Wollega University

School of Veterinary Medicine

Department of Veterinary Medicine
Veterinary Microbiology Laboratory Practical One

Prepared by: - Galana Biratu (VLT) and Dr. Iyob Hirpa(Associate Pro.)

September 2020

Nekemte , Ethiopia

By Galana Biratu
Vet.Microbiology I

1
1 Contents
1. Introduction .......................................................................................................................................... 1
2 This Practical include............................................................................................................................. 1
2.1 General Laboratory Rule’s ............................................................................................................ 2
2.2 Emergency Procedures Including .................................................................................................. 3
2.3 Risk/ hazards/in laboratory........................................................................................................... 3
2.3.1 Different types of hazards..................................................................................................... 3
3 Commonly Used Lab Equipments, Apparatus and Materials ............................................................... 4
4 Specimen collection – microbiology ................................................................................................... 17
4.1 Blood samples ............................................................................................................................. 17
4.2 Ear swabs .................................................................................................................................... 17
4.3 Eye swabs .................................................................................................................................... 18
4.4 Fecal samples .............................................................................................................................. 18
4.5 Fungal samples of hair nail and skin ........................................................................................... 18
5 Media Preparation .............................................................................................................................. 18
5.1 Types of Culture Media .............................................................................................................. 18
5.2 5.2. General purpose media preparation ..................................................................................... 19
5.2.1 Different Types of Oxygen Requirements of Bacteria........................................................ 19
5.2.2 Nutrient Agar Media Preparation ........................................................................................ 20
5.2.3 Nutrient Broth Media Preparation....................................................................................... 20
6 Staining Supplies ................................................................................................................................. 21
6.1 Preparation of staining solutions ................................................................................................ 21
6.2 Common Staining Technique ...................................................................................................... 22
6.3 Kinds Of Stains ............................................................................................................................ 22
6.3.1 Simple Staining ................................................................................................................... 22
6.3.2 Differential Stains ............................................................................................................... 23
6.3.1 Acid-Fast Stain- Principle, Procedure, Interpretation and Examples.................................. 31
6.4 Special Stains............................................................................................................................... 34
7 Biochemical Tests................................................................................................................................ 41
8. Reference ............................................................................................................................................ 62
By Galana Biratu
Vet.Microbiology I

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1. Introduction
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.

General Purpose of the manual:-

 Provides good laboratory technique in practical microbiology.

 Prepare students for confirmatory disease diagnosis.

2 This Practical include

 Laboratory Rules.

 Self Protection in the Laboratory.

 Demonstration and Sterilization of Equipments and Instruments of Microbiology.

 Method of Sample Collection.

 Type of Media and Method of Preparation.

 Staining Supplies.

 Common Staining Technique.

By Galana Biratu
Vet.Microbiology I

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2.1 General Laboratory Rule’s
1. Any individual working in the laboratory should practice safe.
2. Reporting all spills and broken glass ware and proper clean up.
3. Washing prior and following laboratories and at any time contamination is suspected.
4. Never eating or drinking in the lab.
5. Disinfecting lab benches prior to and at the end of each laboratory Work.
6. Never placing objects (fingers, pencils) in the mouth or touching the face.
7. Returning all materials and all chemicals their appropriate places.
8. Proper care and handling of equipments.
9. Keeping the bench top clean of extraneous materials.
10. Typing long hair back.
11. Wearing personal protective equipment like laboratory coats, closed shoes and using
such equipment in appropriate, situations.
12. Always using appropriate pipe ting device and understanding that mouth pipe ting is
forbidden.
13. Read labels carefully.
14. Do not use any equipment unless you are trained and approved as a user by your
supervisor.
15. Wear safety glasses or face shields when working with hazardous materials and/or
equipment.
16. If leaving a lab unattended, turn off all ignition sources and lock the doors.
17. Wash hands before leaving the lab and before eating.
18. Keep hands and other objects away from your face, nose, eyes, ears, and mouth.
19. All unnecessary books, purses, briefcases, etc., must be kept off the counter tops.
20. Label all materials with your name, date, and any other applicable information (e.g.,
media, organism, etc.)
21. When handling chemicals note the hazard code on the bottle and take the appropriate
precautions indicated.
22. Do not pour chemicals down the sink.
23. Flame transfer loops, wires, or needles before and immediately after use to transfer
biological material.
24. Do not work about the laboratory with transfer loops, wires, needles, or pipettes
containing infectious material.
25. Turn off incinerators before leaving the laboratory.
26. If you are injured in the laboratory, immediately contact your course instructor or TA.
27. Any chemical or biological fluid spill or daybreak must be immediately reported to
your course instructor.
28. Follow all instructors given by your course instructor or TA for cleaning up any spills
or broken glass.

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 29. Familiarize yourself with safety equipment in the laboratory and emergency escape
routes.
30. Always wipe and clean the lenses of your microscope before putting it away. Use the
appropriate tissue paper and cleaning solution for this purpose.
31. Use appropriate universal precautions with all biological fluids.
32. Identify and dispose wastes material properly.
33. Do not remove any materials from the laboratory without the written permission of the
course instructor or TA.

2.2 Emergency Procedures Including

1. Properly using emergency equipment
Eye- washes stations,
First – aid kits,
2. Fire extinguishers or co
3. Chemical safety showers
4. Telephones and emergency numbers
5. Following proper steps in the event of an emergency.
6. Never do unauthorized experiments.
7. Never work alone in laboratory.
8. Keep your lab space clean and organized.
9. Do not leave an on-going experiment unattended.
10. Never use open flames in laboratory unless instructed by TA.
11. Do not use corridors for storage or work areas.
12. Do not store heavy items above table height.
13. Any overhead storage of supplies on top of cabinets should be limited to lightweight
items only.
14. Clean your laboratory bench and equipment, and lock the door before you leave the
laboratory.

2.3 Risk/ hazards/in laboratory

2.3.1 Different types of hazards
• An important first step in protecting worker health and safety is recognizing workplace
hazards.
• Most hazards encountered fall into three main categories:
1. Chemical, Cleaning agents and disinfectants, drugs, anesthetic
gases, solvents, paints, and compressed gases are examples of chemical hazards.

Potential exposures to chemical hazards can occur both during use and with poor storage.

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 2. Biological, Biological hazards include potential exposures to allergens, infectious
zoometrics (animal diseases transmissible to humans), and experimental agents such as
viral vectors
3. Physical. Associated with research facilities. The most obvious are slips and falls from
working in wet locations and the ergonomic hazards of lifting, pushing, pulling, and
repetitive tasks.

3 Commonly Used Lab Equipments, Apparatus and Materials

Laboratory Equipments are those that are used in the laboratory for confirmation which
enables for demonstration and identification of micro organisms.
• 1. Autoclave 2. Incubator
• 3. Hot air oven 4. Inoculating loop
• 5. Vortex mixer / shaker 6. Water bath
• 7. Heating mantle 8. Hot plate with magnetic stirrer
• 9. Universal chamber 10. Inoculation chamber
• 11. PH meter 12. Colony counter
• 13. Microscope 14. Refrigerator
• 15. Bunsen burner 16. Spirit lamp
• 17. Micrometer (stage and ocular) 18. Balance (Digital and 4-beam)
• 19. Thermometer 20. Membrane filter set

1. Autoclave

It is a robust, electrically heated steam vessel meant for sterilizing ‘thermostable’

 culture media,
 glassware, and
 Other materials that are not spoiled by moist heat.
• sterilization can be achieved by operating the autoclave at 121°C for 15 minutes

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2. Incubator

 This an insulated, electrically heated cabinet

 It Provide microorganisms with optimum temperature for growth.
 For routine purposes, the temperature is maintained at 28-30°C for bacteria
 25°C for molds
 35-37°C for mesophilic bacteria.
 A temperature as high as 100°C can also be maintained for extremely thermophilic
organisms

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Vet.Microbiology I
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3. Hot air oven

• This is similar to incubator in make except that it can operate temperatures up to 300°C
• Hot air oven is used for sterilization of glassware materials that are spoiled by moist heat
• For routine purpose, sterilization can be achieved by running the equipment at 180°C for
1.5 hours.
• Hot air oven is less effective than autoclave.

1. Inoculatin
g loop
• This is used for transferring and streaking cultures.
• Sometimes, the looped end is straightened out to form what is called inoculating needle.
• Inoculating needles are used for preparing ‘stab ‘Cultures
Loop (wire/ Plastic):- Use for routine inculcation of agars slopes/ deeps Making streak
plates.
Straight wire: - Use for Inoculation from very small Colonies transfer of small Inoculate
from liquid media.

