HAWASSA UNIVERSITY

SCHOOL OF VETERINARY MEDICINE

VETERINARY MICROBIOLOGY I (Vet M 4081) LABORATORY

MANUAL

Mesele Abera (DVM, MSc)

 TABLE OF CONTENT

PAGE

1. SAFETY PROCEDURES IN MICROBIOLOGY LABORATORIES .......................... 1

2. INSTRUMENTS AND EQUIPMENTS IN MICROBIOLOGY LABORATORIES ..... 2
3. STERILIZATION AND DISINFECTION.................................................................... 3
3.1. Methods of sterilization .......................................................................................... 3
4. BACTERIOLOGICAL MEDIA PREPARATION ........................................................ 5
4.1 Types of bacteriological Media .............................................................................. 5
4.2 Preparation of dehydrated artificial media .............................................................. 6
5. INOCULATION OF CULTURE MEDIA .................................................................... 9
5.1. Streaking the agar plates ......................................................................................... 9
5.2. Inoculating slants ................................................................................................. 11
6. BACTERIAL STAINING .......................................................................................... 12
6.1. Preparation of bacterial smears ............................................................................. 12
6.2. Fixing the smears ................................................................................................. 12
6.3. Staining the smears .............................................................................................. 13
6.3.1. Simple staining.............................................................................................. 13
6.3.2. Differential staining ...................................................................................... 13
7. KOH test .................................................................................................................... 17
8. PRIMARY IDENTIFICATION OF BACTERIA ........................................................ 18
8.1. Gram staining ....................................................................................................... 18
8.2. Growth on MacConkey agar ................................................................................. 18
8.3. Motility test .......................................................................................................... 18
8.4. Catalase test ......................................................................................................... 19
8.5. Oxidase test .......................................................................................................... 20
8.6. Oxidation fermentation (O-F) test......................................................................... 20
8.7. Coagulase test ...................................................................................................... 24
9. IMViC test.................................................................................................................. 25
10. TRIPLE SUGAR IRON (TSI) REACTION ............................................................ 27
11. CAMP test .............................................................................................................. 29
12. ANTIMICROBIAL SUSCEPTIBILITY TESTING ................................................ 30

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 PRACTICAL No. 1

1. SAFETY PROCEDURES IN MICROBIOLOGY LABORATORIES

1. Wear protective clothing such as a white coat/gown when working in the laboratory
2. Eating, drinking, smoking or storage of food in the laboratory should not be
permitted where infectious material is used or stored.
3. Avoid mouth pipetting and licking of labels
4. All work should be done on the laboratory table in front of the Bunsen burner and
turn down the Bunsen burner when not in use.
5. Sterilize the inoculating needles before and after use in the Bunsen burner.
6. Petri-dishes containing agar should always be closed on the bench and placed with
the lid down wards.
7. Keep test tubes upright on the rack.
8. Use the microscope properly and leave clean after use.
9. Reagents, stains and laboratory materials should be placed to their original position.
10. Spillages of cultures must be reported immediately to the teacher or technician to be
dealt with quickly and must not be touched with unprotected hands.
11. Observe an appropriate disposal procedure for broken glass if present, if not report to
the teacher or technician in charge.
12. Contaminated clothing should be soaked in disinfectant. Splashes on the skin should
be treated as soon as possible; washing with soap and hot water should be sufficient,
but if necessary the skin can be disinfected.
13. Aerosols: Spillages also carry a risk of generating aerosols (an invisible “mist” of
small droplets of moisture) which may contain microbes and might be inhaled. The
risk of spillages occurring is lessened by using cultures grown on agar instead of in
liquid media whenever possible. Care should also be taken to avoid generating
aerosols during practical work. So please do not inhale culture closely.

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2. INSTRUMENTS AND EQUIPMENTS IN MICROBIOLOGY LABORATORIES

Anaerobic jar
Autoclave
Beaker
Bunsen burner
Flask
Hot air oven
Incubator
Inoculating loop
Inoculating needle
Magnetic stirrer, hot plate
Measuring cylinder (Graduated cylinder)
Microscope
Petri dish
Refrigerator
Safety cabinet
Sensitive balance
Spatula
Test tubes
Water bath
Waterproof marker pens

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 PRACTICAL No. 2

3. STERILIZATION AND DISINFECTION

Sterilization means the complete destruction of all the microorganisms including spores,
from an object or an environment. The criterion of sterilization is the failure of the organism
to grow if a growth-supporting medium is supplied. The limiting requirement of sterilization
is destruction of the bacterial spore.

