FAR 121/4

MICROBIOLOGY FOR PHARMACY

Practical 3: Bacterial Count
Name

LOW KAH JUN (127920)

NAVIN KUMAR A/L THAMILARAJAN (127933)
FOONG PEI PEI (127899)
LYDIA ADILA BINTI ISKANDAR (127339)
NUR KHADIJAH BINTI BASHARUDIN (127946)

Group

Date

19th NOVEMBER 2015

Lecturer

A.P. DR. HJH PAZILAH IBRAHIM

Objectives:
1. To become competent in aseptic techniques
2. To perform bacterial counts
Introduction:
It is often desirable, even 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 has proved to be the most reliable,
giving consistent results with simple apparatus. Essentially, the technique consists of making
a series of dilutions of a sample, plating out, followed by incubation and counting of the
colonies which have grown. From the number of colonies which have grown at the various
dilutions, the total number of cells in the sample can be estimated. In this experiment,
students are introduced to 2 of the above methods 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 of FAR 121/4 Microbiology for Pharmacy (page 10)
Procedure: Refer to practical manual of FAR 121/4 Microbiology for Pharmacy (page 11)

Results:
Dilution

Number of bacteria colonies

Petri Dish 1

Petri Dish 2

Average

10

-4

300

300

300

10-5

300

300

300

10-6

300

215

257.5

41

68

54.5

-7

10

In the pour-plate method, we determine the number of bacteria in 1.0 ml sample of bacteria
culture in the petri dish which has a range of 30 to 300 bacteria colonies. The total number of
viable bacteria for each petri dish is calculated. 10-7 dilution is chosen because it contains
54.5 55 bacteria colonies in average that gives a reliable count of 30 to 300 colonies. It is
assumed that each colony represents one bacterium.
For plating volume = 1.0 ml
Total number of viable bacteria =
Average plate count x Reciprocal of dilution x

1
plating volume

10-7 dilution:
Total number of viable bacteria per ml =
55 x

1
107

1
1.0

= 5.5 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 in the pour plate method, this procedure
may also be used to isolate bacterial cultures.
Materials: Refer to practical manual of FAR 121/4 Microbiology for Pharmacy (page 11)

Method: Refer to practical manual of FAR 121/4 Microbiology for Pharmacy (page 12)
Result:
Dilution

Number of bacteria colonies

Petri Dish 1

Petri Dish 2

Average

10

-4

300

300

300

10-5

300

300

300

10-6

37

24

30.5

-7

33

>300

10

Colony-forming unit (CFU/ml) represents number of bacteria per ml of initial culture.

Formula to calculate CFU/ml: Average number of bacteria on the plate x reciprocal of the dilution x 1/plating volume
For this experiment, we can only calculate the plates with dilution 10-6 only because they fall
in preferable range (30 300 colonies).
So, CFU/ml = [(37 + 24)/2] x 1/10-6 x 1/0.1 ml
= 30.5 x 106 x 10
= 3.05 x 108CFU/ml

Discussion:
Aseptic techniques were used in this experiment to prevent bacterial contamination
and to prevent laboratory infection towards the bacterial cultures. Aseptic is basically the
mindset of keeping things free of contamination. Several assumptions are made for bacteria
viable count that is:
a) 1 colony formed on the agar plate represents 1 bacteria cell
b) Viable bacteria will divide to form visible colonies under suitable conditions
c) The number of visible colony formed is the same as the number of bacteria inoculated
d) Each colony arises from one cell.
Serial dilution is a stepwise dilution of substance in solution. Serial dilution may be
used to reduce the concentration of microscopic microorganisms or cells in a sample. Then, a
sample of the diluted bacterial sample of 1mL is transferred to a petri dish for pour plate

