MIC254 Laboratory Report

NAME OF GROUP MEMBERS:

1. ANISYA SAFFIA BINTI ZORHADI (2022477018)

2. SITI AINATASHA BINTI SHA’ARI (2022698778)
3. SORFINA MIRZA BINTI ZAFIZAL (2022841064)
4. SAFFIYAH ATIQAH BINTI SARMADI (2022696148)
5. ANIS NATASHA SHAHROM BINTI SHAHRIN
(2022894238)

NAME OF EXPERIMENT: ENUMERATION OF E. coli

FROM MEAT PRODUCT
DATE OF LAB: 5/4/2023
LECTURER’S NAME: DR. HIDAYAH
INTRODUCTION:

The epidemiology of food-borne diseases has changed widely in the last decades. New
pathogens have emerged and spread widely, Escherichia coli 0157:H7 is an example. E. coli
is widely distributed in the intestine of humans and warm-blooded animals and is the
predominant facultative anaerobe in the bowel and part of the essential intestinal flora that
maintains the physiology of the healthy host. E. coli is a member of the family
Enterobacteriaceae, which includes many genera, including known pathogens such as
Salmonella, Shigella, and Yersinia. Although most strains of E. coli are not regarded as
pathogens, they can be opportunistic pathogens that cause infections in immunocompromised
hosts. There are also pathogenic strains of E. coli that when ingested, causes gastrointestinal
illness in healthy humans.

In this laboratory experiment, we will be looking at the microflora of meat product. The most
common method of measuring viable bacterial cell numbers is the standard or viable plate
count or colony count. Using this method, a small volume of liquid containing an unknown
number of bacteria is spread over the surface of an agar plate, creating a "spread plate." The
spread plates are incubated for 48 hours. During that time, each individual viable bacterial
cell multiplies to form a readily visible colony. The number of colonies is then counted, and
this number should equal the number of viable bacterial cells in the original volume of
sample, which was applied to the plate.
OBJECTIVE:

 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.

METHODS:

1) Using aseptic technique, 25 g of the meat product was measured into stomacher bag.
2) 225 ml autoclaved distilled water was added into the stomacher bag. This preparation
produced a sample solution with a dilution factor 10^1.
3) The stomacher bag was stomached for three minutes.
4) Three sterile test tubes added with 4.5 ml autoclaved distilled water were prepared.
0.5 ml of sample solution was inserted into the first tube and mixed homogeneously.
The tube was labelled as T2 (dilution factor 10²). The same procedure was done for
the two test tubes and labelled as T3 and T4 (dilution factor 103 and 104), respectively.
5) 100 μL of the homogenized mixture was inoculated (from tubes with dilution factor
10², 103 and 104) into an empty petri dish and molten tryptic soy agar was added. The
plate was swirled gently to let the combinations mix homogenously and the agar was
let solidify.
6) Step 5 was repeated with molten MacConkey agar.
 7) The agar plate was incubated and inverted at 37℃ for 48 h in aerobic condition.
8) The colonies were counted on both types of agar (dark red colonies on MacConkey
agar) and the CFU/ml isolates of the sample were counted.

RESULTS:
A) Enumeration of bacterial colonies from meat product inoculated using tryptic
soy agar.
Dilution Number of colonies

10² 241

103 130

104 41

Number of bacteria in original sample (use the formula C x M)/V) =

C = 241
M = 10²
V = 0.1 ml
Colony forming unit/ml = (C X M)/V)
= (241 x 10²) / 0.1
= 2.41 x 10⁵ CFU/ml
B) Enumeration of bacterial colonies from meat products inoculated using
MacConkey agar.
Dilution Number of colonies

10² Too few to count

103 Too few to count

104 Too few to count

DISCUSSION:
In the enumeration of E. coli from meat product, it is critical that each colony comes from
only one cell, so chains and clumps of cells must be broken apart. However, many bacterial
species grow in pairs, chains, or clusters, or they may have sticky capsules or slime layers,
which cause them to clump together. It is sometimes difficult to separate these into single
cells, which in turn makes it difficult to obtain an accurate count of the original cell numbers.
Therefore, the total number of viable cells obtained from this procedure is usually reported as
the number of colony-forming units (CFUs).