Disinfection involves the destruction of pathogenic organisms associated with inanimate

objects, usually by physical or chemical means. Specific disinfectants at the specified
working strengths are used for specific purposes. The most commonly used disinfectants are
0.25 % chlorine (as sodium hypochlorate) and 70 % ethanol or methylated spirit.

Antisepsis involves the inactivation or destruction by chemical means of microbes associated

with the animal. Antiseptic agents may be bacteriocidal or bacteriostatic.

3.1. Methods of sterilization

1. Autoclave / pressure cooker

The principle of sterilization in an autoclave is the steam under pressure is used to produce a
temperature of 1210C which if held for 15 minutes will kill all microorganisms including
bacterial endospores. Materials that do not resist dry heat such as rubber equipments, plastic
materials, most media etc. are sterilized using this method.

2. Dry heat / Hot air oven

Hot air oven uses a very high temperature (160 to 180 oC) for one hour. Used for sterilization
of flasks, Petri dishes, test tubes and pipettes placed in pipette canisters.

3. Filtration
Filters of very small size are utilized to sterilize substances such as sera, toxins, vitamins,
antibiotic preparations and other labile substances such as sugars.

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4. Tyndalization (steaming)
This process has been replaced largely by filtration. It involves placing the media or
solutions in flowing steam for 1 hour on each of three successive days. The period between
steaming allows the spores to germinate and thus to be killed at the next exposure.
Tyndalization requires a medium or a solution that is sufficiently nutritious to promote
germination of spores in the intervals between steaming. Bacteriological media qualify in
this respect.

5. Irradiation
Rays such as ultraviolet, gamma rays and X- rays damage microorganisms affecting the cell
membranes, essential enzymes or the nucleic acids.

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 PRACTICAL No. 3

4. BACTERIOLOGICAL MEDIA PREPARATION

In the laboratory, bacteria are grown in culture media, designed to provide all essential
nutrients in solution of bacterial growth.
Culture media is any material, solid or liquid that can support the growth of microorganisms.
For bacteriology, culture media can be purchased as dehydrated powder.

Ingredients of bacterial culture media

 Distilled water
 Nutrient that support the growth of bacteria e.g. peptone
 Agar, a solidifying agent
 Buffer, substance added to media to maintain the PH at a suitable range.
Phosphates are usually used as buffer.
 Growth factors, materials that enhance the growth of fastidious organisms. This
includes vitamins, blood, and serum.

4.1 Types of bacteriological Media

Culture media may be classified into several categories depending on their composition or
use.

1. Basic nutritive media: - These are capable of sustaining growth of the less fastidious
bacteria e.g. Nutrient agar.
Nutrient media typically contain peptone, salt, dextrose, water, meat extract, and a
solidifying agent, such as agar or gelatin.
2. Enriched media: - Enriched media are formulated to meet the requirements of the most
fastidious pathogens. They are essentially basic nutrient media with extra nutrients
added, such as blood, serum or egg. Examples include blood agar and chocolate agar.
3. Enrichment media: - Liquid media that favour the growth of a particular group of
organisms. They either contain nutrients that encourage growth of the desired organisms
or contain inhibitory substances that suppress competitors. Examples include
tetrathionate broth and selenite broth.

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4. Selective media: - Is one which has a component added to it which will inhibit or
prevent the growth of certain types or species of bacteria and / or promote the growth of
desired species. These agar media have been made selective for the growth of a
particular bacterium or group of bacteria and are used extensively in diagnostic
bacteriology. They contain inhibitory substances that prevent the growth of unwanted
bacterial species. Many selective media, such as brilliant green and MacConkey agars,
can also be described as indicator media.

MacConkey agar contains the fermentable sugars lactose and neutral red a PH indicator.
Bacteria such as E. coli that ferment lactose produce acid metabolites that change the
colonies and surrounding medium to a pink colour. Salmonella that cannot ferment
lactose will use the peptone in the medium with the production of alkaline metabolic
products and appear as pale colonies.

