Table of Content

1.0 Abstract .............................................................................................................................. 2

2.0 Introduction ........................................................................................................................ 3

2.1 Research Background .................................................................................................................... 3
2.2 Objective ........................................................................................................................................... 4
2.3 Problem Statement .......................................................................................................................... 4

3.0 Literature Review ............................................................................................................... 5

3.1 Food Spoilage .................................................................................................................................. 5
3.2 Causes of Food Spoilage ............................................................................................................... 5
3.3 Food Preservation ........................................................................................................................... 6
3.4 Swabbing Method ............................................................................................................................ 7

4.0 Methodology....................................................................................................................... 8

5.0 Results and Discussion ..................................................................................................... 9

5.1 Results .............................................................................................................................................. 9
5.2 Discussion....................................................................................................................................... 10

6.0 Conclusion and Recommendation...................................................................................11

6.1 Conclusion ...................................................................................................................................... 11
6.2 Recommendation........................................................................................................................... 11

7.0 References.........................................................................................................................12

8.0 Appendices........................................................................................................................13
1.0 Abstract

This experiment was conducted on 12 October 2017. The objective of this experiment is
to isolate microbial colonies from an inoculum by creating areas of increasing dilution on an agar
petri plate and to determine the total aerobic plate count (including yeast and molds) on fresh
bread and spoiled bread by using swabbing method. This experiment also wants to compare the
amount of colony between fresh bread and spoiled bread. The results showed that the number of
bacterias colony in spoiled bread is higher than the fresh bread after 24 hours. In the sample of
spoiled bread 1 and 2, the number of bacteria are 83 and 70 respectively. While in the sample of
fresh bread 1 and 2, the number of bacteria are only 34 and 7 respectively. There are some
precautions that must be followed in this experiment to get the best result. Firstly, before begins
the experiment, our hands must be washed with ethanol to remove all microorganisms or bacteria
that will affect the results obtained. Then, when swabbing the sample on the agar plate, make
sure that the line is not overlap each other to ease the count of colony bacteria and the isolated
bacteria can be seen clearly. Thus, the zigzag pattern is the right technique of the swabbing
process. Last but not least, performed this experiment in incubator and avoid talking when
conducting this experiment to ensure the microbial colonies are not contaminated.

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2.0 Introduction

2.1 Research Background

Food spoilage results when microbiological, chemical or physical changes occurred.

Microbiological food spoilage is caused by the growth of microorganisms which produce enzymes
that lead to unpleasant by-products in the food. Chemical food spoilage occurs when different
components in the food react with each other or with some added component which change the
foods sensory characteristics. Oxidation, enzymatic browning and non-enzymatic browning are
the examples of this cases. Physical food spoilage results when moist foods are excessively
dehydrated or dried foods absorb excessive moisture.

The most important cause of food spoilage is due to the exposed of food to air and oxygen.
78% of air consist of nitrogen, 21% consist of oxygen and another 1% is the mixture of other
gases ("Food Safety Education | For Educators | Competencies | General | Spoilage | Describe
why food spoils.", 2017). Oxygen is very important in living lives including microorganisms.
Oxygen can cause food spoilage by providing conditions that will enhance the growth of
microorganisms, which can damage the foods with the help of enzymes and can cause oxidation.

Other than that, microorganism also is one of the factors that affecting food spoilage.
Oxygen can provide conditions that enhance the growth of microorganisms. Some bacteria
require oxygen for growth (aerobes) while others can grow only in the absence of oxygen
(anaerobes) ("Food Safety Education | For Educators | Competencies | General | Spoilage |
Describe why food spoils.", 2017). Many bacteria can grow under either condition and are called
facultative anaerobes. Molds and most yeast that cause food to spoil require oxygen to grow.
They can often be found growing on the surface of foods when air is present.

