1.

0 Introduction

Microorganisms also known as microbes are organisms that appear microscopic and they are
difficult to be seen with the naked eye. Some microorganisms are pathogenic and are capable
to cause serious illnesses in a host. For example, Escherichia coli and Salmonella typhi. Thus,
it is vital to enumerate microorganisms, especially in particular aspects like the food and
beverages industry to determine the amount and type of microbes present. Enumeration of
microorganisms is important in the food industry to ensure the food meets the
microbiological criteria and is safe to be consumed by the public.

A variety of methods have been employed in the enumeration of microorganisms in

food which include the pour plate method, surface spread plate method, surface drop method
and Petrifilm method. However, these methods are used depending on several factors: type of
sample, characteristics and physiology of microorganism needed, characteristics of specific
media, low limit enumeration, the objective of the test and time limit. Before performing a
specific method to enumerate microorganisms in food, serial dilution is the crucial step that
enables viable microorganisms to be calculated through concentration reduction. The primary
goal of this step is to obtain a reasonable number of colonies for enumeration since it is
commonly known to be impossible to calculate the actual number of microorganisms present
in a sample (University of Vermont, n.d.). Hence, the sample is usually first diluted where it
involves using a capped test tube filled with 9.0 ml of sterile diluent, 1.0 ml of the sample is
pipetted into the first test tube, mixed with a vortex mixer and then transferred to the next test
tube containing another 9.0 ml of sterile diluent, creating a series of decimal dilution
(Inlabtec, n.d.).

The enumeration methods stated above are applicable to microbial enumeration in

food. However, a specific enumeration method for a product may occasionally be required by
the law. For instance, a pour plate method has always been approved to determine the
bacterial counts in pasteurized milk (Mallmann and Broitman, 1956). According to BYJUS
(n.d.), the pour plate method is commonly used to detect obligate and anaerobic bacteria.
Moreover, a successful surface spread plate method is able to show countable bacterial
colonies that are evenly distributed on the plate (Iowa State University, n.d.). Herigstad,
Hamilton and Heersink (2001) mentioned that the surface drop method decreases the time
required for colony counting and increases the accuracy as the sample is distributed in drop

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form. The Petrifilm method is introduced to act as a substitute for the conventional plating
method as it does not require prior agar or medium preparation. Hence, it is designed to save
time, costs and the space that an incubator needs.
The hypothesis of this experiment is: if the food sample or bacterial culture is diluted
to a higher degree of dilution, then the number of viable colony-forming units (CFUs) will
appear in lesser concentration and be more capable to be enumerated.

1.1 Objectives

 To learn different enumeration methods that can be used to determine the total number of
microorganisms present in a sample.
 To determine the total colony forming unit (CFU) per ml or g of bacterial culture
(Escherichia coli) and ready-to-eat food (Bihun).
 To learn and enhance serial dilution and calculation skills.

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2.0 Methods

1. Serial Decimal Dilution

2. Pour Plate Method

3. Surface Spread Plate Method

4. Surface Drop Method

5. Petrifilm Method

2.1 Procedures
2.1.1 Serial Decimal Dilution
For bacteria culture
1. 6 test tubes, each containing 9.0ml of sterile diluent (MRD / 0.1% peptone water)
were placed on a test tube rack. Each test tube was labelled with dilution (10 -1 to 10-6)
using a permanent marker pen. The original bacteria in nutrient broth (NB) were not
diluted, so the dilution is 100.
2. The control culture, E coli in NB was mixed with a vortex mixer and was transferred
aseptically 1.0 ml into a test tube containing 9.0 ml diluent. This is the dilution 10-1.
3. The bacterial culture from dilution 10-1 was mixed with a vortex mixer and was
transferred aseptically 1.0 ml into a test tube containing 9.0 ml diluent by using a
different micropipette tip. This is the dilution 10-2.
4. Step (3) was repeated to serially dilute the culture broth until dilution 10 -6 (total of 6
test tubes).

For ready-to-eat food sample

1. 3 test tubes, each containing 9.0ml of sterile diluent (MRD / 0.1% peptone water)
were placed on a test tube rack. Each test tube was labelled with dilution (10 -2 to 10-4)
using a permanent marker pen.
2. 25 g of food was added with 225 ml of sterile diluent in a sterile Stomacher bag to
prepare a ready-to-eat food homogenate sample. This is the dilution 10-1.

