MIDLANDS STATE UNIVERSITY

P. BAG 9055 Tel. (263) 54 260450/260417

Gweru. 260409/2604972/260632/260596

FACULTY OF SCIENCE AND TECHNOLOGY

APPLIED BIOSCIENCES AND BIOTECHNOLOGY

DEPARTMENT

BACTERIOLOGY PRACTICAL- HABB 202

PRECAUTIONS
1. Be familiar with all laboratory rules and safety precautions to be taken when working
with microorganisms. Regardless of biohazard classification, all materials that come
in contact with microorganisms are considered infectious waste and must be
decontaminated prior to disposal.
2. Follow safety guidelines in accordance with those provided by institutional
Environmental Health and Safety departments, setting up appropriate waste
receptacles for immediate and proper disposal of potentially contaminated materials
(biohazards).
3. Sterilize all instruments, solutions, and media prior to using them for plating
procedures.
4. Clear away all materials cluttering your work area on the laboratory bench.
5. Clean work area with disinfectant to minimize possible contamination.
6. Set up a Bunsen burner and work slowly, carefully, and deliberately within the sterile
field area created by the updraft of the flame.
 If working with BSL-2 organisms, set up your work space in a Biosafety
cabinet. A Bunsen burner cannot be used inside the cabinet because the heat
from the flame disrupts air flow essential to its functionality.
7. Arrange all the supplies needed for the procedure on the laboratory bench near the
sterile field. Make sure all the materials are properly labelled. Organizing the work
area to maximize work efficiency and avoid unnecessary movements will minimize
the exposure time of experimental materials to airborne contaminants.
 Place the Bunsen burner to your right on the bench.
 Place agar plates or Petri dishes to your left.
 Arrange cell cultures, tubes, flasks and bottles in the centre of the bench.
 Loosen the caps of tubes, flasks and bottles so they can be easily opened with
one hand during subsequent manipulations.
8. Wash hands thoroughly with antiseptic soap and warm water before handling
microorganisms.

ASEPTIC TECHNIQUES

Aseptic techniques underpin all work in microbiology. Make sure you are familiar with all
these techniques before embarking on the other microbiology protocols on this website.

Only non-pathogenic cultures should be used in schools – obtained from a recognised

educational supplier. Sterile equipment and media should be used in the transfer and culture
of microorganisms. Aseptic technique should be observed whenever microorganisms are
transferred from one container to another.

It is wise to treat all cultures as potentially pathogenic, because cultures may have been
contaminated, and because mutations to disease-causing forms may occur. The aseptic
techniques described here control the opportunities for contamination of cultures by
microorganisms from the environment, or contamination of the environment by the
microorganisms being handled.

There are some general rules to follow for any aseptic technique.

1. Close windows and doors to reduce draughts and prevent sudden movements
which might disturb the air.
2. Make transfers over a disinfected surface. Ethanol disinfection is recommended
because of its rapid action. If the bench surface is difficult to clean, cover the
bench with a sheet of tough material which is more easily disinfected.
3. Start the operations only when all apparatus and materials are within immediate
reach.
4. Complete all operations as quickly as possible, but without any hurry.
5. Vessels must be open for the minimum amount of time possible.
6. While vessels are open, all work must be done close to a Bunsen burner flame
where air currents are drawn upwards.
7. On opening a test tube or bottle, the neck must be immediately warmed by
flaming (see below) with the vessel held as near to horizontal as possible and so
that any movement of air is outwards from the vessel.
8. During manipulations involving a Petri dish, limit exposure of the sterile inner
surfaces to contamination from the air.
9. The parts of sterile pipettes which will be put into cultures or sterile vessels must
not be touched or allowed to come into contact with other non-sterile surfaces,
such as clothing, the surface of the working area, or the outside of bottles/ test
tubes.
10. All items which come into contact with microorganisms must be sterilised before
and after each such exposure. This could be either by the technical team
preparing for and clearing up after a piece of practical work (for example, in the
case of glassware to be used), or by the worker during the course of the practical
(for example, in flaming a wire loop).
PRACTICAL 1
MICROORGANISMS: SOURCES OF CONTAMINATION
INTRODUCTION

Bacteria and other microorganisms are usually present in very large numbers in the air, skin,
furniture etc. Because of their microscopic size, these organisms are undetectable to the
naked eye. Some microorganisms are pathogenic thus must be handled safely as well as
efficiently. In this experiment, a number of different situations (environments) would be
investigated to see whether microorganisms are present or not. Their presence or absence
depends largely on whether they are able to obtain food materials for growth and
reproduction from these environments. Microorganisms are grown in the laboratory
solutions, which are known as media. Media can either be liquid or solid. Solid media
contain an additional compound called agar. Agar is useful as a gelling agent since it melts
at 1000C and also does not set until the temperature falls to about 420C.

