CPB30103- Biochemical Engineering

Experiment4: Determination of Bacterial Loads

Viable Cell Counts Page

1.0 ABSTRACT

The main objective is to study the types of bacteria and to determine the number of
bacteria in the given sample under various conditions. Other than that, this experiment also
shows the ways of sterile handling the bacteria. Serial dilution was done until the dilution of
10-6. At this stage, two methods was used to determine the number of bacteria. In pour plate
method, 1.0ml of the 10-4 sample was transferred to a sterile empty petri dish. Then 15ml of
hot agar (45C) was pour into the petri dish. 45C is the suitable temperature to avoid the
damaged of the bacterial cells. Make sure the agar and the sample was mixed gently. The
second method was called spread plate method. 100 microlitre of the sample was transferred
into the nutrient agar plate. The diluent was spread all over the petri dish by using the L-
shaped glass rod. Before using the L-shape glass rod, it must be first immersed into 70%
ethanol and put it through the flame. These step was repeated for 10-5 and 10-6 sample. All of
the petri dish was labelled and incubated at 30C for 24 hours. After the incubation period,
the colonies will form and CFU can be calculated.
CPB30103- Biochemical Engineering
Experiment4: Determination of Bacterial Loads
Viable Cell Counts Page

2.0 OBJECTIVES

1. To prepare a series dilution of E.coli of 10-6 to 100 as bacteria sample.

2. To calculate and determine the number of colonies by using pour plate and spread
plate method.
3. To calculate the number of bacteria growth of E.coli by applying technique of pour
plate and spread plate method.
4. To calculate and determine the bacteria count per mL sample (CFU/mL) by using the
formula of CFU.
5. To study the types of bacteria
6. To determine the number of bacteria in the given sample under various condition
7. To learn the ways of sterile handling the bacteria
8. To study pour plate method
9. To study spread plate method

3.0 RESULTS

Table 1.0 : Result of pour plate method

CPB30103- Biochemical Engineering
Experiment4: Determination of Bacterial Loads
Viable Cell Counts Page

Dilution Factor Number of Colonies Bacteria Count per mL Average Count per mL
sample (CFU/mL) sample (CFU/mL)
10-4 50 500 x 10-3 500 x 10-3

10-5 14 1.4 x 10-6 1.4 x 10-6

10-6 2 2000000 2 x 10-6

Sample calculation for Bacteria count per mL sample (CFU/mL) :

Number of colonies
CFU ml 1
Dilution Factor Volume plated (ml)

50
CFU mL-1 4
500000 CFU mL-1
10 1
CPB30103- Biochemical Engineering
Experiment4: Determination of Bacterial Loads
Viable Cell Counts Page

Diagram 1 : The sample that contain the colonies through pour plate method

Table 2.0 : Result of spread plate method

Dilution Factor Number of Colonies Bacteria Count per mL Average Count per mL
sample (CFU/mL) sample (CFU/mL)
10-4 149 14.9 x 10-6 14.9 x 10-6

10-5 14 14 x 10-6 14 x 10-6

10-6 7 70 x 10-6 70 x 10-6

CPB30103- Biochemical Engineering
Experiment4: Determination of Bacterial Loads
Viable Cell Counts Page

Sample calculation for Bacteria count per mL sample (CFU/mL) :

Number of colonies
CFU ml 1
Dilution Factor Volume plated (ml)

149
CFU mL-1 4
14900000 CFU mL-1
10 0.1

Diagram 2 : The sample that contain the colonies through spread plate method
CPB30103- Biochemical Engineering
Experiment4: Determination of Bacterial Loads
Viable Cell Counts Page

4.0 DISCUSSIONS

The main objective of this experiment is to calculate the number of bacteria growth by
applying techniques of pour plate method and spread plate method. The experiment was
conducted by preparing a series dilution of E.coli as bacteria sample which started with the
highest dilution factor from 10-6 to 100. The first one was the pour plate method, where 1 ml
of the sample from dilution factor of 10-4, 10-5, and 10-6 was mixed with molten nutrient agar
before incubated for a period of 24 hours. The second method was the spread plate method,
where 0.1 ml of sample from dilution factor of 10-4, 10-5, and 10-6 was spread on top of the
nutrient agar, then incubated for 24 hours. The major difference between the two methods is
that while the colony will spread on top of agar for the spread plate method, in pour plate
method the colony grows throughout the volume of the nutrient agar (BiteSize Bio 2014).

