UNIVERSITI KUALA LUMPUR

MALAYSIAN INSTITUTE OF CHEMICAL &

BIOENGINEERING TECHNOLOGY
LABORATORY TECHNICAL REPORT
SUBMISSION FORM

To: Mr. Muhammad Sharir bin Abdul Rahman Code Subject: CBQ 10503 Microbiological Food
Safety

From: Student ID. No.:

1. Maryam Bt Amran 1. 55228222027
2. Anis Humaira Bt Abdul Rahman 2. 55228222022
3. Muhammad Aiman Haziq B Zulkifli 3. 55228222016
4. Bat Umar B Bat Kamarulzaman 4. 55228222036

No. of Group: 4 (P2) Date of Experiment: 18/10/2022

Title of Experiment:
TOTAL VIABLE COUNTS OF PERISHABLE FOOD PRODUCTS
Received by: Mr. Muhammad Sharir bin Abdul Date of Submission:
Rahman 12/11/2022

Note: Late submission will not be accepted.

*To be filled by the marker*

VERY VERY
CRITERIA POOR POOR GOOD GOOD EXCELLENT
2 3 5
1 4
1.0 ABSTRACT & OBJECTIVES (TOTAL: 10%) 9
1. State the summary to the experiment conducted
(include introduction, methods performed, results
obtained, conclusion and recommendation, max 2 4 6 8 10
one page).
2. State the objectives of the experiment (point
form)
2.0 PROCEDURES (TOTAL: 5%) 5
1.Complete methodology is presented in suitable
1 2 3 4
and understandable flowchart (extra marks for
diagram).
3.0 RESULTS (TOTAL: 10%)
1.Data are presented as deemed suitable with 8
complete label and units in tables and/or graphs. 2 4 6 10
2. Explanations of the referred tables and/ or
graphs.

4.0 DISCUSSIONS (TOTAL: 15%) 12

1. Compare and discuss the results with
3 6 9 15
references.

5.0 CONCLUSIONS (TOTAL: 5%)

1. Should reflect/ answer the objectives of the 3.5
experiment. 3
2. Include potential recommendation from this 1 2 4 5
experiment (not including the necessity of following
lab manual and importance of aseptic technique,
as these both are compulsory when doing lab
work).

1
 5
6.0 REFERENCES (TOTAL: 5%)
1 2 3 4
1 . Minimum of 4 references.

TOTAL MARKS 42.5 50

1. Abstract & object : TOO LONG

Abstract
STOP USING WE

The purpose of this experiment is to determine the total viable counts of perishable
food products. The determination of the total viable counts in a food product is one of the
most straightforward and frequently employed in microbiological techniques. Before we start
counting, the serial dilution technique needed to be performed. Typically, a sample contains
too many bacteria to count directly. If the sample is serially diluted and then spread out on an
agar surface in a way that allows for the formation of completely separate isolated colonies
by a single isolated bacterium, the quantity of colonies can be used to calculate how many
viable (alive) cells are present in a known dilution. The first method of the experiment is
sample handling which the sample unit was refrigerated (0-5°C) until needed, everything
except shelf- stable products. Before we start preparing the sample, the table top must be
sterilized with sanitizer as well as prevent the cross contamination while doing the
experiment. The solid sample that we use is cabbage that had been cut to pieces and
weighed about 10 g. The 10 g of solid sample was transferred into plastic bag that filled with
90 ml buffer (peptone water). The types of media used for this experiment is plate count agar
(PCA) and potato dextrose agar (PDA), even there are many types of media used in
microbiology the procedures and technique for preparing them are commonly the same
which are weighing, dissolving, sterilizing, pouring, and incubating. The experiment was
conducted by getting the media ready for an hour and a half of autoclaving at 121°C. Next,
the media that had been autoclaved would be poured into 12 sets of Petri dishes. Before we
poured the media into the Petri dishes, we need to label the petri dish first. The first six will be
labelled as plate count agar (PCA) and the other six will be labelled as potato dextrose agar
(PDA). In this experiment we needed to do two plate procedure which is spread plate and
pour plate. So, the first three of plate count agar (PCA) will be labelled as S for spread plate
and the other three will be labelled as P for pour plate, and for potato dextrose agar (PDA) is
vice versa. After prepared the media, we needed to performed the serial dilution technique.
The dilution that was going to prepared is 10-1 until 10-6. The dilution of stock cell suspension
is 10-1 . We needed to transferred 1 ml of stock cell suspension into a sterile, 9 ml in
the second tube and mixed it thoroughly. The first tube had 10-1 and second tube has 10-2 ,
with additional transfers of 1 mL of cell solution from the second tube into the third tube
and so forth, the dilution process can continue forever. The serial dilution technique
would be performed in order to obtain the recommended concentration range of colony
forming unit (CFU) for enumeration of microorganisms. The serial dilution that would be
tested is 10-4 until 10-6. Next, we started to plate the sample by using the first method which
is spread plate for serial dilution 10-4. Agar that we used is plate count agar (PCA) and
2
potato dextrose agar

