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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

It can only in the original container or in hand. To learn how to prepare and dilute a food sample properly. The dilution bottles with the appropriate dilution was obtained and labeled. Never put pipets on the bench top. The last drop was expelled with a pipet aid. 10 and 10−3 . Pipets was get one at a time only when it was needed. . Two Petri plates for each assigned dilution were obtained and each of the duplicate plates was marked with appropriate dilution. Sterile weighing paper. A serial dilution was prepared according to the scheme. To apply the standard rules of counting to various conditions. 2. The aseptic technique was applied. 3. or use the dilution blank or blender directly was obtained. 7. Method Pour plate method 1. 3. each dilution was agitated into each of the appropriately labeled Petri dishes with the beginning at the highest dilution. only a single pipet is required if plating is begun at the highest dilution. 4. −1 −2 2. Each dilution blank was shaken. All data from the product was recorded. 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. 6. When the dilution series has been made. 11g of milk was diluted to consecutive dilution which are 10 . 5. Be able to count colonies according to standard rules of counting. 4. To practice how to pour agar and mix sample correctly and use the Petxrifilm correctly. 1. The weight was tarred on the balance. or aluminum foil. 8. If the excess is replaced in the appropriate blank and the pipet rinsed with the next lower dilution.

or b. The indicated amount of the assigned sample was weighed aseptically. 5. each dilution was agitated into each of the appropriately labeled Petri dishes with the beginning at the highest dilution. Never put pipets on the bench top. When the dilution series has been made. The sample was mixed immediately and the agar medium by rotating each plate on a flat surface first in one direction. The dilution bottles with the appropriate dilution was obtained and labeled. Petrifilm method 1. It can only in the original container or in hand. 11g of milk was diluted to consecutive dilution which are 10 . 7. The last drop was expelled with a pipet aid. The sample was blended under a hood to avoid airborne contamination. and placed on the balance or in the blender or dilution blank with a flamed spatula. The dilution blank was shaking if added directly. Sterile weighing paper was obtained or aluminum foils. 3. a. 6. The plates were laid and the dilution blanks out on the bench top in a pattern corresponding to the scheme. Pipets was get one at a time only when it was needed. then the other in a gentle. 4. Serial dilutions were prepared according to the scheme. . If the excess is replaced in the appropriate blank and the pipet rinsed with the next lower dilution. The aseptic technique was applied. only a single pipet is required if plating is begun at the highest dilution. 10 −3 and 10 . All data was recorded from the product. 11. 8. 12 to 15ml of plate count agar was cooled to 44°C-46°C and poured into each plate within 15 minutes of making original dilution. or use the dilution blankor blender directly. The agar was allowed to solidify and the Petri dishes was inverted and incubated at 35°C for 48±2 hours.9. Two Petrifilm plates for each assigned dilution were obtained and each of the duplicate plates was marked with the appropriate dilution. Each dilution blank was shaken. 10. −1 −2 2.

The agar was allowed to solidify. 12 to 15 ml of plate count agar (PCA) was cooled to 44°C-46°C and poured into each plate within 15 minutes of making the original dilution. . inverted the Petri dishes and incubated it at 35°C for 48±2 hours (or 5 days at room temperature). The sample and the agar medium were mixed immediately by rotating each plate on a flat surface first in one direction. 9. 11. 10. then the other in a gentle. Figure 2: Aerobic pour plates with dilution factor of 10-5. Results and Observations: (i) Pour plate method Figure 1: Aerobic pour plates with dilution factor of 10-4.

0. Count = 106 10-5 x 1.06 x 107 cfu/ml (ii) RIDAfilm method . 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.0 = 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. Table 1. The dilution factor of 10-5 contains 106 colonies.Figure 3: Aerobic pour plates with dilution factor of 10-6. which is within the range of 25-250 colonies.

98 x 107 cfu/ml Count = 28 10-6 x 1.0 = 1.Figure 5: Aerobic RIDAfilms from left are dilution factors of 10-4.0: 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. In pour plate method.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. sample .98 x 107 cfu/ml 10-6 28 2. Table 2. only 10-4 dilution yields 1912 colonies which are out the range of 25 to 250 colonies. 10-6.8 x 107 cfu/ml From table 2. Count = 198 10-5 x 1. 10-5 and 10-6 dilutions yield 198 and 28 colonies respectively. 10-5.0 = 2.0.

