FOOD MICROBIOLOGY

YFS20101P

LAB REPORT 3: QUANTITATIVE ANALYSIS OF MILK BY

STANDARD PLATE COUNT
LAB REPORT 4: ENUMERATION OF BACTERIA IN MILK AND
PRESUMPTIVE TEST FOR COLIFORMS

STUDENT NAME MATRIC ID

012022070917
Dzulfahmi bin Zulkepli

Jiva Tharani A/P Asogan 012023020715

012022090465
Nivetha Sri A/P Krishna Rao

012022091873
Rishabh Bhardwaj

SEMESTER: FEBRUARY 2023

PROGRAMME: BACHELOR IN FOOD SERVICE & TECHNOLOGY
LECTURER: MADAM SASIMALANI
LAB REPORT 3: QUANTITATIVE ANALYSIS OF MILK BY STANDARD PLATE COUNT

INTRODUCTION

Quantitative analysis of milk by standard plate count is a commonly used method for
determining the amount of bacteria present in milk. In standard plate counting, a diluted milk
sample is placed on a solid culture medium and the bacteria grow under specific incubation
conditions.

The resulting colonies on the plate can be counted and used to calculate the number of bacteria
present in the original sample. This method is used to monitor the quality of drinking milk and to
detect harmful bacteria that can cause disease.

The standard bacterial count is a valuable tool for milk producers and for health authorities
regulating the sale and distribution of dairy products. It provides a quantitative assessment of
the total bacterial load in milk and can help identify potential sources of contamination or other
quality problems.

However, it is important to note that the standard plate count only measures the total number of
bacteria present in milk and does not distinguish between beneficial and harmful bacteria.
Therefore, it should be used in conjunction with other tests

OBJECTIVE

To analyze milk samples from microorganisms by standard plate count method.

MATERIALS

• Milk samples (A- pasteurized milk, B- unpasteurized milk)

• Sterile water
• Petri plates
• Pipettes
• Nutrient agar
• Incubator
• Colony counter
• Sticker
Completion time: 2 hours Complications: None

PROCEDURE

1. We procured raw milk samples and labeled them as A and B.

2. We diluted the milk samples in sterile distilled water for the isolation of microorganisms
from water.(Figure A)

3. We transferred 1 ml of milk serially in sterile distilled water to make dilution of 1:100,

1:10 000, and 1: 100 000.

4. We transferred 0.1ml and 1 ml of milk sample from each diluted sample bottle into two
separate Petri dishes, and poured sterilized nutrient agar medium into each plate.

5. After the nutrient agar was solidified, we incubated the plates at 35C for 24-48 hours.

6. As stated in serial dilution technique, we counted the plates containing 30-300 colonies
for calculating the number of organisms per ml of the original milk sample. We then
estimated the CFUs per ml of milk.
RESULTS

TOTAL COUNT PER 0.1 ML 1 ML

PLATE

TEST TUBE 10/1 A

0.1 ML:
The number of
colonies in this plate
was 21 hence the
cfu is labeled as
TFTC.
(too few to count)

1 ML:
The number of 36 colonies= Countable
21 colonies= TFTC
colonies in this plate
was 36 colonies.

36 x 101
1 ml
= 3.6/per ml cfu
TEST TUBE 10/5
A

0,1 ML:
The number of
colonies in the plate
was 42 colonies.

42 x 10 5
0.1

= 2.96/per 0,1 ml cfu 21 colonies= TFTC

42 colonies= countable

1 ML:
The number of
colonies on this plate
was 21, hence
declared TFTC.

TEST TUBE 10/1 B

0.1 ML:
The number of
colonies on this plate
was 92 colonies.

92 x 101
0.1 ml

= 2.90/per 0.1 ml cfu

92 colonies= countable

1 ML: 12 colonies= TFTC

The number of
colonies on this plate
was 12, hence
declared TFTC.

TEST TUBE 10/5

B

0,1 ML:
The number of
colonies on this plate
was 94 colonies.