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3. The food homogenate was mixed by using a Stomacher for 60 seconds, then was
transferred aseptically 1.0 ml into a test tube containing 9.0 ml diluent. This is the
dilution 10-2.
4. The dilution was mixed with a vortex mixer and transferred aseptically 1.0 ml into a
test tube containing 9.0 ml diluent by using a different micropipette tip. This is the
dilution 10-3.
5. Step (4) was repeated to serially dilute the food homogenate until dilution 10-4 (total of
3 test tubes).

2.1.2 Pour plate method

1. The empty plates were labelled with the following dilutions:
for bacterial cultures: 10-6, 10-5, 10-4, 10-3 (each dilution had two plates)
for RTE food samples: 10-6, 10-5, 10-4, 10-3 (each dilution had two plates)
All important information were recorded at the bottom of the plates (e.g. group name,
method, date, etc using a permanent marker pen)
2. 10-6 of bacterial culture dilution and 10 -4 of RTE food dilution were mixed with a
vortex mixer and 1 ml of suspended bacteria were transferred aseptically into a sterile
plate using a micropipette tip for each dilution. Duplicate transfers of the same
dilution were transferred to another plate respectively.
3. Using the same pipette tip, 1 ml of vortexed 10-5 of bacterial culture dilution and 10-3
of RTE food dilution were transferred into another sterile plate aseptically for each
dilution. Duplicate transfers were done to another plate respectively.
4. The same steps were used for 10 -4 and 10-3 of bacterial culture dilutions and 10-2 and
10-1 for RTE food dilutions respectively.
5. One bottle of molten Plate Count Agar (PCA) was taken from the water bath/oven and
about 15 ml agar was poured into the plate until 2/3 was filled under aseptic
condition. The agar was mixed according to the steps shown in Figure 2.1.2.1.
6. The plates were left on the table until the agar hardened. The plates were incubated
upside-down at 37oC for 24 hours.
7. The number of colonies for each dilution were counted using a Colony Counter.
8. Only plates containing 30 – 300 colonies in two consecutive dilutions (e.g. 10-5, 10-6)
or one dilution were used.
9. The total colony forming unit (CFU) per ml or g for the samples tested was
determined using the formula below.

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Formula: cfu/g or ml = (Total number of colony/volume of inoculum transferred) x total
dilution factor

Figure 2.1.2.1 Viable cell count using a pour plating method

2.1.3 Surface Spread Plate Method

1. Two PCA agar plates were prepared for each dilution to be tested (for bacterial
culture: 10-6, 10-5, 10- 4, for RTE food: 10-3, 10-2, 10-1). The bottom of the plates was
labelled.
2. The E. coli culture with the dilution of 10-6 test tube was mixed using a vortex mixer
and 0.1 ml bacterial suspension was transferred aseptically using a micropipette tip to
the centre of the agar surface. This was repeated for a duplicate plate.
3. Step (2) was followed for RTE food starting with the dilution of 10-3.
4. The glass spreader was submerged into a bottle of alcohol and passed through a
Bunsen burner swiftly so that the remainder alcohol catches fire. Alcohol sterilizes the
glass spreader and flaming is to eliminate extra alcohol on the glass spreader. The
glass spreader was not burned for too long to prevent it from breaking. The glass
spreader was cooled before spreading the inoculums. The flammable alcohol was not
too close to the Bunsen burner to prevent it from catching fire.
5. Half of the plates cover was opened, then the bacterial/food suspension was carefully
and evenly spreaded using the glass spreader on the agar surface. The Petri dishes
were turned while spreading. The glass spreader was soaked into the alcohol solution
and passed through the Bunsen burner to evaporate the remaining alcohol.
6. Steps (2), (3), (4) and (5) were repeated for dilution 10 -5 and 10-4 in duplicate for the
bacterial suspension and 10-2 and 10-1 for the RTE food suspension.