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 2. Vortex mixer
 This equipment is used for mixing liquids kept in a test tube.
 It has one or more cup-like depressions at the top to receive the bottom of the test tube.
 When actuated, the machine moves the bottom of the test tube in a gyratory motion.
 Thereby affecting a thorough mixing of the solution.
The speed of the mixer can be varied.

3. Water bath / boiling water bath

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Vet.Microbiology I
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 It is electrically heated and thermostatically controlled.
 It used for heating and melting of:-
• media
• Solutions
• Samples etc. at temperatures below 100°C.
 Several models and types of water bath are available.

4. Heating mantle
 It is an electrically heated and thermostatically controlled unit
 It uses to heat or melt samples and reagents.
 The inner lining is made of asbestos and therefore gives an indirect heat to the materials
to be heated.

5. magnetic stirrer with Hot plate

 This is an electrically powered equipment performs the dual function of heating and
agitation.
 The agitation occurs by magnetic arrangement.

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Vet.Microbiology I
September 2020
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 Any type of glassware can be used for the heating and agitation.
 Magnetic beads are used for the agitation.

6. Inoculation chamber / sterile chamber

 This is an enclosed box in which culture transfers; plating, etc. can be carried out.
 The chamber is equipped with UV lamp for periodic disinfection of the chamber.
 While working, the UV light must be turned off and day-light bulb is turned on.

7. pH meter
 pH meter is an electrical instrument used for measuring hydrogen ion concentration of
solutions and mixtures
 In microbiology lab, it is used for maintaining pH of the medium and diluents.
 The pH meter must be standardized with buffer solutions before operation.
 Since the instrument is very sensitive, it must not be used for stirring and it must not be
dipped in hot or very cold solutions.
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 The electrodes must always be kept immersed in suitable solutions.

8. Colony counter
• It is used for counting microbial colony (bacterial and yeast).
• The instrument is equipped with a backlight source, gridlines and a magnifying lens.
• It also has a sensor for digitally registering the number of colonies counted.

9. Microscope
 It is an instrument for observing microscopic items such as cells, crystals and cell
organelles.
 It has the dual function of magnification and resolution.
 For routine microbiological works, bright field compound microscope with oil immersion
objective is adequate.
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The important microscope parts

1. Oculars lens ( eyepiece) the lens the viewer looks through to see the spacebar
2. Diaper adjustment to correct for any different in vision b/n your two eyes.
3. Body tuba ( Hoed ) connects the eye piece to the objective lose
4. Arm ( frame) connect the body tube to base of microscope
5. Coarse adjustment: - brings the specimen in to general focus.
6. fine adjustment:- fine tunes the focus and increases the detail of the specimen
7. Nose piece: - to select different objective lenses.
8. Objective lenses: - most important part of compound microscope. 4X-100x
9. Stage: - where the slide is placed.
10. Stage clips: - metal clips that hold the slide in place.
11. Stage controller: - move stage left and right or up and place
12. Illumination: - light source for microscope.
13. Iris diaphragm: - Adjust the amount of light the reaches the specimen.

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14. Condenser: - garter and focuses light form the illuminator on the specially baling
viewed.
15. Base: - supports the microscope and it’s where illuminator is located.

10. Refrigerator
• This is common household equipment for keeping foods and beverages cool.
• This equipment is used in microbiology laboratory for storing / preserving cultures,
media, and many sensitive materials

11. Bunsen burner

• Bunsen burner is a common tool used in science
In microbiology lab, it is used for sterilizing inoculating loop, plating out cultures,
transferring cultures, heat-fixing of smears and creating a sterile zone for aseptic
operation.

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Vet.Microbiology I
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 12. Spirit lamp
• The function of spirit lamp is the same as the Bunsen burner but is portable. It uses
rectified spirit as the fuel (produces smoke-free flame). The lamp must be covered with a
lid when not in use to prevent loss of spirit (Fig.

13. Micrometers (stage and ocular)

• These are graduated glass pieces (Fig. 17) used for the measurement of size of the cells.
Stage micrometer is a slide on which etching is done with 0.001 mm spacing. The ocular
micrometer, which is place on the eyepiece, has an arbitrary scale and must be calibrated
against the stage micrometer. During measurements, the ocular micrometer is retained
while the stage micrometer is replaced with the specimen slide.

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Vet.Microbiology I
September 2020
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 14. Balance
• Balance is needed in microbiology lab for weighing chemicals, samples, media, etc.
Digital balances are fast to work with but needs frequent calibration (Fig. 18). The triple-
beam and 4-beam balances are robust equipment that need little care and maintenance.
Beam balances run on mechanical principles while the principles on which electronic
balances run is quite complicated

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Vet.Microbiology I
September 2020
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 15. Thermometer
• Thermometers are required to ensure the heating equipment is running at the correct
temperature. The temperature of the medium, incubator, etc., need to be frequently
checked. Mercury in glass thermometers are standard thermometers, the temperature
measurement is based on the expansion of mercury present in the bulb. Digital
thermometers use probes for measurement of temperatures

16. Coliform membrane filter

• This glass equipment is used for the testing of coliforms in water (Fig. 20). 100 ml of test
water is poured in the funnel and filtered through a special Millipore filter through
external application of suction.
• The filter retains the microorganisms. The filter is then aseptically transferred to a
selective-cum-differential semisolid medium kept in a petri dish. If there are coliforms,
they will appear as pink dots after incubation at 35°C for 22 hrs.

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17. Forceps Metal (Plastic): used for transfer of sterile paper / Antibiotic discs.
18. Pipette (calibrated/dropping glass or Plastic Used for Transfer of measured Volumes/
drops of culture, sterile solution.
19. Teat – Filling and emptying pipettes safely (never pipette by mouth
20. Universal Bottle (wide Nock and McCartney bottle (narrow nock)
21. Use to measure Volumes of liquid and agar media
22. Test tube – Small Volumes (Caps -10cm-3) of liquid med
23. Test tube rack
24. Slide rack
25. Bisow Bottle; - Very small Volumes of up to 3m3 of sterile solution
26. Concel flask; - large Volumes of liquid media for inoculation and liquid for short-
term storage.
27. Prevalent contamination but does not reduce evaporation during long storage.
28. Petridish (plastic/ Glass)
29. Use for sampling and making cultures for sterilization by not air over, e.g paper discs.
30. Marker Penal- to labeling petridish, test tube flasks, and bottle and microscope slides.
31. Personal protective equipments
32. Centrifuges –use to separate fluid or liquid –based on specific suavity.
33. Biker to measure and to transport fluid from place to p lace.

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4 Specimen collection – microbiology
Accurate and rapid identification of significant micro-organisms is vital for
 guiding optimal anti-microbial therapy, and
 Improving outcome from infectious disease.

• Clinicians have responsibility for the collection and safe transportation of samples to the
laboratory.
• Contamination of samples, especially those from normally sterile sites leads to
misleading results.
• Prolonged periods of storage at ambient temperature and delay in transport of specimens
to the laboratory may increase the number of contaminants present.
 It is therefore essential that every effort should be made to avoid these problems.
• All specimens must be clearly labeled next to the sample is taken. to identify their source.

Equipment

This will vary according to the specimen required but must include:
 disposable gloves
 additional personal protective equipment (apron/gown, mask/respirator, visor -
where applicable)
 a protective dish
 a sterile container for the specimen
 appropriate transport medium, if required
 laboratory specimen form
 a polythene transportation bag
 biohazard label, if required

4.1 Blood samples

 Blood sampling should be performed by a veterinarian worker trained and competent in
the procedure.
 Blood collection will be depending on the amount of sample you went (small sample by
capillary tube or slide or large sample by tube with EDTA).

4.2 Ear swabs

 If there is purulent discharge this should be sampled.
 Place a sterile swab into the outer ear and gently rotate to collect the secretions.
 Place swab in transport medium.
 For deeper ear swabbing a speculum may be used.

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  Experienced staff only should undertake this procedure as damage to the eardrum may
occur.

4.3 Eye swabs

 Use a sterile cotton wool swab and gently role the swab over the conjunctiva sac inside
the lower lid.
 Hold the swab parallel to the cornea to avoid injury if the moves.
 Place the swab in the transport medium and then take to lab for diagnosis purpose.

4.4 Fecal samples

 Please specify whether the sample is a routine (admission) screening sample or an
investigation for suspected intestinal infection.
 If viral gastroenteritis is suspected, the feces specimen should be sent to the virology
laboratory.
 To exclude a bacterial cause a second feces specimen can be sent to the microbiology
laboratory.
 Where diarrhoea is present, a small piece of non-absorbent material lining the nappy can
be used to prevent material soaking into the nappy.