Hence. This was because in the absence of anoxic jar. pour plate method favoured the growth of aerobes bacteria. As a result. less visible colonies could be grown on the agar plate due to the competition among each other (Academia. This was because the concentration of bacteria which was within the range of 25-250 colonies only statistically suitable. a ten-fold serial dilution was needed. table 1. we still could observe that there were colonies in the dilution factor of 10-4 and 10-6. leading to fail to survive as no oxygen could diffuse into the agar. This was because in the dilution factor of 10 -5 contained more microorganism. Pour plate method allowed the growth of spoil milk bacteria which was facultative anaerobe. the agar should be leaved to become cooler but still in molten state before pouring onto the bacterial suspension (Academia. while the colonies obtained from the dilution factor of 10-4 and 10-6 were 466 and 11 respectively. For the aerobes bacteria also would be trapped inside the agar. simple and effective method used to detect the presence of bacteria compare with other plating method such as pour plate method (Martin 2012). To overcome the overcrowded problem. the colonies . RIDAfilm method was not statistically different from pour plate method for the enumeration of organisms in spoil milk. Since the warm molten agar was poured onto the bacterial suspension. but both were not suitable to be used to calculate the bacteria counts. From the results. To avoid the damaging or killing of heat-sensitive bacteria. this was a condition of overcrowded happening. Thus. Based on the experiment. there was 10600000 variable cells/ml in the dilution factor of 10-5. no visible colonies could be found. However. 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. some of the obligate aerobes might grow poorly if it was deeply embedded in the agar (David 2015). RIDAfilm method was a convenience. its temperature could damage or kill the heat-sensitive bacteria. which were out from the range of 25-250 colonies.(usually 1ml) was pipetted into the petri dish and mixed with the appropriate molten agar (Adams 2008).0 showed that the bacteria from the spoil milk were higher in aerobic condition. Based on the experiment. However. So.edu 2016). pour plate method was considered as a selective technique. anaerobes bacteria were killed when exposed to oxygen as it must strictly grow under the absence of oxygen.edu 2016).

Doesn’t blank the weighing scale when putting on the sterile weighing paper before adding the sample. Distinguish wrongly between food particles and bacteria Conclusion Throughout the experiments. Tip damaged or broken c. aseptic technique must be practiced to ensure contamination is minimized. The difference of the value might due to experimental error. 4. 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). the colonies counts were affected. b. serial dilution is needed.count for dilution factor of 10-5 by using RIDAfilm method was 1. Sample representation a. which is higher than the count using pour plate method which yielded 1. Pipetting: a. Thus. 2. Human error in calculation of dilution factors. Questions 1. b. For pour plate method.06 x 10 7 cfu/ml. Counting plates a. 5. To getting isolated colonies when you inoculate. Beside. Dilution bottle a. when working with media and reagents used to culture microorganisms. b. Thermal shock to the psychrotrophs might occur when the agar was poured into the petri dish in a hot condition. Miscounting colonies. Amount of the sample does not large enough 3. Inaccurate pipetting. Possibility of an error in my reading the volume accurately or in the volume itself. Sample weighing a. 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. aerobic plate count relies on bacteria growing a colony on a nutrient medium so that the colony becomes visible to the naked eye .98 x 107 cfu/ml. No sterile.

. Adams. Fisheries Processing: Biotechnological applications. 2016.clc. Available from: http://www. Food Microbiology.htm [Accessed 6 June 2016].. 2015. and the number of colonies on a plate can be counted. B. References: Academia. Available from: http://biology.F.edu/fankhauser/Labs/Microbiology/Meat_Milk/Pour_Pla e. Pour Plate Technique [Online]. A. We must follow the standard rules when counting the colonies to avoid sampling error. Martin. 2012. M. 3rd ed.edu. 2008...edu/8826259/LAB_REPORT_OF_MICROBIOLOGY [Accessed 6 June 2016]. APN give a good prediction to how long the food will take to spoil. .academia. LAB REPORT OF MICROBIOLOGY [Online]. David.uc.