method. When using spread plate method; 0.1mL of diluted bacterial sample was spread in
the petri dish. After that, the petri dishes are incubated under 37C for 48 hours. There are
several steps that were followed for the aseptic techniques. These steps were taken to prevent
contamination of the samples:
1. The mouth of the bottle containing the bacteria was flamed before a pipette was
inserted to make sure the pipette is sterile.
2. The tip of the pipette was flamed before inserting it into the bottle containing the
sample.
3. The lid of the petri dish was opened minimally or just enough to allow the pipette or
spreader to enter.
4. The exposure of containers of sterilized culture media to outside air should be
minimized and that flame is used to re-sterilized container lids and rims. The lids of
petri dish should be opened with minimised distance .
5. The pipettes were always changed when needed especially during the serial dilution
process.
In this experiment, we carried out two methods for bacteria count, which are pourplate method and spread-plate method.
7
For the pour plate method, we use 10
as the suitable dilution factor in order to

calculate the total number of viable bacteria per ml of the original sample. The total number
8
of viable microorganism is 5.5 x 10 CFU/ml. During the pour plate method , we have

some precaution regarding to the agar solution should o be completely cooled down before
pouring into the petri dish containing 1mL of diluted bacterial solution to prevent the bacteria
from overheating.
For the spread-plate method, there are obvious of uneven results in terms of
4
7
decreasing colonies formed from 10 to 10 . There are a lot of contaminations

spotted on our plates., we believed that there was a highly chances of contamination of other
bacteria due to not following aseptic technique. There were some members speaking when
transferring the bacteria into the agar plate, there might be saliva containing bacteria
accidentally spit on the nutrient agar plate. We also didnt do the transferring of bacteria near
the Bunsen burner, this can increase the chances of the contamination. Another cause maybe

is, we lift up the lid of the petri dish so wide while spreading the bacterial suspension on the
entire surface of the agar. For that, we have to be really careful and aware and use the aseptic
techniques recommended while conducting our experiment since there a lot of possibilities of
contaminations may occur if poor aseptic techniques being applied.
The uneven result also may be caused by the mistake during our mixing process. We
did not mix our test tubes mixture thoroughly. This mistake can disturb our microbes
distribution inside our test-tubes solution and the sample taken to be spread is not even in
terms of their concentration in each of our test-tube from test-tube labelled
7

10

10

to

.
In this experiment, the number of viable bacteria in 1 ml of the original sample is
8

3.05 10

CFU/mL which means that there are 3.05 10

organisms are transfer onto the

5
agar plate in one mL and 305 colonies are formed with a dilution factor of 10

Pour plate method is commonly used worldwide for viable counting because it is very
practical. It has the advantage of not requiring previously prepared plates, and is often used to
assay bacterial contamination in food. The disadvantage of using pour plate is the assumption
that each colony arises from each cell. The temperature and must be optimised to achieve the
largest colonies possible so that it is easy for counting. The advantage of this method is that
we can observe whether the bacteria is anaerobic, aerobic of facultative anaerobic. In a pour
plate, bacteria will be trapped in the agar and the anaerobe and facultative anaerobe will grow
only within the agar media.
However, there are a few disadvantages of spread-plate method compared to pourplate method for performing viable count of bacteria. Firstly, some of the bacteria may stick
to the spreader and this will reduce the number of bacteria counted, thus the number of
bacteria counted will be inaccurate. Next, some bacteria might be killed by the alcohol on the
spreader. Besides, inappropriate technique of spreading would result in clumping of bacteria
colonies.

Conclusion:

We can estimate the number of viable organisms in the sample given nicely by carrying out
spread plate or pour plate method. The number of bacterial cells per mL of a sample can be
determined by carry out bacterial counts. Both of these methods use the serial dilution
method to reduce the total number of bacterial so that they are countable. The number of
bacteria per ml of sample can be estimated by the following formula:
The number of bacteria = Average plate count x 1/dilution factor x 1/plating
volume
References:
1. J Reynolds. Serial Dilution Protocols. ASMMicrobeLibrary.Last update on 1st April
2013. Retrieved on 31st October 2015. Available from:
http://www.microbelibrary.org/component/resource/laboratory-test/2884-serialdilution-protocols
2. Spread-Plate Technique. Cited on 1st November 2015. Available from:
http://nhjy.hzau.edu.cn/kech/wswx/en/syzd/shiyan/s10.htm
3. Quantifying Bacteria by Spread Plate. Microbiology Techniques. Cited on 1st
November 2015. Available from:
http://www2.hendrix.edu/biology/CellWeb/Techniques/microspread.html