4.5 Fungal samples of hair nail and skin

• Special containers may be obtained from the microbiology department
• Samples of infected hair should be removed by plucking the hair with forceps, lancet or
gloves.
• Samples of the whole thickness of the nail or deep scrapings should be obtained.
• The skin should be cleaned with an alcohol swab.
• Epidermal scales scraped from the active edge of a lesion or the roof of any vesicle
should be obtained.

5 Media Preparation
 Cultural Medias: - prepared in the laboratory from the basic ingredients or for
commercially available dehydrated powders or purchase ready for use .

5.1 Types of Culture Media

1. General purpose media: - support the growth of a broad range of
bacteria.
Example: - Nutrient agar, nutrient broth, trypticase soy agar

2. Reducing media: - used for growing obligate anaerobes. Particularly

contains chemicals (reducing agents) that deplete molecular oxygen.

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 3. Enriched Media: - prepared with special complex nutrients or growth factor.
Example: specific amino acids, Vitamin, sheep or horse blood.
4. Selective Media: - these media are designed to the growth of certain species
of bacteria from a mixed sample.
5. Differential Media: - these media differentiate one species from anther
according to particular biochemist cal traits.

5.2 5.2. General purpose media preparation

 Media can be either liquid (nutrient broth) or solid (agar).
 Media are initially sterilized then inoculated with the bacteria that is of interest and
finally incubated for twenty four hours or more, to encourage bacterial growth.
 Media must be prepared in such a way that it is sterile prior to being inoculated with a
bacterial sample, so that when a particular type of bacteria is cultured (cultivated) on that
medium; it is the only type of bacteria present.
 Growth media must also provide everything the bacterial culture needs to live and grow,
including water, nutrients, and the proper PH.

5.2.1 Different Types of Oxygen Requirements of Bacteria

Unlike animals, bacteria do not all require oxygen.
Some types of bacteria are poisoned by oxygen; others can take it or leave it.
Liquid broth allows bacteria to grow at varying oxygen levels, which decrease as the
depth of the broth increases.
Obligate aerobic bacteria, those that must have oxygen to extract energy from food, will
gather at the top of the test tube, near the broth’s surface, to absorb maximum amount of
oxygen (1 in mage below).
Obligate anaerobic bacteria, those killed by oxygen, will gather at the bottom of the test
tube, staying as far away from oxygen as possible (2).
Facultative bacteria, those that can live with or without oxygen, will gather mostly at the
top, since aerobic respiration is the most energy efficient way to turn food into energy;
but since lack of oxygen does not hurt these microbes, they can survive anywhere in the
broth (3).
Micro aerophiles gather at the upper part of the test tube but not at the top. These
microbes require oxygen, but at concentrations lower than those found in the atmosphere
(4).
Aero tolerant bacteria are not affected at all by oxygen, so they can be found evenly
spread along the test tube (5)

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5.2.2 Nutrient Agar Media Preparation
 Note: Allow 15 cm³ of agar for each Petri dish and 5-10 cm³ of broth for each McCartney
bottle. All cotton wool plugs should be made of non-absorbent cotton wool. Plastic or
metal caps may also be used.
Materials
Nutrient agar, Culture dishes sterilized Water, distilled or deionizer Graduated cylinder,
Autoclave or pressure cooker Stirring rod, Balance, 1 g precision Weighing dish, Beaker,
2L
Procedure
 1. Using a weighing dish and balance mass of nutrient agar you went to prepare.
 2. Transfer the nutrient agar solid to a flask.
 3. Use a graduated cylinder and measure distilled water corresponding to your mass of
agar then transfer the water to the beaker of nutrient agar.
 4. Stir the solution until the solid is evenly distributed.
 5. Place the solution in an autoclave for 15 minutes at 121 °C (15 lbs of pressure).
 6. Allow the solution to cool to 50–55 °C and pour into sterilized culture dishes.

5.2.3 Nutrient Broth Media Preparation

 Suspend grams of the medium in and liter of distilled water.
 Mix well and dissolve by heating with frequent agitation.
 Boil for one minute until complete dissolution. Dispense into appropriate containers and
sterilize in autoclave at 121°C for 15 minutes.
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  The prepared medium should be stored at 2-8°C. The color is amber, slightly opalescent.
 The dehydrated medium should be homogeneous, free-flowing and beige in color. If
there are any physical changes, discard the medium.

6 Staining Supplies
Staining :- is a direct visualization of the morphology of the organisms as well as
their reactions to the chemical is present in stains .
Needed for staining include the following
• Microscope
• Microscope slides
• Cover slips
• loops
• Grams stain kit
• Acid fast stain kit
• Staining rack
• Droppers or squeeze bottles for stain solution.

6.1 Preparation of staining solutions

Grams stain:
Four Contents of the gram stain reagents are:-
1. Crystal violet contents in staining
• Crystal violet 2 gm
• . Ethanol 95% ( vol/vol)___ 20 ml
• . Ammonium oxalate 0.8 gm
• . Distilled water 80 ml
• N.B:- crystal violet powder is dissolved in the ethanol, Ammonium oxalate is dissolved
in distilled water both mixtures are agitated in a bath of hot water than two solutions are
mixed.
2. Grams iodine (Mordant)
• Iodine crystals – 1 gm
• Potassium iodide – 2 gm
• Distilled water – 200 ml
• N.B Iodine and potassium iodide are ground together in a mortar

3. Decolorizer
.Ethanol 95% (Vol/Vol)
4. Safranin solution (counter stain)
• Safranin Powder ------- 4gm
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 • Ethanol/ methanol --------- 200ml
• Distilled water ------------800 ml

6.2 Common Staining Technique

 Microorganisms must be fixed & stained to increase visibility, accentuate specific
morphological features, and preserve them for future use

TERMS RELATED TO STAINING

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.

Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic

image.

Fixation: - Fixation by itself consists of several steps–aims to preserve the shape of the cells or
tissue involved as much as possible.

6.3 Kinds Of Stains

Stains are classified as

• Simple stain

• Differential stain

• Structural or special stains

6.3.1 Simple Staining

Techniques are used to study the morphology better, to show the nature of the cellular contents
of the exudates and also to study the intracellular location of the bacteria.

Commonly used simple stains are

• Methylene blue

• Dilute carbol fuchsin

• Polychrome methylene blue

Methylene blue staining is used to make out clearly the morphology of the organisms

Method of Staining

• Flood the smear with methylene blue on slide

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 • allow for 2 minutes, pour off the stain

• allow the air to dry by keeping in a slanting position and by this the organism will retain
the methylene blue stain

Dilute Carbol Fuchsin Preparation; it stain throat swab from patients of suspected Vincent’s
angina, it is used as a counter stain in Gram stain and to demonstrate the morphology of Vibrio
cholerae (comma shaped)

Method of Staining

• Prepare carbol fuchsin and dilute it to 1/15 using distilled water

• Flood the smear and let stand for 30 seconds, wash with tap water and blot gently to dry

Polychrome Methylene Blue is used to demonstrate Mc Fadyean reaction of B.anthracis and in

this the blue bacilli is surrounded by purple capsular material.

Preparation

• Allow Loeffler’s Methylene blue to ‘ripen’ slowly. Methylene blue stain is kept in half
filled bottles, aerate the content by shaking at intervals, slow oxidation of methylene blue
forms a violet compound and Stain gets polychrome property.

• The ripening nearly takes 12 months and this is hastened by addition of 1% potassium
carbonate.

6.3.2Differential Stains
 Differential Stains use two or more stains and allow the cells to be categorized into
various groups or types.

 Gram staining (or Gram’s method) is an emprical method of differentiating bacterial

species into two large groups

 (Gram-positive and Gram-negative) based on the chemical and physical properties of

their cell wall.

Gram-positive bacteria stain dark blue or violet. Their cell wall is typically rich with
peptidoglycan and lacks the secondary membrane lipopolysaccharide layer found in Gram-
negative bacteria

Gram Staining Technique

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1. Crystal violet acts as the primary stain. Crystal violet may also be used as a simple stain
because it dyes the cell wall of any bacteria.

2. Gram’s iodine acts as a mordant (Helps to fix the primary dye to the cell wall).

3. Decolorizer is used next to remove the primary stain (crystal violet) from Gram Negative
bacteria (those with LPS imbedded in their cell walls). Decolorizer is composed of an organic
solvent, such as, acetone or ethanol or a combination of both.)

4. Finally, a counter stain (Safranin) is applied to stain those cells (Gram Negative) that have lost
the primary stain as a result of decolorization.

