence or absence of oxygen. They do possess enzymes necessary to neutralize toxic
oxygen by-products, but they never use O2 as a final electron acceptor. Facultative
Anaerobes are also able to live either in the presence or absence of oxygen, but they
prefer oxygen so they can carry out aerobic respiration with O2 as final electron acceptor
to maximize ATP yields. These organisms can use other electron acceptors if O2 is not
available, such as fumarate and nitrate. They can also utilize fermentative metabolism in
the absence of oxygen.

Principle of the test

The bacteria present in the agar deep tube will only be able to grow where their oxygen
requirements are met, and will localize to the area(s) of their oxygen requirements in the

47
tube. Based on the above knowledge of oxygen requirements, we can sketch where you
growth would occur in the BHI tube for each oxygen class as follow:

Figure 1. Growth pattern of bacteria based on oxygen requirement

5.5.1 Determine the oxygen requirements of bacteria

Objective
 to determine the oxygen requirements of a given bacterium
Media Needed
 4 Brain Heart Infusion (BHI) deeps
Cultures Needed:
 Staphylococcus xylosus, Mycobacterium smegmatis, Bacillus megaterium,
Micrococcus luteus, Saccharomyces cerevisiae, Clostridium sporogenes,
Enterococcus faecalis, Serratia marcescens
Procedure:
1. Obtain 4 melted BHI tubes from the water bath and label with 4 of the above
bacteria. Each person in the group should be responsible for one BHI tube.
Coordinate with another group so that all microorganisms are tested!!
2. While the BHI tube is still liquefied, add 2 drops of the appropriate inoculum into
the BHI tube using a sterile Pasteur pipette. Alternatively, you may gently

48
 inoculate the liquefied BHI tube using a sterile loop or needle. Place cap loosely
(but securely) on the tube. You need to work quickly so the agar doesn’t solidify!
3. Roll the BHI tube between the palms of your hands to disperse the bacteria
throughout the tube, taking care not to create bubbles or aerate the agar.
4. Place the BHI tube in ice bath until solidified.
5. Remove from ice bath, and place in incubator. Incubate at 37 °C for 48 hours.

Results:
Examine the BHI deep tubes for presence/absence of microbial growth and location of
growth and record the results in the table below.

Species Distribution of growth Oxygen Requirement

Classification

Staphylococcus xylosus
Mycobacterium smegmatis
Bacillus megaterium
Micrococcus luteus
Saccharomyces cerevisiae
Clostridium sporogenes
Enterococcus faecalis
Serratia marcescens

5.5.2 Cultivation of Anaerobic Organisms

Specialized methods are necessary to culture organisms anaerobically. One such method
is the use of fluid thioglycollate broth, which is a reducing medium. It contains sodium
thioglycollate, which reacts with molecular oxygen keeping free oxygen levels low. The
sodium thioglycollate in the broth creates a redox potential in the tube, with higher

49
levels of oxygen at the top of the tube, and a complete absence of oxygen at the bottom of
the tube. Fluid thioglycollate broth also typically contains a redox potential indicator such
as resazurin, which produces a pink coloration in an oxidized environment. As with the
BHI media described above, organisms will only be able to grow where their oxygen
requirements are met, and will localize to the area(s) of their oxygen requirements in the
fluid thioglycollate broth.

A second method used to culture organisms anaerobically is the use of a GasPak Jar.
This is a specialized culture vessel in which an anaerobic environment is generated after
inoculated media are sealed into the chamber. Anaerobic conditions are created by adding
water to a gas generator envelope that is placed in the jar just before sealing. There are
two chemical tablets in the envelope, sodium borohydride and sodium bicarbonate.
Water reacts with these chemicals, producing hydrogen gas from the sodium
borohydride and carbon dioxide from the sodium bicarbonate. The hydrogen gas
combines with free oxygen in the chamber to produce water, thus removing all free
oxygen from the chamber. This reaction is catalyzed by the element palladium (#46 on
the periodic table), which is attached to the underside of the lid of the jar. The carbon
dioxide replaces the removed oxygen, creating a completely anaerobic environment. For
this lab exercise, each group will design and conduct an experiment to determine the
oxygen classification of four different species of bacteria, using the provided media and
equipment.

50
Media Available:
 4 thioglycollate broth tubes
 2 nutrient agar plates
Equipment Available:
 Gas Pak Jar
 Incubators set at 37 °C
Cultures Needed:
Bacillus megaterium, Pseudomonas aeruginosa, Escherichia coli, Clostridium sporogenes
Procedure:

5.6 Bacterial Count

Viable Plate Counts

In microbiological research, it is often necessary to be able to quantify the number of
living bacteria in a particular sample. There are two methods used to count bacteria.
These are total cell count and viable cell count. One of the major ways to do this is using

51
viable plate counts, in which bacterial cells from a liquid culture are spread onto an agar
plate. The plate is incubated, the number of colonies that grow on the plate are counted,
and the number of original bacterial cells in the culture is determined. In most cases,
however, the liquid culture being quantified contains too many cells to be directly plated
onto agar plates – there would be so much growth that it would be impossible to count
individual colonies! Therefore, the liquid culture needs to be diluted, often 1-million-fold,
before it can be plated.

When such a large dilution is required, an accurate dilution cannot be made in a single
dilution step and it is necessary to make serial dilutions. Serial dilutions are a step-wise
set of dilutions which sequentially dilute the bacterial culture. One or more of the
dilutions are then plated on the agar plates to determine the number of colonies present in
the original culture. Only plates containing between 30 and 300 colonies are counted to
ensure statistically significant data. To estimate the number of bacterial in the original
culture, the # of colonies on the plate is multiplied by the total dilution plated. For
example, suppose 0.1 ml of a 10-6 dilution was plated, and 123 colonies were counted
following incubation. The total dilution plated would be 10-7 (since only 0.1 ml was
plated), and the number of bacteria/ml of the original culture would be:
(123) x 1/10-7 = 1.23 x 109 CFU/ml. Note that the results are expressed as “colony
forming units (CFU)” per ml.

52
Objective
 To quantify the number of bacteria in a broth culture.
Media needed:
3 Nutrient agar plates
7 Dilution blanks containing 9 ml water
Cultures Needed:
Overnight broth culture of bacteria (Eg. Escherichia coli)
Procedure:
1. Label the dilution blanks as follows: 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7.
2. Label the agar plates as follows: 10-6, 10-7, 10-8.
3. Using a sterile pipette, transfer 1 ml of the E. coli broth culture into the tube labeled
10-1. Mix thoroughly.
4. Using a new sterile pipette, transfer 1 ml of the 10-1 tube into the tube labeled 10-2.
Mix thoroughly.
5. Using a new sterile pipette, transfer 1 ml of the 10-2 tube into the tube labeled 10-3.
Mix thoroughly.

53
6. Using a new sterile pipette, transfer 1 ml of the 10-3 tube into the tube labeled 10-4.
Mix thoroughly.
7. Using a new sterile pipette, transfer 1 ml of the 10-4 tube into the tube labeled 10-5.
Mix thoroughly.
8. Using a new sterile pipette, transfer 1 ml of the 10-5 tube into the tube labeled 10-6.
Mix thoroughly.
9. Using a new sterile pipette, transfer 1 ml of the 10-6 tube into the tube labeled 10-7.
Mix thoroughly.
10. Using a new sterile pipette, transfer 0.1 ml of the 10-5 tube to the nutrient agar plate
labeled 10-6, and spread the liquid thoroughly and evenly over the surface of the plate
using a sterile disposable spreader. Be careful not to let the spreader dig into the agar!
Note: Since we’re only plating 0.1 ml of the 10-5 dilution, the total dilution plated is
10-6.
11. Using a new sterile pipette, transfer 0.1 ml of the 10-6 tube to the nutrient agar plate
labeled 10-7, and spread the liquid thoroughly and evenly over the surface of the plate
using a sterile disposable spreader.
12. Using a new sterile pipette, transfer 0.1 ml of the 10-7 tube to the nutrient agar plate
labeled 10-8, and spread the liquid thoroughly and evenly over the surface of the plate
using a sterile disposable spreader.
13. After the liquid has absorbed into the plates, tape them closed on both sides. Make
sure your plates are labeled with your name, date, and the organism, and incubate
upside down at 30 °C.
RESULTS:
Examine the nutrient agar plates for growth, and count the number of colonies on each
plate. Remember, the number has to be between 30 and 300 in order to be statistically
accurate. If your plate has fewer than 30 colonies, record the number as “TFTC” for “too
few to count”. If your plate has more than 300 colonies, record the number as “TNTC”
for “too numerous to count”. Then, use the formula on the previous page to determine the
number of CFU/ml of the original broth culture.

54
CHAPTER 6: BIOCHEMICAL TESTS

There are two levels of biochemical tests. The Primary Identification test in which,
once a pure culture is obtained, the results from a few comparatively simple tests can
often identify the bacterium to a generic level. The Secondary Identification of
Bacteria occurs once the bacterium has been identified to a generic level, some of the
tests can be carried out to identify the species. Secondary biochemical tests are:
Fermentation of carbohydrates, Citrate utilization, Decarboxylation of amino acids,
gelatin liquefaction, hydrogen sulfide test, indole test, Methyl red test, nitrate reduction
test, ONPG test, Urease test and Voges Proskauer test. Some of them are describes as
follows.

Catalase Test
Principle
The enzyme catalase produced by some bacteria breaks down hydrogen peroxide
and oxygen. This results in the visible formation of bubbles of oxygen.
Objective
 To determine wither the bacteria produce catalase enzyme

Material
Test tube Method: the more recommended method that can avoid the action of
atmospheric oxygen
a) Small portion of bacterial suspension is placed in test tube,
b) A drop of 3% H2O2 is added to the tube containing bacteria
Result
Positive = presence of bubbles (oxygen) coming out from the bacterial growth;
Negative = no bubbles observed.
Note: Be careful when taking bacterial colonies from media containing blood.
Blood contains the catalase enzyme, and if this media is carried over with the
bacterial colonies, a false-positive result can occur.
Oxidation/ Fermentation Test

55
Principle
This test is used to determine the oxidative or fermentative metabolism of a
carbohydrate by the bacterium in a semi-solid medium which contains glucose as
a test sugar and bromothymol blue as a pH indicator.
Procedure
a) Take two test tubes of OF medium and heat in a beaker of boiling
water immediately before use to remove the any dissolved oxygen.
b) Cool the tubes rapidly under cold running water.
c) Stab inoculates both tubes with the bacteria to be tested.
d) On the top of one tube, cover with sterile paraffin oil to about 1 cm.
e) Incubate at 37oC and examine in 24 hours and then daily for up to 14
days.
Result Open tube Sealed tube
Unreactive Green Green
Oxidation Yellow Green
Fermentation Yellow Yellow
OXIDASE TEST
Principle
Anaerobes are oxidase negative and thus can reduce the dye (tetramethyl-p-phenylene
diamine dihydrochloride).
Objective
 To detects the presence of cytochrome oxidase..
Material
Reagents: Prepare a 1% solution (0.1g in 10 ml of distilled water) of p-
aminodimetylaniline monohydrochloride or 0.5% of N,N,N’,N’-tetramethyl-p-
phenylenediamine dihydrochloride.

Procidure
Method 1:
a) A piece of filter paper is moistened in a Peri dish with 1% aqueous solution of
tetramethyl-p-phenylenediamine dihydrochloride.