2
 (PDA). The aseptic technique needed to be involved to do the spread plate method to
prevent the microbial contamination. Pipetted 0.1 of the respective bacterial culture onto the
center of an agar plate. Dipped the L-shaped glass rod into a beaker of ethanol and then
tapped the rod on the side of the beaker to removed the excess ethanol. Then, briefly passed
the L-shaped rod through the flame to burn off the alcohol, and allowed it to cooled on the
agar surface. The bacteria sample needed to spread evenly over the agar surface with the
sterilized spreader, making sure the entire surface of the plate has been covered. Also make

YO
sure you do not touched the edge of the plate. Immersed the spreader into the ethanol,
tap on the side of the beaker to removed any excess, and reflame the spreader. Repeat
this method to inoculate the remaining two plates which is for serial dilution 10-5 and 10-6.
Next, we applied the second method which is pour plate, Pipetted the diluted sample
about 1 ml into the empty sterile plate. 15 ml of melted agar had been poured into the
sterile plate which had been cooled 45°C. The Petri dish needed to be covered and mixed
thoroughly by gentle tilting and swirling the dish. The agar must not slop over the edge of
the Petri dish. Let the agar cooled and undisturbed to solidify on a flat table top. Repeat
this method to inoculate the remaining two plates which is for serial dilution 10-5 and 10-6.
After that, we incubated the agar, for bacteria we incubated the plates undisturbed in an
upright position at 30-35°C for 48 hour or 2-3 days. The colony had been counted when the
incubation period is expired. After 72 hours, the result for plated count agar (PCA) were
recorded. For spread plate and pour plate method, we founded the Petri dish had growth
of bacteria. This is because, the PCA medium is non-selective and relatively rich in
nutrients, tryptone, vitamin factors from yeast extract and glucose used as an energy
source promote the growth of most bacteria. For yeast and mold, the agar had been
incubated at 22 to 25/ 30°C for 3-6 days. The colony had been counted when the incubation
period is 6 days after. After 6 days, the result for potato dextrose agar (PDA) were
recorded. For spread plate method in serial dilution 10-4 and 10-5 there is no yeast and
mold growth on the petri dish, while serial dilution 10-6 there is one colony that had been
found. This may be due to the condition of the source of agar was dead. Dead bacteria
generally look the same as live bacteria, so you cannot assume that

cells on an agar surface are alive. For pour plate method, they show an amazing result
which we can saw the fungi growth on the petri dish. This is because, the potato infusion in
Potato Dextrose Agar (PDA) offers a nutrient base for the luxuriant growth of most
fungi while dextrose, a carbohydrate source, acts as a growth stimulant. Through this
experiment, we learned the ways to performed the serial dilution technique, ways to
performed colony
counting/ enumeration method for bacteria, yeast and mold, and able to determine total
aerobic microorganism in food sample.

;
 CFU/ ML?

2. Objectives

• Students can perform serial dilution technique

;
 • Students can perform colony counting/ enumeration method for bacteria, yeast
and mould
• Students able to determine total aerobic microorganism in food sample.

3. Methodology

4. Equipment & Materials:-

• Materials:-

i. Cabbage as food sample and cultures of appropriate microorganisms.

ii. Plate count agar (PCA):
1. Tryptone 5.0 g
2. Yeast Extract 2.5g
3. Dextrose 1.0g
4. Agar 15.0g
5. Distilled water 1.0 L
iii. Dissolve and autoclave at 121°C for 15 min and pH 7 ± 0.2. Dispense volume
into Petri dishes and allow to solidify.
iv. Potato dextrose (PDA)
v. Peptone water, 0.1% (PW)

• Equipment:-
i. Sterile glass rod
ii. Stomacher
iii. Colony counting scale
iv. Incubator
v. Autoclave
vi. Weight scale

vii. Sterile utensils

4.1 Procedure
Preparation of sample

4
Preparation for analysis
• Labelling

5
Preparation of Plate Count Agar (PCA)

6
Preparation of Potato Dextrose Agar (PDA)

Preparation of Peptone water (PW)

7
Preparing serial dilution

After that, the same volume was transferred into a second

tube containing 9 mL of solvent form the first tube.