5. Indicator media: - These are particularly useful in diagnostic bacteriology. They are
designed to give a presumptive identification of bacterial colonies due to the
biochemical reaction in the media. Indicator media often contain fermentable sugars
plus a PH indicator that gives a colour change to the media. Examples of indicator
media include XLD agar (hydrogen sulphide production), Edward's media (aesculin
hydrolysis), Blood agar (type of haemolysis) etc.

6. Chemically defined media: - Is a one in which the exact chemical composition is

known. They are used mainly for experimental purposes but citrate broth is an example
of a chemically defined medium that is used in diagnostic bacteriology.

4.2 Preparation of dehydrated artificial media

 The manufacturer's instructions should always be followed

 Use clean glassware rinsed free of detergents and other chemicals. The glassware
need not be sterile unless sterile medium being decanted into it.
 Prepare the medium in a container about twice the final volume of the medium. This
allows adequate mixing and allows for frothing of the medium, during heating.
 Weigh out the appropriate amount of dehydrated medium, place in a flask, and add
distilled water.

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  Media not containing agar can usually be dissolved with gentle agitation, but
dehydrated media containing agar is best dissolved by bringing to the boil with
continuous shaking or stirring using a glass rod or a hot plate that incorporates a
magnetic stirrer system. Caking at the bottom of the flask may occur if stirring is not
adequate.
 Once dissolved, dehydrated media are usually sterilized in an autoclave at 121 oC for
15 minutes. Brilliant green agar is inhibitory to many bacteria and is brought to the
boil only and not autoclaved.
 Agar media should be cooled, preferably in a water bath to 50oC before the plates are
filled. Remember that agar solidifies at 42oC.
 Some additives may not tolerate boiling or autoclaving and so must be added after
the media have been autoclaved and cooled to 50oC. These additives must, of course,
be sterile. Blood and serum are collected aseptically, while certain dyes and sugars
are sterilized by filtration.
 Pour in sterile plates near Bunsen flame. The standard (90 mm) Petri dish should
contain about 15 ml of agar medium, about one-third full. Thus 1 litre of medium
should yield 60 to 70 plates.
 After the poured plates are set, allow them to dry at room temperature or at 37 oC for
a few hours, to ensure that there is no surface moisture when you use them. The
plates are stored, agar-side upwards, in a refrigerator at 4oC.

Agar slants are prepared in a test tubes or universal bottles by allowing sterile molten cooled
medium to solidify in a sloped position.

Preparation of blood agar plates

 The blood agar base is prepared from dehydrated powder and sterilized in
autoclave at 121oC for 15 minutes.
 After the blood agar base has cooled to 50oC, sterile blood at the rate of 5 - 10 %
vol/vol is added and mixed well before the plates are poured. If the sterile blood
has been stored in the refrigerator, it should be warmed to 37oC before being added
to the agar medium to avoid thermal shock to the red cells.
 If bubbles form on the surface of the poured plates, a low Bunsen flame is quickly
passed across the surface of the agar before the agar sets.

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Collecting sterile blood

Bovine or ovine blood is most suitable for veterinary bacteriology. Sterile blood can be
collected from young animal that has no evidence of antibodies to major veterinary
pathogens and has not been treated with antibacterial agents.
A strict aseptic technique must be used. The area over the jugular vein is clipped and shaved.
The area is then cleaned with 70 % ethyl alcohol and allowed to dry through before
venepuncture.

To prevent the blood from clotting one of the following methods may be used:
 Collection into purchased human blood-donor kit
 Collection into a pre-sterilized apparatus consisting of a tube leading into a
conical flask containing glass beads (3 mm). The flask is agitated continuously
during collection and for at least for 5 minutes after obtaining the blood. This
defibrinated blood can be decanted into sterile bottles for storage in a
refrigerator. The glass beads can be recovered from the fibrin clot and reused.
 A sterile anticoagulant solution, such as 0.2 % sodium citrate, can be used either
in a flask, in the place of the glass beads or blood can be collected in a sterile
syringe and immediately added to the anticoagulum.