In this experiment, both pour plate method and spread plate method can be
categorised as direct method under viable plate count. A viable cell count allows one to
identify the number of actively growing or dividing cells in the sample. The plate count
method or spread plate method relies on bacteria growing a colony on a nutrient medium. The
colony becomes visible to the naked eye and the number of colonies on a plate can be
counted. For optimum accuracy of a count, the preferred range for total CFU/plate is between
30 to 300 colonies/plate.
CPB30103- Biochemical Engineering
Experiment4: Determination of Bacterial Loads
Viable Cell Counts Page

In pour plate method, the bacterial suspension was introduced into a Petri dish in 1.0
ml of dilution as a sample of the population. It was then added with 15 ml of the molten
nutrient agar at 45C into the plate. While the spread plate method allows the bacteria grow
on the surface by spread evenly the bacteria suspension over the agar surface with an L-
shaped glass rod. Colonies will grow throughout the agar and on the surface of the agar.
Because of the differences in oxygen availability, colonies that grow within the agar will have
a different appearance than those found on the surface of the agar but are nonetheless counted
as colonies.

The result obtained for both pour plate method and spread plate method were showed
in the result table. For pour plate method, the number of colonies formed in the petri dish
with dilution sample of 10-4, 10-5, and 10-6 was 500 x 10-3, 1.4 x 10-6, and 2 x 10-6respectively.
As we observed, the number of colonies formed decreases as the dilution factor of the sample
increases. As for the spread method, the number of colonies formed in the petri dish with
dilution sample of 10-4, 10-5, and 10-6 was 14.9 x 10-6, 14 x 10-6, and 70 x 10-6 respectively.
Theoretically, the dilution factor of 100 should have the highest colonies formed since it is
pure sample without dilution of distilled water. Among the both method, spread plate method
can be can easily contaminated as it is expose more to the air whereby the pour plate method
limited the exposure to the air.

The viable plate count is a popular method for determining cell number. The
technique is sensitive and has the advantage of only counting living bacteria. Any
concentration of microorganism can be easily counted, if the appropriate dilution is plated. It
is even possible to concentrate a solution before counting, as is often done in water analysis,
where bacterial populations are usually at low density. The equipment necessary for
performing viable plate counts is readily available in any microbiology lab and is cheap in
comparison to the other method.

Despite the advantages, there is one major disadvantage of the viable plate count is
the assumption that the each colony arises from one cell. Great care must also be taking
during dilution and plating to avoid errors. The rate at which bacteria give rise to an
observable colony can also vary. If too short an incubation time is used, some colonies may
be missed. The temperature of incubation and medium conditions must also be optimized to
achieve the largest colonies possible so that they are easily counted. Finally, this technique
CPB30103- Biochemical Engineering
Experiment4: Determination of Bacterial Loads
Viable Cell Counts Page

takes time. Depending on the organism, one day to several weeks might be necessary to
determine the number of CFUs that were present when the experiment started.

5.0 CONCLUSION AND RECOMMENDATIONS

As the conclusion, it can be concluded that the number of bacteria growth can be
determined by applying techniques of pour plate method and spread plate method. Based on
the data obtained recorded in Table 1.0 which shows the result for pour plate method, the
increase the dilution factor, the decrease the number of colonies, the decrease the bacteria
count per mL sample (CFU/mL) and the decrease the average count per mL sample
(CFU/mL) where the dilution factor of 10-4, 10-5, and 10-6 have the number of colonies of 50,
14 and 2 and the bacteria count per mL sample (CFU/mL) of 500 x 10 -3, 1.4 x 10-6 , and 2 x
10-6 , respectively while for in Table 2.0 which shows the result of spread plate method, the
increase the dilution factor starts from 10-6 to 10-4, the decrease the number of colonies, the
decrease the bacteria count per mL sample (CFU/mL) and the increase the average count per
mL sample (CFU/mL) where the dilution factor of 10-4, 10-5, and 10-6 have the number of
colonies of 149, 14 and 7 and the bacteria count per mL sample (CFU/mL) of 14.9 x 10 -6, 14
x 10-6 , and 70 x 10-6 , respectively. The theory is differ from the observation as the plate with
dilution factor of 10-4 shows the overcrowded of the colonies formed compare to the plate
with dilution factor of 10-5 to 10-6. Theoretically, the dilution factor of 10 0 should have the
highest colonies formed since it is pure sample without dilution of distilled water. The spread
plate method can be can easily contaminated as it is expose more to the air whereby the pour
CPB30103- Biochemical Engineering
Experiment4: Determination of Bacterial Loads
Viable Cell Counts Page

plate method limited the exposure to the air which it is requires to take care in every aspect
when handling the microbes because they can grow in the presence or without the oxygen.

