LABORATORY REPORT FOR MIC254

LAB 3: ENUMERATION OF E. coli FROM MEAT

PRODUCT

DATE OF EXPERIMENT: 4TH APRIL 2023

GROUP: AS1162F2
STUDENT NAME NO. MATRIC

NADIA FARHANA BINTI MOHD NAZRI 2022676118

AUFA HALIFAH BINTI MOHD HALIM 2022679844

FARAH HANANI BINTI MOHD JUHARI 2022462572

NURUL HANIM BINTI ZUL HAMIZI 2022809106

SHARIFAH AZIAH BINTI SYED RIDHUAN 2022492798

Objective
1. To determine the presence of E. coli from meat products.

Materials
Molten tryptic soy agar, Molten MacConkey agar, Autoclaved distilled water, sample of meat
products, 70% alcohol, petri dish, micropipette 1000 µL, micropipette tips 1000 µL, test-tube,
test-tube rack, stomacher bag

Introduction
The E. coli bacteria strain in the meat can cause bloody diarrhea and vomiting. A type of
kidney failure called hemolytic uremic syndrome can be caused in more severe cases.
Children under the age of 5, older adults, and those with weakened immunity are more likely
to develop the syndrome. Anyone who bruises quickly, has a pale colour that is alarming,
and urinates less frequently must receive emergency care urgently. To isolate and
enumerate E. coli from meat product, MacConkey Agar method and pour plate method can
be used.
MacConkey Agar (MCA) is a selective and differential medium for the separation and
differentiation of enterics dependent on their lactose fermentation ability. Meanwhile, tryptic
soy agar (TSA) method is usually the method of choice for counting the number of colony-
forming bacteria present in a liquid sample. However, meat is a solid sample, it cannot be
directly isolated to MacConkey Agar and tryptic agar. To turn the solid meat sample into
liquid sample, stomacher is used.

Methods
1. The aseptic technique been using to measure 25 g of the meat product and were put
into the stomacher bag.
2. 225 mL autoclaved distilled water was added into the stomacher bag. This
preparation produces a sample solution with a dilution factor 101 .
3. The sample was stomach for three minutes in stomacher blender.
4. Three sterile test tubes were prepared and were added with 4.5 mL autoclaved
distilled water. 1 mL of sample solution was insert into the first tube and mixed
homogeneously. The tube was label as T2 (dilution factor 102 ). The same procedure
were done for the two other test tubes and were label it as T3 and T4 (dilution factor
103 and 104 ), respectively.
Result
A) Enumeration of bacterial colonies from meat product inoculated using tryptic soy
agar.

Dilution Number of colonies

102 228
103 157
104 104

Number of bacteria in original sample [ (CxM)/V ] =

B) Enumeration of bacterial colonies from meat product inoculated using MacConkey

agar.

Dilution Number of colony

102 TFTC
103 TFTC
104 TFTC
Number of bacteria in original sample [ (CxM)/V ] = TFTC
Discussion
This experiment was made by two types of plate agar, which is tryptic soy agar and
MacConkey Agar to observe the isolation growth of E. coli and enumerate of bacterial
colonies in both sample of agar. After 24 hours, all the plates were observed, and
enumeration of bacteria have been done. Tryptic soy Agar supplemented with glucose was
used to count members of E. coli. We can see the E. coli was shown numerously on tryptic
soy agar and the number of coliforms per ml (CFU/ml) of the sample was calculated using
colony counter after 48 hours. The plates with dilutions 10 2 has the highest number of
colonies which 228 while the plates with dilutions 103 has 157 number of colonies. The plates
with dilutions 104 has the lowest number of colonies which is 104. Based on the results
shown we can observe and enumerate the growth of bacteria colonies which is E. coli on
tryptic agar soy because tryptic soy agar contains pancreatic digest of casein and papaic
digest of soybean meal provides nitrogen, vitamins, and minerals. This makes it suitable for
the growth of a wide variety of fastidious and non-fastidious microorganisms such as E. coli.
We used fresh meat to grow the bacteria because fresh meat is an ideal growth medium for
bacteria because it has high moisture contents, rich in proteins and contains fermentable
carbohydrates. Thus, it is truth that fresh meat sample have been processed in a correct way
to determine the numerous number of E.coli and a large number of colonies formed.

