SCHOOL OF PHARMACEUTICAL SCIENCES

FAR 121/4 Microbiology For Pharmacy

SEMESTER 1 2014/2015

LABORATORY REPORT
PRACTICAL 3:
BACTERIAL COUNTS
GROUP MEMBERS :

1)ABDUL MUHAIMIN SUHAIMI

(123856)

2)ALICE CHONG WAN KIM

(123858)

3)ARVIN KUMAR A/L RAVINDRA

(123862)

4)CHAN ROUYI

(123867)

5)SUTHA A/P KRISHNAN

(123938)

6)TUSVENDRAN PILLAI A/L VEEL PILAY

(123956)

LECTURERS NAME: DR.MEHDI RIAZI

DATE OF SUBMISSION: 10/11/2014

TOPIC TITLE : BACTERIAL COUNT

OBJECTIVES:
1.
2.
3.
4.

To let the students be competent in aseptic technique.

To let the students perform bacterial counts.
To obtain isolated pure colony.
To let the students perform a viable colony count

INTRODUCTION :
It is often desirable, even its essential to know how many living microorganisms are present
in a particular environment. There are various methods which can be used to determine the
number of viable organisms, namely, pour-plate method, spread-plate method, roll-tube
method, `drop-plate method (Miles and Misra), membrane filtration method, etc. the most
widely used technique of viable counting is dilution plating ( the plate count). While it has
several possible sources of error this technique is proved to be the most reliable, giving
consistent results with simple apparatus. Essentially the technique consist of making a series
of dilutions of a sample, plating out, followed by number of cells in the sample can be
estimated. In this exercise, 2 of above methods s introduced to estimate the number of viable
organisms in the sample given.

A. Pour-plate method
This method is commonly used for viable counting because it is very practical. One of the
important factors in carrying out the dilution is to disperse any clumping of cells. In this
method, the temperature and the period of incubation are also important. Petri dishes that
yield 200-300 colonies are considered satisfactory. This method may also be used for
isolating bacterial cultures.
Materials:
Refer to practical manual FAR 121/4 Microbiology for Pharmacy page 10.
Method:
Refer to practical manual FAR 121/4 microbiology for pharmacy page 11.

Results:
A. Pour Plate Method
Serial Dilution
1X10-4
1X10-5
1X10-6
1X10-7

Number of Escherichia coli colonies

Petri Dish 1 (1 mL)
Petri Dish 2 (1 mL)
Average
More than 300
More than 300
More than 300
More than 300
120
136
128
18
14
16

Calculation:
Petri dishes that yield 30-300 colonies are considered satisfactory. Thus, in this experiment,
10-6 dilution has been chosen to determine the total number of viable bacteria in 1mL of the
original sample. This is because it contains 128 bacteria colonies in average. It is assumed
that a colony represents one bacterium.

The total number of viable bacteria in 1mL of the original sample

= average colonies of bacteria x reciprocal of dilution factor x

1
plating volume

For plating volume, 1.0 mL and for 10-6 dilution:

Total number of viable bacteria per mL = 128 x 1/10-6 x 1/1
= 1.28 x 108 CFU/mL

B. Spread Plate Method

One other way to determine a bacterial count is to use the spread-plate method. Nevertheless,
it should be noted that the use of the spreader in this method to spread the organisms could
lead to reduction in the total count as there is a possibility that some bacterial cells might
stick to the spreader. Just as the pour plate method, this procedure may also be used to isolate
bacterial cultures.
Materials:
Please refer to Practical Manual, FAR 121/4, Microbiology For Pharmacy, page 13
Method:
Please refer to Practical Manual, FAR 121/4, Microbiology For Pharmacy, page 13

Results:
(B) Spread Plate Method
Dilution
10-4
10-5
10-6
10-7

Number of Escherichia coli colonies

Petri dish 1 (0.1mL)
Petri dish 2 (0.1mL)
Average
More than 300
More than 300
More than 300
More than 300
235
277
256
10
8
9

Calculation:
In this experiment, 10-6 dilution has been chosen to determine the total number of viable
bacteria in 1mL of the original sample because it contains 256 bacteria colonies in average.

The total number of viable bacteria in 1mL of the original sample

= average colonies of bacteria x reciprocal of dilution factor x

1
plating volume

For plating volume 0.1mL and for 10-6 dilution:

Total number of viable bacteria per mL = 256 x 1/10-6 x 1/0.1
= 2.56 x 109 CFU/mL

DISCUSSION :
Bacteria viable counts are the measure of the number of bacteria cells in the population that
are capable for reproducing.
In this experiment, pour plate method and spread plate method are used to estimate
the number of viable bacteria in the sample given( Escherichia coli) . Serial dilution is first
carried out before the transfer of a known volume of the bacterial culture into the Petri dish is
done. The purpose of doing serial dilution is to separate clumped bacteria cells and by doing
this, the dilution which gives a nice count, avoid bacteria colonies from overlapping each
other and having appropriate number of bacterial cells for easy counting can be chosen.
The sample that produced 30-300 colonies is considered reliable. The sample with
less than 30 colonies represents an unreliably small sample size to estimate the actual number
of bacteria present in the culture. However, in plate with more than 300 colonies, the multiple
colonies will become superimposed (overlapped) on one another and may be mistakenly
counted as a single unit of colony. Besides that, accumulation of metabolic waste in plate
with too crowded colonies will restrict the bacterial growth and increase bacterial death, thus
causing the results of bacterial counts to become inaccurate. Other than that, it is impossible
to count bacteria colonies of more than 300 by sight alone.Hence,that is the reason why our
group wrote down more than 300 in our observation.
The number of colonies formed depends on solubility of the culture medium,
incubation conditions (temperature), rate of growth of organism and the inoculums size.