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 7. When the bacterial or RTE food suspension have absorbed into the plating media
(PCA), The plates were turned upside down and incubated at 37oC for 24 hours.
8. The number of colonies were counted for each dilution using a Colony counter.
9. Only plates containing 30 – 300 colonies in two consecutive dilutions or one dilution
were used.
10. The total colony forming unit (cfu) per ml or g was determined for the samples tested
(N).

2.1.4 Surface Drop Method or Miles and Misra Method

1. PCA plates were labelled at the bottom of the plates.
2. Each PCA plate was divided into four sections and the dilutions were labelled.
(Figure 2.1.4.1)
3. Starting with the highest dilution (e.g.10-6), the bacterial suspensions were mixed with
a vortex mixer.
4. Similarly, with the RTE food homogenate, starting with the highest dilution (e.g.10 -3),
the suspensions were mixed with a vortex mixer.
5. A 10 µl (0.01 ml) volume of the bacterial/ RTE food suspension was drawn up using a
micropipette and sterile tip.
6. The aspirated volume was dispensed perpendicularly onto a sector of the two agar
plates.
7. Step (5) was repeated for the same dilution for another drop in the same sector.
8. Steps (3) to (5) were repeated for the remaining dilutions (bacterial suspension: 10 -5
and 10-4; RTE food suspension: 10-2 and 10-1).
9. The dilution drops were left until they were being absorbed by the agar medium.
10. The plates were turned over and incubated at 37oC for 24 hours.
11. Only plates containing 30 – 300 colonies in two consecutive dilutions or one dilution
were used.
12. The total colony forming unit (cfu) per ml or g was determined for the samples tested
(N).

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 Figure 2.1.4.1: Dividing sectors for surface drop method

2.1.5. Petrifilm Method using Petrifilm® 3M Aerobic Count Plates

1. Petrifilm AC plates were labelled in duplicate for each dilution (6 Petrifilm for each
group). Petrifilms were labelled by writing on the transparent side of the plates.
2. Two Petrifilm AC plates were inoculated for each dilution (10 -6, 10-5 and 10-4 from the
bacterial suspension and 10-3, 10-2 and 10-1 from the RTE food suspension).
3. Petrifilm AC plates were placed on a flat surface. The top film was lifted.
4. Pipette was held perpendicular to the Petrifilm AC plate and 1 ml of the bacterial
inoculum of 10-6 dilution and 10-3 RTE food suspension were carefully dispensed onto
the centre of the bottom film separately on different films.
5. The top film was slowly released onto the inoculum, allowing it to drop. The film was
not rolled down.
6. With the ridge side down, a spreader was placed on top of the film over the
inoculums.
7. The inoculum was evenly distributed using gentle downward pressure on the centre of
the spreader. The spreader was not slided across the film. The spreader was removed,
and the plate was left undisturbed for one minute to permit the solidification of the
gel.
8. Steps (3) to (7) were repeated for the same dilution for the duplicate plate.
9. Steps (3) to (8) were repeated for the other dilutions (10-5 and 10-4 for bacterial
inoculums and 10-2 and 10-1 for the food suspension) in duplicate plates.
10. The Petrifilm AC plates were incubated in a horizontal position, with the clear side up
in stacks not exceeding 20 plates, at a temperature of 37oC for 24-48 hours.

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11. Petrifilm plates were read on a standard colony counter or other magnified light
source. The reduction of the tetrazolium indicator dye will cause the colonies to
become red. All red dots regardless of size or intensity were counted as colonies.
12. The preferable counting range on a Petrifilm AC plate is 30 – 300 colonies. When the
colonies’ numbers are more than 300, the count was estimated. The number of
colonies in four squares (4 cm2) was counted and the average was determined, then
multiplied by 20 to obtain the total count per plate.
13. The circular growth area is approximately 20 cm 2. Estimates can be made on plates
containing greater than 300 colonies by counting a representative number of squares
and multiplying by the appropriate number to obtain an estimated count for the total
20 cm2 growth area. The presence of very high concentrations of colonies on the
plates will cause the entire growth area to become red or pink in colour, record results
as “too numerous to count” (TNTC). Occasionally, on overcrowded plates, the centre
may lack visible colonies but many small colonies will be seen on the edges. When
this occurs, record results as TNTC. Some organisms can liquefy the gel, allowing
them to spread out and obstruct the presence of other colonies. If the liquefy interferes
with counting, an estimated count should be made by counting the unaffected areas.
14. The number of colonies per ml or g of sample cultures/food were reported.