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 PRACTICAL No. 4

5. INOCULATION OF CULTURE MEDIA

If the specimen is a piece of tissue, it is easier to manipulate if it is first placed in an empty

sterile Petri dish. Forceps and scalpel, previously held in 70% ethyl alcohol, are flamed and
allowed to cool. The tissue is scraped, paying particular attention to the edge of an
observable lesion. A little of the tissue scraping is placed at the edge or 'well' of each culture
plate to be inoculated.
When the specimen is liquid or semi-liquid it is best applied to the well of the plate using a
sterile swab.
When either inoculating or streaking culture media plates it is preferable to start with the
non-inhibitory medium, such as blood agar, and then inoculate or streak any inhibitory or
selective medium such as MacConkey agar.

5.1. Streaking the agar plates

The objective of plate streaking is to obtain isolated bacterial colonies that are required for
observing colonial morphology, antibiotic sensitivity testing and for biochemical
identification. The quadrant streak method, using the whole plate, is usually employed for
most diagnostic specimens.
The plate is first labelled, on the agar side, with the type of specimen, date of inoculation and
reference number using a waterproof marker pen. The writing should be kept as near to the
edge of the plate as possible, so that after incubation the bacterial colonies are not obscured.
Two inoculating loops are used so that one can be cooling while the other in use. The loops
should be flamed before starting and after streak 1, 2, 3 and after streak 4 before putting it
down.
The inoculating loop should be kept as nearly parallel to the agar surface as possible to
prevent the loop from digging in to the agar.

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Half-plating or quarter-plating can be employed where the initial inoculum is judged to
contain only a few bacterial cells, such as often occurs in bovine mastitic milk samples.

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 5.2. Inoculating slants

If agar slants are used, only the surface of the slant may be inoculated, or the butt and the
surface may both be inoculated. To inoculate only the surface of the slant, use a straight
flamed wire to obtain a colony of bacteria from the primary isolation plate. Streak the
surface of the slant in the form of an S shape.
To inoculate both the butt and slant, stab the butt of the slant with the tip of the inoculating
wire and carefully withdraw it up the same insertion path. Then streak the surface of the
slant in an S shape. There will be enough bacteria on the wire to inoculate the surface even
after stabbing the butt. Replace the tube's caps loosely.

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 PRACTICAL No. 5

6. BACTERIAL STAINING

6.1. Preparation of bacterial smears

A small amount of the scrapping is placed on the cleaned microscope slide. Another clean
slide is used with a scissor action to prepare a thin smear. With a liquid or semi-liquid
specimens, a little of the sample is placed on the slide with a sterile swab. The content of the
swab are smeared over the surface of the slide, with the aim of having thick and thin areas of
specimen present. The smears are allowed to dry thoroughly before proceeding further.

6.2. Fixing the smears

The reasons for fixing the smears include: killing the vegetative bacteria, rendering them
permeable to the stain and ensuring that the material is firmly fixed to the slide. Fixed and
stained smears should be handled carefully as not all bacteria, especially endospores, may
have been killed. After use, the stained smears should be autoclaved or soaked in a reliable
disinfectant (24-48 hours) before discarding.

For routine staining the smears are fixed by passing the slide, smear side up, quickly through
the Bunsen flame two to three times, taking care not to over heat the smears. This can be
tested on the back of the hand; the slide should feel warm but not hot enough to burn. Dried
smears to be stained by the Giemsa stain are first fixed in absolute methyl alcohol for 3
minutes and then dried.

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 6.3. Staining the smears

The fixed smears are placed on a staining rack over a sink and the staining solutions are
flooded over the entire smear and left on the slide for the appropriate time. Between each
staining reagent the smear is washed under a gently running tap, excess water tipped off and
the next reagent added. Finally the stained smears are washed and dried.

Simple stains involve only one reagent, and stain all bacteria similarly. More complex stains
involve multiple reagents, and are often differential. This means that they stain different
types of bacteria differently.

6.3.1. Simple staining

The use of a single stain to colour a bacterial organism is commonly referred to as simple
staining. Some of the most commonly used dyes for simple staining are methylene blue,
basic fuchsin and crystal violet.

Methylene blue stain

Procedure:
 Cover the bacterial smear with 1 % methylene blue for one minutes
 Wash with water and dry by air or blot dry and examine under oil immersion

Interpretation: - The bacteria appear blue and the shape, arrangements and unstained
inclusions (e.g. bacterial endospores) are observed.

6.3.2. Differential staining

Gram staining

Gram staining is used to categorize bacteria as Gram positive or Gram negative, based on
their cell wall structure.