Materials Required and Reagents

Materials Required:

• Clean glass slides

• Inoculating loop

• Bunsen burner

• Bibulous paper

• Microscope

• Lens paper and lens cleaner

• Immersion oil

• Distilled water

• 18 to 24 hour cultures of organisms

Reagents:

• Primary Stain - Crystal Violet

• Mordant - Grams Iodine

• Decolourizer - Ethyl Alcohol

• Secondary Stain - Safrani

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Procedure:

Part 1: Preparation of the glass microscopic slide

 Grease or oil free slides are essential for the preparation of microbial smears.

 Grease or oil from the fingers on the slides is removed by washing the slides with soap
and water.

 Wipe the slides with spirit or alcohol.

 After cleaning, dry the slides and place them on laboratory towels until ready for use.

Part 2: Labeling of the slides

• Drawing a circle on the underside of the slide using a glassware-marking pen may be
helpful to clearly designate the area in which you will prepare the smear.

• You may also label the slide with the initials of the name of the organism on the edge of
the slide.

• Care should be taken that the label should not be in contact with the staining reagents.

Part 3: Preparation of the smear

• Bacterial suspensions in broth: With a sterile cooled loop, place a loopful of the broth
culture on the slide. Spread by means of circular motion of the inoculating loop to about
one centimeter in diameter. Excessive spreading may result in disruption of cellular
arrangement. A satisfactory smear will allow examination of the typical cellular
arrangement and isolated cells.

• Bacterial plate cultures: With a sterile cooled loop, place a drop of sterile water or
saline solution on the slide. Sterilize and cool the loop again and pick up a very small
sample of a bacterial colony and gently stir into the drop of water/saline on the slide to
create an emulsion.

• Swab Samples: Roll the swab over the cleaned surface of a glass slide.

• Please note: It is very important to prevent preparing thick, dense smears which contain
an excess of the bacterial sample. A very thick smear diminishes the amount of light that
can pass through, thus making it difficult to visualize the morphology of single cells.
Smears typically require only a small amount of bacterial culture. An effective smear
appears as a thin whitish layer or film after heat-fixing.

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Part 4: Heat Fixing

• Heat fixing kills the bacteria in the smear, firmly adheres the smear to the slide, and
allows the sample to more readily take up stains.

• Allow the smear to air dry. After the smear has air-dried, hold the slide at one end and
pass the entire slide through the flame of a Bunsen burner two to three times with the
smear-side up. Now the smear is ready to be stained.

• Please Note: Take care to prevent overheating the slide because proteins in the specimen
can coagulate causing cellular morphology to appear distorted.

Part 5: Gram Stain Procedure

1. Place slide with heat fixed smear on staining tray.

2. Gently flood smear with crystal violet and let stand for 1 minute.

3. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash
bottle.

4. Gently flood the smear with Gram’s iodine and let stand for 1 minute.

5. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash
bottle. The smear will appear as a purple circle on the slide.

6. Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the
alcohol drop by drop for 5 to 10 seconds until the alcohol runs almost clear. Be careful
not to over-decolorize

7. Immediately rinse with water.

8. Gently flood with safranin to counter-stain and let stand for 45 seconds.

9. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash
bottle.

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 10. Blot dries the slide with bibulous paper.

11. View the smear using a light-microscope under oil-immersion.

Typical Gram-positive bacteria:

• Staphylococci such as Staphylococcus epidermidis and Staphylococcus aureus which is

a common cause of boils.

• Streptococci such as the many species of oral streptococci, Streptococcus pyogenes

which causes many a sore throat and scarlet fever and Streptococcus pneumoniae which
causes lobar pneumonia.

• Clostridia such as Clostridium tetani, the causative agent of tetanus (lockjaw).

• Actinomyces such as Actinomyces odontolyticus which is found in mouth.

• Species of the genus Bacillus such as Bacillus subtilis which are common microbes
living in soil.

• Generally cocci are Gram-positive but there are exceptions. The most significant from a
clinical point of view is the gonococcus, Neisseria gonorrhoea which typically appears
as a Gram-negative diplococcus looking very much like a pair of kidney bean.

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Gram-positive vs. Gram-negative Bacteria

• Comparison chart

• Gram-negative Bacteria

• Gram-positive Bacteria

• Gram reaction

• Can be decolourized to accept counter stain (Safranin or Fuchsine); stain red or pink, they
don't retain the Gram stain when washed with absolute alcohol and acetone.

• Retain crystal violet dye and stain dark violet or purple, they remain coloured blue or
purple with gram stain when washed with absolute alcohol and water.

• Peptidoglycan layer

• Thin (single-layered)

• Thick (multilayered)

• Teichoic acids

• Absent

• Present in many

• Periplasmic space

• present

• Absent

• Outer membrane

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• Present

• Absent

• Lipopolysaccharide (LPS) content

• High

• Virtually none

• Lipid and lipoprotein content

• High (due to presence of outer membrane)

• Low (acid-fast bacteria have lipids linked to peptidoglycan)

• Flagellar structure

• 4 rings in basal body

• 2 rings in basal body

• Toxins produced

• Primarily Endotoxins

• Primarily Exotoxins

• Resistance to physical disruption

• Low

• High

• Inhibition by basic dyes

• Low

• High

• Susceptibility to anionic detergents

• Low

• High

• Resistance to sodium azide

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 • Low

• High

• Resistance to drying

• Low

• High

• Cell wall composition

• The cell wall is 70-120 Armstrong thick two layered. The lipid content is 20-30% (High),
whereas Murein content is 10-20% (Low).

• The cell wall is 100-120 Armstrong thick, single layered. The Lipid content of the cell
wall is low where as Murein content is 70-80% (Higher).

• Mesosome

• Mesosome is less prominent.

• Mesosome is more prominent.

• Antibiotic Resistance

• More resistant to antibiotics.

• More susceptible to antibiotics

Bacterial Morphology:

• Bacteria are very small unicellular microorganisms ubiquitous in nature. They are
micrometers (1µm = 10-6 m) in size. They have cell walls composed of peptidoglycan and
reproduce by binary fission. Bacteria vary in their morphological features.

The most common morphologies are:

Coccus (pleural: Cocci):

• Spherical bacteria; may occur in pairs (diplococci), in groups of four (tetracocci), in

grape-like clusters (Staphylococci), in chains (Streptococci) or in cubical arrangements of
eight or more (sarcinae).

• For example: Staphylococcus aureus, Streptococcus pyogenes

Bacillus (pleural: Bacilli):

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 • Rod-shaped bacteria; generally occur singly, but may occasionally be found in pairs
(diplo-bacilli) or chains (streptobacilli).

• For example: Bacillus cereus, Clostridium tetani

Spirillum (pleural: Spirilla):

Spiral-shaped bacteria

• For example: Spirillum, Vibrio, Spirochete species.

Some bacteria have other shapes such as:

Coccobacilli: Elongated spherical or ovoid form.

• Filamentous: Bacilli that occur in long chains or threads.

• Fusiform: Bacilli with tapered ends.

6.3.1 Acid-Fast Stain- Principle, Procedure, Interpretation and Examples

 It is the differential staining techniques which was first developed by Ziehl and later on
modified by Neelsen.

 So this method is also called Ziehl-Neelsen staining techniques.

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  The main aim of this staining is to differentiate bacteria into acid fast group and non-acid
fast groups.

 This method is used for those microorganisms which are not staining by simple or Gram
staining method, particularly the member of genus Mycobacterium those are resistant and
can only be visualized by acid-fast staining.

Principle of Acid-Fast Stain

 When the smear is stained with carbol fuchsin, it solubilizes the lipoidal material present
in the Mycobacterial cell wall

 But by the application of heat, carbol fuchsin further penetrates through lipoidal wall and
enters into cytoplasm,Then all cell appears red.

 Then the smear is decolorized with decolorizing agent (3% HCL in 95% alcohol)

 Then the acid fast cells are resistant due to the presence of large amount of lipoidal
material in their cell wall which prevents the penetration of decolorizing solution.

 The non-acid fast organism lack the lipoidal material in their cell wall due to which they
are easily decolorized, leaving the cells colorless.

 Then the smear is stained with counterstain, methylene blue.

Procedure of Acid-Fast Stain

1. Prepare bacterial smear on clean and grease free slide, using sterile technique.

2. Allow smear to air dry and then heat fix.

3. Cover flooded smear with filter paper

4. Cover the smear with carbol fuchsin stain.

5. Heat the stain until vapour just begins to rise (i.e. about 60 C). Do not overheat

6. Wash off the stain with clean water.

Note: When the tap water is not clean, wash the smear with filtered water or clean boiled
rainwater.