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3.0 Results

Table 3.0.1: Number of Colonies per Plate per Dilution of each enumeration method
Method Sample Plate/ Number of Colonies per Plate per Dilution
Drop 10-1 10-2 10-3 10-4 10-5 10-6
1 TNTC TNTC TNTC TNTC
Pour Bacteria 2
Plate (Escherichia coli)
RTE food (Bihun) 1 TNTC TNTC TNTC 238
2 246
Surface Bacteria 1 TNTC TNTC 86
Spread (Escherichia coli) 2 130
Plate RTE food (Bihun) 1 TNTC TNTC TNTC
2
Bacteria 1 TNTC 49 9
Surface (Escherichia coli) 2 41 5
Drop RTE food (Bihun) 1 TNTC TNTC TNTC
2
Bacteria 1 TNTC TNTC TNTC
Petrifilm (Escherichia coli) 2
Plate RTE food (Bihun) 1 TNTC TNTC TNTC
2
Note: TNTC= Too Numerous To Count

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Table 3.0.2: Colony-forming unit per mL of bacteria suspension and sample

Method Sample Colony-forming unit per mL (CFU/mL)

Pour Bacteria -
Plate (Escherichia coli)
RTE food 2.42 x 106
(Bihun)
Surface Bacteria
Spread (Escherichia coli) 1.08 x 109
Plate RTE food
(Bihun) -

Bacteria
Surface (Escherichia coli) 5.75 x 108
Drop RTE food
(Bihun) -

Bacteria
Petrifilm (Escherichia coli) -
Plate RTE food
(Bihun) -

Note: ‘-’ = CFU was not able to be calculated (TNTC)

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Figure 3.0.1: Results of Food Sample using Pour Plate Method (10-4, 10-3, 10-2 & 10-1)

Notes: Naming of agar plate: Group name, Enumeration method, Dilution factor, Agar plate
number, Type of sample

3.1 Result (Food Sample-Pour Plate Method)

The bacterial cells clump together in the lower dilution agar plates: 10 -3, 10-2 & 10-1. The
number of colonies for 10-3, 10-2 and 10-1 of ready-to-eat food (Bihun) agar plates are more
than 300 hence classified as too numerous to count. Both the agar plates with the highest
dilution of 10-4 are countable. The number of colonies in the first 10 -4 dilution agar plate is

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238 while the second plate is 246. Hence, the average number of colonies that are present in
the ready-to-eat food (Bihun) is 242 which falls in the countable range.

Figure 3.0.2: Results of E.coli using Pour Plate Method (10-6, 10-5, 10-4 & 10-3)

Notes: Naming of agar plate: Group name, Enumeration method, Dilution factor, Agar plate
number, Type of sample

3.2 Result (E. coli-Pour Plate Method)

The bacterial cells in all the agar plates appear tiny in size and do not appear in clump form.
The number of colonies present in all the agar plates are more than 300 colonies hence
classified as too numerous to count.

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Figure 3.0.3: Results of Food Sample using Surface Spread Plate Method (10-3, 10-2 & 10-1)

Notes: Naming of agar plate: Group name, Enumeration method, Dilution factor, Agar plate
number, Type of sample

3.3 Result (Food Sample-Surface Spread Plate Method)

The bacterial cells in the agar plates form aggregates and clump together. The streaks on the
agar plates are uneven making it hard to distinguish and enumerate the colonies. Besides, the
number of colonies present in the agar plates are more than 300 colonies hence they are

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classified as too numerous to count.

Figure 3.0.4: Results of E.coli using Surface Spread Plate Method (10-6, 10-5 & 10-4)

Notes: Naming of agar plate: Group name, Enumeration method, Dilution factor, Agar plate
number, Type of sample

3.4 Result (E. coli- Surface Spread Plate Method)

The number of colonies present in the agar plates with dilutions: 10-5 & 10-4 are more than
300 hence they are classified as too numerous to count. However, the first and second agar
plates with the highest dilution, 10-6 are countable and recorded at 86 and 130 respectively.