56
 b) Streak the test bacterium firmly across the filter paper with a glass rod.
Result: positive = A dark purple colour along the streak line.
Method 2:
a) Equal volumes of 1% alpha-naphthol in 95% ethanol and 1% aqueous solution of
p-aminodimethylaniline oxalate are mixed.
b) Add a drop of the mixture on the surface of a few colonies on a blood agar plate.
Result: Positive = blue within in 10-30 seconds.
 The reagents used in the test should be colorless and stored in a dark bottle at 4oC
Auto-oxidation of the reagents may be retarded by the addition of 1% ascorbic acid.
Overall Result Positive test is indicated by the development of a purple colour.
 Oxidase positive – Pseudomonas, Vibrio, Neisseriae
 Oxidase negative – Salmonella, Shigella
Motility Test
Using Motility Test Medium
Principle
Motility medium is a colorless, semisolid agar medium dispensed in a test tube
that is soft enough that motile organisms can spread through the agar as they
grow.
Objective
Procidure
a. With a needle, pick up some bacterial growth.
b. Stab down the center of the tube but do not penetrate all the way
down into the butt and withdraw in the same line. Leave about one
cm uninoculated.
c. Incubate for 1-2 days.
d. Hold the tube to the light and observe the growth.
Result:
Positive: growth spreads outward from the stab into the medium; Nonmotile =
growth remains in the stab line of inoculation.
TRIPLE SUGAR IRON AGAR (TSI)
Principle

57
 Triple sugar iron (TSI) slant agar is used to determine whether an organism ferments
glucose, lactose, and sucrose. A TSI slant begins as an orange- or red-colored agar at
an alkaline pH. Phenol red is the pH indicator and ferrous sulfate with sodium
thiosulfate detects hydrogen sulphide production. If any of the carbohydrates are
fermented, an acid pH will result, and either the butt or the slant and butt will turn
yellow. In addition to peptone, yeast extract & agar, it contains 3 sugars – Glucose,
Lactose, and Sucrose.
Objective
 To study different properties of a bacterium – sugar fermentation, gas production
and H2S production.
Materials: TSI agar, test bacteria, two types of inoculating needles (straight and wire
loop)
Procedure
a) Inoculate the bottom of the TSI agar with the straight needles.
b) Using the wire loop streak the slant by stabbing to the bottom and then
inoculating the surface of the slant as you withdraw the needle.
c) Cap very loosely and incubate overnight at 37 0C for 24 hours and write
your observations after 24 hours.
Result
Yellow – Acid
Pink (red) - Alkaline
 Yellow slant / Yellow butt (A/A) – Lactose fermenters.
 Pink slant / Yellow butt (K/A) – Non lactose fermenters.
 Pink slant / no colour change (K/K) – Non fermenters
 Black colour – H2S production.
 Gas bubbles or crack in the medium – gas production.
 Lactose Fermentors – E.coli, Klebsiella
 Non Lactose Fermentors – Salmonella, Shigella
 H2S producers- Proteus
INDOLE TEST
Principle

58
Indole-positive bacteria possess an enzyme, tryptophanase, which converts tryptophan
(an amino acid present in peptone water) to indole. Indole combines with
cinnamaldehyde to produce a blue-green compound.
Objective
 To detect indole production by the organism.
Reagents:
 Kovac’s reagent: p-dimethylamino-benzaldehyde 20.0g, Iso-amyl alcohol
300.0 ml, Concentrated hydrochloric acid 100.0 ml
 The aldehyde is dissolved in the alcohol with gentle heat. The acid is
added after cooling. The reagent is stored in a dark bottle at 4oC.
Procedure
Method 1:
a) Incubate the organism in trypticase soy broth containing tryptophan
b) After overnight incubation, a few drops of indole reagent (Kovac’s
reagent) are added.
Result:
Indole Positive: Red (pink) ring at the interface within seconds.
Indole test Negative - yellow ring
Method 2:
a. Place a piece of filter paper in the bottom of a Petri dish.
b. Saturate the filter paper with 1 % p-
dimethylaminocinnamaldehyde. Use a loop or wooden stick to rub
some growth onto the filter paper. The development of a blue color is
a positive test.
Result: Indole Positive: blue-green color
 Indole positive – E.coli
 Indole negative – Klebsiella, Salmonella.

Citrate test
Principle:

59
The purpose of the citrate test is to determine if an organism is capable of
utilizing sodium citrate as a sole source of carbon. If sodium citrate is utilized,
alkaline products including NaOH are produced.
Procedure
1. Inoculate the surface of Simmons citrate slant in a single streak.
2. Cap loosely and incubate 24 hours.
Result: Positive test shows development of deep blue color or visible growth
along with the streak. In some cases re-incubation is necessary for the
development of a blue color.
Positive: Gas production-cracking of SIM media

Urease Test (Urea hydrolysis test)

Principle:
Organisms that possess the enzyme urease hydrolyze urea to form ammonia. The
pH of the medium increases and the indicator, phenol red, turns from yellow to
red.
Procedure
1. Streak the slant portion of a urea agar (Christensen’s medium) slant
2. Cap loosely.
3. Incubate at 35°C.
Result:
Positive: Pink color, often starting at the slant. Rapid urea splitters such as
Proteus produce a positive reaction in 1-2 hours. Slow splitters take 24-48 hours.
Negative: the agar remains yellow
MR-test
Procedure
1 Add 2 drops of MR indicator
2 Observe for an immediate cherry red color that indicates a positive
test. Orange or yellow is considered negative.
Results:
MR positive – red colour

60
MR negative – yellow colour

Voges-Proskauer Test

Procedure
1. Add 0.6ml of 0.5% alpha-naphthol in ethyl alcohol and 0.2ml of 40%
potassium hydroxide (KOH)
2. Shake well for 5-10 minutes
3. A bright orange red color develops and gradually extends throughout the broth
if acetylmethyl-carbinol has been produced.
Alternatively, add 10% KOH Observe for the development of a mahogany red color. The
color development takes 20 minutes or longer.
Note: Be extremely careful with the KOH. It is caustic and may cause burns if it gets on
your skin. The mahogany red color is considered positive.
Result:
Positive: MR = A cherry red color
VP = A red color at the top of the medium

The IMViC Test

This is an acronym for four different tests involved in the differentiation of
Enterobacteria or coliforms. The test involved indole test (I), methyle red (MR) test,
Vages-proskaver (VP) test and citrate utilization test (C). Most of the coliforms are
placed with their relatives in the family Enterobacteriacae. This group is more commonly
known as the enterics. Most of these bacteria exist in the intestines of humans and other
animals. The IMViC tests are good for differentiating between the fecal bacteria; E. coli
and Enterobacter cloacae which can be found in feces.
Materials:
 A 24 hours nutrient culture of enterobacteria such as E. coli, Salmonella,
Klebsiella spp. Etc.
 Different types of media and regents needed for the different tests.
Media:

61
  For indole test, the appropriate media is peptone water which has enough
amount of tryptophan amino acid in it.
 For MR and VP test, the media used is glucose phosphate peptone water.
Chemically it consists of 5gm of peptone, 5gm glucose and 5gm K2HPO4,
in 1000 ml of weter.
 For citrate test, the medium used is simmon’s citrate media and is
composed of 5gm NaCl, 0.2gm MgSO4 1gm of NH4H2PO4 dissolved in
1000 ml of water. To this solution add 2gm of citric acid, 20gm agar, and
40ml of 0.2% bromothymol blue as pH indicator.
Reagents:
 Indole test – Covac’s reagent
 VP test – 5% alcoholic x-naphtol and 40% KOH
 MR test – 1% metyl red
 Citate utilization test – bromothymol blue
Procedure:
1. Inoculate different test tubes with the different test bacteria but at least one
bacterium should be tested in four of the different tests.
2. Incubate the test tube at 37 0C for 48 hours
3. After 48 hours, perform the following tests and write your observations.
Result:
IMViC test result of (indole+/MR+/VP-/Citrate-) is E. coli.

Triple Sugar Iron Slant

Principle
Triple sugar iron (TSI) slant agar is used to determine whether an organism
ferments glucose, lactose, and sucrose. A TSI slant begins as an orange- or red-
colored agar at an alkaline pH. If any of the carbohydrates are fermented, an acid
pH will result, and either the butt or the slant and butt will turn yellow.
Procedure
a) Inoculate the slant with a needle by stabbing to the bottom and then

62
 inoculating the surface of the slant as you withdraw the needle.
b) Cap very loosely and Incubate overnight.
Result:
A/A: ferments glucose (butt) and either sucrose, lactose, or both (slants)
K/A: does not ferment lactose or sucrose; does ferment glucose (alkaline butt,
acid slant)
K/K: a nonfermenter (alkaline butt and slant)
Black precipitate in stab: produces H2S (and ferments glucose)
Coagulase Test
Principle:
Coagulase is an enzyme that causes fibrin in plasma to clot. S. aureus is the only
staphylococcus that produces coagulase. S. aureus may produce two forms of
coagulase. One form, referred to as “bound” coagulase, is located on the exterior
of the cell wall.
Procedure
Free method – for the detection of bound coagulase production
a) Emulsify a small amount of each organism from a slant culture into a
loopful of water on a clear slide to give a heavy homogenous suspension.
b) Add several loopful of rabbit plasma and mix with the loop.
Result: Positive: Clumping in about 10 seconds or less
Negative: No clumping and is always confirmed by the tube test.
Note: Since some bacteria produce fibrinolysins, a clot that has formed by 4
hours may be dissolved after an overnight incubation, resulting in a false-negative
reaction. However, since some strains of S. aureus produce coagulase very
slowly, a culture giving a negative reaction enzyme, and if this at 4 hours should
be reincubated and read again at 16-24 hours

63
CHAPTER 7: ANTIMICROBIAL SUSCEPTIBILITY TESTS

Widespread antibiotic usage exerts a selective pressure that acts as a driving force in the
development of antibiotic resistance which resulted in morbidity and mortality from
treatment failures and increased health care costs. The results of in-vitro antibiotic
susceptibility testing, guide clinicians in the appropriate selection of initial empiric
regimens and, drugs used for individual patients in specific situations. The selection of an
antibiotic panel for susceptibility testing is based on the commonly observed
susceptibility patterns; it became necessary to perform the antimicrobial susceptibility
test as a routine. For this, the antimicrobial contained in a reservoir was allowed to
diffuse out into the medium and interact in a plate freshly seeded with the test organisms.
Even now a variety of antimicrobial containing reservoirs are used but the antimicrobial
impregnated absorbent paper disc is by far the commonest type used.

The disc diffusion method of AST is the most practical method and is still the method of
choice for the average laboratory. Automation may force the method out of the
diagnostic laboratory but in our country as well as in the smaller laboratories of even
advanced countries, it will certainly be the most commonly carried out microbiological
test for many years to come. It is, therefore, imperative that microbiologists understand
the principles of the test well and keep updating the information as and when necessary.

Principle

The techniques involve either diffusion of antimicrobial agent in agar or dilution of

antibiotic in agar or broth.

7.1 Factors Influencing Antimicrobial Susceptibility Testing

A. pH: The pH of each batch of Müeller-Hinton agar should be checked when the
medium is prepared. The exact method used will depend largely on the type of
equipment available in the laboratory. The agar medium should have a pH between 7.2
and 7.4 at room temperature after gelling. If the pH is too low, certain drugs will appear

64
to lose potency (e.g., aminoglycosides, quinolones, and macrolides), while other agents
may appear to have excessive activity (e.g., tetracyclines). If the pH is too high, the
opposite effects can be expected. The pH can be checked by one of the following means:
 Macerate a sufficient amount of agar to submerge the tip of a pH electrode.
 Allow a small amount of agar to solidify around the tip of a pH electrode in a
beaker or cup.
 Use a properly calibrated surface electrode.

B. Moisture: If, just before use, excess surface moisture is present, the plates should be
placed in an incubator (35 0C) or a laminar flow hood at room temperature with lids ajar
until excess surface moisture is lost by evaporation (usually 10 to 30 minutes). The
surface should be moist, but no droplets of moisture should be apparent on the surface of
the medium or on the petri dish covers when the plates are inoculated.

C. Effects of Thymidine or Thymine: Media containing excessive amounts of thymidine

or thymine can reverse the inhibitory effect of sulfonamides and trimethoprim, thus yielding
smaller and less distinct zones, or even no zone at all, which may result in false-resistance

reports. Müeller-Hinton agar that is as low in thymidine content as possible should be used.

To evaluate a new lot of Müeller-Hinton agar, Enterococcus faecalis ATCC 29212, or

alternatively, E. faecalis ATCC 33186, should be tested with
trimethoprim/sulfamethoxazole disks. Satisfactory media will provide essentially clear,
distinct zones of inhibition 20 mm or greater in diameter. Unsatisfactory media will
produce no zone of inhibition, growth within the zone, or a zone of less than 20 mm.

D. Effects of Variation in Divalent Cations: Variation in divalent cations, principally

magnesium and calcium, will affect results of aminoglycoside and tetracycline tests with P.
aeruginosa strains. Excessive cation content will reduce zone sizes, whereas low cation
content may result in unacceptably large zones of inhibition. Excess zinc ions may reduce

zone sizes of carbapenems. Performance tests with each lot of Müeller-Hinton agar must

conform to the control limits.

