FEDERAL UNIVERSITY BIRNIN KEBBI

FACULTY OF SCIENCE
DEPARTMENT OF MICROBIOLOGY
Second Semester 2016/2017 Session
MCB 203 (Basic Techniques in Microbiology)

MCB 203: Basic Techniques in Microbiology

How to Write or Report a Practical

 A brief descriptive title

 Date at which the practical is done
 Purpose of the practical (Aim)
 Materials used for the practical
 The procedures
 Presentation of result (Result)
 Diagram (if any)
 Calculation (if any)
 Discussion
 Conclusion_a brief summary stating the significance of the practical.

Code of Practice

This code is a listing of the most essential laboratory practices and procedures that are used to
develop written practices and procedures for safe laboratory operations.

A. Access
1) The international biohazard warning symbol and sign must be displaced on the doors of
the rooms where microorganisms are handled.
2) Only authorized persons should be accorded to enter the laboratory working areas.
3) Laboratory doors should be kept closed always.
4) Children should not be allowed to enter laboratory working areas.
5) Access to laboratory animals should be specially authorized.
6) No animals should be admitted other than those involved in the work of the laboratory.

B. Personal protection
1) Laboratory coveralls (coat), gowns, or uniforms must be worn at all times for work in the
laboratory.
2) Appropriate gloves must be worn for all procedures that may involve direct or accidental
contact with body fluids and other potentially infectious materials or infected animals.
3) Persons working in the laboratory must wash their hands after handling infectious materials
and animals, and before they leave the laboratory working areas.
4) Safety glasses, face shields (visors) or other protective devices must be worn when it is
necessary to protect the eyes and face from splashes, impacting objects and sources of
artificial ultraviolet radiation.
5) It is prohibited to wear protective laboratory clothing outside the laboratory.
6) Open-toed foot wear must not be worn in the laboratory.
7) Eating, drinking, smoking, applying cosmetics and handling contact lenses is prohibited in
the laboratory working areas.
8) Storing human foods or drinks the laboratory fridge/freezer or anywhere in the laboratory
working areas is prohibited.
9) Protective laboratory clothing that has been used in the laboratory must not be stored in the
same locker or drawer with street clothing.
10) Except in extreme emergency, running or any over-hurried activity should be avoided in
and around the laboratories as should be practical jokes or other irresponsible behaviours.

C. Procedures
1) Pipetting by mouth is strictly forbidden.
2) Materials must not be placed in the mouth.
3) All technical procedures should be performed in a way that minimizes the formation of
aerosols and droplets.
4) The use of hypodermic needles and syringes should be limited. They must not be used as
substitutes for pipetting devices or for any purpose other than parenteral injection or
aspiration of fluids.
5) All spills, accidents and overt or potential exposures to infectious materials must be
reported to the laboratory supervisor/staff.
6) Contaminated liquids must be decontaminated (chemically or physically) before
discharging to the sanitary sewer.
7) A written procedure for the clean-up of all spills must be developed and followed.
8) Written/typed documents that are expected to be removed from the laboratory need to be
protected from contamination while in the laboratory.

D. Laboratory Working Areas

1) The laboratory should be kept neat, clean and free of materials that are not pertinent to the
work.
2) Work surfaces must be decontaminated after any spill of potentially dangerous material
and at the end of the day.
3) All contaminated materials, specimens and cultures must be decontaminated before
disposal or cleaning for re-use.
4) Packing and transportation must follow applicable regulations.
5) Windows should be fitted with arthropod-proof screens.
6) Every person working in the laboratory should ensure that he knows the exits and fire
escape routes and should have free access. The worker should also know the position of
fire extinguishers, fire blanket, fire buckets, and drench showers, and should make sure
he/she knows how to operate them. The person should also know the location of the First
Aids equipment provided for emergency, the position of the necessary telephone and the
numbers of the appropriate medical teams, hospitals.
7) After working hours experiment(s) which must be left running overnight should be sited in
a special overnight room. Clear instructions should be left so that an unqualified person
should not terminate the experiment in an unknowingly. For example, “Please leave it on”
Or “Work in progress”, etc. should be placed alongside any service which is to be left
running (equipment, water, electricity, etc.).
8) Waste disposal-Waste material must never be allowed to accumulate in the laboratory.
These should be removed regularly from the working area for storage in suitable containers
so that it can disposed appropriately. There shall be separate bins with properly fitting lids
for broken glassware and for inflammable materials such as paper or clothes which might
have been used to mop up inflammable liquids. Waste solids should be placed in bins
provided for such, while waste solvents in suitable containers appropriately labeled.
Arrangements by which the accumulated waste materials is disposed-off appropriately
should be made.
9) Accidents such as spilled cultures, cuts and abrasions should be reported immediately.
Existing cuts and abrasions should be adequately covered with a water-proof dressing. If it
occurred in the laboratory, it should be given the suitable first aid treatment as soon as
possible. Spilled cultures should be flushed with suitable disinfectant solution and allowed
to stand for 15-30 minutes before cleaning up. It must be noted that absence of breakages
does not imply the absence of dangers, since the dropping of cultures in a plastic petri dish
can result in the release of microbial aerosol into the atmosphere which can reach a highly
susceptible target-the lungs-undetected where it can produce maximum effect in low
dosage. Therefore, when a culture is dropped on the floor, the area should be treated with
a bactericidal solution and left for some time for a reduction in the concentration of any
aerosol which might have been generated.
10) Labels-All chemicals/reagents, sample bottles and vials must carry a label clearly
indicating the nature and possible hazards. In the case of rends which have lost their labels,
the contents should be positively identified and re-rebelled or disposed for safety if
undefined. Since gummed labels readily dry out and drop off, it is a good idea to seal the
labels with transparent adhering tape. It is also good to write the date of arrival or
preparations on the labels.
 NB: The tradition of excellence in academic pursuits include the performance of students in
practicals. As such, students have to exhibit not only theoretical but practical abilities in a bid
to excel.

BASIC TOOLS/EQUIPMENT USED IN MICROBIOLOGY LABORATORY

The Microscope

Microscope is an instrument used to produce a greatly magnified image of an object which may
be so small as to be invisible to the naked eye. In other-words, the microscope provides the
magnification or apparent enlargement that enables one to see organisms and structures invisible
to the unaided eye. Microscope permit a wide range of magnifications from one hundred times to
hundreds of thousand times. “No microscope, no Microbiology”.

The two categories of microscopes available are the light or optical microscopes and the electron
microscopes. The light or optical microscopes uses light as a radiation source for viewing the
specimen and combinations of lenses (the objective and eyepiece) to magnify the image. These
include the bright-field, the dark-field, the fluorescence and the phase contrast microscopes. The
electron microscopes employ a beam of electrons in place of light waves to produce the magnified
image. These include, the transmission electron microscope (TEM) and the scanning electron
microscope (SEM).

Other types of microscopes are used for research purposes, for procedures in the diagnostic
laboratory and for other special purposes.

In the modern light microscope, light rays from the object on the stage pass through two glass
lenses-the objective and the ocular lenses which depending on their strength give magnifications
of up to 800 times or more. Higher magnifications can be achieved without loss in resolution by
using special objective lens in conjunction with an immersion oil.

The study of the morphology of very small organisms is of such importance that a Microbiologist
must of necessity be a competent in the use of the Microscope. It is important for him/her to obtain
the best possible performance from the use of microscope and for this, a sound knowledge of the
basic optical principles involved is very essential.
Assignment #1

Draw and label a binocular microscope, and give the functions of the labeled parts.

The Autoclave

This is an instrument for sterilization of laboratory equipment/apparatus and media with steam
under pressure. The autoclave uses saturated steam to rapidly and efficiently sterilize articles. It
can be used in hospitals, public health centres, clinics, laboratories and scientific research
institutes, etc.

How to Use the Autoclave:

The operation of an autoclave often vary slightly depending on the type and model. The basic
procedures are however the same:

1. Water: Put 3.5 litres of water into the container sufficient enough to cover the electric tube
(heating element) and just below the container. As each time of sterilization will consume
certain amount of water, add water to the rated amount before using it again, otherwise the
electric tube will be damaged owing to water shortage.
2. Piling: Put the articles/materials to be sterilized on the sieve plate in the sterilizing tank,
properly wrapped up, leaving certain space between the bundles so as to let the steam
penetrate, and thus guaranteeing sterilizing quality.
3. Sealing: Put the container in the sterilizing tank and cover the tank with the lid, then evenly
tighten the wing nuts to closely fit each other.
4. Heating: Plug the power cable and switch on the socket. At this time, heating begins and
so the steam-release valve and the safety valve should be pulled up to let out the cold air.
When fast steam comes out of the two valves, push down the handles on the two valves.
The indicator of the pressure gauge will show the steam pressure in the sterilizatio n tank.
5. Steam pressure: When the steam pressure reaches 0.14 Mpa, the safety valve will work
automatically to ensure constant pressure. Once the temperature reaches 121 0 C and the
pressure of 15 psi (pounds per square inch), exposure continue to the holding time period
of 15 minutes.
6. Cooling: Once the holding period is over, turn off power and allow the
temperature/pressure gauge to return to zero. Open the steam-release valve and the cover.
Remove the material sterilized.