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Figure 3.0.5: Results of Food Sample (10-3, 10-2 & 10-1) and E.coli (10-6, 10-5 & 10-4) using
Surface Drop Method

Notes: Naming of agar plate: Group name, Enumeration method, Dilution factor, Agar plate
number, Type of sample

3.5 Result (Food Sample & E. coli- Surface Drop Method)

The bacterial cells appear in clumps in the agar plate with the food sample. Hence causing the
number of colonies present in the food sample agar plate is unable to count as they are too
numerous.
For the agar plate with E.coli, the number of colonies present in the 10-4 dilution drop

15
could not be determined as they are too numerous. However, the number of colonies present
in the higher dilution drops: 10-5 and 10-6 are countable. The average number of colonies
present in the 10-5 drop is 45 while the average number of colonies present in the 10-6 drop is
7.

Figure 3.0.6: Results of Food Sample using Petrifilm Method (10-3, 10-2 & 10-1)

Notes: Naming of Petrifilm: Group name, Type of sample, Dilution factor, Petrifilm number

3.6 Result (Food Sample-Petrifilm Method)

The colonies appear in red dots and the colonies can be seen appearing in high concentration.
Hence, causing the number of colonies present on the Petrifilm AC plates to be too numerous
to count (>300 colonies).

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Figure 3.0.7: Results of E.coli using Petrifilm Method (10-6, 10-5 & 10-4)

Notes: Naming of Petrifilm: Group name, Type of sample, Dilution factor, Petrifilm number

3.7 Result (E.coli-Petrifilm Method)

The colonies appear in red dots and they can be seen appearing in high concentration. Hence,
causing the number of colonies present on the Petrifilm AC plates to be too numerous to
count.

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3.8 Overall Results

By observing the results of this experiment, the hypothesis made prior to the experiment can
be answered as it can clearly be seen when the concentration of both samples: E.coli and
Bihun are decreased through serial dilution, the amount of colony-forming units (CFU) is
lesser making it possible to be enumerated. From the appearance, agar plates with more
diluted samples have white clearer dots that are observable and capable to be counted while
bacterial cells aggregate and clump together forming unclear larger blots when the sample is
less diluted causing the viable CFUs harder to be enumerated. Based on Table 3.0.1 above,
the number of colonies present in most of the samples except the highly diluted samples are
too numerous to count as they contain more than the countable CFUs range which is more
than 300 CFU/mL.

By comparing the enumeration methods, the pour plate method does not seem reliable
compared to the surface spread plate method and surface drop method as some anaerobes and
facultative anaerobes are able to grow within the media. Hence, the subsurface colonies
might overlap with the surface colonies and cause the agar plates to overcrowd with colonies.
Due to this, the number of colonies are harder to be enumerated as well as the total number of
colony-forming units present in the samples. Although the samples have high dilutions, the
Petrifilm method is unable to provide a countable range of colonies (30-300 colonies) based
on Table 3.0.1 above as the results obtained are more than 300 colonies which are then
classified as too numerous to count. It has a high possibility that the occurrence of this
situation is due to a lack of skill in spreading samples on the Petrifilm AC plates. From the
appearance, most of the colonies appear in tiny red dots and large amounts which is hard to
be enumerated as shown in Figure 3.0.6 and Figure 3.0.7.

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4.0 Discussion

4.1 Pour Plate Method

Advantages, Disadvantages & Errors

One of the advantages of the pour plate method is it is able to detect and enumerate low loads
of bacterial counts (BYJUS, n.d.). Besides, it is more time-saving as it does not require a
prior solidified medium compared to other methods like the surface spread plate method and
surface drop method. Not only that, it also does not require any extra tools for the inoculation
process, hence the time needed to inoculate samples by using the surface spread method can
be eliminated as spreaders are not used in this pour plate method. Since spreaders are not
utilized in this method, the risk of gouging during the inoculation process, for example
spreading and streaking can be excluded. According to Dahal (2022), this method can be used
to isolate facultative anaerobes and anaerobes and further identify the type of bacteria present
whether it is an aerobe, anaerobe or facultative aerobe as anaerobes and facultative anaerobes
as anaerobes and facultative aerobes are able to grow within the media which is the
subsurface of the agar plate. Due to there is less oxygen in the subsurface of the media,
microaerophiles can also be enumerated (StuDocu, n.d.).