Objective:
The practical aims to show some sources of bacterial contamination and to demonstrate a
few techniques available for evaluating environmental contamination.
Sampling various environments to see if microorganisms are present or absent
1. Atmosphere contamination
Expose one plate of nutrient agar (NA) and one plate of malt extract agar (MA) for 30
minutes to the air the lab. Repeat this and expose an NA plate and an MA plate to the
outside air (in the corridor)

2. Surface contamination
a) Aseptically dip a sterile cotton wool swb into sterile quarter strength Ringers solution
and expels excess water against the side of the container.
b) Rub the swab over a 100 cm2 area of your bench surface.
c) Transfer the organisms collected on the swab onto an NA plate by rubbing the swab
sample over the entire surface of the medium.
d) Take a fresh swab, dip it into isopropyl alcohol and swab a similar area of bench
surface adjacent to the area just sampled.
e) When the disinfectant surface is dry use a fresh swab, dipped into sterile quarter
strength Ringer solution, and sample the surface for microorganisms as before, using
a second plate NA.

3. Hand contamination
a) Sample an area from your fingers of one hand by touching them onto the surface of
NA plate.
b) Thoroughly scrub your hands with soap and water, let them air- dry then sample as
before onto fresh plates of NA.

4. Hair contamination
a) Hold your head over an open plate of NA on the bench and run your fingers
vigorously through your hair for 30 seconds.
b) Controls
c) leave one NA and one MA plate untouched as controls
 d) Label all the plates to show where they have been exposed or what they have on
them
e) Invert all the plates and incubate those containing NA at 370C and MA at 25 0C
f) Examine for growth after 2 days and 7 days incubation
g) Describe the size, shape and colour of the colonies. Sketches may help you to do this
accurately
h) Where possible, count the total number of colonies on each plate, noting the
different types of colonies present

Questions
1) What is the definition of Aseptic techniques?
2) Why is it important to prevent any condensation falling on the agar surface?
3) What is the purpose of control plates?
PRACTICAL 2
STAINED PREPARATIONS
A ‘smear’ of bacteria or yeast is made on a microscope slide, fixed, stained, dried and,
without using a cover slip, examined with the aid of a microscope. Aseptic technique must be
observed when taking samples of a culture for making a smear. A smear that is thin and even
enables the shape and arrangement of cells to be clearly seen and ensures that the staining
procedure is applied uniformly.

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

Staining procedure
1. Put the slide with the fixed smear uppermost on a staining rack over a sink or staining tray.
2. Thoroughly cover the smear with stain and leave for, usually, 30 seconds.
3. Hold the slide with forceps (optional but avoids stained fingers), at a 45° angle over the
sink.
4. Rinse off the stain with tap water.
5. Blot-dry the smear with filter/fibre-free blotting paper using firm pressure, but not
sideways movements that might remove the smear.
6. Examine under oil immersion.
7. When finished, dispose of slides into discard jar. Suitable stains include basic dyes (i.e.
salts with the colour-bearing ion, the chromophore, being the cation), such as methylene blue,
crystal violet and safranin.
A DIFFERENTIAL STAIN
GRAM’S STAINING METHOD

Gram-staining is a differential staining technique that uses a primary stain and a secondary
counterstain to distinguish between gram-positive and gram-negative bacteria. It was
developed by Danish microbiologist Hans Christian Gram in 1884 as an effective method to
distinguish between bacteria with different types of cell walls, and even today it remains one
of the most frequently used staining techniques. First, crystal violet, a primary stain, is
applied to a heat-fixed smear, giving all of the cells a purple color. Next, Gram’s iodine,
a mordant, is added. A mordant is a substance used to set or stabilize stains or dyes; Next,
a decolorizing agent is added, usually ethanol or an acetone/ethanol solution. Finally, a
secondary counterstain, usually safranin, is added. This stains the decolorized cells pink
and is less noticeable in the cells that still contain the crystal violet dye.The purple, crystal-
violet stained cells are referred to as gram-positive cells, while the red, safranin-dyed cells are
gram-negative.