Assignment #2
Draw an autoclave and label its parts.

The Incubator

This is an apparatus for maintaining a constant and suitable temperature for the growth of
microorganisms and living cells. Incubators vary in size from small portable to large incubators.
It has a varied temperature range and therefore can be used in the incubation of cultures over a
wide range of temperatures. The temperature should be checked daily and monitored. The
incubation temperature of bacteria and fungi vary.
Assignment #3

Draw an incubator and label its parts.

Hot-Air-Oven

This is an enclosed space equipment for heating objects/materials which can withstand high
temperature for a length of time needed for sterilization by dry heat. This materials are likely to be
affected by contact with steam in autoclave sterilization. The minimum recommended holding
times for sterilization using hot-air-oven is 1600 C for 1-2 hours. Hot-air-oven sterilization is the
best method for sterilization of dry glassware such as test-tubes, glass petri-dishes, flasks, glass
pipettes, and instruments such as forceps, scalpels, scissors, spatula, etc.

An important precaution for achieving satisfactory sterilization is not to pack the load in the oven
too tightly. Vegetative forms of bacteria are killed by dry heat at a temperature just over 1000 C in
1-2 hours. Many of the vegetative forms may die within 1 hour, but increased time is necessary to
ensure complete sterilization. Spores are more resistant to heat thus requiring a temperature of
1400 C for 2-3 hours.
Assignment #4

Draw an hot-air-oven and label its parts.

Wire loops and Straight wire

These are used for inoculation. The original type of wire was made of platinum, but owing to the
high cost of platinum, nichrome resistant wire is now generally used.

One end of the wire is inserted into a glass rod or inserted into a special alluminium holder. The
other end is bent in the form of a loop 2 mm internal diameter (wire loop). The loop must be
completely closed.

Straight wire is similar to the wire loop but without the loop in this case. It is used to stab media
during inoculation and also for picking single colonies.

The wire is sterilized by holding it vertically in a Bunsen flame so that the whole length becomes
red-hot at the same time before and after use.
Anaerobic jar

This is a tightly sealed environmental chamber that can maintain an anaerobic oxygen-free
atmosphere, used for the incubation and storage of anaerobic bacterial cultures. The jar has a
removal lid for the addition or removal of agar petri dishes.

The anaerobic jar is used in a situation where an anaerobic atmosphere is essential for the growth
of strict anaerobes such as Clostridium species.

Several methods are used to obtain anaerobic conditions in the laboratory:

a) The use of anaerobic jar with hydrogen in a cylinder.

b) The use of anaerobic incubator with gas generating units.
c) The use of copper and steel to remove oxygen.
d) The use of sodium thioglycollate to remove oxygen.
e) The use of Gas pack system (H2 + catalyst).

The Centrifuge

This is a laboratory device that applies a relatively high centrifugal force to a liquid specimen
causing its separation to different fractions according to their specific gravities. It allows speedy
separation of microorganisms from the liquid in which they are suspended. The centrifuge is
equipped with a cover that holds cups attached in such a manner that they can swing out and rotate
in a horizontal plane during centrifugation. The tubes are placed in the cups and held at an angle
of 30-500 C. During rotation, the tubes are held in a fixed position. When centrifuging, the units
should always be balanced to prevent breaking of tubes and cause damage to the centrifuge. Tubes
should be placed directly opposite each other, should be of the same weight and should contain
the same amount of liquid.

The device is safe, easy to use, designed for minimum vibration and further clamped by standing
on the rubber feet. It is driven by single motor with up to 2500 revolution per minute, ventilated
and spill proof.

When the tubes rotate, the liquid is subjected to centrifugal force. The particles becomes
compacted at the bottom of the tubes forming a deposit which can then be separated from the
supernatant fluid.

Components of the centrifuge

i. A central shaft or spindle which rotates at high speed.

ii. A head fixed to the shaft with buckets for holding the tubes.
iii. A number of tubes cups which are fixed to the head.
 Assignment #5

Draw a centrifuge and label its parts.

Petri dish

This is a round, shallow, flat-bottomed transparent glass or plastic dish with a slightly large, similar
shaped dish that forms the cover. It is used to hold a solid culture medium for growing cultures. It
is available in glass type, which is re-usable or plastic type, which is mostly disposable.
Assignment #6

Draw a petri dish and label its parts.

Weighing balance

This is an instrument for weighing samples, media, etc. it is a delicate instrument that requires
practical instruction for its correct use. There are different types of weighing balance. The accuracy
of a balance should be checked regularly.

pH meter

The most accurate method of measuring pH is by using a pH meter. This apparatus is therefore a
necessary piece of laboratory equipment where numerous routine determinations of pH are
required. This instrument measures the difference in potential hydrogen ion concentration between
two or more solutions. A pH meter consists of an electrode pair, which is sensitive to hydrogen
ion concentrations.

Water bath

This is a constant temperature device electrically generated and controlled by a thermostat that
holds temperature within required limits. An accurate thermometer should always be immersed to
check the accuracy of the water bath.

Culturing of Microorganisms

Culture Media Preparation and Inoculation

1) Sterilizing glassware in a hot-air-oven at a temperature of 1600 C for 1 hour is required,

timed from when the oven reaches the expected temperature. A cooling time is also
necessary to enable the items in the oven to cool slowly. The oven door must not be opened
until the temperature inside the oven has fallen to about 50 0 C. This will avoid cracking of
the glassware, and contaminated air being drawn into the oven.
 2) Weigh the media to be used according to the manufactures specification and dispense it
into the required amount of distilled water in a conical flask or any other suitable container.
3) Using the magnetic stirrer (or Bunsen burner), boil and stir for proper mixing.
4) Seal the conical flask with cork and aluminium foil.
5) Load the material into the autoclave to sterilize.
6) After autoclaving, allow the media to cool to about 45 0 C in a water bath, before use.
7) Arrange the sterile petri dishes on a level surface.
8) Mix the medium gently by rotating the flask or bottle to avoid forming air bubbles. Flame
the neck of the flask or bottle and pour (15-20 ml) into each petri dish. Gently rotate the
dish on the surface of the bench to ensure an even layer of agar.
9) When the medium has gelled and solidified, inoculate the sample and incubate at 37 0 C for
bacteria and 250 C for fungi.

Inoculation Techniques:

1) Streaking method:
 Heat wire loop red hot in a Bunsen flame, then leave to cool.
 Loosen sample bottle’s lid carefully, then pass the mouth of the bottle through Bunsen
flame. Do not put lid down on the bench.
 Insert the sterilized loop and pick up a loop-full (drop) of the liquid sample.
 Pass bottle neck through flame again before applying lid.
 Loop-full of the liquid may be transferred to agar in a petri dish or broth in universal
bottle.
 Lid of the petri dish needs to be opened at an angle of 45 0 C and the sample streaked
over the agar surface. In case of broth, the loop-full of the sample is transferred directly
into the liquid medium.
 Invert the petri dish so as to prevent condensation droplets from falling onto the agar
surface. Petri dishes are often sealed using two strips of adhesive (masking) tape from
base to lid in order to prevent people who handle them from contamination.
 The inoculated agar plates will then be incubated at 370 C (bacteria) for 18-24 hours.
Bacteria on agar plates becomes visible as distinct circular colonies, each colony
represent an individual bacteria (group of) cell which has divided repeatedly, but being
kept in one place.
 Liquid media such as broth becomes cloudy if there is bacteria growth. This could be
as a result of repeated binary fission of the inoculated bacterium.

2) Pour plate method:

 Liquid sample is introduced into the petri dish using a micropipette before the molten
agar medium is poured on top.
 Gently rotate the petri dish on the surface of the bench to mix properly.
  Allow the agar to solidify. Invert the petri dish so as to prevent condensation droplets
from falling onto the agar surface. Petri dishes are often sealed using two strips of
adhesive (masking) tape from base to lid in order to prevent people who handle them
from contamination.
 Incubate the plates at 370 C (bacteria) for 18-24 hours. Bacteria on agar plates becomes
visible as distinct circular colonies, each colony represent an individual bacteria (group
of) cell which has divided repeatedly, but being kept in one place.

Note: By extension of this method using serial dilutions (in sterile distilled water), the number
of bacteria (colony forming units) can be counted/calculated.

3) Spread plate method:

 Liquid sample is introduced onto the surface of the solid agar in a petri dish using a
micropipette.
 Sterile glass rod is then used to spread the sample evenly.
 Samples collected on sterile swab sticks are inoculated onto the surface of the solid
agar and spread evenly.
 Incubate the plates at 370 C (bacteria) for 18-24 hours. Bacteria on agar plates becomes
visible as distinct circular colonies, each colony represent an individual bacteria (group
of) cell which has divided repeatedly, but being kept in one place.

Note: By extension of this method using serial dilutions (in sterile distilled water), the number
of bacteria (colony forming units) can be counted/calculated.