However, the pour plate method has some drawbacks that should be taken into
account. The pour plate method can be a little time-consuming when it comes to the
processes involved before the inoculation. Terrones-Fernandez et al. (2023) stated that having
to melt the media before inoculation is one of the drawbacks of the pour plate method as
additional time is required. Not only that, dissolving the solid samples and a series of decimal
dilution process before inoculation are also time-consuming. Besides, this method increases
the loss of viable heat-sensitive microbes as the countable microbes must be able to withstand
the heat of the molten agar with a temperature between 43-45˚C. The temperature of the

19
molten agar plays a big role in the enumeration process as molten agar that is too hot can
affect the viable count of the heat-sensitive organisms as well as if it is too cold, it will create
clumps of congealed agar which might be mistaken for colonies. Furthermore, this method is
difficult to isolate and enumerate obligate aerobes as the low oxygen at the bottom of the
plate slows down and inhibits their growth (Dahal, 2022).

There are a few possible errors that might have affected the results and this includes
the temperature of the molten agar. The agar might be too cold and causes the production of
clumps of congealed agar. The clumps of congealed agar might have been mistaken as
colonies and caused the results of the samples to appear as too numerous to count (TNTC) as
seen in Figure 3.0.1. Besides, the results might be affected due to the capillary pipette could
not be emptied completely on the bottom of the agar plate (Van Soestbergen and Lee, 1969).
Some microbes were lost from the plate count due to the minute remnant of fluid that
remained in the pipette tip. This might affect our result for the samples with a dilution of 10 -4
which appeared countable and do not exceed 300 colonies (TNTC).

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4.2 Surface Spread Plate Method

Advantages, Disadvantages & Errors

The main advantage of this method is it is easy to isolate microorganisms as
subsurface colonies are absent in the spread plate (Expertsmind, n.d.). Aerobic organisms are
avoided from getting trapped inside the agar medium which is unlike compared to pour plate
method. Besides, it is more flexible to handle as compared to other methods like the pour
plate method. This is due to the surface spread plate method does not require melting the agar
each time before inoculation. This method also benefits those microbes that are heat sensitive
as it has less interfering effects on temperature-sensitive organisms according to (Thomas et
al., 2015). Microorganisms are not exposed to high temperatures like those inoculated using
the pour plate method. This can provide more reliable results as heat-sensitive microbes are
not killed and can be counted leading to higher viable colony-forming unit counts.

The difficulty to enumerate microorganisms is one of the disadvantages of the surface

spread plate method (Aryal, 2019). Lack of skill in spreading the inoculum on the medium
will cause the microbes to be spread unevenly, causing colonies to crowd and clump together.
There is a higher chance of gouging the media during spreading especially using a metal
spreader by someone who lacks spreading skills and if the media is not properly solidified.
Besides, there is a possible growth of undesired microbes due to accidental cross-
contamination. Cross-contamination might occur if the sterilization of the spreader is not
enough, thus introducing unwanted microbes and resulting in a higher count of colonies. This
method does not facilitate the growth of anaerobes and microaerophiles. It also requires a
specific media pre-solidified before the inoculation. Thus, different types of media are needed
or prior information on the probable microorganisms in the sample needs to be determined
(Dahal, 2022). This method also requires an extra tool compared to other conventional
plating methods which is a spreader. It is used to spread the inoculum on the surface of the
media.