7. Flood slide with acid alcohol (leave 15 seconds). The acid alcohol contains 3% HCl and
95% ethanol, or you can declorase with 20% H2SO4

8. Wash well with clean water.

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 9. Cover the smear with malachite green/Methylene blue stain for 1–2 minutes, using the
longer time when the smear is thin.

10. Wash off the stain with clean water.

11. Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry
(do not blot dry).

12. Examine the smear microscopically, using the 100 X oil immersion objective.

Summary of Acid-Fast Stain

Application of Reagent
Cell color
Summary of Acid-Fast Stain

Acid fast Non-acid fast

Primary dye Carbol fuchsin Red Red

Decolorizer Acid alcohol Red Colorless

Counter stain Methylene blue Red Blue

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Acid fast: Bright red B),Red,straight or slightly curved rods, occurring singly or in small
groups, may appear beaded

Non-acid fast: A Blue color (A)

Examples of Acid-Fast Stain

Acid-fast: Mycobacterium tuberculosis, Mycobacterium smegmatis.

6.4 Special Stains

 Stain for capsules

 Stain for endospore

 Stain for flagella

1. Ca psule staining

• The purpose of the capsule stain is to reveal the presence of the bacterial capsule,

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• the water-soluble capsule of some bacterial cells is often difficult to see by standard
simple staining procedures or after the Gram stain.

• The capsule staining methods were developed to visualize capsules and yield consistent
and reliable results

• Capsule may appear as clear halo When staining- using - India ink

Procedure

1) Place a loop full of India ink on the slide.

2) A small portion of the culture is emulsified in the drop of ink.

3) Place a clean cover slip over the preparation without bubbles and Press down gently.

4) Examine under dry objective.

• Uses

• India ink is used to demonstrate capsule which is seen as unstained halo around the
organisms distributed in a black background.

Endospore Staining

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The endospore stain is a spatial stain used to visualize bacterial endospores.

• Endospores are formed by a few genera of bacteria, such as Bacillus. By forming spores,
bacteria can survive in hostile conditions.

• Endospores can form within different areas of the vegetative cell.They can be at central,
subterminal, or terminal.

 Bacterial endospores are metabolically inactive, highly resistant structures produced by

some bacteria as defensive strategy against unfavorable environmental conditions.

Priniciple

 Primary stain - is malachite green, which stains both vegetative cells and endospores

 Heat is applied to help the primary stain penetrate the endospore.

 Decolorized with water, will removes the malachite green from the vegetative cell but not
the endospore,

 And Safranin – counter stain will stain vegetative cell which have been decolorized.

 Endospores will stain green. Parent cells will stain red.

Procedure

1. Perform a bacterial smear of Bacillus or the organism you want to stain

2. Place a small piece of bibulous paper over the smear. Saturate the paper with malachite
green.
3. Heat the slide gently over the Bunsen burner for 5 minutes.
4. Remove the bibulous paper from the slide, and rinse the slide gently with water.
5. Counterstain with safranin for 2 minutes.
6. Rinse the slide gently with water.
7. Carefully blot the slide dry with bibulous paper.
8. Observe the slide under the microscope, using proper microscope technique.

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 2. Flagella stain

Purpose: To determine the presence/absence and location of flagella on various microorganisms

• Principle: Because bacterial flagella are very thin and fragile a special stain (flagella
stain) is prepared that contains a mordant.

• This mordant allows piling of the stain on the flagella, increasing the thickness until they
become visible. Various arrangements of flagella are seen on different cells.

Procedure:

A. Bacterial Suspension
1. From an agar slant culture:
a. Suspend a loopful of bacteria in 2 ml distilled water to obtain an opalescent
suspension.
b. Allow the suspension to stand undisturbed for 15 to 20 minutes while flagella are
regenerated and extended.

B. Slides
1. Select new or unscratched slides.
2. Clean slides with cleaning powder as directed.
3. Flame the slides and allow it to cool.
4. Make a heavy wax line on the flamed side along the margin of one end, along both
sides to within about one inch of the other end, and across the slide to complete the
rectangle.

5. Handle the slide by the unmarked end only. The wax line creates a retaining wall to
allow a pool of stain to surround the cells during the 15 to 15 minute staining period.

C. Preparation of smear
1. Handling the suspension carefully remove a large loopful of the suspension and place
it at one end of the rectangular area.
2. Tilt the slide to permit the suspension to run down to the other end of the slide. If the
drop fails to run, add another drop.
3. Air dry the film. Do not heat.
4. Place the slide on a horizontal staining rack.

D. Staining procedure
1. Add about 1 ml. of the Flagella Stain solution (one dropper full) to the smear and allow
staining for 10-15 minutes. The solution should not be allowed to flow outside the waxed area.
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 2. Flood off the stain by adding tap water to the slide while it remains on the rack. Do not tip
the slide before this is done.
3. Drain and flood the slide with carbol fuchsin for one minute. Rinse by flooding. Drain and
air dry.

E. Examination of slide
1. With the naked eye identify the line made by the drop as it ran down the slide.
2. Position the slide so that the edge of the run is under the objective.
3. Focus the edge of the run under low power, as oil, and examine it using the oil immersion
objective.
4. Identify cells as distinguished from dye and debris which will adhere to the slide because of
the mordant. Cells will be small rods and have regular outlines. They will be more plentiful
near the lower end of the run and may look larger than usual because of the mordant.
5. Once cells are identified follow along the edge of the run, examining cells until some are
found with flagella attached. Flagella are much longer than the cell and often look like faint
hairs.
6. Ideally, cells should be located which are isolated enough to determine unequivocally the
arrangement of the flagella and, for the Pseudomonas, the number of flagella per cell.
7. Draw the cells and their flagella.

Motility
Test

Procedure of Hanging Drop method to test Bacterial Motility

Hanging drop preparation is a special type of wet mount (in which a drop of medium containing
the organisms is placed on a microscope slide), often is used in dark illumination to observe the
motility of bacteria

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 • In this method a drop of culture is placed on a cover slip that is encircled with
petroleum jelly (or any other sticky material). The cover slip and drop are then
inverted over the well of a depression slide. The drop hangs from the cover slip, and
the petroleum jelly forms a seal that prevents evaporation. This preparation gives
good views of microbial motility.

Materials required:

• Glass slides (glass slide with depression) or Normal glass slide with adhesive or
paraffin ring

• Paraffin wax

• Loop

• Coverslip

• Microscope

• Bunsen burner

Young broth culture of motile bacteria

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Vet.Microbiology I
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Procedure:

1. Take a clean glass slide and apply paraffin ring, adhesive tape ring to make circular
concavity. (This step is not needed if a glass slide with depression is available).

2. Hold a clean coverslip by its edges and carefully dab Vaseline on its corners using a
toothpick.

3. Place a loopful of the broth culture to be tested in the center of the prepared coverslip.

4. Turn the prepared glass slide or concavity slide upside down (concavity down) over the
drop on the coverslip so that the vaseline seals the coverslip to the slide around the
concavity.

5. Turn the slide over so the coverslip is on top and the drop can be observed hanging from
the coverslip over the concavity.

6. Place the preparation in the microscope slide holder and align it using the naked eye so an
edge of the drop is under the low power objectives.

7. Turn the objective to its lowest position using the coarse adjustment and CLOSE THE
DIAPHRAGM.

8. Look through the eyepiece and raise the objective slowly using the coarse adjustment
knob until the edge of the drop is observed as an irregular line crossing the field.

9. Move the slide to make that line (the edge of the drop) pass through the center of the
field.

10. Without raising or lowering the tube, swing the high dry objective into position (Be sure
the high dry objective is clean).

11. Observe the slide through the eyepiece and adjust the fine adjustment until the edge of
the drop can be seen as a thick, usually dark line.

12. Focus the edge of the drop carefully and look at each side of that line for very small
objects that are the bacteria. The cells will look either like dark or slightly greenish, very
small rods or spheres. Remember the high dry objective magnifies a little less than half
as much as the oil immersion objective.

13. Adjust the light using the diaphragm lever to maximize the visibility of the cells.

14. Observe the cells noting their morphology and grouping and determine whether true
motility can be observed.

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 15. Brownian movement should be visible on slides of all the organisms, but there should
also show true motility.

16. Wash the depression slide and after soaking in lysol buckets or discard the prepared glass
slide.

Note: While examining living organism for the property of active locomotion, it is essential
to distinguish true motility, where by the organism move in different directions and change
their positions in the field, from either.