The other factors of food spoilage is enzymes. Oxidizing enzymes is the enzymes that are
naturally present in food. These enzymes have speed up chemical reactions between oxygen and
food components which could leads to food spoilage. For example, catalase and peroxidase
enzymes in vegetables that cause darkening. Besides that, food spoilage also occurs due to the
slight changes in relative humidity. Moisture condensing on a surface of food can also provide a
good environment for bacteria and molds to grow and multiply ("Food Safety Education | For
Educators | Competencies | General | Spoilage | Describe why food spoils.", 2017). Physical

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defects such as cracking, splitting and crumbling occur when excessive moisture is lost from
foods.

2.2 Objective

This experiment was conducted to isolate microbial colonies from an inoculum by creating
areas of increasing dilution on an agar petri plate and to determine the total aerobic plate count
(including yeast and molds) on fresh bread and spoiled bread. Furthermore, this experiment also
wants to compare the amount of colony between fresh bread and spoiled bread.

2.3 Problem Statement

In performing this experiment, 2 conditions of bread are required; fresh bread and spoiled
bread. The area of increasing dilution on an agar petri plate need be created first in order to isolate
microbial colonies from an inoculum and to determine the total aerobic plate count on fresh and
spoiled bread. Swabbing method is used in order to achieve the objective of this experiment. In
microbiology, swabbing is a technique used to obtain the sample by rubbing a sterile swab,
moistened with a non-nutritive medium in several directions over a standardized sample area.

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3.0 Literature Review

This chapter reviews the whole aspects about the experiment conducted in more detailed
knowledge such as the type of food spoilage, how food can spoil and how to measure the spoilage
of food by using swabbing method.

3.1 Food Spoilage

Food is categorized as contaminated when there is existence of unwanted

microorganisms. Usually, the contamination is natural, but sometimes it is artificial. Natural
contamination occurs when the microorganisms attach themselves to foods while the foods are
in their growing stages. For example, fruits are often contaminated with yeasts because yeasts
ferment the carbohydrates in the fruits. Meanwhile artificial contamination occurs when food is
handled or processed, such as when fecal bacteria enter food through improper handling
procedures ("Food Spoilage", 2017).

Food spoilage is an unpleasant change or evacuation from the food's normal state. The
change can be detected with the senses of smell, taste, touch or vision. Changes occurring in
food depend upon the composition of food and the microorganisms present in it and result from
chemical reactions relating to the metabolic activities of microorganisms as they grow in the food.

3.2 Causes of Food Spoilage

Food spoilage and deterioration is no accident. It is a naturally occurring process. To

understand how to keep up the nature of food and prevent spoilage, we need to know what can
cause it. The factors that affect food spoilage are as follows:

i. Microorganism
Many types of microorganisms can cause food problems. The microorganisms that
can cause food-borne illness are called pathogenic microorganisms. These

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 microorganisms grow best at room temperatures (60-90F), but most do not grow well
at refrigerator or freezer temperatures. Pathogenic microorganisms may grow in foods
without any noticeable change in odor, appearance or taste. Spoilage microorganisms,
including some kinds of bacteria, yeasts and molds, can grow well at temperatures as
low as 40F. When spoilage microorganisms are present, the food usually looks or
smells terrible (Network, 2017).
ii. Enzymes
Enzymes are one of the substances naturally present in food that are responsible for
the maturing process in fruits and vegetables. Enzymes are responsible for texture,
color and flavor changes. For instance, as a banana turns from green to yellow to
brown, not only does the color change, but there is also a change in the fruit's texture.
This is the result of enzyme action (Network, 2017).
iii. Air
Oxidation is a chemical process that produces undesirable changes in color, flavor
and nutrient content as a result of when air reacts with food components (Network,
2017).
iv. Temperature
Temperature affects storage time and food deteriorates faster at higher temperatures.
Microorganisms, both spoilage and pathogenic, grow rapidly at room temperature.
food must be store at low temperature to slow down the microbial growth, the
enzymatic and oxidation processes (Network, 2017).