*Make a smear from the colonies formed and Gram stain the slides for microscopic view.

BIOCHEMICAL TESTS

CATALASE TEST

The test is used to differentiate those bacteria that produce the enzyme catalase, such as
Staphylococci, from non-catalase producing bacteria such as Streptococci.

Principle: Catalase acts as a catalyst in the breakdown of hydrogen peroxide to oxygen and water.
An organism is tested for catalase production by bringing it into contact with hydrogen peroxide.
Bubbles of oxygen (O 2 ) are released if the organism is a catalase producer. The culture should not
be more than 24 hours old.

Required: Hydrogen peroxide, 3% H2 O2 (10 volume solution).

Method:

1) Pour 2-3 ml of the hydrogen peroxide solution into a test tube.

2) Using a sterile wooden or a glass rod (not a nichrome wire loop), remove a colony of the
organism and immerse in the hydrogen peroxide solution.
3) Care must be taken when testing an organism cultured on a medium containing blood
because catalase is present in red cells. If any of the blood agar is removed with the
organism, a false reaction may occur.
4) Look for immediate bubbling.

When the rapid slide technique is used, the hydrogen peroxide solution should be added to the
organism suspension after placing the slide in a petri dish. The dish should then be covered
immediately, and the preparation observed for bubbling through the lid.

Results:

Active bubbling……………………………………………….positive catalase test

No bubbles…………………………………………………….Negative catalase test

Controls:

Positive catalase control………………………………………Staphylococcus spp

Negative catalase control……………………………………...Streptococcus spp

OXIDASE TEST

The oxidase test is used to assist in the identification of Pseudomonas, Neisseria, Vibrio, Brucella,
and Pasteurella species, all of which produce the enzyme cytochrome oxidase.

Principle: A piece of filter paper is soaked with a few drops of oxidase reagent. A colony of the
test organism is then smeared on the filter paper (alternatively an oxidase reagent strip can be
used). When the organism is oxidase-producing, the phenylenediamine in the reagent will be
oxidaized to a deep purple clolour.

Required: Oxidase reagent freshly prepared (or an oxidase reagent strip). The strip have a 5 year
shelf-life when stored at 2-80 C.

Fresh reagent method:

1) Place a piece of filter paper in a clean petri dish and add 2 0r 3 drops of freshly prepared
oxidase reagent.
2) Using a piece of stick or glass rod (not an oxidized wire loop), remove a colony of the test
organism and smear it on the filter paper.
3) Look for the development of a blue-purple colour within few seconds (10 seconds).
Results:

Blue-purple colour (within 10 seconds)…………………………….Positive oxidase test

No blue-purple clour (within 10 seconds)…………………………..Negative oxidase test

Note: Ignore any blue-purple colour that develops after 10 seconds.

Oxidase reagent strip method:

1) Moisten the strip with a drop of sterile distilled water.

2) Using a piece of stick or glass rod (not an oxidized wire loop), remove a colony of the test
organism and smear (rub) it on the strip.
3) Look for a red-purple colour within 20 seconds.
*Red-purple colour…………………………………………….Positive oxidase test

COAGULASE TEST

This test is used to identify Staphylococcus aureus which produces the enzyme coagulase.

Principle: Coagulase causes plasma to clot by converting fibrinogen to fibrin. Two types of
coagulase are produced by most strains of the S. aureus:

 Free coagulase which converts fibrinogen to fibrin by activating a coagulase reacting factor
present in plasma. Free coagulase is detected by clotting in the tube test.
 Bound coagulase (clumping factor) which converts fibrinogen directly to fibrin without
requiring a coagulase reacting factor. It can be detected by the clumping of bacterial cells
in the rapid slide test.

A tube test must always be performed when the result of a slide test is not clear, or when the slide
test is negative and Staphylococcus has been isolated from a serious infection.

Required: EDTA (Ethylenediamine tetra-acetic acid) anticoagulated human plasma (preferably

pooled and previously HIV and Hepatitis tested) or rabbit plasma. The plasma should be allowed
to warm to room temperature before being used.

Plasma: Oxalate or heparin plasma can also be used. Do not use citrated plasma because citrate-
utilizing bacteria e.g. Enterococcus, Pseudomonas, and Serratia may cause clotting of the plasma
(in tube test). Occasionally, human plasma may contain inhibitory substances which can interfere
with coagulase testing. It is therefore essential to test the plasma using a known coagulase positive
S. aureus. The plasma can be stored frozen in amounts ready to use.
Slide test method (detects bound coagulase):

1) Place a drop of sterile distilled water on each end of a slide or on two separate slides;
2) Emulsify a colony of the test organism to make two thick suspensions;
3) Add a loop-full (not more) of plasma to one of the suspentions and mix gently, look for
clumping of the organisms within 10 seconds.

Test tube method (detects free coagulase):

1) Take three (3) small test tubes and label: T=Test organism (18-24 hour broth culture);
Pos=Positive control (18-24 hour S. aureus broth culture); Neg=Negative control (sterile
broth). Nutrient broth is suitable;
2) Pipette 0.2 ml of plasma into each test tube;
3) Add 0.8 ml of the test broth culture to tube ‘T’; add 0.8 ml of the S. aureus to the tube
labelled ‘pos’; and add 0.8 ml of sterile broth to the tube labelled ‘Neg’.
4) After mixing gently, incubate the three tubes at 35-370 C. Examine for clotting after 1 hour.
If no clotting has occurred, examine after 3 hours. If the test is still negative, leave the tube
at room temperature overnight and examine again.

Note: When looking for clotting tilt each tube gently.

Results:

Clotting of tube contents or fibrin clot in tube…………………… S. aureus

No clotting or fibrin clot………………………………………….. Negative test

Lactose fermenters (Enteric organisms): Escherichia, Klebsiella, Enterobacter, Citrobacter, etc.

Non-lactose fermenters: Salmonella, Shigella, Proteus, Morganella, Yersinia, Providencia,

Serratia, Edwardsiella, Hafnia, etc.

Bile solubility test

This helps to differentiate Streptococcus pneumoniae, which is soluble in bile and bile salts, from
other alpha-haemolytic streptococci (viridans streptococci) which are insoluble.

Principle

A heavy inoculum of the test organism is emulsified in physiological saline and the bile salt sodium
deoxycholate is added. This dissolves S. pneumoniae as shown by clearing of the turbidity within
10-15 minutes. Viridans and other streptococci are not dissolved and therefore there is no clearing
of the turbidity.

Required:

 Sodium deoxycholate, 100 g/l (10% w/v)

 Physiological saline (sodium chloride, 8.5 g/l)
Tube method

Although the bile solubility test can be performed by testing colonies directly on a culture plate or
on a slide, a tube technique is recommended because the results are easier to read.

1. Emulsify several colonies of the test organism in a tube containing 2 ml sterile

physiological saline, to give a turbid suspension.
2. Divide the organism suspension between two tubes.
3. To one tube, add 2 drops of the sodium deoxycholate reagent and mix.
4. To the other tube (negative control), add 2 drops of sterile distilled water and mix.
5. Leave both tubes for 10-15 minutes at 35-370 C.
6. Look for a clearing of turbidity in the tube containing the sodium deoxycholate.

Results
Clearing of turbidity……………………………………..Probably S. pneumoniae

No clearing of turbidity………………………………….Organism is probably not S. pneumoniae

There should be no clearing of turbidity in the negative control tube to which sterile distilled water
was added.

Note: Some strains of S. pneumoniae are not dissolved by bile salts, and very occasionally some
strains of viridans streptococci give a positive test

Controls

Bile solubility positive control: Streptococcus pneumoniae

Bile solubility negative control: Enterococcus faecalis

Citrate utilization test

This test is one of several techniques used occasionally to assist in the identification of
enterobacteria. The test is based on the ability of an organism to use citrate as its only source of
carbon.
Ways of performing a citrate test

 Using a Rosco citrate identification tablet. This is the most economical method when only
a few tests are performed. The tablets have a long shelf-life and good stability in tropical
climates.
 Using Simmon’s citrate agar but the dehydrated medium is only available in 500 g pack
size from manufacturers. After being opened, the medium does not have good stability in
tropical climates.
Citrate utilization using a Rosco citrate tablet

Citrate identification tablets are available from Rosco diagnostica in a vial of 50 tablets.

1) Prepare a dense bacterial suspension of the test organism in 0.25 ml sterile physiological
saline in small tube.
2) Add a citrate tablet and stopper the tube.
3) Incubate overnight at 35-370 C.

Results

Red colour…………………………………………………………….Positive citrate test

Yellow-orange colour…………………………………………………Negative citrate test

Controls

A positive citrate test reaction is obtained with Klebsiella pneumoniae and a negative reaction with
Escherichia coli.

Citrate method using Simmon’s citrate agar

1) Prepare slopes of the medium in bijou bottles as recommended by the manufacturer (store
at 2-80 C).
2) Using a sterile straight wire, first streak the slope with a saline suspension of the test
organism and then stab the butt.
3) Incubate at 35-370 C for 48 hours. Look for a bright blue colour in the medium.