94 x 105
0.1 ml

= 6.64/ per 0.1 ml 94 colonies= countable

cfu 75 colonies= countable

1 ML:
The number of
colonies on this plate
was 75 colonies.

75 x 105
1 ml

= 1.67/ per ml cfu

DISCUSSION
The hardest part of doing this experiment is measuring the correct amount of solutions and
also sterilising the agar plate. It was also very hard to count the number of colonies in the end
as it was uncountable sometimes and it was very small to even look at it. We had to recount it a
few times to get the average number of colonies from the plate. Extensive statistical analysis is
needed for the quantitative investigation, which might be challenging for researchers without
statistical training. As statistical analysis is based on scientific methodology, it is challenging for
non-mathematicians to complete.

The quantitative analysis quantifies functional information that is classified as quantitative data.
Metrics based on facts and numbers, such as statistics, formulas, and percentages, are used in
quantitative models. The quantitative examination of one of your goods' sales income is an
example. So since we were using the whole dilution instead of just what we used on the plate,
the calculation will be 10 ml of solution instead of the 10/5.

About the plate count in the petri dish, pasteurized milk has more colonies compared to
unpasteurized milk in the results obtained by our group. For A the colonies can be counted but
for b we used the light and took a very long time to count the colonies. We had to count many
times to get the accurate colonies. This is based on the results we obtain but we can come to
the conclusion where unpasteurized milk does not contain colonies. Unpasteurized has more
colonies that cannot be seen and counted due to the one thin layer of the agar. So this is
uncountable colonies which make it TNTC.

So to avoid contamination, we should handle the agar with the proper hygiene and also
cleanliness. We should keep the agar near the fire and also not open the agar plate before
using it. We should also use alcohol to clean up the table first before starting the experiment.
Besides that we should also wash all the test tubes of other materials before using it to avoid
other contamination .
QUESTIONS

1. What do you understand by standard plate count?

SPC which is known as ‘Standard Plate Count’ is a procedure that has been used in
microbiology to estimate the viable microorganisms in a specimen. The plate count requires the
number of colonies in order to determine the level of contamination. There are two special
cases called TNTC and TFTC. Besides, TNTC is Too Numerous To Calculate whereas TFTC is
Too Few To Calculate. TNTC considered as infinite colonies as the formation of colonies are
tremendously more than 300. On the other hand, if the formation of colonies are fewer than 30
colonies it is considered as TFTC.

2. Indicate some possible ways in which foods may become contaminated with enteric
organisms.

In the scenario of horticulture, if human sewage is utilized to fertilize the soil or sewage water is
used to irrigate the crops, enteric pathogen contamination of food may result from the farm.
Such dangers are heightened if the food is handled carelessly during processing and
preparation, when germs may grow rapidly under the right circumstances.

CONCLUSION

In a nutshell, the colony formation of two different samples which are pasteurised and
unpasteurised milk samples were done throughout this practical. The microbial contamination
rate for both samples were observed and calculated the CFU rate accordingly.

REFERENCES
REFLECTION

1. Rishabh- During the time spent doing the experiment and the report, I have learned
about the various factors that could affect a colony count on a plate and the methods
used for calculating serially diluted samples as well as pure samples.
2. Nivetha- during the experiment, I learned how to use the correct way to sterilize nutrient
agar medium into each plate.i also learn on how to do the experiment with having less
contamination with the surroundings. I face some difficulties counting the colonies but at
last I manage to do it . I learned how to be patient during that process. Overall, I had fun
during the class and learned a few theories during the lab section.
3. Fahmi - For this lab session, i learned about new things, such as learn how to use
pipettes in correct way, how to take a sample and many more. I also learn how to
properly make a diluution of a sample. It was fun and thoughtful experience for me. I also
manage to lear how to count a cfu using the correct formula.
4. Jiva Tharani - Throughout this practical, I’ve understood that aseptic technique plays a
vital role in order to obtain ideal results. Other than that, proper handling of specimens
and apparatus should be taken into account to avoid any cross contaminations during
the practical. However, the volume of the specimen to be tested also should be fairly
adequate and free from any contaminations. This is because, a small mistake in sample
or apparatus handling can lead a huge differences in the results.
LAB REPORT 4: ENUMERATION OF BACTERIA IN MILK AND PRESUMPTIVE TEST FOR
COLIFORMS

INTRODUCTION

Coliforms, which are frequently used to check the quality of milk, are not just one kind of
microbe. These are a group of Gram-negative, rod-shaped bacteria with comparable
biochemical properties, including the ability to ferment lactose within 48 hours at 35C while
producing acid and gas, and to grow with or without oxygen. These are typically not in great
abundance in raw milk. Coliform count can be used as a hygienic indicator to reflect the general
microbiological quality in regular tests because it is straightforward and simple to perform.
Coliforms are easily destroyed by heat, making them useful as a heat treatment failure and
post-heat treatment contamination indicator. The variety and quantity of bacteria in milk affect its
quality. However, because milk can become contaminated at any point in the production
process, care must be taken to use sanitary procedures. A total bacterial count and coliform
testing can be used to determine the quality of milk.