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 Possible errors that might affect the result include the temperature of the spreader
being too high. This might cause some of the heat-sensitive microbes to be killed, causing
lower CFUs. Besides, the volume of inoculum transferred for spreading might accidentally
exceeded 0.1ml. Therefore, causing fused colonies and the sample may not be absorbed by
the medium and leaving it to float on the surface of the media. This can be seen in Figure
3.0.3 and Figure 3.0.4.
4.3 Surface Drop Method

Advantages, Disadvantages & Errors

The main advantage of the surface drop method is it requires lesser time and effort when
compared to other conventional plating methods like the surface spread plate method where
dispensing the inoculum is faster and lesser effort is needed than the use of the spreading
method. Besides, colony enumeration is faster and more accurate by dispensing drops of
inoculum on the agar plates (Herigstad, Hamilton and Heersink, 2001). This method is also
cost-effective as it does not require extra tools like the metal spreader to spread the inoculum
instead it only requires dispensing the inoculum in drop form using the micropipette. It is also
less labour-intensive as the dispensing and enumeration processes require less effort. The
enumeration of colonies is faster since the sample is distributed in evenly distributed distinct
spots. According to Hoben and Somasegaran (1982), this method can produce more eight
counts in an agar plate where three to four dilutions could be done at the same time.

Like other methods, this method has its drawbacks which include enumeration being
difficult to perform when bacteria tend to swarm together as some E. coli and Bacillus
subtilis strains are involved due to the smaller size that is covered by the drop. The result
usually exists as undistinguishable clumps of bacteria colonies (Naghili et al., 2013).

This method is also more susceptible to errors like improper pipetting technique.
Based on Figure 3.0.5, the colonies that appear to be merged and clumped together might
result from the poor pipetting technique. Not only that, improper holding of the agar plate
like slanting and also quick dispensing of inoculum may be the possible cause of error made
for this surface drop method in the food sample. To minimize the error, a proper pipetting
technique should be used where the sample should be dropped slowly onto the surface of the

22
media from a lower height angle to produce good droplets. Besides, insufficient or over-
drying the surface of the agar plates before inoculation is also one of the contributors to the
possible errors made during the procedure. The degree of dryness of the agar plates’ surface
may affect the result of the experiment where too wet of a media can cause delayed
absorption of inoculum and slight clouding on the surface of the agar plates while too dry of a
media can decrease the water activity of the microorganisms, thus affecting and slows down
the growth rate of hydrophilic and moisture-dependent microbes.
4.4 Petrifilm Method

Advantages, Disadvantages & Errors

The Petrifilm method is introduced to act as an alternative method to traditional plating

methods. It was produced by the company 3M to serve as a substitute for the pour plate
method and surface spread methods. It was designed to plate 1ml of the sample on the
Petrifilm surface which is roughly the size and volume of the playing card, utilizing dyes like
tetrazolium to show the formation of colonies. The viable cells will reduce tetrazolium
causing the colour to change to red. The red dots present on the Petrifilm indicates the
presence of viable microbes and all the red dots have to be counted as colony cells. The
advantage of the Petrifilm method is it is ready to use. This can eliminate the time required to
prepare the agar. Besides, this method provides consistent and reliable results. Petrifilm has a
compact size thus it does not require large incubation and storage space compared to
traditional agar plates (Ledtechno, 2020). Besides, in comparison to agar plates, Petrifilm
takes up 85% less area, cut waste by 66%, greenhouse gas emissions by 75%, and water
waste by 79% according to Simpson et al. (2022).

However, there are a few disadvantages of this method which are Petrifilm tends to be
overcrowded showing the centre of the Petrifilm with invisible colonies while many small
colonies tend to gather at the edges of the Petrifilm (de Sousa et al., 2005). This may lead to a
high number of errors and eventually cause misinterpretation of results. Besides, the small
volume (1ml per sample) that can be tested is one of the main concerns of this Petrifilm
method (Vail et al., 2003). Furthermore, the Petrifilm method does not have an enrichment
step. Hence, low numbers of Listeria may not be detected in the food sample. This is a
concern in the United States as they have zero tolerance towards Listeria (3M Food Safety
News, 2020).

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 Improper spreading technique is one of the common errors that are encountered using
this Petrifilm method. The inoculum should be distributed evenly using gentle downward
pressure on the centre of the spreader and the spreader should not be slided across the film to
prevent false results. Furthermore, overestimating the number of colonies present is also
another common error made since the Petrifilm has high sensitivity which will lead to a false
enumeration of microbes.