Results
Bright blue…………………………………………………………Positive citrate test

No change in colour of medium.....………………………………..Negative citrate test

DNA-ase test

This test is used to help in the identification of S. aureus which produces deoxyribonuclease (DNA-
ase) enzymes. The DNA-ase test is particularly useful when plasma is not available to perform a
coagulase test or when the results of a coagulase test are difficult to interpret.

Principle

Deoxyribonuclease hydrolyse deoxyribonucleic acid (DNA). The test organism is cultured on a

medium which contains DNA. After overnight incubation, the colonies are tested for DNA-ase
production by flooding the plate with a weak hydrochloric acid solution. The acid precipitates
unhydrolyzed DNA. DNA-ase-producing colonies are therefore surrounded by clear areas due to
DNA hydrolysis.
Required

 DNA-ase agar plate (up to six organisms may be tested on the same plate).
 Hydrochloric acid 1 mol/l (1 N).

Method

1) Divide a DNA-ase plate into the required number of strips by marking the underside of the
plate.
2) Using a sterile loop or swab, spot-inoculate the test and control organisms. Make sure each
test area is labelled clearly.
3) Incubate the plate at 35-370 C overnight.
4) Cover the surface of the plate with 1 mol/l hydrochloric acid solution. Tip off the excess
acid.
5) Look for clearing around the colonies within 5 minutes of adding the acid.

Results

Clearing around the colonies…………………………………………DNA-ase positive strain.

No clearing around the colonies………………………………………DNA-ase negative strain.

Indole test

Testing for indole production is important in the identification of enterobacteria. Most strains of
E. coli, P. vulgaris, P. rettgeri, M. morganii, and Providencia species break down the amino acid
tryptophan with the release of indole.

Principle

The test organism is cultured in a medium which contains tryptophan. Indole production detected
by Kovac’s or Ehrlich’s reagent which contains 4 (p)-dimethylamino-benzaldehyde. This reacts
with the indole to produce a red coloured compound. Kovac’s reagent is recommended in
preference to Ehrlich’s reagent for the detection of indole from enterobacteria.

Ways of performing an indole test

An indole test can be performed:

 As a single test using tryptone water and Kovac’s reagent.

 As a combined beta-glucuronidase-indole test using a Rosco PGUA/Indole identification
tablet and Kovac’s reagent. This is useful when identifying E. coli.
 As a combined lysine dcarboxylase-indole testusing a Rosco LDC/Indole identification
tablet. This is useful in helping to identify Salmonellae and Shigellae.
Detecting indole using tryptone water

1) Inoculate the test organism in a bijou bottle containing 3 ml of sterile tryptone water.
2) Incubate at 35-370 C for up to 48 hours.
3) Test for indole by adding 0.5 ml of Kovac’s reagent. Shake gently. Examine for a red colour
in the surface layer within 10 minutes.

Results

Red surface layer…………………………………………………..Positive indole test

No red surface layer………………………………………………..Negative indole test

Detecting indole using Rosco PGUA/Indole tablet

PGUA/Indole tablets are available from Rosco Diagnostica in a vial of 50 tablets. They have a
long shelf life (3-4 yrs).

1) Prepare a dense suspension of the test organism in 0.25 ml physiological saline in a small
tube.
2) Add a PGUA/Indole tablet and close the tube. Incubate at 35-370 C for 3-4 hours (for
overnight).
3) First read the beta-glucuronidase (PGUA) reaction:
Results
Yellow colour…………………………………………………….Positive PGUA test
Colourless………………………………………………………...Negative PGUA test
4) Add 3 drops of Kovac’s reagent and shake.
5) Wait 3 minutes before reading the indole reaction. Examine the colour of the surface layer.
Results
Red surface layer…………………………………………..Positive indole test
Yellow surface layer……………………………………….Negative indole test

Note: About 94% of E. coli strains are PGUA positive and 99% are indole positive.

Detecting indole using Rosco LDC/Indole tablet

LDC/Indole tablets are available from Rosco Diagnostica in a vial of 50 tablets. They have long
shelf-life (3-4 yrs).

1) Prepare a dense suspension of the test organism in 0.25ml physiological saline in a small
tube.
2) Add an LDC/Indole tablet. Add 3 drops of paraffin oil* and close the tube.
*The oil overlayer provides the anaerobic conditions required for the LDC reaction.

3) Incubate at 35-370 C for 3-4 hours (or overnight).

4) First read the lysine decarboxylase (LDC) reaction.

 Results

Blue/violet colour*……………………………………………..Positive LDC test

Yellow, green or grey colour…………………………………..Negative LDC test

*If examining after overnight incubation, a positive test is indicated by a strong blue or
violet colour.
5) Add 3 drops of Kovac’s reagent and shake.

6) Wait 3 minutes before reading the indole reaction. Examine the colour of the surface layer.

Results

Red surface layer…………………………………………………….Positive indole test

Yellow surface layer…………………………………………………Negative indole test

Litmus milk decolorization test

The test is a rapid inexpensive technique to assist in the identification of enterococci. It is based
on the ability of most strains of Enterococcus species to reduce litmus milk by enzyme action
shown by decolorization of the litmus.
Note: Enterococci can also be identified using aesculin hydrolysis test.

Principle

A heavy inoculum of the test organism is incubated for up to 4 hours in a tube containing litmus
milk. Reduction of the litmus milk is indicated by a change in colour of the medium from mauve
to white or pale yellow.

Required: Litmus milk medium

Method

1) Using a sterile loop, inoculate 0.5 ml of sterile litmus milk medium with the test organism.
Important: A heavy inoculum of the test organism must be used. Scraping the loop three
times across an area of heavy growth is recommended.
2) Incubate at 35-370 C for up to 4 hours, examining at half an hour intervals for a reduction
shown by a change in colour from mauve to white or pale yellow (compare with the positive
control).
Note: The incubation time should not be more than 4 hours because some strains of viridans
streptococci will reduce litmus milk with prolong incubation.
Results

White or pale yellow-pink colour……………………………………Suggestive of Enterococcus

No change or pink colour………………………………………………Probably not Enterococcus

Controls

Positive control: Enterococcus species

Negative control Viridans Enterococcus

Note: Previous work have shown that 83% of Enterococcus could be identified by the rapid litmus
milk reduction test. No false positive reactions were observed. A negative result can be checked
by culturing the organism in aesculin broth and examining daily for up to 7 days for aesculin
hydrolysis shown by blackening in the medium. Enterococci hydrolyze aesculin.

Aesculin hydrolysis test to identify enterococci

This test can be economically performed using a Rosco bile aesculin tablet. The tablets are
available from Rosco Diagnostica in a vial of 25 tablets. They have good stability (3-4 yrs).

The test can be performed by placing a tablet on a blood agar plate inoculated with the test
organism and incubating it at 35-370 C overnight. A positive test is indicated by the tablet and
colonies around it turning black/grey. A negative test is shown by the tablet remaining white and
no change in colour of the colonies. A zone of inhibition may appear around the tablet.

Alternatively, the test can be performed by making a dense suspension in the test organism in 0.25
ml of physiological saline in a small tube, adding a tablet, and incubating at 35-370 C for 4 hours
(for overnight). A positive reaction is shown by black/grey colour in the medium.

Note: An aesculin hydrolysis can also be performed by incubating the test organism on bile
aesculin agar but this medium is expensive.

Urease test

Testing for urease enzyme activity is important in differentiating enterobacteria. Proteus strains
are strong urease producers. Y. enterocolitica also shows urease activity (weakly at 35-370 C).
Salmonella and Shigellae do not produce urease.

Principle

The test organism is cultured in a medium which contains urea and the indicator phenol red. When
the strain is urease-producing, the enzyme will break down the urea (by hydrolysis) to give
ammonia and carbon dioxide. With the release of ammonia, the medium becomes alkaline shown
by a change in colour of the indicator to pink-red.
Ways of performing a urease test

 Using a Rosco urease identification tablet

 Using modified Christensen’s urea broth

Urease test using a Rosco urease tablet

Urease identification tablets are available from Rosco Diagnostica in a vial of 50 tablets. The
tablets have a long shelf-life (3-4 yrs).