65
E. Testing strains that fail to grow satisfactorily: Only aerobic or facultative bacteria
that grow well on unsupplemented Müeller-Hinton agar should be tested on that medium.
Certain fastidious bacteria such as Haemophilus spp., N. gonorrhoeae, S. pneumoniae, and
viridans and ß-haemolytic streptococci do not grow sufficiently on unsupplemented
Müeller-Hinton agar. These organisms require supplements or different media to grow, and
they should be tested on the media described in separate sections.

7.2 Methods of Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing methods are divided into types based on the principle
applied in each system. They include:
Diffusion Dilution Diffusion & Dilution
Stokes method Minimum Inhibitory Concentration E-Test method
Kirby-Bauer method i) Broth dilution

ii) Agar Dilution

7.2.1 Disk Diffusion

Reagents for the Disk Diffusion Test

1. Müeller-Hinton Agar Medium
Of the many media available, Müeller-Hinton agar is considered to be the best for routine
susceptibility testing of nonfastidious bacteria for the following reasons:
* It shows acceptable batch-to-batch reproducibility for susceptibility testing.
* It is low in sulphonamide, trimethoprim, and tetracycline inhibitors.
* It gives satisfactory growth of most nonfastidious pathogens.
* A large body of data and experience has been collected concerning susceptibility
tests performed with this medium.
Although Müeller-Hinton agar is reliable generally for susceptibility testing, results
obtained with some batches may, on occasion, vary significantly. If a batch of medium

66
does not support adequate growth of a test organism, zones obtained in a disk diffusion
test will usually be larger than expected and may exceed the acceptable quality control
limits. Only Müeller-Hinton medium formulations that have been tested according to,
and that meet the acceptance limits described in, NCCLS document M62-A7- Protocols
for Evaluating Dehydrated Müeller-Hinton Agar should be used.

Preparation of Müeller-Hinton Agar

Müeller-Hinton agar preparation includes the following steps.
1. Müeller-Hinton agar should be prepared from a commercially available
dehydrated base according to the manufacturer's instructions.
2. Immediately after autoclaving, allow it to cool in a 45 to 50C water bath.
3. Pour the freshly prepared and cooled medium into glass or plastic, flat-bottomed
petri dishes on a level, horizontal surface to give a uniform depth of
approximately 4 mm. This corresponds to 60 to 70 ml of medium for plates with
diameters of 150 mm and 25 to 30 ml for plates with a diameter of 100 mm.
4. The agar medium should be allowed to cool to room temperature and, unless the
plate is used the same day, stored in a refrigerator (2 to 8C).
5. Plates should be used within seven days after preparation unless adequate
precautions, such as wrapping in plastic, have been taken to minimize drying of
the agar.
6. A representative sample of each batch of plates should be examined for sterility
by incubating at 30 to 35C for 24 hours or longer.

Preparation of antibiotic stock solutions

Antibiotics may be received as powders or tablets. It is recommended to obtain pure

antibiotics from commercial sources, and not use injectable solutions. Powders must be
accurately weighed and dissolved in the appropriate diluents (Annexure III) to yield the
required concentration, using sterile glassware. Standard strains of stock cultures should
be used to evaluate the antibiotic stock solution. If satisfactory, the stock can be aliquoted
in 5 ml volumes and frozen at -20ºC or -60ºC.

67
Stock solutions are prepared using the formula (1000/P) X V X C=W, where P+potency
of the anitbiotic base, V=volume in ml required, C=final concentration of solution and
W=weight of the antimicrobial to be dissolved in V.

Preparation of dried filter paper discs

Whatman filter paper no. 1 is used to prepare discs approximately 6 mm in diameter,

which are placed in a Petri dish and sterilized in a hot air oven.
The loop used for delivering the antibiotics is made of 20 gauge wire and has a diameter
of 2 mm. This delivers 0.005 ml of antibiotics to each disc.

Storage of commercial antimicrobial discs

Cartridges containing commercially prepared paper disks specifically for susceptibility

testing are generally packaged to ensure appropriate anhydrous conditions. Discs should
be stored as follows:
 Refrigerate the containers at 8C or below, or freeze at -14C or below, in a
nonfrost-free freezer until needed. Sealed packages of disks that contain drugs
from the ß-lactam class should be stored frozen, except for a small working
supply, which may be refrigerated for at most one week. Some labile agents (e.g.,
imipenem, cefaclor, and clavulanic acid combinations) may retain greater stability
if stored frozen until the day of use.
 The unopened disc containers should be removed from the refrigerator or freezer
one to two hours before use, so they may equilibrate to room temperature before
opening. This procedure minimizes the amount of condensation that occurs when
warm air contacts cold disks.
 Once a cartridge of discs has been removed from its sealed package, it should be
placed in a tightly sealed, desiccated container. When using a disc-dispensing
apparatus, it should be fitted with a tight cover and supplied with an adequate
desiccant. The dispenser should be allowed to warm to room temperature before

68
 opening. Excessive moisture should be avoided by replacing the desiccant when
the indicator changes color.
 When not in use, the dispensing apparatus containing the discs should always be
refrigerated.
 Only those discs that have not reached the manufacturer's expiration date stated
on the label may be used. Discs should be discarded on the expiration date.

Turbidity standard for inoculum preparation

To standardize the inoculum density for a susceptibility test, a BaSO4 turbidity standard,
equivalent to a 0.5 McFarland standard or its optical equivalent (e.g., latex particle
suspension), should be used. A BaSO4 0.5 McFarland standard may be prepared as
follows:
A 0.5-ml aliquot of 0.048 mol/L BaCl2 (1.175% w/v BaCl2 . 2H2O) is added to 99.5 ml of
0.18 mol/L H2SO4 (1% v/v) with constant stirring to maintain a suspension. The correct
density of the turbidity standard should be verified by using a spectrophotometer with a
1-cm light path and matched cuvette to determine the absorbance. The absorbance at 625
nm should be 0.008 to 0.10 for the 0.5 McFarland standard.
The Barium Sulfate suspension should be transferred in 4 to 6 ml aliquots into screw-cap
tubes of the same size as those used in growing or diluting the bacterial inoculum.

These tubes should be tightly sealed and stored in the dark at room temperature. The
barium sulfate turbidity standard should be vigorously agitated on a mechanical vortex
mixer before each use and inspected for a uniformly turbid appearance. If large particles
appear, the standard should be replaced. Latex particle suspensions should be mixed by
inverting gently, not on a vortex mixer. The barium sulfate standards should be replaced
or their densities verified monthly.

Disc diffusion methods

69
The Kirby-Bauer and Stokes' methods are usually used for antimicrobial susceptibility
testing, with the Kirby-Bauer method being recommended by the NCCLS. The accuracy
and reproducibility of this test are dependent on maintaining a standard set of procedures
as described here.
NCCLS is an international, interdisciplinary, non-profit, non-governmental organization
composed of medical professionals, government, industry, healthcare providers,
educators etc. It promotes accurate antimicrobial susceptibility testing (AST) and
appropriate reporting by developing standard reference methods, interpretative criteria for
the results of standard AST methods, establishing quality control parameters for standard
test methods, provides testing and reporting strategies that are clinically relevant and
cost-effective

Interpretative criteria of NCCLS are developed based on international collaborative

studies and well correlated with MIC’s and the results have corroborated with clinical
data. Based on study results NCCLS interpretative criteria are revised frequently. NCCLS
is approved by FDA-USA and recommended by WHO.

Procedure for Performing the Disc Diffusion Test

Inoculum Preparation
Growth Method
The growth method is performed as follows
1. At least three to five well-isolated colonies of the same morphological type are
selected from an agar plate culture. The top of each colony is touched with a
loop, and the growth is transferred into a tube containing 4 to 5 ml of a suitable
broth medium, such as tryptic soy broth.
2. The broth culture is incubated at 35C until it achieves or exceeds the turbidity of
the 0.5 McFarland standard (usually 2 to 6 hours)
3. The turbidity of the actively growing broth culture is adjusted with sterile saline
or broth to obtain a turbidity optically comparable to that of the 0.5 McFarland
standard. This results in a suspension containing approximately 1 to 2 x 108
CFU/ml for E.coli ATCC 25922. To perform this step properly, either a

70
 photometric device can be used or, if done visually, adequate light is needed to
visually compare the inoculum tube and the 0.5 McFarland standard against a
card with a white background and contrasting black lines.

Direct Colony Suspension Method

1. As a convenient alternative to the growth method, the inoculum can be prepared
by making a direct broth or saline suspension of isolated colonies selected from a
18- to 24-hour agar plate (a nonselective medium, such as blood agar, should be
used). The suspension is adjusted to match the 0.5 McFarland turbidity standard,
using saline and a vortex mixer.
2. This approach is the recommended method for testing the fastidious organisms,
Haemophilus spp., N. gonorrhoeae, and streptococci, and for testing
staphylococci for potential methicillin or oxacillin resistance.

Inoculation of Test Plates

1. Optimally, within 15 minutes after adjusting the turbidity of the inoculum
suspension, a sterile cotton swab is dipped into the adjusted suspension. The
swab should be rotated several times and pressed firmly on the inside wall of the
tube above the fluid level. This will remove excess inoculum from the swab.
2. The dried surface of a Müeller-Hinton agar plate is inoculated by streaking the
swab over the entire sterile agar surface. This procedure is repeated by streaking
two more times, rotating the plate approximately 60 each time to ensure an even
distribution of inoculum. As a final step, the rim of the agar is swabbed.
3. The lid may be left ajar for 3 to 5 minutes, but no more than 15 minutes, to allow
for any excess surface moisture to be absorbed before applying the drug
impregnated disks.
NOTE: Extremes in inoculum density must be avoided. Never use undiluted overnight
broth cultures or other unstandardized inocula for streaking plates.

Application of Discs to Inoculated Agar Plates

71
 1. The predetermined battery of antimicrobial discs is dispensed onto the surface of
the inoculated agar plate. Each disc must be pressed down to ensure complete
contact with the agar surface. Whether the discs are placed individually or with a
dispensing apparatus, they must be distributed evenly so that they are no closer
than 24 mm from center to center. Ordinarily, no more than 12 discs should be
placed on one 150 mm plate or more than 5 discs on a 100 mm plate. Because
some of the drug diffuses almost instantaneously, a disc should not be relocated
once it has come into contact with the agar surface. Instead, place a new disc in
another location on the agar.
2. The plates are inverted and placed in an incubator set to 35C within 15 minutes
after the discs are applied. With the exception of Haemophilus spp., streptococci
and N. gonorrhoeae, the plates should not be incubated in an increased CO2
atmosphere, because the interpretive standards were developed by using ambient
air incubation, and CO2 will significantly alter the size of the inhibitory zones of
some agents.

Reading Plates and Interpreting Results

1. After 16 to 18 hours of incubation, each plate is examined. If the plate was

satisfactorily streaked, and the inoculum was correct, the resulting zones of inhibition
will be uniformly circular and there will be a confluent lawn of growth. If individual
colonies are apparent, the inoculum was too light and the test must be repeated. The
diameters of the zones of complete inhibition (as judged by the unaided eye) are
measured, including the diameter of the disc. Zones are measured to the nearest whole
millimeter, using sliding calipers or a ruler, which is held on the back of the inverted petri
plate. The petri plate is held a few inches above a black, nonreflecting background and
illuminated with reflected light. If blood was added to the agar base (as with
streptococci), the zones are measured from the upper surface of the agar illuminated with
reflected light, with the cover removed. If the test organism is a Staphylococcus or
Enterococcus spp., 24 hours of incubation are required for vancomycin and oxacillin, but
other agents can be read at 16 to 18 hours. Transmitted light (plate held up to light) is

72
used to examine the oxacillin and vancomycin zones for light growth of methicillin- or
vancomycin- resistant colonies, respectively, within apparent zones of inhibition. Any
discernable growth within zone of inhibition is indicative of methicillin or vancomycin
resistance.

