Microbial Assay Comparison of Different Heterotrophic Microorganisms between Bottled

Cola-Cola Beverage and a Soda Fountain Cola Beverage

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 Abstract

The purpose of this experiment was to determine the different kinds of microorganisms,

bacteria, yeast, and coliforms that were present in two Coca-Cola samples that were

obtained in different locations along with one sample being in a bottle while the other

from a self serve fountain dispenser. As well as calculating the cell density of the

samples through using the colony forming units found on the TSA agar plates,

identifying the unknown microbes on the plates using selective and differential agar

such as EMB and PABC was also completed. Biochemical test such as oxidase,

catalase, Rapid ID Yeast Test Kit, and EnteroPluri Test were the different methods used

in order to obtain the results for this experiment. The final results displayed that there

was a large quantity of varying coliforms, yeast, and bacteria existing within one of the

Cola samples. It is important to study the organisms within soft drinks because of its

frequent daily consumption worldwide in order to see if there are any bacteria or yeast

that can cause foodborne illnesses or dangerous health effects to the consumer.

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 Table of Contents

Abstract ii

Figures and Tables iii

1. Introduction 1

1.1. Membrane Filtration Technique: Overview 1

1.2. Application of the Method Theory 1

1.3. Selective and Differential Media Utilized 2

2. Rationale/Justification 3

2.1. Significance of Testing Carbonated Soda Beverages 3

2.2. Gap in Knowledge 3

3. Organisms Used 4

4. Objectives 4

5. Hypothesis 5

6. Materials 5

7. Methodology 6

7.1. Sample/Media Preparation 6

7.2. Membrane Filtration Technique 6

7.3. Biochemical Testing 7

8. Results 7

8.1. Membrane Filtration Cell Density Count 7

8.2. Gram-Staining Analysis 8

 8.3. Catalase and Oxidase Analysis Results 9

8.4. The Rapid Identification Test Analysis 9

9. Discussion 10

10. Conclusion 11

References 12
 Figures and Tables

Table 1. Membrane Filtration Cell Density Count……………………………………...7

Figure 1. Gram Staining of White Colonies……………………………………………...8

Figure 2. Gram Staining of Yellow Colonies…………………………………………….8

Figure 3. Catalase Test for Yeast………………………………………………………...9

Figure 4. Oxidase Test for Yeast…………………………………………………………9

Figure 5. EnteroPluri Test……………………………………………………………….10

Figure 6. RapidID Yeast Plus Test……………………………………………………..10

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1. Introduction

1.1. Membrane Filtration Technique: Overview

The Membrane Filtration Technique has been utilized for years in a variety of

industries that require the isolation of smaller molecules and/or microorganisms for

testing. In microbiology, the testing in our food sources, water systems, and other liquid

beverages is a requirement to utilize the membrane filtration method to indicate any

possible contaminations or to conclude the approval of the safety standards met to

further allow the consumers or residents to ingest (Bikram Gautam & Rameshwar

Adhikari, 2018). Most bacteria, fungi, and molds are filtered through a 0.45 µm pore

sized filter paper and isolated on specific media to a countable range of 20-80 colonies

(Bartram & Ballance, 1996). In addition, Membrane Filtration Technique can also be

used to determine cell density of a given liquid which allows for calculation of cell

number per volume unit of a liquid.

1.2. Application of the Method Theory

Membrane filtration was chosen for this experiment because the volume of the

liquid sample that is being tested for microorganisms is a sizeable amount. In addition,

membrane filtration offers a way to estimate the colony forming units (CFU) more

accurately in larger amounts of volume. Furthermore, two samples from different

environments along with different controls such as sterile water, bottle and cup were

tested, so the filtration process allows for multiple tests to take place at the same time.

Serial dilution along with the spread plate technique were omitted from the methodology

and the experiment, even though a dilution process is widely known to isolate an

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accurate result. These two techniques would not have been suitable for this experiment

because it would decrease the number of microbes in the sample of the bottled Coca-

Cola since there is already a very low number of microbes within the bottle and diluting

that would only decrease it further. As for the self serve fountain sample, it has far too

many microbes existing within it already, therefore, it would take time to dilute the

sample into a reasonable and countable amount. Hence, using membrane filtration finds

a medium between the nature of the two samples allowing both samples to be tested

without taking too much time or further diluting the samples in order to get a rational

amount.

