LAB REPORT

ENVIRONMENTAL MICROBIOLOGY
(NNNE1053)

TITLE:
FOOD MICROBIOLOGY

GROUP MEMBERS:

NAME MATRIC NUMBER

Anis Sofea binti Rosley A180695

Nur Aisyah binti Ayob A180721

Siti Nur Aisyah Binti Hazidin A182354

Nurul Husna Ayuni binti Mohd Azali A181521

LECTURER’S NAME:
Dr. Siti Shahara Zulfakar
RESULT

i- Number of Colonies Per Plate (PCA)

Number of Colonies /Plate Average

Number of
Sample Dilution colonies for two
plates
R1 R2

10-1 (>300) TNTC (>300) TNTC (>300) TNTC

Food Contact 10-2 TNTC TNTC TNTC

Surfaces (cfu/cm²)
10-3 97 97 97

10-4 16 15 15.5

10-5 5 7 6

10-1 TNTC TNTC TNTC

Food Sample (Raw
meat or poultry) 10-2 TNTC TNTC TNTC
(cfu/g)
10-3 TNTC TNTC TNTC

10-4 TNTC TNTC TNTC

10-5 189 159 174

10-1 TNTC TNTC TNTC

Evh Soup (cfu/ml) 10-2 TNTC TNTC TNTC

10-3 TNTC TNTC TNTC

10-4 TNTC TNTC TNTC

10-5 266 204 235

10-6 86 97 91.5
CALCULATION

i. Food Contact surface

Original cell density for 10-3 (dilution factor) = 97 10

×
-3
0.1 ×10 375(cm2)

= 25866.67 CFU/cm2

Original cell density for 10-4 (dilution factor) = 15.5 10

×
-4
0.1 ×10 375(cm2)

= 41333.33 CFU/cm2

Original cell density for 10-5 (dilution factor) = 6 10

×
-5
0.1 ×10 375(cm2)

= 16 × 104 CFU/cm2

ii. Food Sample (Raw meat or poultry)

Original cell density for 10-5 (dilution factor) = 174 91

×
-5
0.1 ×10 91

= 174 × 106 CFU/g

ii. EvH soup

Original cell density for 10-5 (dilution factor) = 235

0.1 × 10-5

= 235 ×106 CFU/ml

Original cell density for 10-6 (dilution factor) = 91.5

0.1 × 10-6

= 915 ×106 CFU/ml

RESULT (OBSERVATION FOR SELECTIVE AGARS)

AGAR SAMPLE EOSIN MANNITOL SALT SALMONELLA

METHYLENE AGAR SHIGELLA AGAR
BLUE AGAR

FOOD SAMPLE Colourless colonies Yellow colonies NO GROWTH

surrounded by yellow
(Salmonella) zone

(Staphylococcus
aureus)

FOOD CONTACT Pink to purple Yellow colonies Colourless colonies

SURFACE colonies with no surrounded by yellow
metallic green sheen, zone (Salmonella)
colourless colonies
(Staphylococcus
(Klebsiella aureus)
pneumoniae &
Salmonella)

EVH SOUP Deep purple colony NO GROWTH NO GROWTH

with green metallic
sheen

(Escherichia coli)
DISCUSSION

Eosin Methylene Blue Agar (EMBA) is a selective stain for tests mainly on the presence
of gram-negative bacteria. The coloured colonies in EMB agar is lactose fermenter while
colourless colonies in EMB agar is non-lactose fermenter. Eosin and methylene blue combine
to form a precipitate at acid pH, thus also serving as indicator of acid production. The test on
the food sample produces colourless colonies which indicate the presence of Salmonella. The
test on the food contact surface produces pink to purple colonies with no metallic green sheen,
which indicates the presence of Klebsiella pneumoniae and colourless colonies which indicate
the presence of Salmonella. The test on EVH soup produces deep purple colonies with green
metallic green which indicates the presence of Escherichia coli.

Mannitol Salt Agar (MSA) is used to test the presence of Staphylococcus aureus. If an
organism can ferment mannitol, an acidic byproduct is formed which cause the phenol red agar
to turn yellow. Both tests on food sample and food contact surface produce yellow colonies
surrounded by yellow zones which indicate the presence of Staphylococcus aureus. In EVH
soup there is no growth of bacteria. It indicates there is no presence of Staphylococcus aureus.

Salmonella Shigella Agar (SSA) is a moderately selective and differential medium for
the isolation, cultivation and differentiation of Salmonella species and some Shigella species.
Differentiation between pathogenic species and coliform is achieved by the colour change of
the pH indicator (neutral red). Non-lactose fermenting species form colourless colonies. In this
experiment, the test on food contact surface produces colourless colonies which indicates the
presence of Salmonella. However, the test on food sample and EVH soup shows no growth of
bacteria. It indicates that there is no Salmonella species presence in the food sample and EVH
soup.
CONCLUSION

Food microbiology is the study of microorganisms that inhibit, create or can

contaminate food. It is also including the study of microorganisms that can cause food spoilage
and also about pathogens that may lead to disease especially if the food is not properly cooked.
In this experiment, we were using chicken, cutting board and EVH soup as our samples. For
the agar, we used Eosin Methylene Blue Agar (EMBA), Mannitol Salt Agar (MSA) and
Salmonella-Shigella agar (SSA).