2. The zone margin should be taken as the area showing no obvious, visible growth that
can be detected with the unaided eye. Faint growth of tiny colonies, which can be
detected only with a magnifying lens at the edge of the zone of inhibited growth, is
ignored. However, discrete colonies growing within a clear zone of inhibition should be
subcultured, re-identified, and retested. Strains of Proteus spp. may swarm into areas of
inhibited growth around certain antimicrobial agents. With Proteus spp., the thin veil of
swarming growth in an otherwise obvious zone of inhibition should be ignored. When
using blood-supplemented medium for testing streptococci, the zone of growth inhibition
should be measured, not the zone of inhibition of hemolysis. With trimethoprim and the
sulfonamides, antagonists in the medium may allow some slight growth; therefore,
disregard slight growth (20% or less of the lawn of growth), and measure the more
obvious margin to determine the zone diameter.

The sizes of the zones of inhibition are interpreted by referring to Tables 2A through 2I
(Zone Diameter Interpretative Standards and equivalent Minimum Inhibitory
Concentration Breakpoints) of the NCCLS M100-S12: Performance Standards for
Antimicrobial Susceptibility Testing: Twelfth Informational Supplement, and the
organisms are reported as either susceptible, intermediate, or resistant to the agents that
have been tested. Some agents may only be reported as susceptible, since only
susceptible breakpoints are given.

7.2.2 Dilution Methods

Dilution susceptibility testing methods are used to determine the minimal concentration
of antimicrobial to inhibit or kill the microorganism. This can be achieved by dilution of

73
antimicrobial in either agar or broth media. Antimicrobials are tested in log2 serial
dilutions (two fold).

Minimum Inhibitory Concentration (MIC)

Diffusion tests widely used to determine the susceptibility of organisms isolated from
clinical specimens have their limitations; when equivocal results are obtained or in
prolonged serious infection e.g. bacterial endocarditis, the quantitation of antibiotic
action vis-a-vis the pathogen needs to be more precise. Also the terms ‘Susceptible’ and
‘Resistant’ can have a realistic interpretation. Thus when in doubt, the way to a precise
assessment is to determine the MIC of the antibiotic to the organisms concerned.
There are two methods of testing for MIC:
(a) Broth dilution method
(b) Agar dilution method.

Broth Dilution Method

The Broth Dilution method is a simple procedure for testing a small number of isolates,
even single isolate. It has the added advantage that the same tubes can be taken for MBC
tests also:

Materials

Sterile graduated pipettes of 10ml, 5ml, 2ml and 1ml Sterile capped 7.5 x 1.3 cm tubes /
small screw-capped bottles, Pasteur pipettes, overnight broth culture of test and control
organisms ( same as for disc diffusion tests), required antibiotic in powder form (either from
the manufacturer or standard laboratory accompanied by a statement of its activity in
mg/unit or per ml. Clinical preparations should not be used for reference technique.),
required solvent for the antibiotic, sterile Distilled Water - 500ml and suitable nutrient
broth medium. Trimethoprim and sulphonamide testing requires thymidine free media or
addition of 4% lysed horse blood to the media. A suitable rack to hold 22 tubes in two
rows i-e 11 tubes in each row.

74
Stock solution

Stock solution can be prepared using the formula

1000
------- x V x C= W
P

Where P=Potency given by the manufacturer in relation to the base

V= Volume in ml required
C=Final concentration of solution (multiples of 1000)
W= Weight of the antimicrobial to be dissolved in the volume V
Example: For making 10 ml solution of the strength 10,000mg/l from powder base whose
potency is 980 mg per gram,the quantities of the antimicrobials required is

W= 1000
------- x 10 x 10=102.04mg
980

Note:the stock solutions are made in higher concentrations to maintain their keeping
qualities and stored in suitable aliquots at -20oC .Once taken out,they should not be
refrozen or reused.
Suggested dilution ranges of some antimicrobials are shown in Annexure II.

Method

Prepare stock dilutions of the antibiotic of concentrations 1000 and 100 µg/L as required
from original stock solution (10,000mg/L). Arrange two rows of 12 sterile 7.5 x1.3 cm
capped tubes in the rack. In a sterile 30ml (universal) screw capped bottle, prepare 8ml of
broth containing the concentration of antibiotic required for the first tube in each row
from the appropriate stock solution already made. Mix the contents of the universal bottle
using a pipette and transfer 2ml to the first tube in each row. Using a fresh pipette ,add 4
ml of broth to the remaining 4 ml in the universal bottle mix and transfer 2ml to the

75
second tube in each row. Continue preparing dilutions in this way but where as many as
10 or more are required the series should be started again half the way down. Place 2ml
of antibiotic free broth to the last tube in each row. Inoculate one row with one drop of an
overnight broth culture of the test organism diluted approximately to 1 in 1000 in a
suitable broth and the second row with the control organism of known sensitivity
similarly diluted. The result of the test is significantly affected by the size of the
inoculum. The test mixture should contain 106 organism/ml. If the broth culture used has
grown poorly, it may be necessary to use this undiluted. Incubate tubes for 18 hours at
37oC. Inoculate a tube containing 2ml broth with the organism and keep at +4 oC in a
refrigerator overnight to be used as standard for the determination of complete inhibition.

Calculations for the preparation of the original dilution.

This often presents problems to those unaccustomed to performing these tests. The
following method advocated by Pamela M Water worth is presented. Calculate the total
volume required for the first dilution. Two sets of dilution are being prepared (one for the
test and one for the control), each in 2ml volumes i-e a total of 4 ml for each
concentration as 4ml is required to make the second dilution, the total requirement is 8ml.
Now calculate the total amount of the antibiotic required for 8ml. For 64 g/l
concentration, 8x64mg/l =512µg in 8 ml. Place a decimal point after the first figure
(5.12) and take this volume in ml (i.e 5.12 ml) of the dilution below 512mg/l and make
up to 8ml with broth. In this example given above, the series has to be started again mid-
way at 2 mg/l which would be obtained in the same way:
8ml of 2mg/l=16µg in 8ml.
1.6 ml of 10 mg/ l + 6.4 ml of broth.

Reading of result
MIC is expressed as the lowest dilution, which inhibited growth judged by lack of
turbidity in the tube. Because very faint turbidity may be given by the inoculum itself, the
inoculated tube kept in the refrigerator overnight may be used as the standard for the
determination of complete inhibition. Standard strain of known MIC value run with the
test is used as the control to check the reagents and conditions.

76
Minimum Bactericidal Concentrations (MBC)
The main advantage of the ‘Broth dilution’ method for the MIC determination lies in the
fact that it can readily be converted to determine the MBC as well.

Method

Dilutions and inoculations are prepared in the same manner as described for the
determination of MIC. The control tube containing no antibiotic is immediately
subcultured (Before incubation) by spreading a loopful evenly over a quarter of the plate
on a medium suitable for the growth of the test organism and incubated at 37 oC
overnight. The tubes are also incubated overnight at 37oC. Read the MIC of the control
organism to check that the drug concentrations are correct. Note the lowest concentration
inhibiting growth of the organisms and record this as the MIC. Subculture all tubes not
showing visible growth in the same manner as the control tube described above and
incubate at 37oC overnight. Compare the amount of growth from the control tube before
incubation,which represents the original inoculum. The test must include a second set of
the same dilutions inoculated with an organism of known sensitivity .These tubes are not
subcultured; the purpose of the control is to confirm by its MIC that the drug level is
correct,whether or not this organism is killed is immaterial.

77
Reading of result

These subcultures may show similar number of colonies- indicating bacteriostasis only.
A reduced number of colonies-indicating a partial or slow bactericidal activity. No growth-
if the whole inoculum has been killed. The highest dilution showing at least 99% inhibition
is taken as MBC.

Micro-broth dilution test

This test uses double-strength Müeller-Hinton broth, 4X strength antibiotic solutions

prepared as serial two-fold dilutions and the test organism at a concentration of 2x106/ml. In
a 96 well plate, 100 l of double-strength MHB, 50 l each of the antibiotic dilutions and
the organism suspension are mixed and incubated at 35C for 18-24 hours. The lowest
concentration showing inhibition of growth will be considered the MIC of the organism.

Reading of result

MIC is expressed as the highest dilution which inhibited growth judged by lack of
turbidity in the tube. Because very faint turbidity may be given by the inoculum itself, the
inoculated tube kept in the refrigerator overnight may be used as the standard for the
determination of complete inhibition. Standard strain of known MIC, run with the test is
used as the control to check the reagents and conditions.

The Agar dilution Method

Agar dilutions are most often prepared in petri dishes and have advantage that it is
possible to test several organisms on each plate .If only one organism is to be tested e.g
M. tuberculosis, the dilutions can be prepared in agar slopes but it will then be necessary
to prepare a second identical set to be inoculated with the control organism. The dilutions
are made in a small volume of water and added to agar which has been melted and cooled

78
to not more than 60oC.Blood may be added and if ‘chocolate agar’ is required, the
medium must be heated before the antibiotic is added.
It would be convenient to use 90 mm diameter petri dishes and add one ml of desired
drug dilutions to 19 ml of broth. The factor of agar dilution must be allowed for in the
first calculation as follows.
final volume of medium in plate = 20 ml
Top antibiotic concentrations = 64mg/l
Total amount of drug = 1280µg to be added to
1 ml of water
2ml of 1280 µg /ml will be required to start the dilution = 2560µg in 2 ml
= 1.28ml of 2000µg /ml
± 0.72 ml of water.
1 ml of this will be added to 19 ml agar.
(Note stock dilution of 2000µg /ml is required for this range of MIC)

The quickest way to prepare a range of dilutions in agar is as follows:

Label a sterile petri dish on the base for each concentration required. Prepare the dilutions
in water placing 1 ml of each in the appropriate dish. One ml water is added to a control
plate. Pipette 19 ml melted agar, cooled to 55oC to each plate and mix thoroughly.
Adequate mixing is essential and if sufficient technical expertise is not available for the
skilled manipulation, it is strongly recommended that the agar is first measured into
stoppered tubes or universal containers and the drug dilution added to these and mixed by
inversion before pouring into petri dishes. After the plates have set they should be well
dried at 37oC with their lids tipped for 20 to 30 minutes in an incubator. They are then
inoculated either with a multiple inoculator as spots or with a wire loop or a platinum
loop calibrated to deliver 0.001ml spread over a small area. In either case the culture
should be diluted to contain 105 to 106 organisms per ml. With ordinary fast growing
organisms, this can be obtained approximately by adding 5 µl of an overnight broth
culture to 5ml broth or peptone water.

79
It is possible to test spreading organism such as P. mirabilis by this method either by
cutting ditches in the agar between the inocula, or by confining each with small glass or
porcelain cylinders pressed into the agar. Although swarming of P. mirabilis can be
prevented by the use of higher concentration of agar in the medium, this is not
recommended for determination of MIC because of the difficulty of ensuring adequate
mixing of the drug with this very viscous medium. Selective media should not be used
and electrolyte deficient media will give false results because of the effect of variation in
the salt content on the action of many antibiotics.

Reading of results

The antibiotic concentration of the first plate showing  99% inhibition is taken as the
MIC for the organism.

7.2.3 Dilution and Diffusion

E test also known as the epsilometer test is an ‘exponential gradient’ testing

methodology where ‘E’ in E test refers to the Greek symbol epsilon ().The E test(AB
Biodisk) which is a quantitative method for antimicrobial susceptibility testing applies
both the dilution of antibiotic and diffusion of antibiotic into the medium.. A predefined
stable antimicrobial gradient is present on a thin inert carrier strip. When this E test strip
is applied onto an inoculated agar plate, there is an immediate release of the drug.
Following incubation, a symmetrical inhibition ellipse is produced. The intersection of
the inhibitory zone edge and the calibrated carrier strip indicates the MIC value over a
wide concentration range (>10 dilutions) with inherent precision and accuracy.

E test can be used to determine MIC for fastidious organisms like S. pneumoniae,
ß-hemolytic streptococci, N.gonorrhoeae, Haemophilus sp. and anaerobes. It can also be
used for Nonfermenting Gram Negative bacilli (NFGNB) for eg-Pseudomonas sp. and
Burkholderia pseudomallei.

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Resistance of major consequence may be detected for e.g., the test is very useful in
detecting glycopeptide resistant Enterococci (GRE) and glycopeptide intermediate
S.aureus (GISA) and slow growing pathogens such as Mycobacterium tuberculosis.
Further it can be used for detection of extended spectrum beta lactamases (ESBL). In
conclusion E test is a simple, accurate and reliable method to determine the MIC for a
wide spectrum of infectious agents.

