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1Experiment 4 - Determination of Bacterial Load: Viable Cell Counts

ABSTRACT

The objectives for the experiment are to determine the amount of bacteria in a colony and
to distinguished the difference between pour plate and spread plate method in calculation.
Both pour plate and spread method are significant for an easier counting the number of viable
bacteria. Generally, the Colony Forming Units (CFU) then are determined from the plate
count. However, only living cells can be count. Among of both methods, pour plate method is
better than spread plate method where the pour plate has lesser exposure to the air than spread
plate method. Thus, the pour plate methods have a little risk for the contamination. As an
addition, in this experiment, the experimenter also experienced in sterile handling to ensure
an experiment done can growth the bacteria and to ensure less in contaminant,

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2Experiment 4 - Determination of Bacterial Load: Viable Cell Counts

OBJECTIVES

The main objective of the experiment is to determine the amount of bacteria are in a
colony

To distinguished the difference between pour plate and spread plate method in
calculating the bacterial

To practice a right way in sterile handling

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3Experiment 4 - Determination of Bacterial Load: Viable Cell Counts

RESULT

Both Table 1 and Figure 1 show the result obtained from the pour plate method.

Table 1: The Amount of Bacteria in Colony Obtained Through Pour Plate Method

Dilution Factor Number of Colonies


Colony-forming unit per mL sample (CFU/ml)
10-4 91
9100000
10-5 2
2000000

10-6 1 10000000

Example of calculation for CFU/mL sample:

CFU mL-1 =

CFU mL-1 = = 9100000

10-4 10-5 10-6

Figure 1: The Sample Obtained Through Pour Plate Method

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4Experiment 4 - Determination of Bacterial Load: Viable Cell Counts

Table 2 and Figure 2 show the result obtained from the spread plate method.

Table 2: The Amount of Bacteria in Colony Obtained Through Spread Plate Method

Dilution Factor Number of Colonies


Colony-forming unit per mL sample (CFU/ml)
10-4 8
80000
10-5 0
0

10-6 0 0

Example of calculation for CFU/mL sample:

CFU mL-1 =

CFU mL-1 = = 80000

10-4 10-5 10-6

Figure 2: The Sample Obtained Through Spread Plate Method

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5Experiment 4 - Determination of Bacterial Load: Viable Cell Counts

DISCUSSION

The main objective of the experiment is to determine the amount of bacteria are in a
colony, to distinguished the difference between pour plate and spread plate method in
calculating the bacterial. To begin the experiment, a series dilution of E.Coli were prepared.
The dilution factor starting with 10-1, 10-2,10-3, 10-4, 10-5 and 10-6. From the six diluted sample,
the three most diluted sample is taken which is 10 -4, 10-5 and 10-6. The three sample is taken
as it is more dilute compared to the first three batch that have been prepared. It is easier to be
count as is more dilute. The two sample were count using two method which is using spread
plate method, and using pour plate method. In pour plate method, the sample were taken in a
plate and mixed with molten nutrient agar. The mixture were then incubated for 24 hours. For
spread plate method, the sample were spread on top of nutrient agar and incubated for 24
hours. Basically, the difference between this two method is in the way on how the bacteria
cultivated. In spread method, the bacteria growth and spread on top surface of the nutrient
agar, while in pour plate method, the bacterial tend to growth through out the volume of the
molten nutrient agar.

According to Boundless Microbiology, 2017 , both method spread and pour plate
method is considered as viable cell count method. This method allows ones to count the cell
in the sample. The colony which growth able ones to count with naked eyes.

Theoretically, upon dilution, the concentration of bacterial become lower. Meaning


that, the highest the dilution factor, the lower the number of bacterial will be. From the result
obtained in figure 1, it showed the result of pour plate method. In the table, it showed that the
number of colonies increases as the dilution factor decrease. From the result, it showed that,
at dilution factor 10-6, the number of colonies is 1, while at 10-5 the number of colony is 2 and
at dilution factor 10-4, the number of colonies is 91.

In spread plate method, based on table 2, it showed that, at dilution factor of 10 -4, the
number of colonies present is 8 while there is no colonies present at dilution factor 10 -5 and
10-6. This might due to remaining two samples are contaminated before the result can be
taken.

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6Experiment 4 - Determination of Bacterial Load: Viable Cell Counts

Among both method, the spread plate method is easily contaminated as it is exposed to
more air compared to pour plate method. While in pour plate method, it allowed the growth
and quantification of micro-aerophiles because there is less oxygen than on the agar surface.
It is also identifiable whether bacteria is an anaerobe, aerobe or a facultative aerobe because
the anaerobe and facultative anaerobe will grow within the media. Also in pour plate method,
more surface area is covered as the sample is spread throughout the media whereas in Spread-
plate the sample is spread only to a limited to the surface of the agar(Quora,2017).

Nevertheless, viable count via spread and pour plate method is important. It enable ones
to count living bacteria. Not only that, these simple method requires less equipment to
perform the viable plate counts. However the disadvantages are only living cells develop
colonies that are counted. Besides, clumps or chains of cells develop into a single colony and
colonies develop only from those organisms for which the cultural conditions are suitable for
growth. Also, it is very sensitive with the plating conditions ("Growth of Bacterial
Populations", 2017).

Overall, all objective were achieve. From the experiment, it can be concluded that, as
the dilution factor increase, the lower the number of colonies will be. It was also showed that
the spread method is more difficult compared to pour plate method. Thus, it is undeniable that
pour plate method is better compared to spread plate method.

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7Experiment 4 - Determination of Bacterial Load: Viable Cell Counts

References

 Counting Bacteria | Boundless Microbiology. (2017). Courses.lumenlearning.com.


Retrieved 9 October 2017, from https://courses.lumenlearning.com/boundless-
microbiology/chapter/counting-bacteria/

 (2017). Retrieved 9 October 2017, from https://www.quora.com/What-is-the-


difference-between-the-pour-plate-method-and-the-spread-plate-method-in-isolation-
of-bacterial-colonies

 Growth of Bacterial Populations. (2017). Textbookofbacteriology.net. Retrieved 9


October 2017, from http://textbookofbacteriology.net/growth_2.html

 (2017). Retrieved 9 October 2017, from https://www.quora.com/What-is-the-


difference-between-the-pour-plate-method-and-the-spread-plate-method-in-isolation-
of-bacterial-colonies

 Pour Plate Method for Isolating Bacteria - Membrane Solutions. (2017). Membrane-
solutions.com. Retrieved 9 October 2017, from https://www.membrane-
solutions.com/News_976.htm

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