Method:
1. Put the slide with the fixed smear uppermost on a staining rack over a sink or staining tray.
2. Thoroughly cover the smear with crystal violet solution and leave for 1 minute.
3. Hold the slide with forceps (optional but avoids stained fingers), at a 45° angle over the
sink.
4. Pour off the stain, wash off any that remains (and any on the back of the slide) with iodine
solution.
5. Put the slide back on the staining rack.
6. Cover the smear with iodine solution and leave for 1 minute. Iodine solution acts as a
‘mordant’ (a component of a staining procedure that helps the stain to adhere to the
specimen), a crystal violet–iodine complex is formed and the smear looks black.
7. Hold the slide with forceps at a 45° angle over the sink and wash off the iodine solution
with 70% alcohol until the washings are pale violet.
8. Rinse immediately with tap water.
9. Put the slide back on the staining rack.
10. Cover the smear with the counterstain, e.g. aqueous safranin solution, 0.5 % (w/v), for 30
seconds.
11. Rinse off the stain with tap water.
12. Blot dry the smear with filter/fibre free blotting paper using firm pressure but not
sideways movements that might remove the smear.
13. Examine under oil immersion.
14. When finished, dispose of slides into discard jar
PRACTICAL 3
BIOCHEMICAL TESTS

Use of various biochemical reagents and tests to get closer to the identification of bacteria.
There are many biochemical tests available for bacterial identification. Few of them are
required to be carried out depending upon the bacteria. The commonly used biochemical tests
are: Catalase test, Coagulase test, Oxidase test, Sugar fermentation test, Indole test, Citrate
test and Urease test.

(a) Catalase test

The catalase test facilitates the detection of the enzyme catalase in bacteria.

Procedure
1. Place a microscope slide inside a petri dish.
2. Using a sterile inoculating loop or wooden applicator stick, collect a small amount of
organism from a well-isolated 18- to 24-hour colony and place it onto the microscope
slide.
3. Using a dropper or Pasteur pipette, place 1 drop of 3% H 2O2 (Hydrogen peroxide)
onto the organism on the microscope slide. Do not mix.
4. Immediately cover the petri dish with a lid to limit aerosols and observe for
immediate bubble formation (O2 + water = bubbles).
5. Observing for the formation of bubbles against a dark background enhances
readability.

b) Coagulase test
The coagulase test differentiates strains of Staphylococcus aureus from other coagulase-
negative species. S. aureus strains are capable of coagulating plasma in the tube test and will
produce clumps of cells in the slide test. Clumping or clots of any size indicate a positive
response.

Procedure

The slide test is performed by preparing a suspension of bacterial cells mixed into a drop of
rabbit plasma on a microscope slide. If bound coagulase is present on the bacterial cells, then
the presence of plasma will cause the bacterial cells to clump.

The tube test is performed by

1. Mixing bacterial cells into a larger volume of plasma in a small test tube.
2. Incubate overnight
3. Formation of a clot will be noted within 24 hours for a positive response.

c) Oxidase test
The oxidase test is a biochemical reaction that assays for the presence of cytochrome oxidase,
an enzyme sometimes called indophenol oxidase. In the presence of an organism that
contains the cytochrome oxidase enzyme, the reduced colorless reagent becomes an oxidized
colored product.

Procedure
Filter Paper Test Method
1. Soak a small piece of filter paper in 1% Kovács oxidase reagent and let dry.
2. Use a loop and pick a well-isolated colony from a fresh (18- to 24-hour culture) bacterial
plate and rub onto treated filter paper.
3. Observe for color changes.
4. Microorganisms are oxidase positive when the color changes to dark purple within 5 to 10
seconds.
5. Microorganisms are oxidase negative if the color does not change or it takes longer than 2
minutes.