Serial dilutions with dilution factor 10-1, 10-2,10-3,10-4,10-5 and, 10-6 (for EVH soup
only) is used to decrease the bacterial concentration in the samples and bacterial density of the
samples was determined by calculations using the average number of colonies for two plates.
Original cell density for food contact surfaces with dilution factor 10-3 is 25866.67 CFU/cm2,
for dilution factor 10-4 is 41333.33 CFU/cm2 and for dilution factor 10-5 is 16x104 CFU/cm2.
Original cell density for food sample with dilution factor 10-5 is 174x106 CFU/g. Lastly,
original cell density for EVH soup with dilution factor 10-5 is 235x106 CFU/ml and for dilution
factor 10-6 is 915x106 CFU/ml. Viable plate count was used to determine bacterial populations
quantitatively as it shows the number of bacterial colonies growing on an agar. Using this
method, the sample need to be diluted first so that the bacteria can be counted accurately.

In this experiment, For SS agar, the test on food contact surface produces colourless
colonies which indicates the presence of Salmonella. However, the test on food sample and
EVH soup shows no growth of bacteria. It indicates that there is no Salmonella species presence
in the food sample and EVH soup. For EMB agar, the test on the food sample produces
colourless colonies which indicate the presence of Salmonella. The test on the food contact
surface produces pink to purple colonies with no metallic green sheen, which indicates the
presence of Klebsiella pneumoniae and colourless colonies which indicate the presence of
Salmonella. The test on EVH soup produces deep purple colonies with green metallic green
which indicates the presence of Escherichia coli. Lastly, for MSA agar, both tests on food
sample and food contact surface produce yellow colonies surrounded by yellow zones which
indicate the presence of Staphylococcus aureus. In EVH soup there is no growth of bacteria
which indicates there is no presence of Staphylococcus aureus.
REVIEW QUESTIONS

1. Why is a standard plate count performed on food products and food contact surfaces?

The standard plate count is one way to determine bacterial populations quantitatively. It can
also indicate the number of bacterial colonies growing on a non-specific solid nutrient agar
(medium) after a given period of incubation. This count can sometimes be used to indicate the
microbial quality and spoilage level of food products and the hygiene of food contact surfaces.
This altogether could help the food that we consume to be less contaminated by pathogen that
can cause foodborne disease.

2. What is the purpose of using the set of differential and selective media in this experiment?
Explain the differences between each selective media used in this experiment.

Selective media for the most part chooses for the growth of a desired life form, stopping the
growth of or altogether murdering non-desired organisms. Differential bacteria helps to
distinguish colonies of different bacteria by observing the color changes in the medium and
color of the colonies.
i) Eosin Methylene Blue agar (EMBA) is a selective, differential agar medium used for
isolation of gram negative rods in a variety of specimen types.
ii) Mannitol Salt agar (MSA) is selective for Gram-positive bacteria (Staphylococcus) because
it contains high concentration of salt (NaCl).
iii) Salmonella Shigella agar (SS) is used as a selective and differential medium for isolation
of Salmonella species. It is used for the isolation, cultivation and differentiation of gram-
negative enteric microorganisms isolated from both clinical and non-clinical specimens such
as from feces, urine, and suspected food items (fresh and canned foods).

3. Why are 30-300 colonies used for the calculations?

A plate having 30-300 colonies is chosen because this range is considered statistically
significant. If there are less than 30 colonies on the plate, small errors in dilution technique or
the presence of a few contaminants will have a drastic effect on the final count. Furthermore,
in case there are more than 300 colonies, the microbes is too crowded and not allow it to create
distinct colonies.
4. Explain the limitations of plate count method in determining contamination of food products
and food contact surfaces.

The limitations of plate count methods should have between 30 and 300 colonies because this
range is considered statistically significant. If the colonies are fewer than 30 colonies, it is not
acceptable for statistical reasons since there are too few colonies which may not be
representative of the sample. The colonies that consist of more than 300 colonies on a plate are
likely to produce colonies that are too close to each other. It makes it harder to be distinguished
as distinct colony-forming units (CFU). Limitations of plate count methods are being used to
ensure we get the suitable amount of colonies to determine the contamination of food products
and food contact surfaces.

5. How does performing this experiment affect how you handle the food that you are serving
at home?

This experiment exposed us to the importance of high standards of sanitation, cleanliness and
the knowledge of good hand washing practices. Preparing food at home in a safe manner should
be practiced to stop the spreading of bacteria through cross contamination. For instance, do not
wipe hands on the clothing as this can easily transfer microbes and bacteria. Good
housekeeping also should be maintained at all times to make sure the environment is safe and
clean for the food preparation.
DO(S) AND DON’T(S)
DO(S)
● Sanitize all the surfaces on the weight
scale
● Make sure to swab the entire surface
on the cutting board
● Label all agar plates and test tube
● Use aseptic technique throughout the
entire lab procedure
● Spread the solution immediately after
adding it onto the agar surface
DON’T(S)
● Do not wear accessories such as
watch while conducting the
experiment
● Do not blow flame on the hockey
stick
● Do not remove any equipment or
chemicals from the laboratory
● Do not put the chicken sample
directly onto weight scale without
any container to avoid contamination
● Do not let the tip box left open while
doing spread plating as bacteria from
air might contaminate it