1.3. Selective and Differential Media Utilized

In this experiment, the following media were used: Eosin Methylene (EMB);

and Tryptic Soy Agar (TSA). EMB is a selective and differential media that is used to

identify gram negative bacteria, fecal coliforms and coliforms. It is differential because

the coliforms isolated will display a metallic green colour due to the methylene blue

which is a pH indicator dye (Jackson, et al., 2013). This media was used to confirm if

any coliforms were present in any of the Coca-Cola samples. TSA is a general growth

media that is neither a differential nor selective media, therefore, various kinds of

coliforms and bacterial flora will flourish on it (Zimbro & Power 2009). This is why the

TSA media was used to measure the microbiological load of both the bottled Coca-Cola

and self serve fountain Coca-Cola. One main crucial ingredient in TSA is soybean meal

which assisted in increasing the amount of bacteria growth on the agar plates.

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2. Rationale/Justification

2.1. Significance of Testing Carbonated Soda Beverages

Carbonated soft drinks are indulged daily worldwide in a vast amount; therefore, it

is very important to study the health issues surrounding the consumption of these

drinks. Carbonated soft drinks are made up of solely water, but also contain

preservatives and carbon dioxide that would prevent microorganism such as, bacteria,

to proliferate (Ogueri & Vincent, 2017). The causation of contamination can be due to

poor manufacturing processes: poor hygiene, poor sterilization of microbiological

equipment, poor in-store cleaning regulations (soda fountain machines), or the raw

material itself (Ogueri & Vincent, 2017). The average pH of the Coca-Cola beverage is

about 2.4 to 3.2 with a sugar concentration of around 0-10% and contains a high

amount of carbonation levels (Shankar et al., 2021), which signifies that yeast and

certain fecal/coliform bacteria can proliferate. The extreme acidity of Coca-Cola aligns

with the range of the acidity (1.5 to 3.5) that already exists within the stomach;

therefore, it can damage the membrane surrounding the stomach and cause potential

health issues if consumed in large quantities frequently. Some microorganisms can

survive in these extreme conditions with the possibility of causing human illness which

is why it is important that this experiment was performed in order to isolate, identify, and

confirm if there were any pathogenic organisms present in the two samples.

2.2. Gap in Knowledge

The antimicrobial properties of the acidic Coca-Cola beverages lack peer-

reviewed articles that showcases how drinking an excess of acidic carbonated drinks

can destroy the normal flora of humans in the intestinal tract. E. coli is a normal floral

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bacterium living in the intestines that helps with food digestion (Şeker Dağ et al., 2015),

which is certain that this microorganism will be a target for this experiement.

Biochemical tests were performed to determine the identity of the unknown microbes

found on each of the agar plates in order to verify if any of the species are pathogenic or

can cause harm to humans.

3. Organism Used

The microorganisms used in this experiment would be based on unknown

identifications; therefore, no isolated colonies were used during experiment, but there

are expected commonly found microorganisms that can be identified in soda

carbonated beverages. Types of coliforms or fungi, such as yeast, can be commonly

found in these drinks, which would be an expected isolation. These microorganisms are

known to be contaminants in carbonated soda beverages because of its capabilities to

survive in acidic, high sugar, and/or high carbonation environments, heterotrophic

microorganisms (Ogueri & Vincent, 2017). Saccharomyces cerevisiae, also known as

yeast, are very common fungi utilized or found, but the proliferation of yeast causes the

spoilage rate to increase in soft drinks (Ogueri & Vincent, 2017). E. coli is a common

pathogenic bacterium that can be found as a contaminant in cola soft drinks for a more

than a day time span (Shankar et al., 2021).

4. Objectives

1) To investigate and analyze the microbial quality between a Coca-Cola sample from

a soda fountain dispenser in a fast-food establishment with a bottled Coca-Cola

sample for comparison utilizing the membrane filtration technique.

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2) To determine the colony forming units (CFU) of the sample solutions to conclude if

the soda beverage samples are contaminated.

3) To utilize different biochemical testing such as, gram staining and the catalase test,

to further conclude the present microorganisms isolated.

5. Hypothesis

1) The Coca-Cola obtained from the self-serve fountain dispenser will contain a higher

concentration of coliforms, yeast, and/or Listeria due to the microbiological build-up

of flora in the machine.

2) The Standard Plate Count of the self-serve soda fountain sample will have more

colony growth than the bottled sample.