(d) Indole test

Purpose
The indole test screens for the ability of an organism to degrade the amino acid tryptophan
and produce indole.

Procedure
1. Inoculate the tube of tryptone broth with a small amount of a pure culture.
2. Incubate at 37°C for 24 to 48 hours.
3. To test for indole production, add 5 drops of Kovác's reagent directly to the tube.
4. A positive indole test is indicated by the formation of a pink to red color (“cherryred
ring”) in the reagent layer on top of the medium within seconds of adding the reagent.
5. If a culture is indole negative, the reagent layer will remain yellow or be slightly
cloudy.
6. A positive diagnostic test rests on the generation of alkaline by-products of citrate
metabolism and is done on Simmons citrate medium which are in test tubes.
7. The citrate test is used to identify gram-negative pathogens and environmental
isolates.

a) Citrate test
Procedure
1. Use a fresh (16- to 18-hour) pure culture as an inoculation source.
2. Pick a single isolated colony and lightly streak the surface of the slant using a needle
3. Avoid using liquid cultures as the inoculum source.
4. Citrate utilization requires oxygen and thus screw caps, if used, should be placed
loosely on the tube.
5. Incubate at 35oC (+/- 2oC) for 18 to 48 hours. Some organisms may require up to 7
days of incubation due to their limited rate of growth on citrate medium.
6. Citrate positive: growth will be visible on the slant surface and the medium will be an
intense Prussian blue.
 7. 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.

b) Urease test
Purpose
The urease test identifies those organisms that are capable of hydrolyzing urea to produce
ammonia and carbon dioxide. It is primarily used to distinguish urease-positive bacteria from
other Enterobacteriaceae.

Procedure
1. Using Christensen’s Urea Agar a heavy inoculum from an 18- to 24-hour pure culture
to streak the entire slant surface. Do not stab the butt as it will serve as a color control.
2. Incubate tubes with loosened caps at 35oC.
3. Observe the slant for a color change at 6 hours, 24 hours, and every day for up to 6
days.
4. Urease production is indicated by a bright pink (fuchsia) color on the slant that may
extend into the butt.
5. Note that any degree of pink is considered a positive reaction.
6. To eliminate protein hydrolysis as the cause of a positive test, a control medium
lacking urea should be used.
7. The culture medium will remain a yellowish color if the organism is urease negative.

c) Mixed acid fermentation

Some bacteria can ferment glucose to a mixture of the following organic acids: formic acid,
acetic acid and lactic acid. This is called mixed acid fermentation and it causes highly
decreased pH in the medium. Mixed acid fermentation can, therefore, be detected by addition
of the pH indicator methyl red (MR). The test method is sometimes called the MR test.
1. Suspend a bacterial colony from a pure culture in the MR/VP medium.
2. Incubate at 30°C during 3 days or at 37°C during 48 h.
3. Add 2-3 drops of a solution of methyl red.

 Positive test result: red colour change

 Negative test result: no colour change.
PREPARATION OF CULTURE MEDIA

1. Rehydrate tablets or powder according to manufacturer’s instructions.

2. Before sterilisation, ensure ingredients are completely dissolved, using heat if
necessary.
3. Avoid wastage by preparing only sufficient for either immediate use (allowing extra
for mistakes) or use in the near future.
4. Normally allow 15–20 cm3 medium per Petri dish.
5. Dispense in volumes appropriate for sterilisation in the autoclave/pressure cooker.
6. Agar slopes are prepared in test tubes or Universal/McCartney bottles by allowing
sterile molten cooled medium to solidify in a sloped position.
7. Bottles of complete, sterile media are available from suppliers but are expensive

USE OF THE AUTOCLAVE

Sterilisation is achieved in an atmosphere of steam under pressure. Usual conditions are not
less than 15 minutes exposure to pure steam at a pressure of 103 kPa (kN m2) or 15 lbf in–2,
producing a temperature of 121 °C.