Next, for the MacConkey agar plate, 0.1mL sample of the diluents was pipetted into the petri
dish and pour approximately 15mL of MacConkey Agar. MacConkey Agar supplemented
with glucose was used to count members of Enterobacteriaceae. After 24 hours, after 48
hours, the number of coliforms per ml (CFU/ml) of the sample was calculated and it shows a
very low number of colonies and some did not have colonies. All the plates with dilutions 10 2,
103 and 104 doesn’t contain any number of colonies. we can’t observe and enumerate the
growth of bacteria colonies which is E. coli. This is because the number of colonies is Too
Few to Count (TFTC). Minced meat contains a low number of colonies because it is a
processing food while fresh meat becomes spoilt after a few hours or days due to
microorganism’s contamination like bacteria and fungi as fresh meat is an ideal growth
medium for bacteria because it has high moisture contents, rich in proteins and contains
fermentable carbohydrates. By this result, it can be found that the minced meat sample have
been processed in a correct way and the hygiene is well care thus a smaller number of
colonies formed.
Conclusion
In conclusion, the objective of the experiment were proven which to determine the presence
of E. coli from meat products. There are two ways to isolate E. coli from meat product which
are tryptic soy agar and MacConkey agar. Based on the results shown we can observe and
enumerate the growth of bacteria colonies which is E. coli on tryptic agar soy because tryptic
soy agar contains pancreatic digest of casein and papaic digest of soybean meal provides
nitrogen, vitamins, and minerals. This makes it suitable for the growth of a wide variety of
fastidious and non-fastidious microorganisms such as E. coli because Tryptic soy agar is
nonselective, meaning that it supports the growth of a broad range of bacteria while
MacConkey agar is a selective and differentiating agar that only grows gram-negative
bacterial species so that’s why we can’t observe and enumerate the growth of bacteria
colonies.

Discussion question
1. Discuss why MacConkey media is categorized as a differential and selective media.
Based on the ability to ferment lactose, different species will yield colonies in varying
appearance on a MacConkey medium. This gives McConkey agar its differentiating property.
MacConkey agar is a selective and differentiating agar that only grows gram-negative
bacterial species; it can further differentiate the gram-negative organisms based on their
lactose metabolism.

2. Predict the similarities and/or differences in the observations if the plate are
incubated at 30°C.
There is no different between the two plates. Similar result is expected. This is because
there is not much difference between the temperature of incubation in practical (37°C) and
the one stated here (30°C). Both temperatures still same as the room temperature and
normal temperature for the growing of bacteria.

3. Discuss the difference between result observed using tryptic soy agar and
MacConkey agar.
Tryptic soy agar is nonselective, meaning that it supports the growth of a broad range of
bacteria while MacConkey agar inhibits the growth of Gram-positive bacteria and is thus a
selective medium. Gram-negative bacteria growing on the media are differentiated by their
ability to ferment the sugar lactose. so, only fewer bacteria can grow in MacConkey agar.
References
1. Jung, B. (2022, September 26). MacConkey Medium. StatPearls - NCBI Bookshelf.
https://www.ncbi.nlm.nih.gov/books/NBK557394/#:~:text=Based%20on%20the
%20ability%20to,McConkey%20agar%20its%20differentiating%20property.
2. Loqman, B. (n.d.). Tryptic Soy Agar TSA | Principle | Preparation | Interpretation.
https://microbiologie-clinique.com/trypticase-soy-agar-principle-interpretation.html
3. MacConkey agar plates protocols - american society for microbiology. (n.d.).
Retrieved April 7, 2023, from
https://asm.org/ASM/media/Protocol-Images/MacConkey-Agar-Plates-Protocols.pdf?
ext=.pdf
4. Aryal, S. (2022, May 3). Tryptic Soy Agar- Composition, Principle, Preparation,
Results, Uses. Microbe Notes. https://microbenotes.com/tryptic-soy-agar/