OBJECTIVE

To count the number of bacteria in milk and the presumptive test for coliforms.

PROCEDURE
1.procure raw milk samples and labled as A and B.
2. The milk was diluted in distilled water as illustrated in the figure (a) as the isolation of the
microorganisms from water.
3.1ml of milk serially transferred in sterile distilled water to make dilution of 1:10 ,1:100 and
1:1000.
4.1ml of milk samples were transferred from each diluted sample bottle into the petri dish, and
then it was poured, melted, and cooled tryptone glucose yeast extracted the agar medium into
each plate.
5. The plates were incubated at 35 C for 24-48 hours after the agar had solidified.
6.CFU per ml of milk we counted.
7.then 1ml from the highest dilution we transferred into the MacConkey broth tube for production
of acid and gas.
8.MacConkey were transferred into tubes for production of acid and gas.
9.A single Durham tube was placed in an inverted position inside the broth containing tube
answers incubated for 24 hours.
10 The observations were recorded with the presence of acid and gas production.

RESULTS

Labeling Result Calculation

pasteurized milk in agar The number of colony is

plate too few to count hence the
exact number of colony
cannot be find therefore
cannot do the cfu
calculation.

unpasteurized milk in agar The number of colony is

plate too numerous to count
hence the exact number of
colony cannot be find
therefore cannot do the cfu
calculation.
pasteurized milk in
macconkey

pasteurized milk in
macconkey
DISCUSSION

In this particular test, the contamination rate of pasteurised milk is compared to that of
unpasteurized milk. Milk that has been pasteurised generally has a reduced risk of
contamination than milk that has not been pasteurised. It is because of the heat treatment that
has been applied to the milk that the bacteria in the milk will finally be killed. By the end of the
experiment, the agar plate that had been inoculated with unpasteurized milk tended to produce
an excessive number of colonies, while the agar plate that had been inoculated with pasteurised
milk did not produce any colonies. This demonstrates that the test results match up with what
might be expected from a perfect enumeration method.

On the other hand, presumptive tests for coliforms are essentially performed to find the
presence of faecal Coliforms like E. coli, Klebsiella, Enterobacter, and others. It is possible to
define faecal coliforms as faecal oral contaminated bacteria. Although coliforms are almost
always present in raw milk, the number of coliforms can be kept to an extremely low level with
good production practices. These organisms can be present in milk and milk products if they
were produced in an unhygienic environment or handled improperly.

The transformation of the broth's colour from violet to yellow is seen to be a potential
good outcome of this practical. It shows the presence of coliforms in the sample which can be
also explained as faecal oral contaminated sample. Because of the fermentation of lactose,
which results in the production of acid and gas, the colour of the broth changes. This will cause
the broth to turn acidic. Observing the Durham tube floating with some bubble development in it
is another way to assess the gas production. While the negative results would be without any
changes in the broth colour and to the Durham tube in the broth remains at the bottom of the
tube. Comparatively, the results that have been obtained from the practical were negative for
pasteurised milk sample and positive for unpasteurised milk sample. Moreover, this result
defines that the pasteurised milk sample is free from the Coliform contaminations while the
unpasteurised milk sample consists of Coliforms bacteria.
QUESTIONS

1. Explain why it is not advisable to thaw and then refreeze food products without having
cooked them.

It is not advisable to thaw and refreeze food without cooking it because when food is frozen, it
prevents the growth of bacteria and other microorganisms that can cause spoilage or disease.
However, when the food is thawed, these microorganisms can grow again. Re-freezing these
foods does not kill these bacteria, but only creates the conditions for their growth.

In addition, each time food is frozen and thawed, its texture and taste change and its quality
decreases. When refreezing, the texture may change further, and the food may become dry or
develop a bad taste.