1) Prepare a dense ‘milky’ suspension of the test organism in 0.25 ml physiological saline in
a small tube.
2) Add a urease tablet, close the tube and incubate at 35-370 C (preferably in a water bath for
a quicker result) for up to 4 hours or overnight. Proteus and M. morganii organism give a
positive reaction within 4 hours.
Results
Red/purple colour………………………………………………….Positive urease test
Yellow/orange……………………………………………………..Negative urease test

Urease test using Christensen’s (modified) urea broth

1) Inoculate heavily the test organism in a bijou bottle containing 3 ml sterile Christensen’s
modified urea broth.
2) Incubate at 35-370 C for 3-12 hours (preferably in a water bath for a quicker result).
3) Look for a pink colour in the medium.
Results
Pink colour……………………………………………………….Positive urease test
No pink colour…………………………………………………...Negative urease test

Carbohydrate Fermentation Tests

Principle

These are used to test the ability of an organism to ferment a specific carbohydrate (glucose,
lactose, maltose or sucrose) and to produce acid, or acid and gas. Indicator: Andrade’s indicator (a
solution of acid fuchsin to which is added sodium hydroxide), Bromocresol purple, Bromothymol
blue or phenol red is used. The medium change in colour as a result of lowering of the pH due to
acid production by fermentation of carbohydrates. Colour change only occurs when sufficient
amount of acid is produced, as bacteria may utilize the peptone producing alkaline by product.
Small inverted Durham’s tube immersed in the medium shows gas production (hydrogen or
carbondioxide), if present. If the test organism produce gas, the gas displace the media present
inside the Durham’s tube and get trapped above producing a visible air burble. The term
fermentation is often used to describe the breaking down or catabolism of carbohydrate under
anaerobic conditions. Therefore, bacteria capable of fermenting a carbohydrate are usually
facultative anaerobes. It is used to identify gram-negative bacilli such as Escherichia coli,
Klebsiella species, Salmonella species, Proteus species, etc.

 All members of Enterobacteriaceae family are glucose fermenters (they can metabolize
glucose anaerobically)
 Maltose fermentation differentiates Proteus vulgaris (positive) from Proteus mirabilis
(negative).
 Both Neisseria gonorrhoeae (gonococci) and Neisseria meningitides (meningococci)
ferments glucose, but only meningococci ferments maltose.
 Rapid carbohydrate utilization test can be performed to identify Corynebacterium
diphtheriae and other Corynebacterium species.

Common end-products of bacterial fermentation include lactic acid, formic acid, acetic acid,
butyric acid, butyl alcohol, ethyl alcohol, acetone, carbondioxide and hydrogen.

Procedure

1) Prepare a broth medium of peptone water (or phenol red carbohydrate broth) and add single
carbohydrate source based on your requirement (preferred carbohydrate concentration is
1%).
2) Fill 13 x 100 mm test tube with 4-5 ml of the peptone broth.
3) Insert a Durham’s tube to detect gas production.
4) Autoclave the prepared test media (at 1210 C for 15 minutes) to sterilize. The sterilization
process will also drive the broth into the inverted Durham’s tube. [Note: When using
arabinose, lactose, maltose, salicin, sucrose, trehalose, or xylose, autoclave at 121 0 C for 3
minutes as these carbohydrates are subject to breakdown by autoclaving.
5) After autoclaving allow the medium to cool to 45 0 C and inoculate each test tube with 18-
24 hours culture of the test organism.
6) Incubate tubes at 35-370 C for 18-24 hours. Longer incubation period may be required to
confirm a negative result.

Result

Change in colour of the medium…………………………………………Acid production: Positive

No change in colour of the medium……………………………………..Acid production: Negative

Bubble (small or big space above in the inverted tube)………………...Gas production: Positive
No bubble (or space in the inverted tube)………………………………Gas production: Negative
Triple Sugar Iron (TSI) Agar

Principle: By the aid of a composite medium, e.g., TSI agar medium, different different properties
of a bacterium can be studied in a single medium by incorporating specific carbohydrates in it with
or without the production of gas, along with the determination of possible hydrogen sulphide (H2 S)
production. It is composed of glucose, sucrose, lactose, ferric citrate and phenol red indicator are
added for testing H2 S production.

Procedure

1) Prepare the medium in the form of a butt and slant in the test tube.
2) Inoculate the medium using straight wire by first stabbing the butt and then streaking the
slope in a zig-zag pattern.
3) Incubate at 35-370 C for 24-48 hours.
Result

H2 S production is indicated by blackening of the medium.

Possible combination of different TSI reactions (remember that the slant reaction is folloed by butt
reaction):

i) K/A (red/yellow)-Glucose only fermented

ii) A/A (yellow/yellow)-Glucose fermented, lactose and/or sucrose fermented.
iii) K/K (red/red)-Neither glucose, lactose, nor sucrose fermented.

*Key: (K-Alkaline; A-Acidic).

Methyl Red-Voges Proskauer Test

Principle

Methyl Red (MR) test determines whether the microbe performs mixed acids fermentation when
supplied with glucose. Mixed acid fermentation is one of the two broad patterns, 2-3-butanediol
fermentation being another. In mixed acid fermentation, three acids (acetic, lactic and succinic)
are formed in significant amounts. The mixed acid pathway gives 4 mol of acetic acid products
(mainly lactic and acetic acid), 1 mol of neutral fermentation product (ethanol), 1 mol of CO 2 , and
1 mol of H2 per mol of glucose fermented. The large amounts of acid results in significant decrease
in the pH of the medium below 4.4. This is visualized by using pH indicator, methyl red (p-
dimethylaminobenzene-O-carboxylic acid), which is yellow above pH 5.1 and red at pH 4.4 and
below. The pH at which methyl red detects acid is considerably lower than the pH for other
indicators used in bacteriology culture media. Thus, to produce a colour change, the test organism
must produce large quantities of acid from carbohydrate substrate being used.
Voges-Proskauer is a double eponym named after two microbiologists. They first observed the red
colour reaction produced by appropriate culture media after treatment with potassium hydroxide.
It was later discovered that the active product in the medium formed by bacterial metabolism is
acetyl methyl carbinol, a product of the butylenes glycol pathway. Pyruvic acid, the pivotal
compound in the fermentative degradation of glucose, is further metabolized through various
metabolic pathways, depending on the enzyme systems possessed by different bacteria. One such
pathway result in the production of acetoin (acetyl methyl carbinol), a neutral-reacting end product.
Organisms such as the members of the Klebsiella-Enterobacter-Hafnia-Serratia group produce
acetoin as the chief end product of glucose metabolism and form smaller quantities of mixed acids.
In the presence of atmospheric oxygen and 40% potassium hydroxide, acetoin is converted to
diacetyl, and alpha-naphthol serves as a catalyst to bring out a red complex.
Procedure:

 Inoculated a colony of the test organism into 5ml of MR-VP broth and incubated for 48-
72hours at 370 C.
 After this period of incubation, transfer 2 ml of the inoculated broth to a sterile test tube.
 To this small quantity, add 2-3 drops of methyl red.
 Development of a red colour on the addition of the indicator (methyl red) signified positive
methyl red (MR) test.
 A yellow colour signified a negative test.
 To the rest of the inoculated broth in the original test tube, add 3 drops of 5% α-Naphthol
reagent, followed by 40% KOH and shaken vigorously losing the cap of the test tube and
placed in a sloping position.
 The development of red colour starting from the liquid air interphase within 1 hour
indicates a VP positive test.
 No colour change occurs signified a VP negative test.

Control

MR Positive control: Escherichia coli

MR Negative control: Enterobacter aerogenes

VP Positive control: Enterobacter aerogenes

VP Negative control: Escherichia coli

GRAM STAIN

SAFETY CONSIDERATIONS
Be careful with the Bunsen burner flame. Do not use volatile and flammable liquids near an open
flame. If the stains used in this experiment get on your clothing, they will not wash out. Discard
slides in a container with disinfectant. Hold all slides with forceps or a clothespin when heat-fixing.
Gram crystal violet, safranin, and iodine can cause irritation to the eyes, respiratory system and
skin. Avoid contact with skin and eyes. Do not breathe spray. Wear suitable protective gloves.
Always keep the containers tightly closed.

Objective
The major objective of this exercise is to enable the student to correctly use the Gram stain to
differentiate a mixture of bacteria into gram-positive and gram-negative cells.

Medical Application
Gram staining is the single most useful test in the clinical microbiology laboratory. It is the
differential staining procedure most commonly used for the direct examination of specimens and
bacterial colonies because it has a broad staining spectrum. The Gram stain is the first differential
test run on a bacterial specimen brought into the laboratory for specific identification. The staining
spectrum includes almost all bacteria, many fungi, and parasites such as Trichomonas,
Strongyloides, and miscellaneous protozoan cysts. The significant exceptions include Treponema,
Mycoplasma, Chlamydia, and Rickettsia, which are too small to visualize by light microscopy or
lack a distinct cell wall.