1 mL of 10-1 was transferred into the next bottle (10-2)

8
Plating
• Spread plate procedure

9
• Pour plate procedure

10
Incubating

11
 5. Result

Calculation of PDA, PCA& Peptone Water:-

• Potato Dextrose Agar

39g/x = 1000mL/300mL
39/x = 3.33g
X = 39/3.33
X= 11.71g PDA powder needed

• Plate Count Agar

17.5g/x = 1000mL/300mL
17.5/x = 3.33g
X = 17.5g/3.33g
X = 5.26g PCA powder needed

• Buffered Peptone water

20g/x = 1000mL / 200mL

20g/x = 5
X = 20/5
X = 4g of peptone broth powder

Number of colonies
Dilution Spread plate Pour plate
PCA

10-4 112 41
10-5 70 133
10-6 84 49
PDA

10-4 0 4
10-5 0 3
10-6 1 2

Table 1: Number of colonies in each petri dish.

Calculation of CFU/mL
Dilution
PCA
10 (Spread)
-4
112 x 10 x 1/0.1
4

= (1.12x102) x 104 x 101

= 1.12 x 107 #

10-5 (Pour) 133 x 105 x 1/1

12
 = (1.33 x 102) x 105 x 1

= 1.33 x 107 #

Table 2: Calculation of Cfu/mL

Observation of petri dish

PCA SPREAD
Before After
Observation after 24 hours, there is no microbial growth for both method

10-4 10-4

10-5
10-5

10-6 106
PCA POUR

10-4 10-4

USE SUPERSCRIPT
13
13
 10-5 10-5

10-6 10-6
Observation of petri dish
PDA SPREAD
Before After
Observation after 24 hours, there is no microbial growth for both method

10-4 10-4

10-5
10-5

10-6
10-6
PDA POUR

10-4 10-4

14
 10-5 10-5

10-6
10-6

15
 Observation of petri dish
PDA SPREAD
Before After
Observation after 24 hours, there is no microbial growth for both method

10-4 10-4

10-5
10-5

10-6
10-6
PDA POUR

10-4 10-4

10-5 10-5

10-6
10-6

16
16
 Observation of petri dish
PCA & PDA
Observation after 48 hours are forgotten to capture while observing in the lab, but based on
our observation there still no sign of growth.

Observation of petri dish

PCA SPREAD
Observation after 7 2 hours.

10-4

10-5

10-6

PCA POUR

10-4

17
 10-5

10-6

Observation of petri dish

PDA SPREAD
Observation after 7 2 hours.

10-4

10-5

10-6
PDA POUR

18
 10-4

10-5

10-6

Observation of petri dish

PCA SPREAD
Observation after 6 days (25.10.2022: Tuesday)

10-4

10-5

19
 10-6
PCA POUR

10-4

10-5

10-6

Observation of petri dish

PDA SPREAD
Observation after 6 days (25.10.2022: Tuesday)

10-4

10-5

10-6
PDA POUR

WHY IS THERE NO DESCRIPTION?

20
 10-4

10-5

10-6

6. Discussion

The purpose of this experiment is to determine the total viable counts of perishable
food products. The determination of the total viable counts in a food product is one of the
most straightforward and frequently employed in microbiological techniques (F. Diez-Gonzalez,
2014). Before we start counting, the serial dilution technique needed to performed. By counting the
number of colonies which were cultured from succeeding dilutions of the sample, the serial dilution
method aims to estimate the concentration (number of colonies, organisms, bacteria, or
viruses) of an unknown sample. The measured counts are then used to calculate the
unknown concentration
(Avishai Ben-David et al, 2014).

In this experiment, there were 12 sets of petri dish that needed to determined the total
viable counts. They were two types of medium that we use to detect the microbe in food sample
which is plate count agar (PCA) and potato dextrose agar (PDA). According to (Sagar
Aryal,2022), a bacteriological substrate called plate count agar (PCA) is used to count all the
aerobic, living bacteria in a sample. The medium is not selective. Colony-forming units per
gramme (CFU/g) and colony- forming units per milliliter (CFU/ml) are used to measure the
number of bacteria in solid and liquid samples, respectively. There will be 6 sets of petri dish that
would be used plate count agar (PCA) to determined the growth of bacteria. In this experiment, we
need to do two types of procedure which is spread plate and pour plate. The first three set of petri
dish would do the spread plate method and the other three would be pour plate. After that, we
incubated the agar, for bacteria we incubated the plates undisturbed in an upright position at 30-
35°C for 48 hour or 2-3 days. After 72 hours, the result for plated count agar (PCA) were
recorded. For serial dilution 10-4 spread plate, we detected about 112 of colonies on agar
surface. This is because the PCA medium is non-selective and relatively rich in nutrients,
21
tryptone, vitamin factors from yeast extract and glucose used as an energy
source promote the growth of most bacteria (PCA agar| Principle| Preparation| Interpretation, n,d.).