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Reagents
Crystal violet:
Crystal violet 2g
Ethanol 95 % (vol/vol) 20.0 ml
Ammonium oxalate 0.8 g
Distilled water 80.0 ml
The crystal violet is first dissolved in the ethanol, and then the ammonium oxalate dissolved
in the distilled water. The two solutions are added together. To aid the dissolving process,
both mixtures are agitated in a bath of hot water.

Gram's iodine (mordant):

Iodine crystals 1.0 g
Potassium iodide 2.0 g
Distilled water 200 ml
The iodine crystal and the potassium iodide are ground together in a mortar and distilled
water is added slowly. If necessary the mixture can be agitated in a bath of hot water to aid
dissolution.

Decolourizer:
Ethanol 95 % (vol/vol)

Counter stain
 Dilute carbol fuchsin
Cocentrated carbol fuchsin 10 ml
Distilled water 90 ml
 Safranin (stock solution)
Safranin 2.5 g
Ethyl alcohol (95 %) 100 ml
To prepare the working solution, the stock solution is diluted 1:4 with distilled water.

Procedure:
 Place the slide on the staining rack over a sink
 Pour crystal violet solution on to the smear for 30 seconds
 Rinse gently with tap water
 Pour iodine solution onto the smear for 30 seconds
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  Rinse gently with water
 Wash the smear with decolourizer (95 % ethanol) for 10 seconds
 Rinse with water
 Pour basic fuchsin or safranin for 30 seconds
 Rinse with water
 Air dry or blot and examine under oil immersion

Interpretation: - Gram-positive bacteria retain the iodine-bound crystal violet and stain
purple. Gram-negative bacteria are stained with safranin dye and appear pink. As a general
rule, organism that gives a doubtful reaction is Gram-positive. Aging Gram-positive cells
become Gram-negative because autolytic enzymes attack the cell wall.

Ziehl-Neelsen or Acid-fast stain

This stain is used to detect mycobacteria (Mycobacterium bovis, M. avium, M.

paratuberculosis) and Nocardia.

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Reagents
Concentrated carbol fuchsin:
Basic fuchsin 1.0 g
Ethanol 95 %(vol/vol) 10.0 ml
Phenol 5g
Distilled water 100.0 ml
The basic fuchsin is 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 in to a clean container.

Acid-alcohol decolourizer:
Concentrated HCl 8.0 ml
Ethanol 95 % (vol/vol) 97.0 ml
The acid should be added to alcohol.

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
If Loeffler's methylene blue is used the potassium hydroxide can be omitted.

Procedure:
 Flood the slide with carbol fuchsin stain and heat over the flame until the stain
steams. Remove the slide from the heat and let it set for 5 minutes.
 Rinse with water
 Decolourize with acid-alcohol for 1 to 2 minutes until the red colour is gone
 Rinse with water
 Counter stain with methylene blue for 30 seconds
 Rinse with water
 Blot dry and examine under oil immersion
Interpretation: - Mycobacterium species resist decolourization with the acid-alcohol
solution and retain the carbol fuchsin stain and appear red, whereas non-acid fast bacteria
are decolourized and stain blue.

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7. KOH test

This test is performed to detect whether the bacteria is Gram-positive or Gram-negative.

Procedure:
 A loopful of the culture is taken from non-selective medium (blood agar) and
mixed with an equal amount of 3 % KOH on a clean microscope slide
 Mix thoroughly with the loop and lift the loops at intervals to see whether the gel
is formed or not

Interpretation: - If the bacterium is Gram-negative a viscous gel forms within 60 seconds

while no gel is formed if the bacterium is Gram-positive.

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 PRACTICAL No. 6

8. PRIMARY IDENTIFICATION OF BACTERIA

Once pure culture is obtained, the results from a few comparatively simple tests can often
identify the bacterium to generic level by:
 Gram-staining
 Growth or absence of growth on MacConkey agar
 Motility test
 Catalase test
 Oxidase test-
 Oxidation-fermentation (O-F) test

8.1. Gram staining

A Gram- stained smear from the culture will establish the Gram-reaction (Gram-positive or
Gram-negative), cellular morphology (coccus or rod) and cellular arrangements (pair,
cluster, chain etc.).