• Passive drifting of the organisms in the same direction in a convectional current in the
fluid or

• Brownian movement, which is an oscillatory movement about a nearly fixed point

possessed by all small bodies suspended in fluid and due to irregularities in their
bombardments by molecules of water.

7 Biochemical Tests
Biochemical Tests can be used to differentiate even closely related organisms.

 These various tests were designed to identify various metabolic properties of different
bacterial species.

 Catalase Test:-Metabolic reactions that occur in the presence of water and oxygen often
result in the formation of hydrogen peroxide (H2O2).

This compound is toxic to cells. Therefore, most organisms that can grow in the presence of
oxygen possess catalase, an enzyme that converts hydrogen peroxide to water and oxygen.
2H2O2 + catalase --> 2H2O + O2

Procedure

a. Place a small amount of growth from your culture onto a clean microscope slide. If using
colonies from a blood agar plate, be very careful not to scrape up any of the blood agar— blood
cells are catalase positive and any contaminating agar could give a false positive.

b. Add a few drops of H2O2 onto the smear. If needed, mix with a toothpick. DO NOT use a
metal loop or needle with H2O2; it will give a false positive and degrade the metal.

c. A positive result is the rapid evolution of O2 as evidenced by bubbling.

d. A negative result is no bubbles or only a few scattered bubbles.

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e. Dispose of your slide in the biohazard glass disposal container. Dispose of any toothpicks in
the Pipet Keeper.

• Test Staphylococcus aureus and Streptococcus pyogenes.

Triple Sugar Iron Agar (TSI) Tests

Composition of Triple Sugar Iron Agar (TSI)

• 0.1% Glucose: If only glucose is fermented, only enough acid is produced to turn the butt
yellow. The slant will remain red

• 1.0 % lactose/1.0% sucrose: a large amount of acid turns both butt and slant yellow, thus
indicating the ability of the culture to ferment either lactose or sucrose.

• Iron: Ferrous sulfate: Indicator of H2S formation

• Phenol red: Indicator of acidification (It is yellow in acidic condition and red under
alkaline conditions).

• It also contains Peptone which acts as source of nitrogen. (Remember that whenever
peptone is utilized under aerobic condition ammonia is produced)

 TSI is similar to Kligler’s iron agar, except that Kligler’s iron agar contains only two
carbohydrates: glucose (0.1%) and lactose (1%).

Materials Required:

• Culture:
• 24 hour typticase soy broth culture.
• Media:
• Triple sugar-iron agar slants.
• Equipments:
• Bunsen burner
• Inoculating needle
• Test tubes
• Marking pen

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Procedure:-

1. Sterilize the inoculating needle in the blue flame of the bunsen burner till red hot and then
allowed to cool.

2. From the rack, take the Trypticase soy broth tube containing the 24-48 hour culture,
remove the cap and flame the neck of the tube.

3. Using aseptic technique, take the culture of the organism from the TSB (tryptic soy
broth) tube with the needle.

4. Again flame the neck of the tube and replace the tube in the test tube rack.

5. Take a sterile TSI slant tube from the rack, remove the cap and flame the neck of the
tube.

6. Stab the needle containing the pure culture into the medium, up to the butt of the TSI
tube, and then streak the needle back and forth along the surface of the slant.

7. Again flame the neck of the TSI tube, cap it and place it in the test tube rack.

8. Incubate at 37oc for 18 to 24 hours.

Expected Results:

1. Alkaline slant (red) and acid butt (yellow) with or without gas production (breaks in the
agar butt):

Only glucose fermentation has occurred. The organisms preferentially degrade glucose
first. Since this substrate is present in minimal concentration, the small amount of the acid
produced on the slant surface is oxidized rapidly. The peptones in the medium are also used in
the production of alkali. At the butt, the acid reaction is maintained because of the reduced
oxygen tension and slower growth of the organisms.

2. Acid slant (yellow) and acid butt (yellow) with or without gas production:

Lactose or sucrose fermentation has occurred. Since these substances are present in higher
concentrations, they serve as substrates for continued fermentative activities with maintenance of
an acid reaction in both the slant and the butt.

3. Alkaline slant (red) and alkaline butt (red) or no change (orange-red) butt:

No carbohydrate fermentation has occurred. Instead; peptones are catabolized under anaerobic
and /or aerobic conditions resulting in alkaline pH due to production of ammonia. If only aerobic
degradation of peptones occurs, the alkaline reaction is evidenced only on the slant surface. If
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 there is aerobic and anaerobic utilization of peptone, the alkaline reaction is present on the slant
and the butt.

4. Hydrogen sulfide (H2S) production:

Some bacteria utilize thiosulfate anion as a terminal electron acceptor, reducing it to sulfide. If
this occurs, the newly-formed hydrogen sulfide (H2S) reacts with ferrous sulfate in the medium
to form ferrous sulfide, which is visible as a black precipitate. The blackening of the medium is
almost always observed in the butt (bottom) of the medium.

5. Carbon dioxide (CO2) production:

It is recognized simply as bubbles of gas between the agar and the wall of the tube or within the
agar itself. The carbon dioxide production is sufficient to split the agar into two or more sections.
To obtain accurate results, it is absolutely essential to observe the cultures within 18-24 hours
following incubation. This will ensure that the carbohydrate substrates have not been depleted

and that degradation of peptones yielding alkaline end products has not taken place.

Result (slant/butt) Symbol Interpretation

1..Red/Yellow K/A Glucose fermentation only, peptone catabolized.

2 .Yellow/Yellow A/A Glucose and lactose and/or sucrose fermentation.

3. Red/Red K/K No fermentation, Peptone catabolized.

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 Glucose and lactose and/or sucrose fermentation,
4. Yellow/Yellow with bubbles A/A,G
Gas produced.

5. Red/Yellow with bubbles K/A,G Glucose fermentation only, Gas produced.

6. Red/Yellow with bubbles and black Glucose fermentation only, Gas produced, H2S
K/A,G,H2S
precipitate produced.
7. Yellow/Yellow with bubbles and black Glucose and lactose and/or sucrose fermentation,
A/A,G,H2S
precipitate Gas produced, H2S produced.

8. Red/Yellow with black precipitate K/A,H2S Glucose fermentation only, H2S produced.

Glucose and lactose and/or sucrose fermentation,

9. Yellow/Yellow with black precipitate A/A,H2S
H2S produced.

Some example of Triple Sugar Iron (TSI) Agar Reactions

Name of the organisms Slant Butt Gas H2S

Escherichia, Klebsiella, Acid (A) Acid (A) Pos (+) Neg (-)
Enterobacter

Shigella, Serratia Alkaline (K) Acid (A) Neg (-) Neg (- )

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Salmonella, Proteus Alkaline (K) Acid (A) Pos (+) Pos (+)

Pseudomonas Alkaline (K) Alkaline (K) Neg (-) Neg (-)

• The oxidase test detects the presence of a cytochrome oxidase system that will
catalyse the transport of electrons between electron donors in the bacteria and a
redox dye- tetramethyl-p-phenylene-diamine.

Principle

• Cytochrome containing organisms produce an intracellular oxidase enzyme. This oxidase

enzyme catalyzes the oxidation of cytochrome c. Organisms which contain cytochrome c
as part of their respiratory chain are oxidase-positive and turn the reagent blue/purple.

• Organisms lacking cytochrome c as part of their respiratory chain do not oxidize the
reagent, leaving it colorless within the limits of the test, and are oxidase-negative.

Procedure: There are many method variations to the oxidase test.

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These include, but are not limited to, the filter paper test, direct plate method, swab
method, impregnated oxidase test strip method and test tube method.

Positive Result

• Development of a deep purple-blue/blue colour indicates oxidase production within 5-10

seconds.

Negative Result

• No purple-blue colour/No colour change.

Indole Test

This test demonstrate the ability of certain bacteria to decompose the amino acid tryptophane to
indole, which accumulates in the medium.

Principle

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. Indole is generated by reductive deamination from tryptophan via the intermediate molecule
indolepyruvic acid. Tryptophanase catalyzes the domination reaction, during which the ammonia
(-NH2) group of the tryptophan molecule is removed.

• Final products of the reaction are indole, pyruvic acid, ammonium (NH4+) and energy.
Pyridoxal phosphate is required as a coenzyme.

Two methods are in used;

1. a spot indole test, which detects rapid indole producing organisms and

2. a conventional tube method requiring overnight incubation, which identifies weak indole
producing organisms.

Procedure

1. Conventional tube method requiring overnight incubation, which identifies weak indole
producing organisms

 Take sterilized test tubes containing 4 ml of tryptophan broth.

 Inoculate the tube aseptically by taking the growth from 18 to 24 hrs culture.