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CHAPTER 8: COMMON SEROLOGICAL TESTS

8.1 Principle of Antigen Antibody Interactions

Antigen - antibody reactions are the methods by which antigens and antibodies are
measured. Antibodies can be detected by various methods from the serum. Many types of
immunoglobulin molecules can be produced in response to a single antigen and detected
either total Ig or Ig classes, as IgM, IgG and IgA. Infectious diseases can be definitively
diagnosed in only three ways:
7. By documenting the presence in the patient of an agent known to cause the disease,
either by visualizing the agent directly in clinical material obtained from the
patient, by detecting antigens or genetic material specific for the agent, or by
cultivating the agent in the lab.
8. By detecting a specific product of the infectious agent in clinical material obtained
from the patient, a product that could not be produced without the agent´s
presence.
9. By detecting an immunological response specific to the infecting agent in the
patient´s serum.

Antigen - antibody reaction can be visualized in different ways according to the type of
the antigen, conditions of the reaction and the medium the reaction takes place in.
 When an antibody combines with a corpuscular antigen (forming part of a cell -
e.g. bacteria, virus, blood cell or inert part with bound antigen) the
cells agglutinate that means they form clumps.
 When an antibody combines with a noncorpuscular antigen (toxin, enzyme,
microbial extract) a precipitate is formed, antigen - antibody complex is thrown
out of solution.
 When an antibody combines with an antigen which forms part of the surface of
certain cells (e.g. a few species of gram negative bacilli and red blood cells) and
provided complement is also present the cells are lysed, that means they are
dissolved.

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  Other types of serological reactions used for antigen or antibody detection are
used in microbiology - e.g. immunofluorescent test, EIA, ELISA (enzyme
immunosorbent assays), RIA (radioimmunoassay), which use specific detection
systems. We shall talk about them during the special microbiology lectures.

Serological tests are used for indirect diagnosing the infectious diseases and for
prevention and prophylaxis of them as well. Generally, serological tests can be used in
two ways:
1. a known antibody can be used to detect and measure an unknown antigen
2. a known antigen can be used to detect and measure an unknown antibody.

Factors Affecting Antigen Antibody Reactions: Many factors can affect the interaction
between antigen and antibody. These include specificity, cross reactivity, temperature
PH, ionic strength, concentration, and intermolecular specificity.

Antibody production is a dynamic process changing during the disease. Therefore

examination of the single serum sample is not sufficient. To diagnose acute infectious
disease the first blood sample must be taken in the beginning of the disease, the second
one after 10 or 14 days. Both blood samples are examined at the same time under the
same laboratory conditions.

Dilution is the act of making a weaker solution from a stronger one. This is usually done
by adding a water or saline, which contains none of the material being diluted. Dilution is
usually expressed as one unit of the original solution to the total number of units of final
solution. Serial dilution means decreasing the volume of serum progressively by
maintaining a constant volume of fluid most commonly, serial dilutions are two fold, that
is, each dilution is half as concentrated as the preceding one (or ten-fould dilutions). The
total volume in each tube is the same.

Serologic tests are performed as: (a): qualitative tests – performed to find out presence
of antibodies or antigens in the serum or (b): quantitative tests - performed more often.

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Quantitative results are normally expressed in terms of the titre of the serum, the
highest dilution of the serum at which a particular effect can be demonstrated. For
example a titer of 1:128 means that in the reaction being studied the serum shows the
effect when is diluted 1 in 128. The titre is thus a measure of the amount of antibody in a
unit volume of the original serum. Acute infectious disease is diagnosed by appearance of
four-fold increase of antibody titre in the second serum sample (e.g.titer 1:8 in the first
serum sample and the titer 1:32 in the second one).

8.2 Collection, Preparation and Preservation of Specimen for Serologic Tests

Specimens that are used for serologic test include: serum, plasma & cerebrospinal fluid.
Serum or plasma sample could be obtained from venous blood, which can be performed
by the laboratory personnel however. Cerebrospinal fluid should be collected by a
physician or a trained nurse. For serum or plasma sample, first 2-3 ml of venous blood is
collected using sterile syringe and needle from a patient. If serum is required, allow the
whole blood to clot at room temperature for at least one hour and centrifuge the clotted
blood for 10 minutes at 2000 rpm. Then transfer the serum to a labeled tube with a
pasture pipette and rubber bulb. Plasma sample is obtained by treating fresh blood with
an anticoagulant, centrifuge and separates the supernatant. The specimen should be free
from hemolyzed blood. Finally, seal the specimen containing tube; the tube should be
labeled with full animal’s identification (Age, Sex, code no, etc). The test should be
performed with in hours after sample collection, if this could not be done preserve it at-
200c.

8.3 Materials Necessary for Basic Serologic Tests

As discussed above, serologic techniques are used to detect either an antibody or antigen.
For the detection of this unknown substance from patient’s specimen, the specimen
should be collected and prepared appropriately. In addition the equipment that is used for
testing should be free from any contaminants so as to get true result. The following are
some of the equipment used in routine serology.
 Glassware like Test tube, Glass slides and Serologic pipette with a size of l0ml,
5ml, 2ml&1ml.

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  Constant temperature device like Incubator and water bath are usually used in
serologic tests.
 Rotating machines like shaker are required to facilitate antigen antibody
reactions.

Components in the serological reactions.

a. Serum: is taken after the blood coagulation into the sterile tube. The full blood can
coagulate itself or can be centrifuged 10 minutes at 800g. Serum can be exhausted after
blood coagulation by Pasteur pipette or by plastic tip on the automatic pipette. Serum
samples can be examined either native or are taken in a special tubes in the deep-freeze at
-20oC. In some reactions the serum must be inactivated 30 min at +56oC before
examination. Inactivation removes the serum complement.

b. Antigen: (1) Corpuscular antigens are mostly suspensions of inactivated bacteria

taken from the solid culture medium. (2) Non-corpuscular antigens are the filtrates of
bacterial cultures taken from the fluid culture medium. They can be also taken from the
bacteria destroyed by sonication or by repeating freezing and thawing.

c. Vehiculum: Serologic reactions are carried out in a special medium, either in solution
or agar or under an optimum temperature.

d. Incubation time: varies from a few minutes (serotypization of bacteria) to several

days (agar precipitation).

Formerly the serologic tests were performed in the tubes. Now most of the tests are
performed as micromodifications. Instead of the tubes the plastic microtubes are used for
storage, inactivation or predilution of the sera, then the sera are tested in the wells of a
microplate. 25 l, 50 l, 100 l or 200 l of a serum is used. The other laboratory
equipment being used for microtests are automatic micropipettes with plastic tips,
automatic dilutors, automatic washers and readers. In several laboratories the readers are

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in line with computers and at the end of the reaction it is possible to get the final result
printed on the laboratory slip.

8.4 Common Types of serologic tests.

There are different types of antigen antibody interaction these include:

A. Agglutination reaction
B. Precipitation reaction
C. Complement fixation reaction
D. Enzyme Immuno Assay (EIA)
E. Radio Immuno Assay (RIA)

I. Agglutination
A corpuscular antigen - agglutinogen - is agglutinated when the specific antibody -
agglutinin - is added. Agglutination can be read either visually or in the microscope.
Presence or absence of clumping is noted.

a. Direct agglutination: It tests the presence of antibodies in the serum. It is used most
often as Widal reaction for diagnosing typhus and paratyphus. Direct agglutination is
also used for detecting antibodies in tularemia, brucellosis (Wright reaction),
listeriosis, rickettsial disease (Weil-Felix reaction).
b. Indirect agglutination: It tests the presence of unknown microbial antigen structure
with a diagnostic serum. Slide agglutination is most often used method for the
identification of the bacteria by means of specific diagnostic sera, especially in
enterobacteria. Specific antisera are prepared by immunization of animals with
bacterial strain.
c. Coagglutination: Specific immunoglobulin G (e.g. against Streptococcus spp.,
Haemophilus influenzae, Neisseria meningitidis) is bound with Fc fragment to protein
A which is present on the surface of bacterial cell wall of Staphylococcus aureus,
strain Cowan I. After addition of the antigen the clumps are formed on the slide within

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 1 minute. This reaction is used for identifying serological types of Streptococcus spp.,
Neisseria meningitidis and Haemophilus influenzae.
d. Direct haemagglutination: It is an agglutination of red blood cells.
Haemagglutinaytion can be caused by antierythrocytic antibodies, by several viruses
(e.g. myxoviruses, paramyxoviruses) and bacteria (Bordetella pertussis) which contain
antigen called haemagglutinin. Haemagglutination is caused after their binding on the
receptors present on the erythrocytal surface. Direct haemagglutination is used for the
diagnosing of infectious mononucleosis (Paul-Bunnel reaction), for the detection of
cold agglutinins in atypical pneumonia caused by Mycoplasma
pneumoniae (agglutination of human group 0 red blood cells at low temperatures).
e. Haemagglutination - inhibition test: It is used in virology. Many viruses agglutinate
red blood cells because of containing haemagglutinin. Since the process is specifically
inhibited by antibodies against the virus, haemagglutination inhibition can be used as a
test for identifying viruses and measuring antibodies. Disadvantage of this technique
include: time consuming, & subjective bias in the interpretation for results. Negative
results do not always indicate the absence of antibody. In some case false negative
results can occur from a low titer of antibody. Nonspecific inhibitors can cause false
positive results.
f. Indirect (passive) haemagglutination: Red blood cells which are first treated with
tannic acid or formalin are the carriers of soluble antigens. Antigen is either adsorbed
or bound on the erythrocyte surface. The method is sensitive and is used to detect
antibodies against enterobacteria, Clostridium tetani, Treponema pallidum (TPHA
test) and against some tissue antigens (e.g. thyroid gland antigens).
g. Latex agglutination: Inert latex particles are the carriers of antigens. There are many
commercial latex particle tests, e.g. for grouping streptococci, detecting bacterial
antigens from cerebrospinal fluid, for detecting rheumatoid factors, for detecting some
viruses from obtained samples (rotaviruses and adenoviruses from the stools). Also
antibodies against rubella can be detected by latex particle test.

II. Precipitation

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When a specific antibody - precipitin combines with a coloidal antigen-
precipitinogen in solution or in gel the antigen - antibody complex is thrown out of
solution - precipitate. The precipitate is most heavy in the equivalence zone, when
antigen and antibody are fully combined. In some tests optimum relation between antigen
and antibody must be kept up to carry out the reaction - the so
called flocculation. Flocculation test is used for the quantitative measurement of toxin,
toxoid or antitoxin. Precipitation reactions may be carried out in various ways:

a. Ring precipitation test: A solution of antigen is layered on the surface of the

antibody in a small tube or capillary tube. A narrow ring of precipitate occurs near
the junction of two fluids. The result can be read visually. The concentration of
immunoprecipitate is possible to be measured by this method or by laser
nephelometry. This type of test is used for grouping streptococci (according to C
polysaccharide), for determining unknown proteins in forensic medicine.
b. Slide precipitation: It is carried out on a slide and the occurrence of precipitate is
detected in the light microscope. This type of precipitation test is used for diagnosing
of lues (quick reagin reaction).
c. Gel - diffusion precipitation: Antigen and antibody meet in an agar medium and a
thin line of precipitate is produced there (antigen - antibody complex).
1. Single diffusion
Antigen diffuses in the agar medium (antibody is homogenously spread in the agar).
It is carried out either in the tubes - single gel - diffusion by Oudin or on the slide -
single radial immunodiffusion by Mancini.
The principle of the reaction: The antigen is placed in a well cut in an agar gel
containing suitable diluted antibody. A ring of precipitate forms where the reactants
meet in optimal proportions. The higher is the concentration of the examined
antigen, the greater is the diameter of the ring. According to the diameter of the ring
it is possible to count the concentration of the examined antigen.This type of
immunodiffusion is used for quantitative determination of immunoglobulins (IgM,
IgG, IgA and IgD), complement components and other serum proteins.
2. Double immunodiffusion by Ouchterlony

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 is used more often. Antigen and antibody are allowed to diffuse towards each other
in an agar medium, e.g. from separate wells cut in an agar plate or in a Petri dish.
When antigen and antibody meet in optimal proportions they produce a thin line of
precipitate. Position of the precipitate line depends on concentrations of both
antigen and antibody and on their diffusion coefficient. This reaction is used for
diagnosing various bacterial, viral, fungal and autoimmune diseases, for
recognizing toxin production by Corynebacterium diphtheriae.
d. Immunoelectrophoresis: It is a combination of electrophoresis and gel - diffusion
precipitation. Antigens (most usually serum proteins) are first divided by
electrophoresis according to their electric charge (albumins are directed towards the
anode and globulins towards the cathode) on an agar coated slide. After
electrophoresis is finished the longitudinal troughs are cut in the agar parallel to the
axis of electrophoresis and filled with antibody. Diffusion then takes place. When
antigen and antibody meet precipitate lines of single immunoglobulin classes occur.
The lines are read after staining by amidoblack dye. Immunoelectrophoresis is a
delicate technique for analyzing complicated mixtures of antigens and antibodies,
e.g. serum immunoglobulins.

e. Countercurrent immunoelectrophoresis: It is a rapid and more sensitive variant of

double diffusion method in which an electric current is used to drive the antigen
towards the antibody in negatively charged gel. This method was used to detect
hepatitis B surface antigen. It is used for the rapid detection of bacterial antigens in
clinical specimens, alfa-1-fetoprotein, etc. This method is being replaced by the
ELISA methods.