3.3 Food Preservation

Food preservation is the process of treating and handling food to stop or slow down food
spoilage, loss of quality, edibility or nutritional value and thus allow for longer food storage.
Preservation usually involves preventing the growth of bacteria, fungi (such as yeasts) and other
micro-organisms as well as retarding the oxidation of fats which cause rancidity. Food
preservation can also include processes which inhibit visual deterioration, such as the enzymatic
browning reaction in apples after they are cut, which can occur during food preparation.

Many processes designed to preserve food will involve a number of food preservation
methods. Below are some of the preservation methods.

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 i. Fermentation
ii. Canning
iii. Pickling
iv. Drying and freeze-drying
v. Irradiation
vi. Pasteurization
vii. Addition of chemical additives.

3.4 Swabbing Method

Swabbing method is always used on flat surfaces. It provided a template that is used to
define the sample size. Two types of swabs that may be used for the swabbing technique include
cotton, synthetics and calcium alginate materials with the appropriate diluent ("A Surface
Monitoring Refresher: Contact Plates and Swabs", 2017).

The swab method involves obtaining the sample by rubbing a sterile swab, moistened with
a non-nutritive medium in several directions over a standardized sample area. The swab is then
placed into a specified amount of rinse solution and agitated to transfer the microorganisms
present on the swab into the solution ("A Surface Monitoring Refresher: Contact Plates and
Swabs", 2017). The collection medium may be tested by a most probable number method,
membrane filtration method or direct plating method.

Figure 3.1: Swabbing process on agar plate

The disadvantages of swab method are the sampling and technique can affect the results
and the method requires manipulation to culture the sample. Swabs are designed for hard-to-
reach places and can fit easily into equipment recesses, nooks and crevices.

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4.0 Methodology

The experiment was conducted by using swabbing method. Two samples of fresh bread
and two samples of spoiled bread were prepared. Agar plate is used in this experiment determine
the total aerobic plate count on bread samples. Firstly, spoiled bread was prepared by left it
opened in a room temperature for two days to make sure it is exposed to air and microorganisms
before conducting experiments. The experiment must be carried out by wearing glove to prevent
our hands from exposed to any chemical used and to get an accurate result on total aerobic plate
count of the prepared sample. Before starting the experiment, some ethanol was sprayed to our
hands (cover by gloves) to prevent the infection of bacteria. Then, the sample is taken by using
cotton swab by swabbing it on the surface of the bread. After that, the cotton swab is swab gently
onto the surface of agar plate in a zigzag pattern line without double the line to prevent the
microorganisms from overlap. The length of time of swabbing process was minimize to prevent
agar plate from exposed too long to the environment. The agar plate with sample was leave in
the incubator for 24 hours before the colony of bacteria can be counted. This experiment was
repeated by using fresh bread as the sample. After 24 hours, the agar plate is put in colony counter
and start to count the colony appeared in food sample.

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5.0 Results and Discussion

5.1 Results

Table 5.1: Number of colony of bacteria in fresh and spoiled bread

Food Sample Fresh Bread Spoiled Bread

1 2 1 2
Colony bacteria count 34 7 83 70

GRAPH OF NUMBER OF BACTERIA'S

COLONY FOR FRESH AND SPOILED BREAD
90

80
83
70
70
60

50

40

30 34
20

10
7
0
Fresh Bread 1 Fresh Bread 2 Spoiled Bread 1 Spoiled Bread 2

Figure 5.1: Graph of Number of Bacteria's Colony for Fresh and Spoiled Bread

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5.2 Discussion

For solid sample such as bread, the swabbing method is used in order to isolate the
microbial colonies and to determine the total aerobic plate count. The colonies of microbiological
culture that has been taken from the samples was grown on a new plate with agar that is used for
the surface growth of microorganisms in order to observe colony appearance, so that the
organism can be identified.