4.5 Overall Errors

Sampling, dilution, plating, counting and calculation errors might be present in this
experiment. Sampling error includes the random distribution of microbes throughout the
samples. Some parts of the food sample are more highly contaminated than other parts.
Improper weighing of the sample will also affect the result of the experiment as the
concentration of microbes in the inoculum is adjusted to a denser level. Dilution error might
also occur especially improper handling of the pipette. For instance, the capillary pipette is
not emptied completely on the bottom of the agar plate (Van Soestbergen and Lee, 1969).
Some microbes were lost from the plate count due to the minute remnant of fluid that
remained in the pipette tip. This might affect our result for the samples as the number of
colonies is underestimated. For plating error, the degree of dryness and coolness of the agar
media may also affect the result of the experiment as too wet of a media can cause delayed
absorption of inoculum and slight clouding on the surface of the agar plates while too dry of a
media can decrease the water activity of the microorganisms, thus affecting and slows down
the growth rate of hydrophilic and moisture-dependent microbes. During the plating process,
the improper dispensing techniques used produce cloudy colonies causing misinterpretation
of the result. As for the counting process, miscounting and overcounting may occur as
coalescence may occur in which two or more adjacent colonies merge into one countable
colony. Calculation errors might also occur when calculating the total number of colony-
forming units using the formula.

4.6 Overall Applications

The results show that the number of colonies present in the food sample which is Bihun are
mostly too numerous to count (TNTC) or has a high number of colonies which is 2.42 x 106

24
CFU/ml. This might increase the risk of food poisoning if the food is consumed. The food
sample used in this experiment has been left out at room temperature for quite some time
causing the temperature of the food to reach the temperature danger zone and this is
dangerous as most of the bacteria grow and multiply rapidly within the temperature danger
zone which is from 5˚C to 60˚C to an unsafe level which will cause food poisoning.
Therefore, the results can help to raise awareness among the public regarding the
consequences of consuming food that has been time-temperature abused. Consumers will
think more wisely before they choose their food. They will opt for food that is freshly cooked
and has not been temperature abused especially when they eat out to prevent themselves from
getting food poisoning.
The four enumeration methods are commonly employed in various industrial
applications. For example, wastewater treatment plants use the pour plate method to detect
microbial and chemical contamination of treated water. Besides, it is also utilized in
pharmaceutical companies. They perform the pour plate method to determine the level of
microbial contamination of newly developed medications during the production,
transportation and storage stages. By sampling the medications at different stages and then
further plating the samples using the pour plate method, microbial loads that are
contaminating the drugs can be easily determined (MN Editors, 2021). The Petrifilm method
is normally used for citizen-based and educational monitoring since it is easy to use and does
not require prior preparation of the agar plates. It is also employed as a preliminary screening
method to locate problem sites (Vail et al., 2003).

5.0 Conclusion

 Sampling of the food sample is required before the serial dilution process.
 Serial dilution must be performed prior to the inoculation and plating process to decrease
dense microorganisms to a countable concentration through a series of decimal dilutions
for a specific method: 30-300 colonies for the pour plate method, surface spread plate
method and Petrifilm method and 3-30 colonies for surface drop method. The more the
sample is diluted, the lower the number of viable colony-forming units (CFUs). This can
answer the hypothesis.
 Pour plate method involves the pouring of molten agar into the petri dish containing the
inoculum and mixing. Colonies are found in the subsurface of the agar causing them to

25
 overlap with the colonies present at the bottom of the agar. This causes the colonies to be
too numerous to count (TNTC).
 Surface spread plate method involves the usage of a glass spreader to spread the inoculum
evenly on the surface of the agar. Colonies only appear on the surface of the agar and
most of the obligate aerobes can be identified.
 Surface drop method involves the dispensing of inoculum onto a sector of the agar plate
perpendicularly using the micropipette. This method can only be used for samples with
low concentrations of microbes and swarming bacteria cannot be isolated using this
method as coalescence would occur.
 Petrifilm method requires even distribution of inoculum using a gentle downward
pressure on the centre of the spreader and the spreader should not be slided across the
film. This method can only test a small volume of sample (1ml per sample) hence large
samples with a low number of microbes cannot be isolated using this method.
 Surface drop method is the best enumeration method in this experiment as the
enumeration of colonies is faster since the sample appears in evenly distributed distinct
spots. It is also less time-consuming compared to the pour plate method and surface
spread plate method.

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