III. Complement Fixation Reaction

Complement fixation is a classic method for demonstrating the presence of antibody in

serum. This method consists of two components. The first component is an indicator
system consisting of a combination of sheep red cells; complementfixing antibody
produced against the sheep red cells in another animal, and an exogenous source of

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complement, usually guinea pig serum. When these three components are combined in an
optimal concentration, the antisheep cell antibody, hemolysin, can bind to the surface of
the red cells. Complement can subsequently bind to this antigen antibody complex and
cause cell lysis. The second component consists of a known antigen and patient serum,
which are added to a suspension of sheep erythrocytes, hemolysin, and a complement.

The two components of the complement fixation procedure are tested in sequence.
Patient serum is first added to the known antigen, and complement is added to the
solution. If the serum contains antibody to the antigen, the resulting antigen antibody
complexes will bind all of the complement. Sheep red cells and hemolysin are then
added. If complement has not been bound by an antigen antibody complex formed from
the patient serum and known antigen, it is available to bind to the indicator system of
indicates both a lack of antibody and a negative complement fixation test. If the patient’s
serum does contain a complement fixing antibody appositive result will be demonstrated
by the lack of haemolysis.

IV. Immunofluorescent Test (IFT)

The fluorescent techniques are extremely specific and sensitive. This technique consists
of labeling antibody with fluorescein isothiocyantate, a fluorescent compound with an
affinity for proteins to form a complex, conjugate. The fluorescent assay includes: direct
immunofluoresent assay and indirect immunofluoresent assay.
a. Direct Immunofluorescent assay: In this technique, Fluorescein- conjugated
antibody is used to detect antigen- antibody reactions. This method can be applied
to the detection of hepatitis B virus & chlamydia. A fluorescent microscope is
required to observe the production of color; fluorescein gives a yellow- green
light.
b. Indirect Immunofluorescent Assay (IFA): This method is based on the fact that
antibodies not only react with homologous antigens but can act as antigens and
react with antibody.

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In the indirect immunofluorescent assay, the antigen source to the specific antibody being
tested is fixed to the surface of a microscopic slide. The patent’s serum is diluted and
placed on the slide to cover the antigen source. If antibody is present in the serum, it will
bind to its specific antigen unbound antibody is then removed by washing the slide,
finally antihuman globulin conjugated to a fluorescent substance that will fluoresce when
exposed to a fluorescent substance that will fluoresce when exposed to ultraviolet light is
placed on the slide. This conjugated marker of human antibody will bind to the antibody
already bound to the antigen on the slide and will serve as a marker for the antibody
when viewed under a fluorescent microscope

V. Enzyme Immuno Assay (EIA)

An enzyme labeled antibody or enzyme labeled antigen conjugate is used in immunologic

assays for detection of antigens or antibodies, in a patient’s serum e.g. HIV antibody,
HIV antigen, hepatitis A antibody.

Various enzymes are employed in enzyme immunoassay. The most commonly used
enzymes are peroxidase and alkaline phosphatase. In EIA, a plastic bead or plastic plate
is coated with antigen. The antigen reacts with antibody in the patient serum. The bead or
plate is then incubated with an enzyme-labeled antibody conjugate, if antibody is present
on the bead or plate. The enzyme activity is measured spectrophotometrically after the
addition of the specific chromogenic substrate. Test result is calculated by comparing the
spectrophotometer reading of patient serum to that of a control serum.

VI. Radio Immunoassay (RIA)

In radioimmunoassay, radioisotopes can be used to measure the concentration of antigen

or antibody in serum sample. If antibody concentration is being measured, radioactive
labeled antibody competes with patient unlabeled antibody for binding sites on a known
amount of antigen. The main advantage of the radioimmunoassay method is the extreme
sensitivity and ability to detect trace amounts of antigen or antibody. In addition, a large
number of tests can be performed in a relatively short time period. The disadvantage is
the hazards and instability of isotopes.

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CHAPTER 9: FUNGAL CULTURE AND TESTS

Fungi are found in varied environments like tropical forests, oceans, and deserts. Various
fungi adapt to PH extremes and are found in hot and cold climates, although they are not
as heat or cold resistant as prokaryotes. The fungi are saprophytic (derives nourishment
from dead organic matter) and parasitic eukaryotic organisms. Although formerly
considered to be plants, they are now generally assigned their own kingdom, Mycota.
They resemble plants because they have rigid cell walls and are non-motile. Unlike
plants, the fungi lack chlorophyll and are unable to photosynthesize. The fungi also lack
the multicellular complexity and organization of most animals

The biological kingdom of the fungi is composed of approximately 70,000 known species
of fungi. Fewer than 300 species of fungi have been implicated directly as agents of
human or animal disease, and fewer than a dozen of these species cause about 90% of all
fungus infections. Opportunistic invasive fungal infections remain an important cause of
morbidity and mortality. Fungi have been implicated directly as agents of human or
animal disease, and fewer than a dozen of these species cause about 90% of all fungus
infections. Early and accurate diagnosis of these infections is important for several
reasons, including timely institution of anti-fungal therapy and to decrease the
unnecessary use of toxic antifungal agents.

Historically, the fungi were regarded as relatively insignificant causes of infection. It is

now well documented that the fungi are the common cause of infection, particularly in
immunocompromised patients. The most common fungi that cause disease in transplant
recipients and other immunocompromised patients are Candida and Aspergillus
species.4There are several approaches to manage these infections, including prevention,
anti-fungal prophylaxis, and empiric therapy. The first essential component of the
treatment is diagnosis. The diagnosis of deep seated Candidiasis is difficult and often
depends primarily on the clinical picture, with definitive diagnosis established by
histopathology of visceral tissues.

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9.1. Characteristic Features of Fungi

Fungi seen in the clinical laboratory can generally be separated into two groups based on
the appearance of colonies formed. The yeasts produce moist, creamy opaque or pasty
colonies on media, whereas the filamentous fungi or molds produce fluffy, cottony,
wooly or powdery colonies. Several pathogenic species of fungi that exhibit either yeast
(or yeast like) and filamentous form are referred to as being dimorphic. The dimorphism
is temperature dependent; the fungi are designated as thermally dimorphic.

Morphology/Structure of The Fungi: With the notable exception of yeasts, fungi

consist of masses of intertwined filaments of cells called hyphae. In many species of
fungi, individual cells are separated by cross-wall, or septa, such fungi are described as
septate. The septa are incomplete, however, and pores allow adjacent cytoplasm to mix.
In other fungal species, the cells have no septa, and the cytoplasm and organelles of
neighboring cells mingle freely. These fungi are said to be coenocytic. The common
bread mould Rhizopus stolonifer is coenocytic, while the blue-green mould that produces
penicillin, penicillim notatum, is septate.

The hypha is the morphological unit of fungus and is visible only with the aid of a
microscope. Hyphae have broad diversity of forms and many hyphae are highly branched
with reproductive structures. A thick mass of hyphae is called a mycelium. This mass is
usually large enough to be seen with the unaided eye, and generally it has a rough cottony
texture. The study of fungi is called mycology, and a person who studies fungi is called a
mycologist.

Yeasts: Yeasts are unicellular organisms that are round to oval and range in size from 2
to 60μm.The microscopic morphologic features usually appear similar for different
genera and are not particularly helpful in their separation. Fungi grow in two basic
morphologic forms, as yeasts or moulds.

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Molds (Moulds): The mold (or mould) form of growth refers to the production of
multicellular, filamentous colonies. These colonies consist basically of branching
cylindrical tubules varying in diameter from 2 to 10 am and termed hyphae. Hyphal
growth occurs by apical elongation. The mass of intertwined hyphae that accumulates
during active growth in the mold form is called a mycelium.

Molds are usually identified by observation of their morphology. Macroscopic

examination of a mold isolate should include notation of such characteristics as the rate
of growth, topography (e.g., glabrous, verrucose), surface texture (e.g., velvety, cottony,
powdery) and any pigmentation (surface, reverse, or diffusible into the medium).
Microscopically the types of spores or other reproductive structures (pigment, size, shape,
mode of attachment) and their ontogeny are characteristic for each species.

9.2. General Considerations for the Lab Diagnosis of Fungal Infections

There is remarkable change in the pattern of pathogens responsible for serious human
infections. The frequency of invasive fungal infections has risen dramatically in recent
years. Early and accurate diagnosis of these infections is important for several reasons,
including timely institution of anti-fungal therapy and to decrease the unnecessary use of
toxic antifungal agents. In addition the availability of accurate and timely diagnosis could
reduce the use of empirical anti-fungal therapy, thereby reducing antifungal resistance.

Collection and Transport of the Clinical Specimen: The diagnosis of fungal infections
is dependent entirely on the selection and collection of an appropriate clinical specimen
for culture. Many fungal infections are similar clinically to mycobacterial infections, and
often the same clinical specimen is cultured for both fungi and mycobacteria. It is
common for many years, such as Candida species to be recovered in routine bacteriology
media and fungal culture media.

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Specimens Include: Skin scrapings, Blood, Hair and nails, Respiratory tract secretions,
cerebrospinal spinal fluid, Mouth and vagina, Urine, Pus, Ocular specimen, Tissue and
Bone marrow.

9.3. Direct Examination of Clinical Specimen

Direct microscopic examination of fungal cells within the clinical specimen is a valuable
diagnostic procedure for the following reasons:

 In many instances, a tentative or even a definitive diagnosis can be made before the
growth of fungal cells would be apparent in culture.

 Observing fungal cells in a clinical specimen may be more valuable as a criterion for
diagnosis than isolating in a culture.

Preparations for direct examination of clinical specimen include KOH, India ink, and
Calcoflour white; in addition, a few staining techniques such as Giemsa and Periodic
acid-Schiff are effective.

9.4 Fungal Smear and Staining Technique

Wet Smears
Preparations With KOH (Unstained): Patches from the mucous membrane of the mouth,
vagina, skin, or nails scrapping, sputum etc are collected in a sterile container. These are
examined in a KOH wet mount or gram stain. Yeast cells of 4-8μm with budding mixed
pseudohyphae are seen. The presence of pseudohyphae shows colonization and tissue
invasion and so their demonstration is significant. For detection of Candida, wet smear
microscopy has been positive in the majority, but not in all cases with positive culture.

Stained Preparations

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Preparations with Potassium Hydroxide (KOH): The specimen should first be
examined microscopically for necrotic, purulent, bloody, or caseous areas. Because these
are the areas most likely to yield evidence of fungal growth, they are selected for direct
examination. Preparation with KOH clears the tissue and cellular debris from all types of
clinical specimens without damaging the fungal cells. This clearing process requires only
5 to 10 min, after which one can observe the fungal morphology as well as the pigment of
the fungal cell wall.

