Introduction

Aerobic plate count (APC), also known as standard plate count or the total viable
count is one of the recommended tests applied to identify the presence of microbes in
the food. Some food industry applies APC to ensure the quality of food is suitable for
human consumption.

APC measures only that microbial cell able to grow to visible and separate colonies.
Standardized test condition is provided for APC which is the sample used must
incubated for a sufficient period of time in an aerobic atmosphere and at a suitable
temperature. Besides, the sample microbial cell for APC is present in an analytical
unit which the colonies are mixed with agar containing appropriate nutrients. This
analytical unit can be referred as colony forming units (CFU). In fact, CFU is more
preferable by Microbiologist or Scientist instead of total bacteria counts in a sample.
This is because the dead bacteria and the bacteria which cannot replicate under
conditions being tested can be ignore.

Futhermore, a new method, Petrifilm Aerobic Count is also used by food industry
nowsaday. Petrifilm plates are thin film, sample-ready, dehydtated versions of
conventional Petri agar plate. To improve its visibility, there is a special medium on
the film. The medium is containing a colourless tetrazolium salt which bacteria use as
an electron acceptor reducing the compound to a coloured product, tetrazolium
formazan. Hence, Petrifilm are ready to use immediately after taking out of their
packet.

Objective
 1. To learn how to prepare and dilute a food sample properly.
2. To practice how to pour agar and mix sample correctly and use the Petxrifilm
correctly.
3. Be able to count colonies according to standard rules of counting.
4. To apply the standard rules of counting to various conditions.

Method

Pour plate method

1. All data from the product was recorded.

1 2
2. 11g of milk was diluted to consecutive dilution which are 10 , 10 and

103 .

3. The dilution bottles with the appropriate dilution was obtained and labeled.

4. Two Petri plates for each assigned dilution were obtained and each of the
duplicate plates was marked with appropriate dilution.

5. Sterile weighing paper, or aluminum foil, or use the dilution blank or blender
directly was obtained. The weight was tarred on the balance.

6. The indicated amount of the assigned sample was weighed aseptically and
placed on the balance or in the blender or dilution blank with a flame spatula.

7. A serial dilution was prepared according to the scheme. Pipets was get one at a
time only when it was needed. Never put pipets on the bench top. It can only in the
original container or in hand. The aseptic technique was applied. Each dilution blank
was shaken.

8. When the dilution series has been made, each dilution was agitated into each
of the appropriately labeled Petri dishes with the beginning at the highest dilution.
The last drop was expelled with a pipet aid. If the excess is replaced in the appropriate
blank and the pipet rinsed with the next lower dilution, only a single pipet is required
if plating is begun at the highest dilution.
9. 12 to 15ml of plate count agar was cooled to 44C-46C and poured into each
plate within 15 minutes of making original dilution.

10. The sample was mixed immediately and the agar medium by rotating each
plate on a flat surface first in one direction, then the other in a gentle.

11. The agar was allowed to solidify and the Petri dishes was inverted and
incubated at 35C for 482 hours.

Petrifilm method

1. All data was recorded from the product.

1 2
2. 11g of milk was diluted to consecutive dilution which are 10 , 10

3
and 10 .
3. The dilution bottles with the appropriate dilution was obtained and labeled.
4. Two Petrifilm plates for each assigned dilution were obtained and each of the
duplicate plates was marked with the appropriate dilution. The plates were laid
and the dilution blanks out on the bench top in a pattern corresponding to the
scheme.
5. Sterile weighing paper was obtained or aluminum foils, or use the dilution
blankor blender directly.
6. The indicated amount of the assigned sample was weighed aseptically, and
placed on the balance or in the blender or dilution blank with a flamed spatula.
a. The sample was blended under a hood to avoid airborne
contamination, or
b. The dilution blank was shaking if added directly.
7. Serial dilutions were prepared according to the scheme. Pipets was get one at a
time only when it was needed. Never put pipets on the bench top. It can only
in the original container or in hand. The aseptic technique was applied. Each
dilution blank was shaken.

8. When the dilution series has been made, each dilution was agitated into each
of the appropriately labeled Petri dishes with the beginning at the highest
dilution. The last drop was expelled with a pipet aid. If the excess is replaced
in the appropriate blank and the pipet rinsed with the next lower dilution, only
a single pipet is required if plating is begun at the highest dilution.
 9. 12 to 15 ml of plate count agar (PCA) was cooled to 44C-46C and poured
into each plate within 15 minutes of making the original dilution.

10. The sample and the agar medium were mixed immediately by rotating each
plate on a flat surface first in one direction, then the other in a gentle.

11. The agar was allowed to solidify, inverted the Petri dishes and incubated it at
35C for 482 hours (or 5 days at room temperature).

Results and Observations:

(i) Pour plate method

Figure 1: Aerobic pour plates with dilution factor of 10-4.

Figure 2: Aerobic pour plates with dilution factor of 10-5.

Figure 3: Aerobic pour plates with dilution factor of 10-6.

Table 1.0: Aerobic pour plates with different dilution factors

Dilution Petri Dish 1 Petri Dish 2 Average count Count per unit
Factor volume / weight
10-4 412 520 466 TNTC

10-5 111 101 106 1.06 x 107 cfu/ml

10-6 9 13 11 TFTC

Based on the table 1.0, the colonies obtained from the dilution factor of 10-4 and 10-6
are 466 and 11 respectively, which are out from the range of 25-250 colonies. The
dilution factor of 10-5 contains 106 colonies, which is within the range of 25-250
colonies.

