CPB 30103 Biochemical Engineering

EXPERIMENT 4:
DIRECT MEASUREMENTS OF MICROBIAL
GROWTH: VIABLE COUNTS

● Apply the techniques of bacterial count using the pour plate and the spread plate methods.
● Calculate the cell numbers in CFU/mL (colony forming units).

INTRODUCTION

As part of daily routine, the laboratory microbiologist often has to determine the number of
bacteria in a given sample as well as having to compare the amount of bacterial growth under
various conditions. Enumeration of microorganism is especially important in dairy microbiology,
food microbiology, and water microbiology.

THEORY

The number of bacteria in a given sample is usually too great to be counted directly. However, if
the sample is serially diluted and then plated out on an agar surface in such a manner that single
isolated bacteria from visible isolated colonies, the number of colonies can be used as a measure
of the number of viable (living) cells in that known dilution. However, keep in mind that if the
organism normally forms multiple cell arrangements, such as chains, the colony forming units
may consist of a chain of bacteria rather than a single bacterium. Therefore, when doing the plate
count technique, we generally say we are determining the number of Colony Forming Units
(CFUs) in that known dilution. By extrapolation, this number can in turn be used to calculate the
number of CFUs in the original sample.

Normally, the bacterial sample is diluted by factors of 10 and plated on agar. After incubation,
the number of colonies on a dilution plate showing between 30 and 300 colonies is determined.
A plate having 30-300 colonies is chosen because this range is considered statistically
significant. If there are less than 30 colonies on the plate, small errors in dilution technique or the
presence of a few contaminants will have a drastic effect on the final count. Likewise, if there are
more than 300 colonies on the plate, there will be poor isolation and colonies will have grown
together.

For more accurate count it is advisable to plate each dilution in duplicate or triplicate and then
find an average count.

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LABORATORY PROCEDURE

Apparatus and Reagent

1. Test tube containing 9 ml of sterile distilled water
2. Universal bottle containing 5 ml of bacterial cell suspension (E. coli)
3. Petri dishes containing nutrient agar – spread
4. Sterile empty Petri dish – pour plate
5. Molten agar
6. Pipette with 1.0 ml tips and 0.1 ml sterile tips
7. L-shaped glass rod placed in a 70% ethanol solution
8. Bunsen burner
9. Laminar flow cabinet and incubator
10. Autoclave
11. Marker pen

Procedure

A. Preparation of the 10 fold sample dilution

a) Arrange the test tubes containing sterile distilled water and aseptically transfer 1.0 ml of the
cell suspension into the first test tube using the pipettes with sterile tips. Discard the tips and
label the first dilution tube as 10-1.
b) Using the first sterile tips, mix the content of the first dilution tube thoroughly. Withdraw 1.0
ml of the content of the first tube and transfer into the second tube and label as 10-2.
c) Prepare the same manner as described above for further dilution (till 10-6).

B. Pour plate method

a) Transfer 1.0 ml of the known dilution of the cell suspension into a sterile empty Petri dish.
Make it duplicate so that we can get the average in the results later.
b) Add 10ml of the molten nutrient agar at 45 ͦ C into the plate and mix the agar and the samples
by a combination of horizontal and circular movement for 5-10 seconds. The procedure
consists of 5 right angle movements, 5 clockwise circular movements and 5 anticlockwise
circular movements. The procedure will ensure complete dispersal of the sample in the agar.
Take care not to splash the agar.
c) Allow the Petri dishes to set, then invert and incubate at 30 ͦ C for 24 h.
d) Determine the number of colonies formed for each dilution and calculate the cell number.

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C. Spread plate method

a) Transfer 0.1 ml of the known dilution of the cell suspension into a sterile Petri dish containing
nutrient agar. Make it duplicate so that we can get the average in the results later.
b) The sample is then spread over the surface of the agar using an L-shaped glass rod evenly
spread the sample.
c) Allow the sample to dry, and incubate the plates in the inverted position and at 30 ͦ C for 24 h.
d) Determine the number of colonies formed for each dilution and calculate the cell number.

Results/Discussion

☼ Build an appropriate table to record the data collected (Tube dilution; Number of colonies,
Bacterial count per ml of sample; Average count per ml of sample).
☼ For the pour plate method, the number of colonies multiply with the dilution factor will give
the number of cell expressed as CFU/ml.
☼ In the case of the spread plate method, the number of colonies multiplies with the dilution
factor and divide with 0.1 ml will give the CFU/ml of the cell suspension.
☼ Take photo of all samples.
☼ Compare and discuss the easiness and reliability of the two methods.

Tutorial:
1. What are the advantages and disadvantages of the serial dilution – agar plate procedure?
2. What is the major disadvantage of microbial counts performed by methods other than the
serial dilution – agar plate procedure?
3. Distinguish between dilution and dilution factor.