Principles
Simple staining depends on the fact that bacteria differ chemically from their surroundings and
thus can be stained to contrast with their environment. Bacteria also differ from one another
chemically and physically and may react differently to a given staining procedure. This is the
principle of differential staining. Differential staining can distinguish between types of bacteria.
The Gram stain (named after Christian Gram, Danish scientist and physician, 1853–1938) is the
most useful and widely employed differential stain in bacteriology. It divides bacteria into two
groups— gram negative and gram positive.
The first step in the procedure involves staining with the basic dye crystal violet. This is the
primary stain. It is followed by treatment with an iodine solution, which functions as a mordant;
that is, it increases the interaction between the bacterial cell and the dye so that the dye is more
tightly bound or the cell is more strongly stained. The smear is then decolorized by washing with
an agent such as 95% ethanol or isopropanol-acetone. Gram-positive bacteria retain the crystal
violet-iodine complex when washed with the decolorizer, whereas gram-negative bacteria lose
their crystal violet-iodine complex and become colorless. Finally, the smear is counterstained
with a basic dye, different in color than crystal violet. This counterstain is usually safranin. The
safranin will stain the colorless, gram-negative bacteria pink but does not alter the dark purple
color of the gram-positive bacteria. The end result is that gram-positive bacteria are deep purple
in color and gram-negative bacteria are pinkish to red in color. The Gram stain does not always
yield clear results. Species will differ from one another in regard to the ease with which the crystal
violet-iodine complex is removed by ethanol. Gram-positive cultures may often turn gram negative
if they get too old. Thus, it is always best to Gram stain young, vigorous cultures rather than older
ones. Furthermore, some bacterial species are gram variable. That is, some cells in the same
culture will be gram positive and some, gram negative. Therefore, one should always be certain to
run Gram stains on several cultures under carefully controlled conditions in order to make certain
that a given bacterial “strain” is truly gram positive or gram negative. Indistinct Gram-stain results
can be confirmed by a simple test using KOH. Place a drop of 10% KOH on a clean glass slide
and mix with a loopful of bacterial paste. Wait 30 seconds, then pull the loop slowly through the
suspension and up and away from the slide. A gram-negative organism will produce a mucoid
string; a gram-positive organism remains fluid. In most introductory microbiology laboratories,
the bacteria that are used in staining exercises are normally relatively small gram-negative or gram-
positive cocci and rods. One usually does not have the opportunity to observe larger bacteria or
those with differences in morphology and reproduction.

Procedure for Gram-Stain Technique

1. Make a smear from the colonies formed on your culture plates (24 hours culture) and allow
it to air-dry (smears should be spread evenly covering an area of about 15-20 mm diameter
on a slide);
2. Fix the dried smear with the Bunsen flame. Note that when smear is for the detection of
gonococci or meningococci, it should be fixed with methanol for 2 minutes (to avoid
damaging pus cells);
3. Cover the fixed smear with crystal violet stain for 30-60 seconds;
4. Rapidly wash off the stain with clean water. [Note: when the tap water is not clean, use
filtered water or clean boiled rainwater];
5. Tip off all the water, and cover the smear with Lugol’s iodine for 30-60 seconds;
6. Wash off the iodine with clean water;
7. Decolorize rapidly (few seconds) with acetone-alcohol. Wash immediately with clean
water. [Caution: Acetone-alcohol is highly flammable therefore use it away from an open
flame];
8. Cover the smear with neutral red (or safranin) stain for 60 seconds;
9. Wash off the stain with clean water; and
10. Blot dry with bibulous paper or air dry and examine under oil immersion. Gram-positive
organisms stain blue to purple; gram-negative organisms stain pink to red. There is no need
to place a coverslip on the stained smear.
Control
It is very important that controls be included in each staining run, preferably on the same slide
using Staphylococcus aureus (ATTC 25923) and Escherichia coli (ATCC 25922). Both of these
are also available from Difco as Bactrol™ Disks.

HINTS AND PRECAUTIONS

(1) Don’t make your smears too thick.
(2) Thick smears will require more time to decolorize than thin ones.
(3) Decolorization has occurred when the solution flows colorlessly from the slide. If you cannot
tell accurately when the solution becomes colorless, try decolorizing with isopropanol-acetone
mixture for about 30 to 40 seconds.
(4) Some common sources of Gram-staining errors are:
(a) the inoculating loop was too hot,
(b) excessive heat was used during the heat-fixing procedure, and
(c) the decolorizing alcohol was left on the slide too long.

Procedure for Isolation of Pure Cultures and Their Maintenance

1. With a wax pencil, label the agar slants with the names of the respective bacteria. Do the same
for the broth tubes. Add your name and date.
2. Using aseptic technique, select a well-isolated colony for each of the three bacteria and pick off
as much of the center of the colony as possible with an inoculating loop. It may be necessary to
obtain material from more than one colony. Prepare a slant culture and broth tube for each of
the bacteria. If screw-cap tubes are used, they must be loosened slightly before incubation to
keep the slant aerobic.
3. After incubating for 24 to 48 hours, you should have three pure slant and three pure broth stock
cultures.
4. Observe the broth cultures while taking care not to agitate them. Record your observations in
the report book.
5. Place the pure cultures in the refrigerator for later use.

Transferring Techniques
 Stab technique for transferring bacteria. Notice that the inoculating needle is moved into
the tube without touching the walls of the tube, and the needle penetrates medium to its
depth.
 Technique for streaking the surface of a slant with a loop is in zigzag manner.

A pure culture—one containing a single kind of microorganism - can be isolated from an

enrichment culture in many ways. Common isolation procedures include the streak plate, the agar
dilution, and liquid dilution. For organisms that form colonies on agar plates, the streak plate is
quick, easy, and the method of choice; if a well-isolated colony is selected and re-streaked several
successive times, a pure culture can usually be obtained. With proper incubation facilities (for
example, anoxic jars or anoxic chambers for anaerobes, it is possible to purify both aerobes and
anaerobes on agar plates by the streak plate method.

HINTS AND PRECAUTIONS

(1) When pipetting, always position your eyes so that they are horizontal with the top of the fluid
column in the pipette. This avoids parallax (an apparent displacement of position of an object
due to change in the observer’s position) errors that can occur from misalignment of the
meniscus with the graduated line on the pipette. Hold the pipette vertical and use your
forefinger to control the flow. Remember to always use a pipetting aid to fill the pipette and
do not pipette by mouth.
(2) Media containing fermentable carbohydrates should be avoided for the maintenance of
cultures.
(3) Selective media should never be used.
(4) Cultures should not be allowed to dry out; tightly closed screw-cap tubes should be used for
storage.
(5) Be sure to flame and cool needles between all inoculations to avoid incidental cross-
contamination of cultures.

ENUMERATION OF MICROORGANISMS
DETERMINATION OF BACTERIAL NUMBERS

SAFETY PRECAUTIONS
No mouth pipetting. Be careful with the Bunsen burner flame and water baths.

Medical Application
In the clinical laboratory and in research laboratories, it is frequently necessary to have an accurate
count of living (viable) cells in a given culture. If done properly, counting procedures can produce
very accurate results.

Principles
Many studies require the quantitative determination of bacterial populations. The two most widely
used methods for determining bacterial numbers are the standard, or viable, plate count method
and spectrophotometric (turbidimetric) analysis. Although the two methods are somewhat
similar in the results they yield, there are distinct differences. For example, the standard plate count
method is an indirect measurement of cell density and reveals information related only to live
bacteria. The spectrophotometric analysis is based on turbidity and indirectly measures all bacteria
(cell biomass), dead or alive. The standard plate count method consists of diluting a sample with
sterile saline or phosphate buffer diluent until the bacteria are dilute enough to count accurately.
That is, the final plates in the series should have between 25 and 250 colonies. Fewer than 25
colonies are not acceptable for statistical reasons, and more than 250 colonies on a plate are likely
to produce colonies too close to each other to be distinguished as distinct colony-forming units
(CFUs). The assumption is that each viable bacterial cell is separate from all others and will
develop into a single discrete colony (CFU). Thus, the number of colonies should give the number
of live bacteria that can grow under the incubation conditions employed. A wide series of dilutions
(e.g., 10–4 to 10–10 ) is normally plated because the exact number of live bacteria in the sample is
usually unknown. Greater precision is achieved by plating duplicates or triplicates of each dilution.
Increased turbidity in a culture is another index of bacterial growth and cell numbers (biomass).
By using a spectrophotometer, the amount of transmitted light decreases as the cell population
increases. The transmitted light is converted to electrical energy, and this is indicated on a
galvanometer. The reading indirectly reflects the number of bacteria. This method is faster than
the standard plate count but is limited because sensitivity is restricted to bacterial suspensions of
107 cells or greater.

Dilution Ratios
According to the American Society for Microbiology Style Manual, dilution ratios may be reported
with either colons (:) or shills(slash) (/), but note there is a difference between them. A shill
indicates the ratio of a part to a whole; for example, 1/2 means 1 of 2 parts, with a total of 2 parts.
A colon indicates the ratio of 1 part to 2 parts, with a total of 3 parts. Thus, 1/2 equals 1:1, but 1:2
equals 1/3.