In this experiment, two samples of fresh bread and spoiled bread were used. For the fresh
bread, the results after left for 24 hours in incubator showed the difference number of bacteria
which was 34 and 7 for fresh bread 1 and fresh bread 2 respectively. Based on study conducted
by the American Chemical Society's Journal of Agricultural and Food Chemistry, carbohydrate in
the bread boosted Lactobacillus, a group of good bacteria, located in our intestines. This type of
bacteria is good for warding off sicknesses such as the flu, diarrhea and other related health
issues (Bachai, 2017). When there is not enough of this bacteria in a persons gut, they become
more prone to disease.

This experiment also conducted by using spoiled bread from the same brand of fresh
bread (gardenia). After left for 24 hours in the incubator, the sample of spoiled bread 1 and spoiled
bread 2 showed the number of colonies bacteria of 83 and 70 respectively. Total number of
bacteria found in both spoiled bread was higher compared to fresh bread. This spoiled bread was
exposed to light and air for two days. This condition will enhance the growth of microorganism
and can cause oxidation. The exposure of foods to light can cause deteriorative of food
constituents. In most solid foods, the light only penetrates the outer layer of the product and
photodegradation occurs in this surface layer. Discoloration on the surface of foods can certainly
affect consumer acceptance of these products.

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6.0 Conclusion and Recommendation

6.1 Conclusion

As a conclusion, the number of bacteria in spoiled bread is much higher than in the fresh
bread which are 83 and 70 for spoiled bread 1 and spoiled bread 2, compared to 34 and 7 in fresh
bread 1 and fresh bread 2. This shows that microorganisms are easily to growth on spoiled food,
especially food that is exposed to air and light. Some other factors that might cause food spoilage
are temperature around the food, moisture of food and its environment, enzymes that contain in
the food and others. In order to prevent food from spoiled and keep the food long lasting,
preservation techniques can be used. Some of the techniques are canning, pickling, fermentation
and drying.

6.2 Recommendation

There are a few recommendations and precautions that have been considered while
conducting the experiment in order to get an accurate result.

Firstly, before begins the experiment, our hands must be washed with ethanol to remove
all microorganisms or bacteria that will affect the results obtained.

Then, when swabbing the sample on the agar plate, make sure that the line is not overlap
each other to ease the count of colony bacteria and the isolated bacteria can be seen clearly.
Thus, the zigzag pattern is the right technique of the swabbing process.

Last but not least, performed this experiment in incubator and avoid talking when
conducting this experiment to ensure the microbial colonies are not contaminated.

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7.0 References

A Surface Monitoring Refresher: Contact Plates and Swabs. (2017). Controlled Environments
Magazine. Retrieved 26 October 2017, from https://www.cemag.us/article/2003/01/surface-
monitoring-refresher-contact-plates-and-swabs

Bachai, S. (2017). White Bread Found To Boost Good Gut Bacteria, But Still Linked To Weight
Gain And Obesity. Medical Daily. Retrieved 28 October 2017, from
http://www.medicaldaily.com/white-bread-found-boost-good-gut-bacteria-still-linked-weight-
gain-and-obesity-288144

Food Safety Education | For Educators | Competencies | General | Spoilage | Describe why food
spoils.. (2017). Foodsafetysite.com. Retrieved 23 October 2017, from
http://www.foodsafetysite.com/educators/competencies/general/spoilage/spg1.html

Food Spoilage. (2017). Cliffsnotes.com. Retrieved 25 October 2017, from

https://www.cliffsnotes.com/study-guides/biology/microbiology/food-microbiology/food-
spoilage

Network, U. (2017). How Food Spoils | UNL Food. Food.unl.edu. Retrieved 24 October 2017,
from https://food.unl.edu/how-food-spoils

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8.0 Appendices

Colony Counter

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Samples of Bread on Agar Plate before 24 hours

Sample of Fresh Bread 1 Sample of Fresh Bread 2

Sample of Spoiled Bread 1 Sample of Spoiled Bread 2

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Samples of Bread on Agar Plate after 24 hours

Sample of Fresh Bread 1 Sample of Fresh Bread 2

Sample of Spoiled Bread 1 Sample of Spoiled Bread 2

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