1. Do not overload and fill vessels to no more than 2/3 capacity and loosen caps of
screw-capped vessels to allow for expansion.
2. Protect cotton wool plugs from becoming wet and losing their ability to function as a
barrier to the passage of airborne microbes by covering with either greaseproof paper
or aluminium foil.
3. Ensure that there is more than enough water to generate steam throughout the
sterilisation cycle and to prevent boiling dry.
4. Use either distilled or deionised water to prevent corrosion.
5. Allow several minutes of vigorous release of steam to ensure complete expulsion of
air otherwise the required temperature will not be achieved.
6. Begin timing the holding time when air has been completely expelled and the heat has
penetrated throughout the materials to be sterilised.
7. Ensure that steam continues to escape throughout the holding time.
8. After completion of the holding time, allow the autoclave to return atmospheric
pressure and temperature before starting the opening procedure.
9. Never attempt to reduce either the cooling period with cold water or the time taken for
pressure reduction by premature opening of the air cock.

POUR PLATE
This method often is used to count the number of microorganisms in a mixed sample, which
is added to a molten agar medium prior to its solidification. The process results in colonies
uniformly distributed throughout the solid medium when the appropriate sample dilution is
plated. This technique is used to perform viable plate counts, in which the total number of
colony
 forming units within the agar and on surface of the agar on a single plate is enumerated.
Viable plate counts provide scientists a standardized means to generate growth curves, to
calculate the concentration of cells in the tube from which the sample was plated, and to
investigate the effect of various environments or growth conditions on bacterial cell survival
or growth rate.

Procedure
1. Collect one bottle of sterile molten agar from the water bath.
2. Hold the bottle in the right hand; remove the cap with the little finger of the left hand.
3. Flame the neck of the bottle.
4. Lift the lid of the Petri dish slightly with the left hand and pour the sterile molten agar
into the Petri dish and replace the lid.
5. Flame the neck of the bottle and replace the cap.
6. Gently rotate the dish to ensure that the medium covers the plate evenly.
7. Allow the plate to solidify.
Note: The base of the plate must be covered, agar must not touch the lid of the plate and the
surface must be smooth with no bubbles. The plates should be used as soon as possible after
pouring. If they are not going to be used straight away they need to be stored inside sealed
plastic bags to prevent the agar from drying out.

STREAK PLATE PROCEDURE

The streak-plate procedure is designed to isolate pure cultures of bacteria, or colonies, from
mixed populations. A mixture of cells is spread over the surface of a semi-solid, agar-based
nutrient medium in a Petri dish such that fewer and fewer bacterial cells are deposited at
widely separated points on the surface of the medium and, following incubation, develop into
colonies.
Procedure
1. Insert the loop into the culture broth or agar and withdraw some bacteria
2. Partially lift the lid of the Petri dish containing the solid medium.
3. Hold the charged loop parallel with the surface of the agar; smear the inoculums
backwards and forwards across a small area of the medium
4. Remove the loop and close the Petri dish.
5. Flame the loop and allow it to cool. Turn the dish through 90° anticlockwise.
6. With the cooled loop streak the plate from area ‘1 across the surface of the agar in
three or four parallel lines (‘2’). Make sure that a small amount of culture is carried
over.
7. Remove the loop and close the Petri dish. 16. Flame the loop and allow to cool. Turn
the dish through 90° anticlockwise again and streak from ‘2’ across the surface of the
agar in three or four parallel lines (‘3’).
8. Remove the loop and close the Petri dish. 18. Flame the loop and allow to cool. Turn
the dish through 90° anticlockwise and streak loop across the surface of the agar from
‘3’ into the centre of the plate (‘4’).
 9. Remove the loop and close the Petri dish. Flame the loop.
10. Seal and incubate the plate in an inverted position.

Figure 1. Streak-plate technique using the quadrant method.