Procedure
Standard Plate Count
1. With a wax pencil, label the bottom of six petri plates with the following dilutions: 10 –4 , 10–5 ,
10–6 , 10–7 , 10–8 , and 10–9 . Label four bottles of saline or phosphate buffer 10 –2 , 10–4 , 10–6 , and
10–8 .
2. Using aseptic technique, the initial dilution is made by transferring 1.0 ml of liquid sample or 1
g of solid material to a 99 ml sterile saline blank. This is a 1/100 or 10 –2 dilution. Cap the bottle.
3. The 10–2 blank is then shaken vigorously 25 times by placing one’s elbow on the bench and
moving the forearm rapidly in an arc from the bench surface and back. This serves to distribute
the bacteria and break up any clumps of bacteria that may be present.
4. Immediately after the 10–2 blank has been shaken, uncap it and aseptically transfer 1.0 ml to a
second 99 ml saline blank. Since this is a 10 –2 dilution, this second blank represents a 10 –4
dilution of the original sample. Cap the bottle.
5. Shake the 10–4 blank vigorously 25 times and transfer 1.0 ml to the third 99 ml blank. This third
blank represents a 10–6 dilution of the original sample. Cap the bottle. Repeat the process once
more to produce a 10–8 dilution.
6. Shake the 10–4 blank again and aseptically transfer 1.0 ml to one petri plate and 0.1 ml to another
petri plate. Do the same for the 10–6 and the 10–8 .
7. Remove one agar pour tube from the 48° to 50°C water bath. Carefully remove the cover from
the 10–4 petri plate and aseptically pour the agar into it. The agar and sample are immediately
mixed by gently moving the plate on the tabletop. Repeat this process for the remaining five
plates.
8. After the pour plates have cooled and the agar has hardened, they are inverted and incubated at
35°C for 24-48 hours.
9. At the end of the incubation period, select all of the petri plates containing between 25 and 250
colonies. Plates with more than 250 colonies cannot be counted and are designated too
numerous to count (TNTC). Plates with fewer than 25 colonies are designated too few to
count (TFTC). Count the colonies on each plate. If at all possible, a special counter such as a
Quebec colony counter should be used. Your instructor will demonstrate how to use this counter
or a handheld counter.
10. Calculate the number of bacteria (CFU) per milliliter or gram of sample by dividing the number
of colonies by the dilution factor. The number of colonies per ml reported should reflect the
precision of the method and should not include more than two significant figures. For example,
suppose the plate of the 10–6 dilution yielded a count of 130 colonies. Then, the number of
bacteria in 1 ml of the original sample can be calculated as follows:
Bacteria/ml = (130) ÷ (10–6 ) = 1.3 x 108 or 130,000,000.

Types of Colony Counter

a) The Leica Dark-field Quebec Colony Counter. The counter illuminates the petri plate uniformly
from the side, and the plate is magnified for easier counting of small colonies. The exclusive
dark-field design provides even, glare-free illumination. Contrasted against dark-field
background, colonies are bright—readily distinguished from other structures in agar. In this
model, an electric probe is touched to each colony to record the count automatically.
b) An electronic handheld colony counter. This model is a combination marker and counter. When
the petri plate is touched, it is marked by the pen and counted at the same time. An electronic
beep verifies each entry. A cumulative total appears on an easy-to-read LCD.

Turbidimetry Determination of Bacterial Numbers

1. Put one empty tube and four tubes of the sterile broth in a test-tube rack. With the exception of
the empty tube, each tube contains 3 ml of sterile broth. Use four of these tubes (tubes 2 to 5)
of broth to make four serial dilutions of the culture.
2. Standardize and use the spectrophotometer as follows:
a. Turn on the spectrophotometer.
b. Set the monochromator dial so that the correct wavelength in nanometers (550 to 600 nm) is
lined up with the indicator in the window adjacent to this dial. (Your instructor will inform you
which wavelength to use.)
c. When there is no cuvette in the cuvette holder, the light source is blocked. The pointer should
thus read zero transmittance or infinite absorbance. This is at the left end of the scale. Turn knob
until the pointer is aligned with the left end of the scale.
d. Place in the cuvette holder the cuvette that contains just sterile broth. This tube is called the
blank because it has a sample concentration equal to zero. It should therefore have an
absorbance of zero (or a transmittance of 100%). This is at the right end of the scale. Set the
pointer to the right end of the scale using knob.
e. Place the other cuvettes, which contain the diluted bacterial suspension, in the cuvette chamber
one at a time. Repeat steps c and d between experimental readings to confirm settings.
f. Close the hatch and read the absorbance values of each bacterial dilution, and record your values.
Remember to mix the bacterial suspension just before reading its absorbance.
g. Record your values in the report book. Using the plate count data, calculate the colony forming
units per milliliter for each dilution.

HINTS AND PRECAUTIONS

When mixing dilution tubes, do not use a vortex mixer.

ENUMERATION OF SOIL MICROORGANISMS

SAFETY CONSIDERATIONS
Be careful with the Bunsen burner flame. No mouth pipetting.

Principles
Actinomycetes (including actinoplanetes, nocardioforms, and streptomycetes), other bacteria, and
filamentous fungi (Rhizopus, Mucor, Penicillium, and Aspergillus) are all important members of
the soil microbial community. Protozoa, algae, cyanobacteria, nematodes, insects and other
invertebrates, and viruses are also important members but will not be studied in this exercise. Each
gram of rich garden soil may contain millions of these micro- and macroorganisms. Since soils
vary greatly with respect to their physical features (e.g., pH, general type, temperature, and other
related factors), the microorganisms present will also vary. For example, acid soils will have a
higher number of fungi compared to alkaline soils, and rich garden soil will contain more
actinomycetes than either the other bacteria or fungi. Not surprisingly, no single technique is
available to count the microbial diversity found in average garden soil.
Thus, in this exercise, each group of students will try to determine only the relative number of
fungi, actinomycetes, and other bacteria in a sample of garden soil using the serial dilution agar
plating procedure covered.
To support the three different groups of microorganisms, you will use three types of media:
(1) Sabouraud dextrose agar for the isolation of fungi,
(2) Glycerol yeast agar for the isolation of actinomycetes, and
(3) Nutrient agar for the isolation of other bacteria.
Procedure
1. Place 1 g of garden soil in a 99 ml sterile water blank. Mix the soil and water thoroughly by
shaking the water-soil mixture vigorously for 3 minutes, keeping your elbow on the lab table.
Transfer 1 ml of this mixture to the second water blank and mix as above. Transfer 1 ml of this
mixture to the third water blank and mix as above.
2. Using a wax pencil, label three sets of four petri plates each as follows: actinomycetes (10 –3 , 10–
4 , 10–5 , and 10–6 ), fungi (10–2 , 10–3 , 10–4 , and 10–5 ), and other bacteria (10–4 , 10–5 , 10–6 , and 10–
7 ). Be sure to use the correct medium for each type of microorganism.

3. Set the pipette at 1 ml, then using aseptic technique, distribute the proper amount of each soil
dilution to the respective petri plates.
4. Remove the melted glycerol yeast agar from the water bath and pour 15 ml into each of the
actinomycetes petri plates. Mix on a flat surface, using a circular motion, and allow to solidify.
Do the same for the Sabouraud dextrose and the nutrient agars.
5. Invert the petri plates and incubate for 3 to 7 days (for fungi plates) and between 24-48 hours at
370 C for the bacteria plates. Observe daily for the appearance of colonies. Count the plates with
fewer than 250 colonies but more than 25. Designate plates with over 250 colonies as too
numerous to count (TNTC) and those with less than 25 colonies as too few to count (TFTC).
Record your data in the report book.
6. Determine the number of respective microorganisms per milliliter of original culture (gram of
soil) as follows:
Microorganisms per gram of soil = count/plate dilution used
For example, if 200 colonies were present on the 10 –7 plate, the calculation would be
Microorganisms per gram of soil = 200/10–7 = 2.0 x 109 colonies/gram

HINTS AND PRECAUTIONS

Optimal results in this exercise depend on thoroughly mixed soil samples containing evenly
distributed microorganisms.

BACTERIAL COUNT OF A FOOD PRODUCT

SAFETY CONSIDERATIONS
In this experiment, students will be taking unknown samples and growing them to large
concentrations. Any of these samples could contain human pathogens; thus, extreme caution
should be taken when working with and disposing of the final product. No mouth pipetting. Always
handle raw meat with caution.

Principles
The sanitary control of food quality is primarily concerned with testing food products for the
presence of specific microorganisms. Food products are the primary vehicle responsible for the
transmission of microbial diseases of the gastrointestinal system. For this reason, food products
are routinely examined for the presence of bacteria. The heterotrophic plate count can be used
to determine the number of viable bacteria in a food sample. The larger the count, the greater the
likelihood that specific pathogens capable of causing disease will be present and also that the food
will spoil. Normally, raw hamburger should not contain over 10 6 bacteria per gram. One of the
limitations of the heterotrophic plate count is that only bacteria capable of growing in the culture
medium under the environmental conditions provided will be counted. As a result, a medium that
supports the growth of most heterotrophic (requiring organic carbon) bacteria is commonly used.