Count = 106
10-5 x 1.0
= 1.06 x 107 cfu/ml

(ii) RIDAfilm method

Figure 5: Aerobic RIDAfilms from left are dilution factors of 10-4, 10-5, 10-6.

Table 2.0: Aerobic RIDAfilms with different dilution factors

Food Dilution factor Average count Count per unit

volume / weight
-4
Milk 10 1912 TNTC
10-5 198 1.98 x 107 cfu/ml
10-6 28 2.8 x 107 cfu/ml

From table 2.0, only 10-4 dilution yields 1912 colonies which are out the range of 25
to 250 colonies. 10-5 and 10-6 dilutions yield 198 and 28 colonies respectively.

Count = 198
10-5 x 1.0
= 1.98 x 107 cfu/ml

Count = 28
10-6 x 1.0
= 2.8 x 107 cfu/ml

Discussion:

Pour plate method was a method used to detect the presence of viable
bacterium and amplify itself to form visible colonies. In pour plate method, sample
(usually 1ml) was pipetted into the petri dish and mixed with the appropriate molten
agar (Adams 2008). Pour plate method allowed the growth of spoil milk bacteria
which was facultative anaerobe.

Based on the experiment, table 1.0 showed that the bacteria from the spoil
milk were higher in aerobic condition. This was because in the absence of anoxic jar,
anaerobes bacteria were killed when exposed to oxygen as it must strictly grow under
the absence of oxygen. Thus, pour plate method favoured the growth of aerobes
bacteria. However, some of the obligate aerobes might grow poorly if it was deeply
embedded in the agar (David 2015). From the results, there was 10600000 variable
cells/ml in the dilution factor of 10-5, while the colonies obtained from the dilution
factor of 10-4 and 10-6 were 466 and 11 respectively, which were out from the range of
25-250 colonies. This was because in the dilution factor of 10 -5 contained more
microorganism, this was a condition of overcrowded happening. So, less visible
colonies could be grown on the agar plate due to the competition among each other
(Academia.edu 2016). To overcome the overcrowded problem, a ten-fold serial
dilution was needed. However, we still could observe that there were colonies in the
dilution factor of 10-4 and 10-6, but both were not suitable to be used to calculate the
bacteria counts. This was because the concentration of bacteria which was within the
range of 25-250 colonies only statistically suitable.

Since the warm molten agar was poured onto the bacterial suspension, its
temperature could damage or kill the heat-sensitive bacteria. As a result, no visible
colonies could be found. For the aerobes bacteria also would be trapped inside the
agar, leading to fail to survive as no oxygen could diffuse into the agar. Hence, pour
plate method was considered as a selective technique. To avoid the damaging or
killing of heat-sensitive bacteria, the agar should be leaved to become cooler but still
in molten state before pouring onto the bacterial suspension (Academia.edu 2016).

RIDAfilm method was a convenience, simple and effective method used to

detect the presence of bacteria compare with other plating method such as pour plate
method (Martin 2012). A fluid food product or a dilution of a solid food was placed to
the dry culture medium overlaid with polyethylene film coated with water soluble
gelling agent. RIDAfilm method was not statistically different from pour plate method
for the enumeration of organisms in spoil milk. Based on the experiment, the colonies
count for dilution factor of 10-5 by using RIDAfilm method was 1.98 x 107 cfu/ml,
which is higher than the count using pour plate method which yielded 1.06 x 10 7
cfu/ml. The difference of the value might due to experimental error. Thermal shock to
the psychrotrophs might occur when the agar was poured into the petri dish in a hot
condition. Thus, the colonies counts were affected.

RIDAfilm method was easier to identify the presence of bacteria as red dye is
used and the available of grid make counting of bacteria easier. For pour plate
method, if the colonies grow was too crowded and was likely to result in many
colony-forming colonies which eventually lead to underestimate of counting the
presence of the bacteria (Martin 2012).

Questions

1. Pipetting:
a. Possibility of an error in my reading the volume accurately or in the
volume itself.
b. Tip damaged or broken
c. No sterile.
2. Sample representation
a. Amount of the sample does not large enough
3. Sample weighing
a. Doesnt blank the weighing scale when putting on the sterile weighing
paper before adding the sample.
4. Dilution bottle
a. Human error in calculation of dilution factors.
b. Inaccurate pipetting.
5. Counting plates
a. Miscounting colonies.
b. Distinguish wrongly between food particles and bacteria

Conclusion

Throughout the experiments, when working with media and reagents used to
culture microorganisms, aseptic technique must be practiced to ensure
contamination is minimized. To getting isolated colonies when you inoculate,
serial dilution is needed. Beside, aerobic plate count relies on bacteria growing a
colony on a nutrient medium so that the colony becomes visible to the naked eye
 and the number of colonies on a plate can be counted. We must follow the
standard rules when counting the colonies to avoid sampling error. APN give a
good prediction to how long the food will take to spoil.

References:

Academia.edu., 2016. LAB REPORT OF MICROBIOLOGY [Online]. Available

from:
http://www.academia.edu/8826259/LAB_REPORT_OF_MICROBIOLOGY
[Accessed 6 June 2016].

Adams. M., 2008. Food Microbiology, 3rd ed.

David, B.F., 2015. Pour Plate Technique [Online]. Available from:

http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Meat_Milk/Pour_Pla
e.htm [Accessed 6 June 2016].

Martin, A., 2012. Fisheries Processing: Biotechnological applications.