8.2. Growth on MacConkey agar

MacConkey agar inhibits the growth of majority of Gram-positive bacteria, but it will
support the growth of all members of the Enterobacteriaceae but is selectively inhibitory to
the other Gram-negative bacteria.

8.3. Motility test

'Hanging-drop' method

Procedure:
 A small amount of Vaseline is placed near each corner of cover glass with a tooth
pick
 A loopfuls of the broth culture is placed in center of cover glass
 Depression slide is pressed against Vaseline on cover glass and quickly inverted
 The completed preparation can be examined under oil immersion

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Semi-solid motility medium

SIM medium can be used to detect motility and will also indicate indole and hydrogen
sulphide production.

Procedure:
 Tubes of the medium are stab inoculated using a straight wire
 Incubate at 37oC for 24 hours
 Examined for motility

Interpretation: - If there is a diffuse growth throughout the medium, the bacterium is

motile. The growth of non-motile bacterium is confined to the stab line.

8.4. Catalase test

Principle: - To detect the presence of the enzyme catalase. Catalase enzyme is found in
most bacteria and it catalyses the breakdown of hydrogem peroxide (H 2O2) with the release
of free Oxygen. This test is done in the identification of Gram-positive cocci and small
Gram-positive bacilli.
Catalase
H2 O2 H2 O + O2

Procedure:
 A loopful of the bacterial growth is taken from the top of the colonies (avoiding
the blood agar medium) and placed on a clean slide.
 A drop of 3 % hydrogen peroxide is added (The reagent 3 % hydrogen peroxide
should be stored at 4oC in dark bottle).

Interpretation: - An effervescence of oxygen gas, within a few second, indicates a positive

reaction.

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 8.5. Oxidase test

Principle: - To detect the presence of cytochrome oxidase, an enzyme which is able to

oxidize the substrate tetramethyl-p-phenylenediamine dihydrochloride, forming a coloured
end product, indophenol. Oxidase test is used primarily in the identification of aerobic
Gram-negative bacteria.

Procedure:
 A piece of filter paper is moistened in a Petri dish with a 1 % tetramethyl-p-
phenylenediamine dihydrochloride fresh reagent.
 The test bacterium is streaked firmly across the filter paper with a glass rod.
The reagent used in this test should be colourless and stored in a dark bottle.

Interpretation: - A dark purple colour along the streak line within 10 seconds indicates a
positive reaction. Pseudomonas aeruginosa and E. coli can be used as positive and negative
control respectively.

8.6. Oxidation fermentation (O-F) test

Principle: - This test is used to determine the oxidative or fermentative metabolism of a

carbohydrate by the bacterium.

Procedure:
 Prepare O-F base medium and when the O-F base has cooled to 50oC add 20 ml of
sterile glucose solution into 200 ml of O-F base (1 %) and dispense into tubes.
 Before use, two tubes of O-F medium, with loosened caps, should be heated in a
beaker of boiling water to drive off dissolved oxygen.
 Then the tubes are cooled rapidly under cold running water.
 Both tubes of the media are stab inoculated with the test organism, using a straight
wire.
 Immediately a layer of paraffin oil should be added to a depth of about 1 cm on the
top of one of the tubes (sealed).
 Incubate at 37oC for up to 14 days, though 24 to 48 hours is usually sufficient.

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Interpretation: - The PH indicator in the medium is usually bromothymol blue. This gives
the un inoculated medium a green colour, turning yellow when acid is produced by the
organism's attack on the glucose in the medium.
Fermentative Oxidative Unreactive
Tubes with out oil yellow yellow green
Tubes with oil yellow green green
Examples E. coli P.aeruginosa R. equi

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Primary Identification of Gram positive bacteria (Cat = catalase; Ox = oxidase; + ve=
positive reaction, -ve = negative reaction; + = variable; * = motile; ** = anaerobic)

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Primary Identification of Gram negative bacteria (Cat = catalase; Ox = oxidase; + ve=
positive reaction, -ve = negative reaction; + = variable; * = motile)

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 PRACTICAL No. 7

8.7. Coagulase test

Principle: - Rabbit plasma contains fibrinogen that is converted to fibrin by the

staphylococcal coagulase enzymes.
As some pathogenic staphylococci can be negative to the slide coagulase test but positive to
the tube test, some laboratories perform only the tube coagulase test. 'Bound' coagulase is
detected by the slide test and 'free' coagulase by the tube test.