 Incubate the tube at 37°C for 24-28 hours.

 Add 0.5 ml of Kovac’s reagent to the broth culture.

 Observe for the presence or absence of ring.

2. spot indole test, which detects rapid indole producing organisms

 Place several drops of Indole Spot Reagent on a piece of filter paper.

 With an inoculating loop or wooden applicator stick, pick a portion of an 18-24 hour
isolated colony from a non-selective media and rub it onto the reagent saturated area of
the filter paper.

 Examine immediately

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 Result Interpretation of Indole Test

Positive: Formation of a pink to red color (“cherry-red ring”) in the reagent layer on top of the
medium within seconds of adding the reagent.

Negative: No color change even after the addition of appropriate reagent

Citrate Utilization Test

This test is among a suite of IMViC Tests (Indole, Methyl-Red, Vogues-Proskauer, and Citrate)
that are used to differentiate among the Gram-Negative bacilli in the family Enterobacteriaceae.

Principle:

• When an organic acid such as citrate (Remember Krebs cycle) is used as a carbon and
energy source, alkaline carbonates and bicarbonates are produced ultimately. In addition,
ammonium hydroxide is produced when the ammonium salts in the medium are used as
the sole nitrogen source.

• Utilization of exogenous citrate requires the presence of citrate transport proteins

(permeases). Upon uptake by the cell, citrate is cleaved by citrate lyase to
oxaloacetate and acetate. The oxaloacetate is then metabolized to pyruvate and CO2.

• Citrate = oxaloacetate + acetate

• oxalacetate = pyruvate + CO2

Citrate Utilization Test

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 • Further metabolic breakdown is dependent upon the pH of the medium.

A. Under alkaline conditions, pyruvate is metabolized to acetate and formate.

pyruvate = acetate + formate

B. At pH 7.0 and below, lactate and acetoin are also produced.

pyruvate = acetate + lactate + CO2
pyruvate = acetoin + CO2
The carbon dioxide that is released will subsequently react with water and the sodium ion in the
medium to produce sodium carbonate, an alkaline compound that will raise the pH. In addition,
ammonium hydroxide is produced when the ammonium salts in the medium are used as the sole
nitrogen source.

Growth usually results in the bromothymol blue indicator, turning from green to blue. The
bromothymol blue pH indicator is a deep forest green at neutral pH. With an increase in medium
pH to above 7.6, bromothymol blue changes to blue.

Procedure of citrate utilization test:

• Inoculate Simmons Citrate Agar lightly on the slant by touching the tip of a needle to a
colony that is 18 to 24 hours old.

• Incubate at 35oC to 37oC for 18 to 24 hours. Some organisms may require up to 7 days of
incubation due to their limited rate of growth on citrate medium.

• Observe the development of blue color; denoting alkalinization.

Citrate positive: growth will be visible on the slant surface and the medium will be an intense
Prussian blue. The alkaline carbonates and bicarbonates produced as by-products of citrate
catabolism raise the pH of the medium to above 7.6, causing the bromothymol blue to change
from the original green color to blue .

Citrate negative: trace or no growth will be visible. No color change will occur; the medium
will remain the deep forest green color of the uninoculated agar. Only bacteria that can utilize
citrate as the sole carbon and energy source will be able to grow on the Simmons citrate medium,
thus a citrate-negative test culture will be virtually indistinguishable from an uninoculated slant.

• Quality Control strains used in citrate utilization test

Positive: Klebsiella pneumoniae

Negative: Escherichia coli

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List of Bacteria which give positive citrate utilization test

• Klebsiella pneumoniae

• Enterobacter species (minority of strains gives negative result)

• Citrobacter freundii

• Salmonella other than Typhi and Paratyphi A

• Serratia marcescens

• Proteus mirabilis (minority of strains gives negative result)

• Providencia

Citrate Test Variable (different strains give different results)

• Proteus vulgaris

• Vibrio cholerae

• Vibrio parahaemolyticus

• Citrate test Negative

• Escherichia coli

• Shigella spp

• Salmonella Typhi

• Salmonella Paratyphi A

• Morganella morganii

• Yersinia enterocolitica

• Although uncommon, natural E. coli variants that are citrate positive have been isolated.
Citrate-negative strains of E. aerogenes have also been found.

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 • Uses of Citrate Utilization Test
Citrate utilization test is often part of a battery of tests used to identify gram-
negative pathogens of Enterobacteriaceae family and environmental isolates. For
instance, test kits such as the API-20E and Enterotube II include citrate utilization
medium as one of the diagnostic tests.

Citrate utilization test (Image source: Microbe library)

A: Negative
B: Positive

Voges–Proskauer (VP) Test-

Principle of Voges–Proskauer (VP) Test

• The Voges-Proskauer (VP) test is used to determine if an organism

produces acetylmethyl carbinol from glucose fermentation.

• If present, acetylmethyl carbinol is converted to diacetyl in the presence of ∝- naphthol,

strong alkali (40% KOH), and atmospheric oxygen the ∝-naphthol was not part of the
original procedure but was found to act as a color intensifier by Barritt and must be added
first.

• The diacetyl and quanidine-containing compounds found in the peptones of the

broth then condense to form a pinkish red polymer.

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 • 2 pyruvate = acetoin + 2CO2
acetoin + NADH + H+ = 2,3-butanediol + NAD+

Media used in Voges–Proskauer (VP) Test

• MRVP broth (pH 6.9)

Ingredients per liter of deionized water:
buffered peptone= 7.0 gm
glucose= 5.0 gm
dipotassium phosphate= 5.0 gm

Reagents used in Voges–Proskauer (VP) Test

• Voges-Proskauer Reagent A: Barritt’s reagent A

Alpha-Naphthol, 5% 50 gm

Absolute Ethanol 1000 ml

• Voges-Proskauer Reagent B: Barritt’s reagent B

Potassium Hydroxide 400 gm

Deionized Water 1000 ml

Procedure of Voges–Proskauer (VP) Test

1) Prior to inoculation, allow medium to equilibrate to room temperature.

2) Using organisms taken from an 18-24 hour pure culture, lightly inoculate the medium.

3) Incubate aerobically at 37 degrees C. for 24 hours.

4) Following 24 hours of incubation, aliquot 2 ml of the broth to a clean test tube.

5) Re-incubate the remaining broth for an additional 24 hours.

6) Add 6 drops of 5% alpha-naphthol, and mix well to aerate.

7) Add 2 drops of 40% potassium hydroxide, and mix well to aerate.

8) Observe for a pink-red color at the surface within 30 min. Shake the tube vigorously
during the 30-min period.

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Coblentz Method for Streptococci

1) Use 24-hour growth from blood agar plate to heavily inoculate 2 mL of MRVP broth.

2) After 6 hours of incubation at 35°C in ambient air, add 1.2 mL (12 drops) of solution A
(alpha-naphthol) and 0.4 mL (4 drops) solution B (40% KOH with creatine).

3) Shake the tube and incubate at room temperature for 30 minutes.

Result Interpretation of Voges–Proskauer (VP) Test

• Positive Reaction: A pink-red color at the surface

Examples: Viridans group streptococci (except Streptococcus vestibularis), Listeria,
Enterobacter, Klebsiella, Serratia marcescens, Hafnia alvei, Vibrio eltor, Vibrio
alginolyticus, etc.

• Negative Reaction: A lack of a pink-red color

Examples: Streptococcus mitis, Citrobacter sp., Shigella, Yersinia, Edwardsiella,
Salmonella, Vibrio furnissii, Vibrio fluvialis, Vibrio vulnificus, and Vibrio
parahaemolyticus etc.

• A copper color should be considered negative. A rust color is a weak positive reaction.

Methyl Red (MR) Test

 Some bacteria have the ability to utilize glucose and convert it to a stable acid like lactic
acid, acetic acid or formic acid as the end product.

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  These bacteria initially metabolise glucose to pyruvic acid, which is further metabolized
through the ‘mixed acid pathway to produce the stable acid.

 The type of acid produced differs from species to species and depends on the specific
enzymatic pathways present in the bacteria.

 The acid so produced decreases the pH to 4.5 or below, which is indicated by a change in
the color of methyl red from yellow to red.

 In the methyl red test (MR test), the test bacteria is grown in a broth medium containing
glucose.

 If the bacteria has the ability to utilise glucose with production of a stable acid, the color
of the methyl red changes from yellow to red, when added into the broth culture.

 The mixed acid pathway gives 4 mol of acidic products (mainly lactic and acetic acid), 1
mol of neutral fermentation product (ethanol), 1 mol of CO2, and 1 mol of H2 per mol of
glucose fermented. The large quantity of acids produced causes a significant decrease in
the pH of the culture medium.

