Faculty of Fisheries and Food Science

University Malaysia Terengganu

Program: Food Science (Foodservice & Nutrition)
Semester I 2022/2023

Lab Report 5 & 6

Subject Code:STM3013
Subject title: Food Microbiology
Academic Session: Semester I 2022/2023
Lecturer ‘s Name: Dr.Tuan Zainazor Bin Tuan Chilek
Lab Topic: LAB 5: Enumeration of the microorganisms
                LAB 6: Enumeration of bacteria, yeast, and molds. 

Student Name  Matric Signature Date

Number 

NURUL AUNI SYAZANA BINTI MOHD S63620 AUNI 13/12/2022

RIDZWAN
OBJECTIVES
The objective is 
 To learn dilution and plating for microbial enumeration.
 To determine and compare the Total Plate Count of different foods.
 To evaluate the factors that influence the TPC determination.
 To practice applying colony-counting rules.

INTRODUCTION

The two widely used in microbiology to determine the bacterial population is the
viable/ standard plate method and the spectrophotometric turbidimetric analysis. In
this practical we used Standard plate count (TPC) as a method which is a direct
measurement of cell density and reveals the population of living bacteria using a
spread plate method or pour plate method. The spectrophotometric analysis is based
on turbidity that indirectly measures all the bacteria, dead or alive cells in the
microbes. This laboratory exercise is to train the students to analyse solid foods for
total count and compare different methods of homogenizing and plating food
samples. Food with inherently different microbial profiles and population size will be
analysed. 

MATERIALS AND METHODS

 
Overview of the TPC procedure
Spread plate method

1. Weigh 25g chicken 2. Add the 225 ml 3. Then do the

meat samples and put of the sterile homogenise
in the stomacher bag saline using the
stomacher.
Then do the
dilution until 10-6

4. Select the highest 5. Spread the 7. Determine the

dilution samples and solution using L Cfu/ml.
pipette 1.0ml sample spreader.
dilution in the 3 6. Dry it and
different plate. (PCA, incubate at 37
EMB, BPA) degrees Celsius

Pour plate method

1. Weigh 25g fruit samples 2. Add the 225 ml 3. Then do the

and put in the stomacher of the sterile homogenise
bag saline using the
stomacher
Then do the
serial dilution
until 10-5

4. Then select 3 highest 5. Pour the PCA 6. Wait until it

dilutions. Pipette 1.0ml and DRBC into becomes solid
sample dilution into the the petri dish and incubate at
petri dish  that containing 37 degrees
the samples. Celsius for 24
Swirl the petri hours. 
dish like 8
number  7. Determine the
Cfu/ml.

COUNTING RULES

1- Plates with colonies in the range of 30-300 colonies

2- Plate with 1-20 colonies
3- Plate with no colonies
 4- Plate with greater colonies

RESULT

Spread plate method

Type of agar Dilution factor No of colony Cfu/g

medium

PCA 10-4 70 6.4x 105

10-5 2

10-6 1

EMB 10-4 9 9x 105

10-5 1

10-6 0

BPA 10-4 4 4x104

10-5 2

10-6 1
Pour plate method

Sample Dilution Type of agar No of colony

factor
mango 10-3 PCA TNTC

DRBC Moulds 30

Yeasts 77

10-4 PCA TNTC

DRBC Moulds 6

Yeasts 21

10-5 PCA 643

DRBC Moulds 2

Yeasts 5

Type of agar Dilution factor Cfu/g

PCA 10-5 3.00X108 (6.43X108)

DRBC (YEAST) 10-3 7.7X105

DRBC (MOULD) 10-3 3.0X105

DISCUSSION 

In this experiment, we used viable/ standard plate count frequently in the

enumeration of microorganism populations. There are two method that we can used
which is spread plate method and pour plate method. We use colony forming unit
(CFU) in the calculation of enumeration the microorganism. However, this method
has error as viable/ standard plate count just for the visible colony in enumeration.
We need to wait until the colonies to grow and only count the visible one. There are
also some errors to count if have many colonies on the plate.

For this solution, we do a serial dilution to overcome it. We used the highest dilution
in this experiment. We used dilution 10 -4 until 10-6 for the spread plate method and
10-3 to 10-5 in the spread plate method. So we reduce the sample size in the
enumeration as it is easy to determine and counting the colonies. Although dilution
is used to minimize sample size for ease of enumeration, microbes can multiply very
quickly until there are too many visible colonies. Because the margin of error is
based on the sample size, the higher the sample size, the smaller the error.
However, if the sample size is too huge, the result is more difficult to handle and
calculate. So, we only use colony-forming units (CFU) between 30-300 to compute
microorganism concentration. If the colonies are more than 300, we will not count it.

In spread plate method, this method allows the microorganism to growth on the
surface as we spread it using L spreader. It will error if the L spreader were not
sterile carefully as it will get contamination to the plate. So, all the tools use for the
spread plate method also to avoid the contamination. When doing the serial dilution,
the sample must dilute enough so the viable microorganism is load between the
range of 30-300 cfu/g. The process of this method must be repeated if the range of
the bacteria is not between 30-300 cfu/g. We also can avoid the damage or dead of
heat sensitive microorganism.

In the pour plate method, we are using the hot agar’s temperature (between 45°C
and 50°C). The possibility to kill the microorganism is higher than the spread plate
method. So, the heat sensitive organisms that come into contact with hot agar will
lose the viability. The error can happen in the counting of the colony. We must
ensure that the temperature of the agar must be between 45°C and 50°C before
pouring into the plate.

The nutrient agar that we used is different which are Dichloran Rose Bengal
Chloramphenicol Agar (DRBC), Eosin-Methylene Blue (EMB) and Baird-Parker Agar
(BPA) for the selective agar. For the non-selective agar, we use Plate Count Agar
(PCA). Selective agar used to grow a specific microorganism.
CONCLUSION 
In conclusion, we have learned that to determine the microorganism we have many
kinds of method that we can use. I learned to do a serial dilution for the culture to
do an enumeration of microorganism. We also know how to compare the total plate
count in different food categories like meat and food. We used the total plate count
to determine the number of aerobic and facultative anaerobic, mesophilic bacteria
per unit volume or weight of the food samples.
We used chicken and mango to determine the microorganism. We know how to
count the microorganism in the plate and learn the calculation using colony counting
rules.

REFERENCE
Tankeshwar A. (2022, October 4). Pour plate method: procedure, uses and
disadvantage. From
https://microbeonline.com/pour-plate-method-principle-procedure-uses-dis-
advantages/
Dahal P, (2022, August 26). Spread plate method- Definition, principle, procedure
and use. From https://microbenotes.com/spread-plate-technique/
Petersen J and Mclaughlin S. (2021, June 2). Introduction to enumeration of
bacteria. From https://bio.libretexts.org/Courses/North_Carolina_State_University/
MB352_General_Microbiology_Laboratory_2021_(Lee)/
05%3A_Enumeration_of_Bacteria/
5.01%3A_Introduction_to_Enumeration_of_Bacteria
Onyeaka, H. N., & Nwabor, O. F. (2022). Enumeration of foodborne microorganisms.
Food Preservation and Safety of Natural Products, 39-49.
https://doi.org/10.1016/B978-0-323-85700-0.00001-0
APPENDICES
SPREAD PLATE METHOD

PCA

EMB

BPA
Pour plate method

PCA

DRBC