SPREAD-PLATING WITH A TURNTABLE GLASS OR METAL ROD

This technique typically is used to separate microorganisms contained within a small sample
volume, which is spread over the surface of an agar plate, resulting in the formation of
discrete colonies distributed evenly across the agar surface when the appropriate
concentration of cells is plated.
1. Place agar into petri dish
2. Pipette 0.1 to 0.2 ml aseptically onto the centre of the plate using a micropipette.
3. The spreader is sterilized by dipping it into a beaker of ethanol and flaming it
4. Spreader should be cooled by touching it to the agar near the rim of the plate.
5. The spreader is gently moved back and forth through the sample across the plate
while the turntable is slowly spinning.
6. This action allows gradual but even spreading of the sample across the agar surface.
7. After closing the lid, the plate should be set on the bench top undisturbed for at least 5
minutes to allow the sample to absorb completely into the agar
8. Invert the plate for incubation.
SPREAD-PLATE TECHNIQUE WITH GLASS BEADS (COPACABANA METHOD)
1. Glass beads that have been pre-sterilized in an autoclave are poured onto the surface
of an agar plate sitting on the bench top.
2. A small volume of sample (100 to 150 μl) is dispensed aseptically onto the center of
the agar using a micropipette.
3. With the lid of the plate closed, a horizontal shaking motion is used to gently move
the beads back and forth across the plate 6 to 7 times, spreading the sample.
4. This action is repeated after rotating the plate 60°. The shaking motion is repeated a
third time following another 60° rotation. Once the sample has completely absorbed
into the agar medium, the beads are poured off into a beaker containing 10% bleach.
The plates then are inverted for incubation.
USING A WIRE LOOP

Wire loops are sterilised using red heat in a Bunsen flame/gas burner before and after use.
They must be heated to red hot to make sure that any contaminating bacterial spores are
destroyed.

1. The handle of the wire loop is held close to the top, as you would a pen, at an angle
that is almost vertical.
2. This leaves the little finger free to take hold of the cotton wool plug/screw cap of a
test tube/bottle.
3. Position the handle end of the wire in the light blue cone of the flame. This is the cool
area of the flame.
4. Draw the rest of the wire upwards slowly up into the hottest region of the flame,
(immediately above the light blue cone).
5. Hold there until it is red hot. Ensure the full length of the wire receives adequate
heating. Allow to cool then use immediately.
6. Do not put the loop down or wave it around.
7. Re-sterilise the loop immediately after use.

USING A PIPETTE

Sterile graduated or dropping (Pasteur) pipettes are used to transfer cultures, sterile media and
sterile solutions.

1. Remove the pipette from its container/ wrapper by the end that contains a cotton wool
plug, taking care to touch no more than the amount necessary to take a firm hold.
2. Fit the teat.
3. Hold the pipette barrel as you would a pen but do not grasp the teat. The little finger is
left free to take hold of the cotton wool plug/cap of a test tube/ bottle and the thumb to
control the teat.
4. Depress the teat cautiously and take up an amount of fluid that is adequate for the
amount required but does not reach and wet the cotton wool plug. 5.
5. Return any excess gently if a measured volume is required. The pipette tip must
remain beneath the liquid surface while taking up liquid to avoid the introduction of
air bubbles which may cause ‘spitting’ and, consequently, aerosol formation when
liquid is expelled.
6. Immediately after use put the now contaminated pipette into a nearby discard pot of
disinfectant. The teat must not be removed until the pipette is within the discard pot
otherwise drops of culture will contaminate the working surface.
Hints: A leaking pipette is caused by either a faulty or ill-fitting teat or fibres from the cotton
wool plug between the teat and pipette.
SERIAL DILUTION

Involves taking a sample and diluting it through a series of standard volumes of sterile
diluent, e.g. distilled water or 0.9 % saline. Then a small measured volume of each dilution is
used to make a series of pour or spread plates. By diluting the sample in this controlled way
it is possible to obtain an incubated plate with an easily countable number of colonies (30–
100) and calculate the number of microbes present in the sample. Culture of bacteria or yeast
or sample of natural material 6 sterile test tubes containing 9 cm3 sterile diluent, fitted with a
cap or cotton wool plug, labelled 1→6 and with the dilution factor as shown in the diagram
12 sterile, plugged Pasteur pipettes 1 cm3 syringe barrel fitted with rubber tubing (see page 9)
Pot of Virkon disinfectant