There were several possible errors that may occur while conducting the experiment
and here some precaution may be recommended. As the recommendation, in order to obtain
more accurate and desirable result and data of this experiment in the future, it is necessary to
the bottle neck must be immediately warmed by passing the bottle neck through flame in
Bunsen burner when opening a bottle which it ensures that no microorganisms enter the
mouth of the vessel to contaminate the culture or the medium. Next, limit exposure of the
sterile inner surface to contamination from the air during manipulation of Petri dish. Lastly,
make sure all the work area and hands are wiping with 70% ethanol before handling any
apparatus where the sterile handling provides a barrier between the microorganisms in the
environment and the sterile culture. As its recommendation, the microscope can also be used
instead of viable plate count which viable plate count takes some time for the visible colonies
to grow and too many colonies could cause error in the count. Thus, microscope could be the
best instrument used for viewing very small objects.

6.0 TUTORIALS

1. Discuss the importance in determining the microbial loading in a food sample.

The importance in determining the microbial loading in a food sample is the presence
of microbes on foods specified to be commercially sterile needs to be detected which are a
total load below a specified limit needs to be ascertained for other types of foods and the
absence of pathogens on foods or surfaces in the processing area must be ensured. Next,
microbial loading in a food sample is based on the determination of bacterial ATP after
separation from ATP endogenous to the food. Lastly, immunoassays are used now and
immunosensors which functions in the future in latter application where the total microbial
load, rapid methods have been developed which it is based on the monitoring of the
impedance of a culture broth inoculated with the sample.
CPB30103- Biochemical Engineering
Experiment4: Determination of Bacterial Loads
Viable Cell Counts Page |

2. You are a process engineer and have developed a bioreactor for microbiological production
of bioplastic by using bacterial cells, Pseudomonas sp. The determination of microbial
growth gives detailed insight to monitor and optimize the bioreactor so that the bioreactor
produces the maximum amount of bioplastics. Explain how you can determine the growth of
Pseudomonas sp. in your bioreactor for a period of 48 hours.

As a process engineer and have developed a bioreactor for microbiological production

of bioplastic by using bacterial cells, Pseudomonas sp., I can determine the microbial growth
of Pseudomonas sp. in my bioreactor for a period of 48 hours so that the bioreactor produces
the maximum amount of bioplastics. Firstly,these microbes have the potent procedures of
PHB due to their high adaptability in various extreme environmental conditions. Next, the
parameter in a bioreactor is the pH and the dissolved oxygen where for pH, nutrient agar of
several different pH can be made and the number of colonies after 48 hours of incubation can
be counted and the bacteria requires oxygen to grow within the agar rather than at the surface.
Lastly, the temperature and nutrients required can be tested by varying the nutrient compound
and the temperature inside the molten agar,

7.0 REFERENCES

1. Rajiv Dutta (2008) Fundamentals of Biochemical Engineering, Ane Books India

2. Microbial food safety in animal agriculture: current topics by Mary E. Torrence,
Richard E. Isaacson June 2003, Wiley-Blackwell page 36
3. Viable Cell Counting - Boundless Open Textbook. (n.d.). Retrieved December 8,
2016.
4. Making a pour plate. (2011, October 1). Retrieved December 10, 2016.
CPB30103- Biochemical Engineering
Experiment4: Determination of Bacterial Loads
Viable Cell Counts Page

8.0 APPENDICES

Sample calculation for Bacteria count per mL sample (CFU/mL) :

Number of colonies
CFU ml 1
Dilution Factor Volume plated (ml)

50
CFU mL-1 4
500000 CFU mL-1
10 1

Sample calculation for Bacteria count per mL sample (CFU/mL) :

Number of colonies
CFU ml 1
Dilution Factor Volume plated (ml)

149
CFU mL-1 4
14900000 CFU mL-1
10 0.1
CPB30103- Biochemical Engineering
Experiment4: Determination of Bacterial Loads
Viable Cell Counts Page |