Slide coagulase test

Procedure:
 A loopful of the staphylococcal culture is emulsified in a drop of water on a
microscope slide.
 A loopful of rabbit plasma is added and mixed well with bacterial suspension.
 Then the slide is gently rocked

Interpretation: - Clumping within one or two minutes indicates a positive reaction.

Tube coagulase test

Procedure:
 0.5 ml of rabbit plasma is placed in a small (7 mm) test tube.
 Two drops of an over night broth culture of the Staphylococcus or a heavy
suspension made from the culture on the agar plate in a sterile water, are added.
 The tube is rotated gently to mix the contents and then incubated at 37 oC,
preferably in a water bath.

Interpretation: - A positive test, with clotting of the plasma, can occur in 2 to 4 hours.
However, many weak coagulase-positive strains will coagulate the plasma only after
overnight incubation.

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 PRACTICAL No. 8

9. IMViC test

Indole test

Principle: - Indole positive bacteria possess an enzyme, tryptophanase, which converts

tryptophan (an amino acid) to indole. When indole reacts with the aldehyde group of p-
dimethyl aminobenzaldehyde (Kovac’s reagent) a red colour complex is formed.

Kovac’s reagent
P-dimethyl amino-benzaldehyde 20.0g
Iso-amyl alcohol 300.0ml
Concentrated hydrochloric acid 100.0ml

The aldehyde is dissolved in the alcohol with gentle heat. The acid is added after cooling.
The reagent is stored in dark bottle at 4oc.

Procedure:
 Prepare Tryptone water or peptone water medium in a test tube
 Inoculate the medium with the test organism and incubate at 37 oC for 24 hours.
 Add 0.5ml of Kovac’s reagent to the medium and shake.
 If SIM medium is used, add 0.2ml of Kovac’s reagent to the tube and stand for
10min.

Interpretation: - The development of a bright red colour at the interface of the reagent and
the broth with in seconds after adding the reagent is indicative of the presence of indole and
is a positive test.

Methyl (MR) test

Principle: – Methyl red test is a quantitative test for acid production; requiring positive
organisms to produce strong acids (Lactic, acetic, formic) form glucose through the mixed
fermentation pathway.

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Methyl red reagent
Methyl red 0.1g
95% ethyl alcohol 300.0ml
Distilled water 200.0ml

The methyl red is dissolved in the alcohol and the solution is diluted to a total volume of
500ml with distilled water.

Procedure:
 Prepare MR-VP medium in a test tube
 Inoculate MR-VP broth with the test bacterium and incubate at 37oC for 2 days
 Add 5 drops of MR reagent to the medium

Interpretation: - The development of a stable red colour in the surface of the medium
indicates sufficient acid production (PH 4.4 – 6.0) and is a positive test.

Voges–Proskauer test

Principle: - Some organisms produce acetoin as the chief end product of glucose
metabolism and form less quantity of mixed acids. In the presence of atmospheric oxygen
and 40% KOH acetoin is converted to diacetyl and -naphtol serves as a catalyst to bring out
a red colour complex.

Voges-Proskaver reagent
-naphtol (5%)
-naphtol 5g
absolute ethyl alcohol 100ml

Potassium hydroxide (40%)

Potassium hydroxide 40g
Distilled water 100ml
*Be extremely careful with the KOH, it is caustic and my cause burns if it gets on your
skin.

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Procedure:
 Prepare MR-VP medium in a test tube
 Inoculate a tube of MR-VP broth with the test organism and incubate at 37 oc for 24
hours
 Add 3ml of 5% alphanaphthol in absolute ethyl alcohol and then 1ml of 40% KOH.
 Shake and leave for 5 minutes

Interpretation: - A positive test is represented by the development of a red colour.

Citrate utilization test

Principle: - To determine if an organism is capable of utilizing citrate as sole carbon source

for metabolism with resulting alkalinity. Bacteria that can utilize citrate can also extract
nitrogen from ammonium salt, with the production of ammonia leading to alkalinzation of
the medium (PH change).

Procedure:
 Prepare citrate agar slant
 Inoculate the slant surface of citrate agar with the test bacterium and incubate at
37oC for 48 hrs.

Interpretation: - A positive test is indicated by the development of deep blue colour with in
24 to 48 hours.