Procedure
First Period
1. Weigh out 20 g of raw hamburger or chicken or any other solid food sample.
2. Blend the 20 g of the meat in a blender with 180 ml of sterile distilled water for 5 minutes. This
gives a 1/10 dilution.
3. Make dilutions from 10-2 to 10-6 .
4. Using the wax pencil, label the petri plates with your name, date, and dilution.
5. Using aseptic technique, pipette 1 ml aliquots from the dilution blanks to the petri plates.
6. Melt the plate count agar (PCA) pours in a water bath and cool to about 48° to 50°C. Add the
cooled PCA agar to the plates. Gently swirl on a flat surface and allow to solidify.
7. Incubate the plates in an inverted position for 24 to 48 hours at 37°C.
Second Period
1. Arrange the plates in order of lowest to highest dilution.
2. Count the number of colonies on the plates that falls within the range of 25 to 250 colonies.
Designate plates with fewer than 25 colonies as too few to count (TFTC) and plates with more
than 250 colonies as too numerous to count (TNTC).
3. Calculate the average number of bacteria per gram of hamburger (or chicken, or any other solid
food sampls) as follows:
Number bacteria/gram = number of colonies x 1/dilution factor x 1/weight of sample
For example, if 200 colonies were counted from a
10-3 dilution of a 20 gram sample:
Number bacteria/gram = 200 x 1/10-3 x 1/ 20g = 10,000 cfu/per gram.

AGGLUTINATION REACTIONS: BLOOD GROUPS

SAFETY CONSIDERATIONS
Human blood may be a vehicle for the transmission of blood-borne diseases such as AIDS and
hepatitis. At a minimum, precautions should include:
1. The use of disposable gloves by the students.
2. Procedures for the prevention of accidental needle or lancet sticks.
3. The use of appropriate containers for the disposal of lancets, cotton, slides, toothpicks, and
gloves. Immediately report any blood that has been dropped on the lab table, bench, or floor to
your instructor.
Principles
When agglutination reactions involve the clumping of red blood cells, they are termed
hemagglutinations. Hemagglutination occurs because of the ability of antibodies to cross-link red
blood cells by binding to surface antigens. Hemagglutination microscopic-slide tests are routinely
used in blood typing. The four major human blood types (A, B, AB, and O) are genetically
determined. Individuals with blood types A, B, or AB have antigens on the surface of their red
blood cells. These antigens, known as agglutinogens, correspond to the specific group (e.g., type
A has agglutinogen A). An individual having blood type O lacks these agglutinogens. These
surface antigens are also referred to as isoantigens (antigens that exist in alternative forms in a
species and which are capable of causing an immune response in genetically different individuals
of the same species, but not in individuals who carry them). The ABO blood grouping system is
unique because individuals who have isoantigens on their red blood cells will have the opposite
isoantibodies (agglutinins) in their serum. For example, persons with type A red blood cells have
anti-B isoantibodies.

Antigens and Antibodies in Human ABO Blood Groups

Blood Antigen present on Antibody in Can receive Can give blood

Type Erythrocyte plasma blood from to
Membranes
A A Anti-B A and O A and AB
B B Anti-A B and O B and AB
AB A and B Neither Anti-A nor AB; A; B; O AB Only
Anti-B
O Neither Anti-A and Anti-B O O; A; B; AB.

Transfusion reactions, or incompatibility reactions, generally result when blood typing is done
incorrectly. Such a reaction is caused by the transfusion of the wrong ABO group. This is due to
the fact that the blood of a person receiving a different blood type from his/her own will agglutinate
these cells. The ABO antibodies will cause rapid destruction of the transfused blood, and the
clumped erythrocytes may block blood vessels. In this exercise, students will use antibodies
against type A and B antigens to determine their own (or commercially prepared and tested) blood
type, using microscopic-slide agglutination reactions. In addition, data will be pooled for the whole
class in order to determine blood group frequencies. (If a slide-warming, blood-typing box is
available, your instructor may also want to demonstrate and include Rh typing data.)

Procedure
1. Obtain a microscope slide and wash it thoroughly in the grease-cutting detergent. With the wax
pencil, write the letters A and B on each side of the slide. Draw two separate circles next to the
letters, about 1.5 cm in diameter.
2. Obtain dropper bottles containing commercially prepared antibody solutions and place a drop
of the antibody to a particular blood type within the circle near the corresponding letter.
3. To obtain some of your blood, clean the tip of your finger by rubbing it with an alcohol saturated
cotton swab or gauze pad with iodine and again with alcohol.
4. Obtain a new and clean sterile lancet from the package and jab the point into your finger. If an
automatic lancet device is available, your instructor will demonstrate how to use it.
Never use a lancet that someone else has used because viral diseases such as AIDS and
hepatitis can be transmitted in this way.
5. Carefully massage your finger, and work out several drops of blood. Discard the first two drops
since they represent mostly tissue fluid. Let the third or fourth drop fall into the anti-A antibody
on the slide and the next two into the anti-B antibody circle. (Do not touch your finger to the
slide.) Dispose of the lancet in the proper SHARPS container.
6. Stir the antibody and the blood in each circle immediately using a rotary motion.
7. After donating blood, wipe your finger once more with an alcohol pad or gauze pad.
8. Observe the two mixtures against a light background with bright light, watching for the
appearance of red granules, which indicate that the red blood cells are agglutinating or
clumping. If you cannot easily see differences between the two samples, study the slide with
your microscope. Use the low-power objective with the sub-stage condenser closed to increase
contrast.
9. Determine your blood type according to the following chart:

Antisera Blood Group

A B
No clumping No clumping O
Clumping No clumping A
No clumping Clumping B
Clumping Clumping AB

HINTS AND PRECAUTIONS

(1) You should massage your finger after using the lancet and then release pressure to allow several
drops of blood to flow freely and evenly. Discard the first few drops of blood as they may
contain tissue fluid.
(2) If you are using an Autolet automatic lancet or a similar device to draw blood, carefully remove
and discard both the lancet and the platform after every use. NEVER reuse the platform or you
may transfer infected blood to another person.
(3) Usually it is less painful if you draw blood from the side of the fingertip rather than from the
tip itself.
URINE ANALYSIS

Possible pathogens

Bacteria:
Gram positive Gram negative

Staphylococcus Escherichia coli

Saprophyticus Proteus species

Haemolytic Streptococci Pseudomonas aeruginosa

Klebsiella strains

Salmonella typhi
Salmonella paratyphi

Neisseria gonorrhoeae

The species are not primarily pathogens of the urinary tract, but may be found in urine. Also,
Mycobacterium tuberculosis, Leptospira interrogans, Chlamydia, Mycoplasma, and Candida
species.

Parasites:
Schistosoma haematobium,

Trichomonas vaginalis, and

Occasionally Enterobius vermicularis, Wuchereria bancrofti and Onchocerca volvulus.

*Finding intestinal parasites in urine indicates faecal contamination.

Collection of urine

Whenever possible, the first urine passed by the patient at the beginning of the day (early morning
urine) should be sent for examination. This specimen is the most concentrated and therefore the
most suitable for culture, microscopy, and biochemical analysis.

Description of the urine specimen

Report:

 Colour of specimen
 Whether it is clear or cloudy (turbid)
 Appearance Possible cause
Cloudy (urine usually has an unpleasant smell Bacterial urinary infection
and contains WBCs)
Red and cloudy (due to red cells) Urinary Schistosomiasis
Bacterial infection
Brown and cloudy (due to haemoglobin) Malaria haemoglobinuria
Other conditions that cause intravascular
haemolysis
Yellow-brown, or green-brown (due to Acute viral hepatitis
bilirubin) Obstructive jaundice
Yellow-orange (due to urobilin, i.e. oxidized Haemolysis
urobilinogen) Hepatocellular jaundice
Milky-white (due to chyle) Bancroftian filariasis
Pale-yellow Normal

Preparation and examination of wet preparation

1) Aseptically transfer 5-10 ml of well mixed urine into a labelled test tube;
2) Centrifuge at 500-1000g for 5 minutes. Pour the supernatant fluid (by completely inverting
the tube) into a second container not the original one. This can be used for biochemical
tests;
3) Remix the sediment by tapping the bottom of the tube. Transfer one drop of the well-mixed
sediment to a slide and cover with cover slip (glass). [Do not discard the remaining
sediment because this may be needed to prepare Gram smear if WBCs and, or bacteria are
seen in the wet preparation];
4) Examine the preparation microscopically using the 10x and 40x objective with the
condenser iris closed sufficiently to give good contrast.

Urine is examined microscopically as a wet preparation to detect:

 Significant pyuria, i.e. WBCs in excess of 107 WBC/l of urine.

 Red cells
 Casts
 Yeast cells
 T. vaginalis motile trophozoites
 S. haematobium eggs
 Bacteria (provided the urine is freshly collected, usually seen as rods or cocci)

Some clinical Biochemical (chemical) tests include:

 Protein
 Glucose
  Ketones
 Bilirubin
 Urobilinogen
 Haemoglobin & white blood cells
 Nitrite & leukocyte esterase
 Mass density (specific gravity)

Reference

Cheesbrough, M (2010). District Laboratory Practice in Tropical Countries. Second edition update.
Cambridge University Press. Pp; 45-70, 105-115.

Harley, J. P. and Prescott, L. M (2002). Laboratory Exercises in Microbiology. Fifth edition. The
McGraw-Hill Companies. Pp; 31-138.

Chakraborty, P. and Pal, N. K (2008). Manual of Practical Microbiology and Parasitology. New
Central Book Agency (P) Ltd. Pp; 9-135.