The disadvantage of using KOH stems from its reaction with pus, sputum, and skin; in
these instances, artifacts can be produced that superficially resemble hyphae or budding
forms of fungi. The number of these artifacts increases as time after preparation elapses,
and experience is required in interpreting the results. KOH can damage the microscope
stage if the slide overflows. In addition, crystals can form on standing so that reading of
smear becomes difficult.

The slide is examined under a phase-contrast or bright-field microscope, using low-power

followed by high-power objectives. With a bright-field microscope, one can detect
hyphae and yeast cells more readily by closing the aperture of the iris diaphragm to
reduce the intensity of light. Brown-walled hyphal cells can be detected without a
reduction in light.

To highlight the fungal cell walls, parker super chrome blue-black ink can be
incorporated into the KOH preparation. Adding the dye however will mask the cell-wall
pigment of dermatiaceous fungi.

Tentative diagnosis can be derived from the presence of fungal elements compatible to
the etiologic agents of aspergillosis, mucormycosis, dermatophytosis, candidiasis,
sporotrichosis, or cryptococcosis. To confirm such a diagnosis, however, cultural proof is
necessary.

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Preparations with Calcoflour White and KOH: Calcoflour White is used as a
whitening agent in the textile and paper industry. The dye is useful for demonstrating the
presence of fungal cells in clinical specimens because it binds to β 1-3, β 1-4
polysaccharides. The dye then fluoresces as it is exposed to the shorter wavelengths of
ultraviolet light. A Fluorescence microscope is needed for detecting fungal cells prepared
with Calcoflour White.

Yeast cells, pseudohyphae and hyphae display a chalk-white or brilliant apple-green

fluorescence, depending on filters used, that readily differentiates them from background
material. The disadvantages of using Calcoflour are the need for a Fluorescence
microscope, the inability of the dye to detect the endospores within a spherule of
Coccidioides immitis, and the difficulty in interpreting vaginal secretions.

Preparation with INdia Ink: India ink is useful for indicating the presence or absence of
extra cellular polysaccharide capsules of fungal cells. The technique is particularly
helpful for detecting Cryptococcus neoformans in CSF. Because India ink serves as a
negative stain, the encapsulated yeast cells can readily be detected against the dark back
ground. The ink should be free from artifacts or granular carbon particles to ensure a
good preparation.

The presence of encapsulated yeast cells in CSF in almost always an indicator of

Cryptococcal meningitis. Making the distinction between encapsulated yeasts and
artifacts requires considerable experience. Because tests for antigen also have false-
positive results, the tests can be used to complement one another. Every spinal fluid that
is India ink positive is also antigen positive, although the converse is not true. Antigen
may be detected in absence visible Cryptococci on India ink smear. There are reports of
Cryptococcal species other than neoformans in CSF.

Preparations With Periodic Acid-schif (PAS) Stain: The PAS stain one of the most
widely used stains for fungal histopathology. In a direct examination of clinical
specimen, PAS stain is sometimes used when a KOH preparation do not reveal fungi that
are suspected to be present.

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PAS preparation requires 20 to 25 minutes depending on the weather counterstaining is
employed. Light green is preferred as a counter stain because the fungus appears deep
purplish red against the contrasting background color. For the counter staining process
the slides should be placed in light green stain for 5 sec and washed for 5 to 10 sec
between steps.

The PAS reaction stains certain polysaccharides found in the fungal cell wall. For good
results, both the periodic acid solution and the sodium Meta bisulphate solution should be
fresh and protected from light. For laboratories with a fluorescent microscope, calcoflour
white is preferred over PAS.

Acid-Fast Stain Procedure for Nocardia (modified Kinyoun Method):

Acid-fast staining is useful for detecting Nocardia species and for differentiating them
from other aerobic actinomycetes. Some of the filaments stain red with carbol- fuschin
staining, while others may appear blue because of counterstaining effect. The slides are
then examined under a bright field microscope with oil immersion objective. Viewing
tissue homogenate specimens with this method is difficult because of background
staining.

Grams's Stain: It is effective for some pathogens but not for others. In general, the
procedure is more suited to sections than smears.

Gormori's Methenamine Silver Stain (Grocott's Modification):

It is based on the liberation of aldehyde groups and their subsequent identification by the
reduced silver method. It is used for demonstration of polysaccharide content on the
fungus in tissue sections. The aldehydes reduce the methenemine silver nitrate complex,

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resulting in brown black staining fungal cell wall due to the deposition of reduced silver
wherever aldehydes are located.

9.5 Culture Medias and Culturing technique

All fungi require several specific elements for growth and reproduction. The
requirements for growth are generally less stringent than for sporulation, so it is often
necessary to try several types of media when attempting to identify a fungus in culture.

Common media for primary fungal isolation include Sabouraud dextrose agar and brain-
heart infusion agar, either in petri dishes or screwtop tubes. The media may be enriched
with 5% to 10% sheep blood to support the growth of certain fungi. Specimens that are
likely to be contaminated with other microorganisms, such as urine or sputum, are set up
on agar media containing antimicrobials. Chloramphenicol, streptomycin, or penicillin
are incorporated into the agar to inhibit the growth of bacteria, and cycloheximide is used
to inhibit the growth of contaminant.

Most fungi also thrive on Potato Dextrose Agar (PDA), but this can be too rich for many
fungi, so that excessive mycelial growth is obtained at the expense of sporulation.

Constituents of Media: Media generally contain a source of carbon, nitrogen and

vitamins. Glucose (dextrose) is the most widely utilizable carbon source, and hence is the
most commonly used in growth media. Fructose and mannose are the next most
commonly utilized sugars by fungi and are found in media from natural sources. Sucrose
(table sugar) may be used in some media. Nitrogen sources include peptone, yeast
extract, malt extract, amino acids, ammonium and nitrate compounds. Casamino Acids, a
Difco product, is acid-hydrolyzed casein, a mixture of amino acids. It is a good general
source of nitrogen but is vitamin-free. Bacto-Peptone, another Difco product, contains
nitrogen and a high peptone and amino acid content. Salts, including Fe, Zn and Mn, are
often added to ‘defined’ media, but are usually not added to the common media used for
routine culture. Fungi have natural deficiencies for vitamins that are satisfied at mM to

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nM concentrations. The most common naturally occurring vitamin deficiencies are
thiamin and biotin. Deficiency of both is quite common among the Ascomycota. Other
organic nutrients such as glucose are often contaminated with vitamins sufficient to
supply the growth requirements of fungi.

Culture media used for fungal growth: The most commonly used cultures media used
for fungal growth are as follows: Sabouraud agar, Hay infusion agar, Potato dextrose
agar, Potato Dextrose Broth, Yeast Agar, Yeast Broth, Mycological Agar, Malt extract
agar, Malt Extract Broth, Mycological Agar, Soy Peptone Yeast Extract Agar, Water
Agar (WA), Antibiotic Agar (AA), Acidified Cornmeal Agar (ACMA), Cornmeal Agar
(CMA) and Potato Carrot Agar (PCA).

Special Tests:

Chlamydospore Formation For Candida

 Inoculate suspected Candida on plates or slides with Chlamydospore agar. Inoculate
by cutting angles into medium. Known culture of Candida is used as culture, and
multiple cultures can be tested on the plate.

 Incubate at room temperature for 48 to 72 hrs. Examine slides, plates or slide mounts
from plates, under microscope for presence of Chlamydospore and characteristic
growth of mycelia. C albicans produces abundant chlamydospores in the lateral
terminal.

 Inoculate as cut streaks on Eosin Methylene Blue Agar (EMBA) plates and incubate
at room temperature for 24 to 48 hrs 10.

Germ Tube Formation (Reynauld-Braude Phenomenon)

Germ Tube Formation (Reynauld-Braude Phenomenon). The serum germ tube test is a
rapid “presumptive test” for C. albicans. A light inoculum of cultured yeast is incubated
in bovine serum for 2-3 hours at 37oC. The test is positive if there are short hyphae
without a constriction where the hypha joins the parent cell. Not all C. albicans isolates

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are germ tube positive and false-positive results are a possibility, especially with C.
tropicalis. Carbohydrate assimilation tests are commercially available. Rapid
identification tests based on preformed enzymes are particularly good for confirmation of
a germ-tube positive organism as C. albicans, but C. dubliniensis is positive too.

Principle: Strains of C. albicans produce germ tubes from their yeast cells when placed
in a liquid nutrient environment and incubated at 35° C for 3 hours 16 Characteristics
 It is one of the most common phenomenons in all the kingdom fungi. However,
this occurs over the course of 1 to 3 hrs for Candida albicans, so used as rapid
presumptive identification test.

 It is accepted method for rapid identification for C. albicans but it is recognized

that sensitivity and specificity is not absolute.

 It is a simple, efficient and economic test for screening and identification of C.

albicans.

 True germ tube is defined as hyphal projections from the germinating yeast cell,
lacking any constriction at the point of origin.

 Inoculate lightly the test strain of Candida and culture (control) into 2 separate
tubes containing 0.5 ml each of same batch of human serum. Incubate at 37°C.
Examine first the control C. albicans for satisfactory formation of germ tubes, this
is seen as an outgrowth from the cell with no constriction at the base resembling a
ladies hand mirror. The width of the outgrowth is less than half the width of the
yeast and length of the outgrowth 2-3 times that of the yeasts cell.

 Old cultures, heavy inoculum and cultures from SAB can give negative results.

 It is important to read the test within 3 hrs as other Candidal species will also
form germ tubes in serum after this time period

 In conclusion germ tube appears to be the cheapest test, but it is time consuming
and laborious.

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Adhesive (scotch Tape Method)

 Touch the adhesive side of a small length of transparent tape to the surface of the
colony.

 Adhere the length of tape to the surface of a microscope slide to which a drop of
lactophenol cotton or aniline blue has been added.

 Observe microscopically for the characteristic shape and arrangement of the spores.
The transparent adhesive tape preparation allows one to observe the organism
microscopically approximately the way it sporulates in culture. The Spores are usually
intact, and the microscopic identification of an organism can be made easily. Some
laboratories prefer to use the microslide culture for making the microscopic identification
of an organism.

Biochemical Tests
A. Carbohydrate Fermentation

Yeasts are able to metabolize some foods, but not others. In order for an organism to
make use of a potential source of food, it must be capable of transporting the food into its
cells. It must also have the proper enzymes capable of breaking the food’s chemical
bonds in a useful way. Sugars are vital to all living organisms. Yeast is capable of using
some, but not all sugars as a food source. Yeast can metabolize sugar in two ways,
aerobically, with the aid of oxygen, or anaerobically, without oxygen. 21

The fermentation of glucose can be described by the following equation:

C6Hl2O6 2 CH3CH2OH + 2 CO2 + energy

Glucose ethyl alcohol carbon dioxide.

B. Carbohydrate Assimilation

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 This test is used for definite speciation of Candida and few other fungal micro
organosms. Carbohydrate assimilation test (Modified Wickerham Method):- The
carbohydrates used are Glucose, Maltose, Lactose, Sucrose, Galactose, Xylose,
Trehalose, Cellibiose.

Rapid Urease Test: This test is used to detect presence of urease enzyme produced by
different Candida species. Christensens urea agar slants are used. Conversion of the
yellow slope to pink or red is considered positive. A negative test is reported when there
is no color change observed.

Other Techniques:

 Microslide Culture

 Hair Preformation Test

 In Vitro COnversion Of Dimorphic Molds

 Exo-Antigen Test

 Nucleic Acid Probe Testing

Newer Diagnostic Techniques:

 CT Scanning MRI Techniques

 Nuclear Magnetic Resonance (NMR)

 Immundiagnostic Techniques

 PCR Techniques

 Serologic Test

 Latex Agglutination Test

 Immunfluoresce

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CHAPTER 10: CULTIVATION OF VIRUSES

Specimens for Viral Diagnosis

The likelihood of making a specific viral diagnosis depends largely on the quality of the
specimen that is received in the laboratory. Important variables include the timing of the
specimen in relation to the patient's illness, the type of specimen, the quality and amount
of specimen material obtained, and the time and conditions of transport to the laboratory.