Therefore, it is highly recommended to cook the food after thawing and eat it immediately
instead of refreezing it. In this way, you can ensure the safety and quality of the food you
consume.

2. Would detection of E.coli in meat be indicative of contamination or spoilage of the

product? Explain.

Detection of E. coli in meat would indicate contamination rather than spoilage of the product. E.
coli is a bacterial species commonly found in the digestive tracts of animals and humans and
may be present on the surface of meat products. E. coli can cause foodborne illness and can be
harmful to humans, especially those with weakened immune systems.

Spoilage of meat can also cause illness when consumed, but is usually caused by other
bacteria that cause a change in the taste, texture, and odor of the meat. The presence of E. coli
in meat is more likely to indicate a contamination problem, such as improper handling or
processing of the meat. To prevent foodborne illness, it is important to ensure that food is
handled and cooked properly.
CONCLUSION
A color shift is brought on by the presence of an acid product. Mac Conkey broth agar
changes color from purple-violet to yellow. There is no petrol in existence. Hence, the test for
coliforms is presumed to be negative.Multiple tube test for the alleged coliform count. The test is
referred to as presumptive because the reaction seen during it occasionally may be caused by
the presence of another organism, and because it is important to confirm the hypothesis that the
reaction is caused by a coliform bacteria.A positive total coliform sample should be taken as a
sign that your well is contaminated even though total coliforms can come from sources other
than feces. Positive fecal coliform readings, particularly positive E. Coli results should be taken
as a sign that your well is contaminated with faeces.Coliforms are measured for the aim of
assessing the efficacy of water treatment and the structural integrity of the distribution network.
The RTCR's main provisions are: In order to safeguard against potential fecal contamination,
maximum contaminant level goals (MCLG) and maximum contaminant levels (MCL) for E. coli
were established.Coliform bacteria are characterized as facultatively anaerobic, Gram-negative,
non-spore-forming rods that vigorously ferment lactose to acid and gas at 35 2 °C within 24 or
48 hours.The test's core tenet is that if lactose is present along with bile salts and basic dyes,
members of this group can produce acid and gas. Coliform contamination is indicated when
typical coliform colonies are found in Petri plates.
REFERENCES

Download citation of An Assessment of In-situ Water Quality Parameters and its

variation with Landsat 8 Level 1 Surface Reflectance datasets. (n.d.).

ResearchGate.

https://www.researchgate.net/publication/353549142_An_Assessment_of_In-situ

_Water_Quality_Parameters_and_its_variation_with_Landsat_8_Level_1_Surfac

e_Reflectance_datasets/citation/download

Experiment 22A MTFM | Lab22 | Virtual Edge | Molb 2021 | College of Agriculture and
Natural Sciences. (n.d.).
http://www.uwyo.edu/molb2021/virtual-edge/lab22/exp_22a_mtfm.htm

Lesson 5 Water Testing. (n.d.). Www.youtube.com. Retrieved March 27, 2023, from
https://www.youtube.com/watch?v=y0NdZRC3uTQ

‌
REFLECTION

1.Nivetha= for this experiment, i learned a few things such as measuring in the correct way and
also observing the colonies plus the color change from purple to yellow. I also learned the way
to pasteurize properly with less contamination. Even though it was challenging, I had so much
fun while conducting that activity.

2. Fahmi = For this lab session I have a great experience because all of the experiments are
done as expected. All the results that we got were within our expectations. It is because we do it
in the proper ways to the experiment. All was thoughtful and meaningful to us as a BFSTt
student.

3. Jiva Tharani - In the bacterial enumeration practical session, I’ve learnt that the usage of ‘L
stick’ should be very gentle while spreading the sample all around the plate. This is because the
agar tends to tear with vigorous handling of specimens or apparatus. Meanwhile, as for the
presumptive test for Coliforms the Durham must be used wisely and carried carefully as it is a
tiny tube it has the higher fragility. Lastly, labeling also is very important while conducting a
science practical to avoid any confusions of the samples to be tested.

4. Rishab - I was unable to attend this particular lab session, though according to my friends
they found this practical to be fun and easier to conduct compared to the previous lab session.
This is because they had understood the flow of the previous procedures well. Besides, they
had also understood the importance of meniscus usage in order to measure the solution levels.
This helps to fill in the adequate amount of solutions or specimens needed.