Media used in Methyl Red (MR) Test

• MRVP broth (pH 6.9)

Ingredients per liter of deionized water:
buffered peptone= 7.0 gm
glucose= 5.0 gm
dipotassium phosphate= 5.0 gm

.Reagents used in Methyl Red (MR) Test

Methyl red solution, 0.02%

a. Dissolve 0.1 g of methyl red in 300 ml of ethyl alcohol, 95%.
b. Add sufficient distilled water to make 500 ml.
c. Store at 4 to 8 degree C in a brown bottle. Solution is stable for 1 year.

Procedure of Methyl Red (MR) Test

1. Prior to inoculation, allow medium to equilibrate to room temperature.

2. Using organisms taken from an 18-24 hour pure culture, lightly inoculate the medium.

3. Incubate aerobically at 37 degrees C. for 24 hours.

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Vet.Microbiology I
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 4. Following 24 hours of incubation, aliquot 1ml of the broth to a clean test tube.

5. Reincubate the remaining broth for an additional 24 hours.

6. Add 2 to 3 drops of methyl red indicator to aliquot.

7. Observe for red color immediately.

Result Interpretation of Methyl Red (MR) test

• Positive Reaction: A distinct red color (A)

Examples: E. coli, Yersinia sps, etc.

• Negative Reaction: A yellow color (B)

Examples: Enterobacter aerogenes, Klebsiella pneumoniae, etc.

• A weak positive is red-orange. If an orange color is seen, incubate the remainder of the
broth for up to 4 days and repeat the test after further incubation. In this case it may also
be helpful to set up a duplicate broth at 25C.

Coagulase Test

Principle of Coagulase Test

• Coagulase is an enzyme-like protein and causes plasma to clot by converting fibrinogen

to fibrin.

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Vet.Microbiology I
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 • Staphylococcus aureus produces two forms of coagulase: bound and free.

Bound coagulase

 (clumping factor) is bound to the bacterial cell wall and reacts directly with
fibrinogen.

 This results in an alternation of fibrinogen so that it precipitates on the staphylococcal

cell, causing the cells to clump when a bacterial suspension is mixed with plasma. This
doesn’t require coagulase-reacting factor.

Free coagulase involves the activation of plasma coagulase-reacting factor (CRP), which is a
modified or derived thrombin molecule, to from a coagulase-CRP complex. This complex in turn
reacts with fibrinogen to produce the fibrin clot.

Procedure and Types of Coagulase Test

• Slide Test (to detect bound coagulase)

1) Place a drop of physiological saline on each end of a slide, or on two separate slides.

2) With the loop, straight wire or wodden stick, emulsify a portion of the isolated colony in
each drop to make two thick suspensions.

3) Add a drop of human or rabbit plasma to one of the suspensions, and mix gently.

4) Look for clumping of the organisms within 10 seconds.

5) No plasma is added to the second suspension to differentiate any granular appearance of

the organism from true coagulase clumping.

Tube Test (to detect free coagulase)

1. Dilute the plasma 1 in 10 in physiological saline (mix 0.2 ml of plasma with 1.8 ml of
saline).

2. Take 3 small test tubes and label as T (Test), P (Positive Control) and N (Negative
Control). Test is 18-24 hour broth culture, Positive control is 18-24 hr S. aureus broth
culture and Negative control is sterile broth.

3. Pipette 0.5 ml of the diluted plasma into each tube.

4. Add 5 drops (0.1 ml) of the Test organisms to the tube labelled “T”, 5 drops of S. aureus
culture to the tube labelled “P” and 5 drops of sterile broth to the tube labelled “N”.

5. After mixing, incubate the three tubes at 35-37 Degree Celsius.

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 6. Examine for clotting after 1 hours. If no clotting has occurred, examine at 30 minutes
intervals for up to 6 hours.

Interpretation of Coagulase Test

(Picture Source: University of Malta, Msida MSD 2080, Malta)

Fibrin Clot of any size- Positive

No Clot- Negative

• Clumping in both drops of slides indicates that the organism auto agglutinates and is
unsuitable for the slide coagulase test. All the negative slide test must be confirmed using
the tube test.

• During slide test, there may be chance to false positive results in case of citrate utilizing
bacteria ( Enterococcus and Pseudomonas). In this case also, tube test should be
performed and confirmed.

Examples

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 • Coagulase Positive Organisms: Staphylococcus aureus and other animal host bacteria like
S. pseudintermedius, S. intermedius, S. schleiferi, S. delphini, S. hyicus, S. lutrae, S.
hyicus

• Coagulase Negative Organisms: Staphylococcus epidermidis, S. saprophyticus, S.

warneri, S. hominis, S. caprae, etc.

• Coagulase Test- Principle, Procedure, Types, Interpretation and Examples

• Rated 4.9/5 based on 8 revi

Urease Test

• Principle of Urease Test

• Urea is the product of decarboxylation of amino acids. Hydrolysis of urea produces

ammonia and CO2. The formation of ammonia alkalinizes the medium, and the pH shift
is detected by the color change of phenol red from light orange at pH 6.8 to magenta
(pink) at pH 8.1. Rapid urease-positive organisms turn the entire medium pink within 24
hours.

• Weakly positive organisms may take several days, and negative organisms produce no
color change or yellow as a result of acid production.

Rapid Urease Test (RUT)

• The rapid urease test (RUT) is a popular diagnostic test for diagnosis of Helicobacter
pylori. It is a rapid, cheap and simple test that detects the presence of urease in or on the
gastric mucosa. It is also known as the CLO test (Campylobacter-like organism
test). This test uses a procedure called gastric endoscopy and biopsy to collect stomach
lining cells.

• The test is performed at the time of gastroscopy. A biopsy of mucosa is taken from the
antrum of the stomach, and is placed into a medium containing urea and an indicator such
as phenol red. The urease produced by H. pylori hydrolyzes urea to ammonia, which
raises the pH of the medium, and changes the color of the specimen from yellow
(NEGATIVE) to red (POSITIVE).

• The test can also be used to provide an informal assessment of the accuracy of the
histopathology result and discrepancies should prompt a review of the histopathology and
discussions with the pathologist.

Urea Breath Test Urea breath test is a common non-invasive test to detect Helicobacter
pylori also based on urease activity. This is highly sensitive and specific test.
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 • Patient ingests radioactively labeled Urea (either radioactive carbon-14 or non-
radioactive carbon-13). If infection is present, the urease produced by Helicobacter pylori
hydrolyzes the urea to form ammonia and labeled bicarbonate that is exhaled as CO2.
The labeled CO2 is detected either by a scintillation counter (Carbon-14) and a isotope
ratio mass spectrometry or by mass correlation spectrometry (Carbon-13).

Media used in Urease Test

• Christensen’s Urea Agar
• Composition
• Ingredients per liter of deionized water:*
• Urea 20.0 gm
• Sodium Chloride 5.0 gm
• Monopotassium Phosphate 2.0 gm
• Peptone 1.0 gm
• Dextrose 1.0 gm
• Phenol Red 0.012 gm
• Agar 15.0 gm
• Final pH 6.7 +/- 0.2 at 25 degrees C.

Preparation
1. Dissolve the ingredients in 100 ml of distilled water and filter sterilizes (0.45-mm pore
size).
2. Suspend the agar in 900 ml of distilled water, boil to dissolve completely.
3. Autoclave at 121 degree C and 15 psi for 15 minutes.
4. Cool the agar to 50 to 55 degree C.
5. Aseptically add 100 ml of filter-sterilized urea base to the cooled agar solution and mix
thoroughly.
6. Distribute 4 to 5 ml per sterile tube (13 x 100 mm) and slant the tubes during cooling
until solidified.

Procedure of Urease Test

I. Streak the surface of a urea agar slant with a portion of a well-isolated colony or
inoculate slant with 1 to 2 drops from an overnight brain-heart infusion broth culture.

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 II. Leave the cap on loosely and incubate the tube at 35°-37°C in ambient air for 48 hours to
7 days.

III. Examine for the development of a pink color for as long as 7 days.

Result Interpretation of Urease Test

• Positive Reaction: Development of an intense magenta to bright pink color in 15 min to

24 hr.
Examples: Proteus spp, Cryptococcus spp, Corynebacterium spp, Helicobacter
pylori, Yersinia spp, Brucella spp, etc.

• Negative Reaction: No color change.

Examples: Escherichia, Shigella, Salmonella,etc.

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Vet.Microbiology I
September 2020
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 8. Reference
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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