TABLE 17.1 Instructions for obtaining and transporting specimens for viral culture

SpecimenInstructions
Blood Usual volume required is 3-10 mL. The appropriate tube is determined by the test to be
performed, the blood component on which the test is performed (whole blood,
leukocytes (18), plasma, or serum), and the preference of the laboratory. Whole blood,
leukocytes, and plasma require collection into a tube containing an anticoagulant. The
most commonly used anticoagulants are ethylenediaminetetraacetic acid
(EDTA_(purple top tube), heparin (green top tube), acid-citrate-dextrose (yellow top
tube). Note: Heparin is inhibitory for some polymerase chain reaction (PCR)
procedures, especially those performed on plasma. Serum specimens require collection
into a tube that does not contain an anticoagulant (red top tube). Blood specimens do
not have to be placed on ice if transport requires less than 1 day.
Swab Swabs with Dacron or rayon tips are preferred. If available, swabs designed specifically
for collection of viral specimens should be used. Swabs can also be placed in viral
transport media. In this case, the shaft of the swab may have to be cut to allow the top
of the transport vial to be securely closed. The swab or tube of transport media should
be placed on ice if transport will require more than 1 hour.
Fluid Place in a sterile container.
Tissue Place in a sterile container with a small amount of viral transport media or sterile saline
or phosphate-buffered saline to maintain moisture.
Stool Obtain at least 4 g of stool. Place in a clean or sterile container.
Specimens other than blood should be transported on ice if more than 1 hour is required for
arrival in the laboratory. If a delay of more than 24 hours is anticipated, specimens should be

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frozen at -70°C and transported on dry ice. Note: Some viruses such as respiratory syncytial
virus (RSV) and CMV are unlikely to retain viability with freezing. Specimens should not be
frozen at -20°C.

10.1. Cultivation of Viruses in Cell Cultures

Virus Detection
Principle:
A determination of whether a causal relationship exists between the virus and the illness
requires consideration of several factors. Two of the most important are (a) the nature of
the virus-host interaction and (b) whether the virus is known to cause the disease
manifestations that the patient is experiencing. If the virus is one that is known only to
cause acute infection, detection of that virus is likely to be associated with a current
illness.

10.1.1 Methods Used in Diagnostic Virology: Viral Culture

Viral culture is the procedure that sets diagnostic virology apart from other areas of
diagnostic microbiology. Because viruses require cellular machinery for replication,
living systems must be used. Historically, inoculation of animals and eggs was used to
cultivate and propagate viruses, but this technique has been supplanted by cell culture in
virtually all clinical laboratories. Viral culture has important advantages and
disadvantages compared with other diagnostic methods. By its very nature, culture is an
amplification method that increases the amount of the putative pathogen, facilitating
detection and characterization. Viral culture is unique among detection methods in that it
provides an isolate of viable virus that can be further characterized as necessary, and can
be stored for future studies. Another important feature is that culture methods allow the
detection of many different viruses, including ones not suspected at the time the culture is
established. Even viruses not previously known can be discovered. This is in contrast to
immunologic or nucleic acid-based diagnostic tests, which ordinarily detect only the

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specific virus to which the diagnostic reagent is directed. Viral culture also has significant
disadvantages as a diagnostic method, including requirement for specialized facilities and
expertise, expense, relatively prolonged time to detection, and the relatively limited range
of viruses that can be detected.

Growth of Cell Cultures

Cells are grown in the laboratory using the appropriate nutrient media, temperature, and
atmosphere of incubation. A number of defined basal media are commercially available,
and are often supplemented with additives such as glutamine, fetal bovine serum, and
antibiotics to counteract bacterial or fungal contamination. Most cell cultures used in
diagnostic virology laboratories grow as cell monolayers that are adherent to glass or
plastic surfaces. Most commonly, cultures are planted in individual glass tubes, but other
vessels, including plastic flasks and microtiter trays, can be used. Some cell culture types
do not adhere to surfaces and are grown as suspension cultures. No one type of cell
culture can grow all medically important viruses and, therefore, diagnostic virology
laboratories must use several different types of cell culture. Cell cultures can be
maintained and propagated within the laboratory or, alternatively, can be purchased from
commercial suppliers who maintain supplies of a wide variety of cell culture types.

Types of Cell Culture

Cell cultures can be divided into four distinct categories. Primary cell cultures are derived
directly from the source animal. Examples include primary monkey kidney cells and
primary rabbit kidney cells. Viruses endogenous to the source animals occasionally can
be propagated in these cells, and care must be taken to distinguish these viruses from
viruses derived from the clinical specimen. Diploidor or semicontinuous cells are capable
of a limited number of passages before undergoing senescence. The most widely used
semicontinuous cells are human fibroblast cell cultures such as MRC-5 and WI-38 cells.
Continuous or transformed cell lines are immortalized cells that can be passaged without
limit. Examples include HEp-2, HeLa, A549, and Madin-Darby canine kidney cells.

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Hematopoietic cells, such as peripheral blood mononuclear cells or umbilical cord
mononuclear cells, are required for the growth of viruses such as EBV, HHV, and HIV.
These cultures are grown in suspension and must be set up from the source material
within a few days before inoculation of the specimen. Although necessary for growth of
the viruses mentioned, hematopoietic cell culture systems are not used routinely in most
diagnostic virology laboratories. Table 17.2 lists commonly used cell cultures and the
medically important viruses that grow in those cultures.
Determining which cells to use for a given specimen is based on the viruses likely to be
present in that specimen. Thus, communication between clinicians and the laboratory is
essential to maximize yield. When specific communication does not take place, the
laboratory staff select types of cell cultures based on the viruses likely to be present in a
given specimen type.

10.1.2 Specimen Processing and Inoculation for Isolation of Virus

Performing a viral culture requires the following steps: processing of the specimen and
inoculation onto the cell culture, maintenance of the inoculated cell culture, and detection
of viral growth. Normally, sterile body fluids (e.g., CSF) can be inoculated directly onto
cell cultures. Urine specimens should have the pH adjusted toward neutrality before
inoculation. Specimens from body sites typically contaminated with bacteria (e.g.,
respiratory or genital specimens) are treated with antibiotics before inoculation to prevent
bacterial or fungal overgrowth. After inoculation, cultures are incubated at 35 °C to 37 °C
and inspected periodically (e.g., once daily or every other day). Respiratory cultures
directed at detection of influenza or rhinoviruses can be incubated at 33 °C.

TABLE 17.2 Cell culture types used to detect medically important viruses

Influenza Viruses, Parainfluenza Viruses,

Primary Monkey kidney Enteroviruses
Rabbit kidney Herpes simplex virus

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 Human embryonic kidney Adenovirus, enteroviruses
Diploid Fibroblasts (e.g. MRC-5,CMV, VZV, HSV, rhinovirus, enteroviruses
Wi38) (some), adenovirus, RSV
Continuous HEp-2 RSV, adenovirus, HSV, parainfluenza viruses
(some), enteroviruses (some)
A549 HSV, adenovirus, enteroviruses
MDCK Influenzaviruses
LLC-MK2 Parainfluenza viruses, HMPV
Rhabdosarcoma (RD) Echoviruses
Buffalo green monkey Coxsackievirus
CMV, cytomegalovirus; HMPV, human metapneumovirus; HSV, herpes simplex virus;
RSV, respiratory syncytial virus; VZV, varicella zoster virus.

Detection of Viral Replication

Cytopathic Effect: The growth of viruses in cell culture is often (but not always)
associated with microscopically visible morphologic changes in the infected cells,
referred to as cytopathic effect (CPE). The features of the CPE may allow recognition of
the infecting virus. Important characteristics of CPE include (a) which cell culture types
are affected, (b) the timing and rate of progression, (c) the distribution (focal, diffuse,
limited to the margins of the cell culture), and (d) the nature of the morphologic changes.
Several examples of CPE caused by common viruses are shown in Figure 17.1. The CPE
may be sufficiently distinctive to allow unequivocal identification of the virus, but in
other cases, techniques such as FA staining performed on cells scraped from the cell
culture, are used to confirm the presence of the suspected virus.

Hemadsorption: Paramyxoviruses, including influenza, parainfluenza, and mumps, may

produce only minimal CPE and, instead, are routinely detected by testing the cultures for
their ability to bind red blood cells, a process termed hemadsorption. This occurs because
certain viruses express on the surface of infected cells viral proteins that bind to
erythrocyte membranes. The binding is specific for the erythrocytes of certain species,

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such as guinea pig, rat, or monkey. To test for hemadsorption, a suspension of guinea pig
erythrocytes is introduced into the cell culture previously inoculated with a clinical
specimen. After a 30-minute incubation period, the culture is inspected microscopically
to detect attachment of the erythrocytes onto the cells in culture. If hemadsorption is
detected, the causative virus can be identified by FA staining of cells from the culture.

Interference: The interference assay is based on the phenomenon that a cell culture
infected by one virus may be rendered resistant to infection by other viruses to which it
was originally sensitive. To carry out this assay, a culture previously inoculated with a
specimen is challenged with a test virus to which it is ordinarily susceptible. A
companion culture of the same type that was not inoculated with a clinical specimen is
challenged simultaneously to demonstrate viability of the challenge virus. Interference is
present if the challenge virus grows in the companion tube but does not grow in the tube
inoculated with the clinical specimen. When interference is demonstrated, the virus
responsible can be identified by FA staining of cells from the culture.

Antigen or Nucleic Acid Detection: When growth of a specific virus is suspected by

virtue of the CPE it provokes or by the presence of hemadsorption, the presence of that
virus can be specifically confirmed by antigen detection techniques such as FA staining
using virus-specific antibodies or by nucleic acid detection using techniques such as PCR
with specific probes and primers.

Electron Microscopy: Viruses can be demonstrated in diagnostic specimens by electron

microscopy. This method can be used not only to recognize mixed viral infections but
also to detect viruses which cannot be grown in vitro. When growth of a virus is
suspected, but the identity is unknown or specific reagents are unavailable, material from
the culture can also be evaluated by electron microscopy. Visualization of viral particles
confirms viral growth, and a description of the nature of the particles may provide an
indication of the virus family. This technique was used in the discovery of the severe
acute respiratory syndrome (SARS) coronavirus during the outbreak of SARS in Hong
Kong in 2003.

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A number of methods are used for the preparation of samples for examination. When
preparing fluid samples, low speed centrifugation to remove large particulate debris is
followed by ultracentrifugation to sediment virus particles. Negative staining with heavy
metal compounds such as phosphotungstic acid or uranyl acetate increases contrast so
that the brighter virions stand out against a dark background. The addition of antiserum to
the specimen in immmunoelectron microscopy enhances the sensitivity of the procedure
by clumping virus particles and facilitating their recovery by centrifugation.
Alternatively, antiserum, when applied to the copper grid used for examining the
specimen, can aggregate virions in the specimen.

For diagnosis of viral infection the common serological tests like ELISA, CFT, HAI, IFT
and SNT can be used. Additionally molecular tests like PCR, RT-PCR, Western blotting
(immunoblotting) and other hybridization methods can also be used. Some of the
commonly used diagnostic techniques are described below.

Serum neutralization test

This test is highly specific and sensitive for viruses which produce cytopathic effects
(CPE). It is considered to be the definitive standard against which other serological tests
are compared. Neutralizing antibodies usually correlate closely with immune protection.
In this test, which is typically carried out in microtitre plates, a constant amount of stock
virus is added to doubling dilutions of a test serum. Cells susceptible to the virus are
added to the wells. The presence of neutralizing antihodies in the serum prevents
infection of the cells and CPE. The titre of the serum is the highest dilution at which the
virus is neutralized. The neutralizing effect of test serum can also be evaluated in
susceptible experimental animals and in chick embryos. Neutralizing antibodies tend to
persist in recovered animals for long periods, often for many years.

10.2 Cultivation of Viruses in Natural Hosts and Laboratory Animals

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CHAPTER 11: REFERENCES

Cain D., Hanks H., Weis M., Bottoms C and Lawson J. (2013): Microbiology Laboratory
Manual (Biol 2421L). Collin County Community College.
Castro, A. E and Heuschele, W. P. (1992): Veterinary diagnostic virology, A practioner’s
guide. St. Louis, MO: Mosby-Year Book, Inc.
Microbiology Laboratory Manual (Bio 204): Morgan Community College.
OIE (2008): Office International des Epizooties: Terrestrial Manual on Biosafety and
Biosecurity: In the Veterinary Microbiology Laboratory and Animal Facilities.
WHO (2004): Laboratory biosafety manual, 3rd eds., World Health Organization Geneva.

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