21
This experiment’s hypothesis should state that the lowest dilution factor gives the higher number
of colonies. If we look at the serial dilution 10-5 pour plate method, the higher dilution factor gives
the higher number of colonies than the lower dilution factor, so we report as LA (laboratory
accident). According to (Microbiologics dilutions Guide,n.d.), dilution is the process of making a
solution weaker or less concentrated. In terms of microbiology, serial dilutions are used to
decrease a bacterial
concentration for a specific test, method, or to a concentration which is easier to count when plated
to an agar plate. This laboratory accident happens when we do not do the aseptic technique. As we
all know, using the aseptic procedure can aid in preventing the contamination of crucial equipment
or media because germs like bacteria can be found in many different areas. To
decrease contamination from germs, aseptic technique uses target-specific practices and
procedures under adequately controlled settings. To perform laboratory research in the area of
microbiology, you must
have certain skills (Siddiquee S, 2017).

Following that, the hypothesis can be clarified by stating that there is a possibility growth
of fungi in the potato dextrose agar (PDA) when performing spread plate and pour plate method.
This is because, the potato infusion in Potato Dextrose Agar (PDA) offers a nutrient base for the
luxuriant growth of most fungi while dextrose, a carbohydrate source, acts as a growth stimulant
(Sagar Aryal, 2022). The result shows that serial dilution of 10-4 and 10-5 spread plate method there
is no growth of yeast and mold, even we incubated them for 6 days. Some of the possible
reasons is a essential
nutrient might not be present in the culture media, the culture medium itself might be poisonous, or
other bacteria in the sample might be producing chemicals that are suppressive to the target

organism. Furthermore, we are aware that bacteria can rely on one another to grow (Wade, 2002).
BUT THERE WAS NO BACTERIAL GROWTH AT ALL FOR THAT PLATE
HO
At the end of this experiment, we can conclude that performed the serial dilution technique
is important in general work. This technique is to estimate the concentration (number of
colonies, organisms, bacteria, or viruses) of an unknown sample by counting the number of
colonies cultured from serial dilutions of the sample, and then back track the measured
counts to the unknown concentration. Moreover, we also learn to determine the total viable count
of microorganism in food product, to assess and control safety hazards, the potential for spoilage
or to ensure correct product
characteristics.

7. Conclusion & Recommendations

In conclusion, the objective of this experiment to ensure that students will be able to

22
performed the serial dilution technique and apply this technique in general work. The serial
dilution technique had been performed within the technician's instructions as well follow
the lab experimental manual requirements. The next objective is students able to
performed
colony counting/enumeration method for bacteria, yeast and mold. Students also can

22
 determined the number of bacteria, yeast and mold on the agar to make sure that the food
product is in a good quality. Despite the fact that the objectives were met, they were also lab
accidents that had been involved in this experiment. There are a few ways to overcome this
problem. To begin, we need to make a simple jotter note from the lab manual that had been
read, to make sure the experiments run smoothly and followed the instruction precisely.
HOW CAN YOU ACTUALLY FORGET TO APPLY ASPETIC TECHNIQUE?

8. References

Avishai Ben-David, C. E. D. (Ed.). (2014). Estimation method for serial dilution experiments
(Vol. 107). Elsevier.

Diez-Gonzalez, F. (Ed.). (2014). TOTAL VIABLE COUNTS | Specific Techniques.

SCIENCEDIRECT.https ://www .sciencedirect .com/topics /immunolo gy-and-
microbiology/total-viable-count
Aryal, S. (2022). Plate Count Agar (PCA)- Composition, Principle, Preparation, Results,
Uses. Microbe Notes.https://microbenotes.com/plate-count-agar-pca/

Microbiologics Dilutions Guide. (n.d.). Lets Get Growing.

https://www.microbiologics.com/core/media/media.nl?id=1758034&c=915960&h=fc555
e1fdf60b6138d07&_xt=.pdf

Aryal, S. (2022a). PDA- Composition, Principle, Preparation, Results, Uses. Microbe Notes.
https://microbenotes.com/potato-dextrose-agar-pda/

WHY ARE NOT ALL CITED REF WRITTEN HERE?

WHERE'S WADE 2002?

23