Only a few of the plates following incubation will contain a suitable number of colonies to
count. Those plated from low dilutions may contain too many colonies to count easily while
those plated from high dilutions may contain too few colonies or none. So, the number of
colonies for dilution factor 10² in tryptic soy agar is higher compared to dilution factor 104.
As stated in our results, the colonies that have been counted for 10² dilution factor is 241 and
for 104 is 41. This is because the 10² plate have densely packed colonies, are too numerous to
count, and most likely more than 300 CFUs. On the other hand, 104 plate has a countable
number which is between 30 to 300 CFUs. However, we found that the number of colonies
from meat products inoculated using MacConkey agar is too few to count for dilution factor
10², 103, and 104. This is because MacConkey is a selective medium that inhibits the growth
of Gram-positive bacteria only.
CONCLUSION:
In conclusion, the objective of this experiment has been achieved. It was found that the
number of colonies from meat product inoculated using tryptic soy agar are higher and more
visible to count compared to using MacConkey agar. This is because MacConkey agar
contains a limited amount of nutrients which may limit the growth of some bacterial species.
The agar may not support the growth of some fastidious bacteria, which require specific
nutrients or growth conditions. As for tryptic soy agar, it is a universal medium because it
supports the growth of different types of microorganisms including Gram positive and gram
negative. This is one of the factors on why the number of colonies in tryptic soy agar is
higher compared to MacConkey agar.

DISCUSSION QUESTIONS:

1) Discuss why MacConkey media is categorized as a differential and selective

media.
MacConkey is a selective medium that inhibits the growth of Gram-positive bacteria
due to the presence of crystal violet and bile salts. But gram-negative bacteria grow
well on MacConkey. This medium differentiates or distinguishes between groups of
bacteria on the basis of a color change reaction. MacConkey contains two additives
that make it differential which are neutral red (pH indicator) and lactose (a
disaccharide).

2) Predict the similarities and/or differences in the observations if the plates are
incubated at 30℃.

The similarity in the observation is the bacteria can grow on the both temperature.
Meanwhile the difference is the number of colonies for temperature in 30 degree
celsius are less than the number of colonies for temperature in 37 degree celsius. This
is because the bacteria need the suitable temperature which is 35 degree celsius. to
optimize.
3) Discuss the difference between results observed using tryptic soy agar and
MacConkey agar.

The difference between results observed using tryptic soy agar and MacConkey agar
is the number of colonies. We observed that the number of colonies in tryptic soy agar
are larger than in MacConkey agar. It is because MacConkey agar only grows gram-
negative bacteria, and those bacteria will appear differently based on their lactose
fermenting ability as well as the rate of fermentation and the presence of a capsule or
not. Meanwhile, Tryptic Soy Agar is used as a general growth medium for the
isolation and cultivation of microorganisms. They are general-purpose, non-selective
media providing enough nutrients to allow for a wide variety of microorganisms to
grow. So, the bacterial colonies in meat are more likely to in tryptic soy agar than
MacConkey agar.
REFERENCES:
 Peter Feng (ret.), Stephen D. Weagant (ret.), Michael A. Grant (dec.). (2020). BAM
Chapter 4: Enumeration of Escherichia coli and the Coliform Bacteria. U.S. Food
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BAM Chapter 4: Enumeration of Escherichia coli and the Coliform Bacteria | FDA

 Joan Petersen, Susan McLaughlin. (2021). Introduction to Enumeration of Bacteria.

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https://bio.libretexts.org/Courses/North_Carolina_State_University/MB352_General_Microbi
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 MacConkey’s Agar (MAC): Differential & Selective Bacterial Growth Medium. (n.d.).

https://www.scienceprofonline.com/microbiology/macconkeys-agar-mac-differential-
selective-bacterial-growth-medium.html

 Acharya Tankeshwar. (2018). MacConkey Agar: Composition, Uses, Colony

Characteristics. Microbe Online. Retrieved April 12, 2023.

https://microbeonline.com/macconkey-agar-mac-composition-preparation-uses-and-colony-
characteristics/