The number of bacteria is counted by using the colony-forming unit (CFU) technique.
The theory behind the technique of CFU is to establish that a single bacterium can grow and
become a colony, via binary fission. Wherever a single living bacterium is deposited on an
agar plate, it will divide to form a colony. Each bacterium represents a colony-forming unit
(CFU).
In order to make the calculation easier, several assumptions were done:
Viable bacteria will divide to form visible colony under suitable conditions.
1 colony formed on the agar plate represents 1 bacteria cell.
Number of visible colony formed is the same as the number of bacteria inoculated.
However, this will make the number of bacteria counted to be less than the actual one.
This is because one colony of bacteria may contain hundreds or thousands of bacteria.
The formula for bacteria count is as follows:
Number of bacteria colonies per ml of the initial culture
=Average plate count x reciprocal of the dilution x 1/plating volume
Unit for colony is CFU/ml.

In the pour-plate method, the number of viable bacteria in 1mL of medium is 1.28 108
CFU/mL whereas in the spread-plate method, the number of viable bacteria in 0.1mL of
medium is 2.56 109 CFU/mL.
In fact, there was a difference between the number of bacteria count in the duplicated
plates. This is because in pour plate method, some of the bacteria cells may be killed by the
hot liquid agar. While in spread plate method, bacteria cells might stick on to the spreader
used. Moreover, the alcohol which was not let to evaporate completely, after the spreader was
immersed in 70% of alcohol solution for sterilizing purpose, might kill some of the bacteria
cells. Nevertheless, spread plate method seems to yield better result than pour plate method.
The followings are the similarities and differences of pour-plate and spread plate method.
Pour-plate method

1.
2.
3.
4.

Spread-plate method

Similarities:
Both methods have to perform serial dilution first.
Both methods use agar as medium.
The samples from both methods are being incubated for 24 48 hours.
The temperature for incubation is 37C

Differences:
Pour-plate method

Spread-plate method

1. The colonies grow in the medium

and on the surface of the medium.
2. Enable anaerobe and facultative
anaerobe to grow in the medium.
3. Difficult to count, as the colonies
dont only grow on the surface of the
agar medium.
4. Useful for enumerating the
population of microbes in the
sample.

1. The colonies grow only on the

surface of the medium.
2. Enable aerobe to grow on the
surface of the medium.
3. Easier to count, the colonies grow
only on the surface of the agar
medium.
4. Ideal for purification of mixed
bacteria.

5. Large amount of the sample is

5. Small amount of the sample is used

needed (1.0ml)
Advantages:
Risk of contamination is reduced.
There would be less overlapping of
bacteria colonies.

(0.1ml)
Advantages:
Bacteria colonies are easier to be
observed.
Morphology of the colonies can be
observed.

Disadvantages:
Parallax may occur while counting
bacteria colonies growing inside the
agar medium.
Some bacteria might be killed by the
hot agar liquid.

Disadvantages:
Some bacteria stick to the spreader,
thus reducing the number of bacteria
counted.
Some bacteria might be killed by the
alcohol on the spreader.
Inappropriate technique of spreading
would result in clumping of bacteria
colonies.

PRECAUTIONS :
1. Lab coats must be worn in lab at all times
2. Wash hands before and after the experiment
3. No mouth pipetting
4. Aseptic technique must be practiced when dealing with the bacterial culture in
order to reduce contamination.
5. Disinfection of working areas, minimizing possible access by bacteria from the air
to exposed media, by handling the experiment near flames( within a 20cm radius).
The purpose is to kill bacteria which might enter vessels as they are opened.
6. Invert the Petri dishes during incubation, to prevent condensation droplets from
falling onto the surface of the agar.
7. Evenly spread inoculums, on agar surface and must not touch the lid of the plate
and the surface must be smooth with no bubbles.

8.

For pour-plate, swirl the molten agar carefully on the bench after you add the
dilution of bacteria, to get even distribution of colonies on a plate.

CONCLUSION :
1. By using the pour-plate method, the number of viable bacteria in 1mL of medium is
1.28 108 CFU/mL.
2. By using the spread-plate method, the number of viable bacteria in 0.1mL of medium is
2.56 109 CFU/mL.
3. As a whole, the spread-plate method yields a better accuracy than the pour-plate method.

REFERENCE:
1) Microbiology For Pharmacy Practical Manual
2) Microbiology: An Introduction 11th Edition