Procedure
1. Take a sterile pipette.
2. Place the syringe onto the plugged end of the pipette (see page 9).
3. Draw up 1 cm3 of a well mixed sample/ culture into the pipette.
4. Add this sample to the first tube. The volume of this tube is now 10 cm3 . This provides an
initial dilution of 10–1.
5. Mix the dilution thoroughly, by emptying and filling the pipette several times.
6. Discard this pipette into the pot of disinfectant, but keep the syringe for making the next
dilution.
7. Take a new pipette, fit it to the syringe and draw up a 1 cm3 sample of the 10–1 dilution
and place it in the second tube.
8. Mix well as before. This gives a 10–2 dilution.
9. Discard the pipette in disinfectant.
10. Repeat this for the remaining tubes, removing 1cm3 from the previous dilution and
adding it to the next 9 cm3 of diluent. If 6 tubes are used, the final dilution for the bacteria
will be 10–6 (1 in 1,000,000). Plating and counting procedure Use a known volume of each
dilution to make either pour plates or spread plates
14). By starting with the highest dilution, the same pipette may be used throughout. For
statistical purposes, replicate plates should be prepared.
After incubation the plates will show a range of numbers of colonies.
Choose the plate that has an easily countable number (about 30–100) and carefully count
every colony. Using a marker pen helps to avoid counting the same colony twice. Then
calculate the number of micro-organisms in the sample: Number of microbes/cm3 = number
of colonies × dilution of sample.

DETERMINATION OF MICROBIAL NUMBERS

Dilutions are usually expressed as negative exponents rather than as fractions (i.e., 10-5
rather than 1/100,000). The population number is usually recorded as the number of cells or
colony forming units (cfu) per milliliter of liquid being examined e.g water, milk.
 1. Using a sterile pipette, stir your bacterial culture to mix it thoroughly, and then
transfer 0.1 ml of culture media to the water blank labeled 10-3.
2. Note: If you wish to obtain maximal accuracy, you may also add 0.9 ml of sterile
water to the water blank, bringing its volume to exactly 100 ml.
3. Close the inoculated water blank and shake it vigorously (about 25 times) to
thoroughly mix the sample and to break up any clumps of bacteria.
4. Using a second sterile pipette, transfer 1 ml of the solution from the sample labeled
10-3 into the water blank labeled 10-5 .
5. Mix this sample thoroughly as in step 5 above. 8.
6. Using a third sterile pipette, transfer 1 ml of the solution from the sample labeled 10 -5
into the water blank labeled 10-7. (Note - If you carefully slip the pipette used for this
step into its sterile plastic cover, you will be able to use it again in step
10. Mix this sample thoroughly as in step 5 above.
11. Using a fourth sterile pipette (or the pipette retained from step 8), transfer 1 ml of the
solution labeled 10-5 onto the agar plate labeled 10 -5 and then transfer 0.1 ml from the
same water bottle onto the plate labeled 10-6 .
7. Using a fifth sterile pipette, transfer 1 ml of the solution labeled 10 -7 onto the agar
plate labeled 10-7.
8. Starting with the most dilute sample (10 -7) and working backward, spread the
inoculum evenly over the entire surface of each plate using a sterile glass spreader as
demonstrated.
9. If handled properly, the same spreader can be used on all three plates without
sterilizing it between them.
10. There will be some excess water on the agar surface of some of your plates.
11. Tape the plates together in a stack of three, and then invert them quickly so the water
is not allowed to run to one side.
12. Incubate all plates at 37oC overnight.
13. Count the colonies present on each plate containing between 20 and 200 colonies.
Calculate the plate count (i.e. the number of bacteria contained in 1ml of the original
bacterial sample) by multiplying the number of colonies counted times the dilution
factor expressed as a positive exponent.
Example: If you count 50 colonies on the plate labeled 10 -7 , you will multiply 50 times 10 -7 ,
so the number of viable cells or colony-forming units (cfu) in the original culture was 5 x 10 -8
cfu/ml. (Note - When numbers are expressed in scientific notation there may be only one
digit to the left of the decimal point. As the decimal point is moved one place to the left, the
exponent is increased by one.) This means the original sample contained 500,000,000 cells or
cfu per ml.

Questions:
1. Why would a scientist/technician want to know how many viable bacterial cells are
present in a sample?
2. Give one example of a situation in which this information would be important to
know.
3. If the initial inoculum used at step 4 above was 1.0ml instead of 0.1ml, what degree of
dilution would be reached in the third water blank?
4. Approximately how many viable bacterial cells or colony forming units were present
in 1ml of your original bacterial culture?
5. Do all bacterial cultures (in the stationary phase of growth) contain similar numbers
of cells? What explanation might be given for the variation in population observed
6.