10. TRIPLE SUGAR IRON (TSI) REACTION

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 – coloured 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.

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Procedure:
 Prepare TSI agar slant
 Touch the top of the isolated colony with the tip of the inoculating wire
 Stab the butt of the slant with the tip of the inoculating wire and carefully with
draw it up and streak the surface of the slant in S-shape
 Cap very loosely and incubate overnight at 37oC

Interpretation:
 Alkaline (red) slant and acid (yellow) butt (R/Y): glucose fermentation only
 Acid (yellow) slant and acid (yellow) butt (Y/Y): lactose and / or sucrose attacked as
well as glucose
 Blackening of the medium: Hydrogen sulphide Production (H 2S+)

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 PRACTICAL No. 9

11. CAMP test

Principle: Group B-Streptococci produces beta hemolysin that reacts synergistically

with the staphylococcal beta toxin produced by Staphylococcs aureus. Streptococci
are inoculated perpendicularly to streaks of a beta toxin producing S. aureus on sheep
blood agar. The production of “arrow head” of hemolysis indicates a positive test and
is presumptive identification for group B streptococci.

Procedure:
a) Streak the beta-hemolytic S. aureus in a straight line across the center of a
Sheep Blood Agar plate. At a right angle to (but not touching) the inoculum,
streak the streptococcus to be tested.
b) Incubate at 35°C overnight.

Interpretation:
Positive = “arrowhead” of hemolysis at the junction of the two streaks = group B
streptococcus; Negative = no enhancement of hemolysis where the two meet = beta-
streptococcus, not group B.

Staphylococcus aureus

CAMP positive
Streptococcus agalactiae

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12. ANTIMICROBIAL SUSCEPTIBILITY TESTING

This is a test to determine the most suitable antibiotic for the effective treatment of a given
disease. The test can be conducted on isolates from clinical cases.

There are several laboratory methods for measuring the in vitro susceptibility of bacteria to
antimicrobial drugs such as the agar dilution methods, broth dilution method, disc diffusion
technique etc. However, the most commonly used and standardized method is the disc
diffusion method.

Standard Method
(1) Select 4-5 colonies of the same morphological type and transfer to a tube containing
4-5 ml of tryptone soya broth or alternatively, into 4-5 ml of saline.
(2) Incubate at 35-37oC for 2-8 hours or until slight visible turbidity appears.
(3) Adjust to 0.5 McFarland Turbidity standard
(4) A swab is dipped into the standardized suspension of bacteria and excess fluid is
expressed by pressing and rotating the swab firmly against the inside of the tube
above the fluid level.
(5) Streak the swab over the entire surface of the agar with the objective of obtaining a
uniform inoculation. Unless otherwise indicated, blood should not be included in the
medium.
(6) Let the plates stand for 3-5 minutes and apply the antimicrobial drugs.
(7) Apply the discs with an applicator.
(8) Invert the plate and incubate at 35oC for 16-18 hours (24 hours for staphylococci).
(9) Measure the diameter of inhibition to the nearest mm with a ruler or calipers.

Interpretation:
Compare with the standard and record the results as susceptible, intermediate or resistant.
Note: The standard is edited regularly and we can get a copy from the NCCLS.

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Factors affecting the size of the zone of inhibition
1. The size of the inoculum: adjust the concentration to 0.5 McFarland opacity
standard.
2. The test medium: The Mueller-Hinton medium or its modification is the best for
routine tests. Because it is reproducible, is low in sulphonamide, trimethoprim and
tetracycline inhibitors, and the most pathogens grow satisfactorily.
3. The antimicrobial agent and its concentration in the disc: The concentration of the
antimicrobial agents in the discs is chosen to give zone sizes that correlate with
achievable serum levels in the patient.
4. Incubation conditions: under aerobic incubation at 35oC for 16-18 hours and 24
hours for the staphylococci.
5. The test bacterium:

31
Zone size interpretation chart (modified from NCCLS)

32
Routine isolation of important members of the Enterobacteriaceae and their presumptive
identification on colonial morphology and/or biochemical tests.
+ = positive reaction, - = negative, ‘IMVIC’ = indole, methyl red, Voges-Proskauer and citrate tests, TSI = triple sugar iron
agar, lysine = lysine decarboxylase test

33
Isolation of Salmonellae from clinical specimens

34