6. Materials

 2 Coca-Cola Samples:

o 1 from the bottled package

o 1 from the self-serve fountain machine

 Yeast Rapid ID Kit

 Entero Pluri Test

 Oxidase Test Kit

 Catalase Test Kit

 Sterile Water

 3 Sterile Membrane Filtration Kits (with filter papers)

 6 TSA plates

 2 EMB plates

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 Microscope

 Inoculation loop

 Gram-Staining Kit

7. Methodology

7.1. Sample/Media Preparation

Two Coca-Coca solutions were bought in different enclosed environments: One

was dispensed from a fast-food soda dispenser from a restaurant, and the other was

from a bottled package purchased in a grocery store. Both samples were then brought

to the laboratory for testing. A TSA powder was obtained to prepare the plates needed

for this experiment.

7.2. Membrane Filtration Technique

Three Membrane Filtration Kits were obtained for 3 experiments: one for the

control, one for the soda dispensed sample, and one for the bottle sample. The control

cup that was obtained at the fast-food store was analyzed by adding sterile water and

ran through a membrane filtration unit. The filter paper was then removed from the

filtration kit with a sterile forceps and placed in a prepared TSA plate. Next, the plate

was incubated for 24 hours at 35oC. Then, the results of the control were determined if

there were any contamination present on the cup carrying the sample. A 5mL, 2.5mL,

and 1mL of the Coca-Cola sample from the self-serve soda dispenser was measured

and added into a new sterile membrane filtration kit which contained a new sterile filter

paper. After filtration, the filter paper was placed on TSA plates. Next, all the

plates were incubated for 24 to 48 hours at 25oC. Then, a 5mL, 2.5mL, and 1mL of the

Coca-Cola sample from the bottled package was put through the same membrane

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filtration process using a different membrane filtration kit. The bottle itself, was also

tested as a control using sterile water poured into it. Then it was filtered through a

different membrane filtration kit and placed on TSA agar plates. Lastly, it was incubated

at 25oC for 24 to 48 hours.

7.3. Biochemical Testing

After the plates were all incubated, the process of gram staining was done from a

colony on the TSA plates to conclude the microorganism present using the Microscope

at 1000x magnification. Next, the colonies were streak plated on EMB and PABC

selective and differential media. Then, all colonies on TSA and EMB were oxidase and

catalase tested along with running an E. coli rapid ID test kit in order to determine the

identity of the unknown bacteria.

8. Results

8.1. Membrane Filtration Cell Density Count

The fountain cola sample, the bottled coke sample, and controls were tested

through Membrane Filtration. The volumes filtered were 5mL, 2.5mL, and 1mL using

different filtration kits for different samples. The filter paper used was a 0.45μm pore

size and place in TSA plates incubated for 48 hours at 23oC. The isolated colonies were

counted to confirm the proper range of 20-80 colonies and the cell density was

calculated.

Cell Density (cfu/mL)

Sample Type
1mL 2.5mL 5mL
Fountain 111cfu/mL 77 cfu/mL 8 cfu/mL
Bottle 0 cfu/mL 0 cfu/mL 2 cfu/mL
Control 0 cfu/mL

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Table 1 - The filtration results showcasing the cell density (cfu/mL) of the fountain,
bottle, and control sample isolated on the TSA plate incubated for 48 hours at 23 oC.

The fountain sample produced more colonies on the TSA filter paper than the

bottle sample. The 2.5mL fountain sample was the only plate in the 20-80 colony range.

The control of didn’t produce any colonies on the filter paper itself, but the sterile water

control grew possible contamination on the agar away from the filter.

8.2. Gram-Staining Analysis

The gram-staining procedure was done on two possible colonies that grew on the

TSA plate: there were white colonies and yellow colonies. The colonies were analyzed

using a microscope at 1000x magnification with oil immersion.

Figure 1 - The gram staining of the Figure 2 - The gram staining of the
white colonies: gram-positive, yellow colonies: gram-negative,
oval-shaped, large colonies. diplococcus, small colonies.

The gram-staining in Figure 1 shows a gram-positive reaction, but due to its large

oval shape, the white colonies confirmed a possibility of a yeast microorganism. The

gram staining in Figure 2 shows a gram-negative reaction, but the diplococcus shape

confirmed a possibility that a possible error has occurred during the gram-staining

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procedure. Further biochemical testing was done to confirm the possible error and to

identify the yeast species isolated.

8.3. Catalase and Oxidase Analysis Results

The catalase test and the oxidase were performed on both yeast and the gram-

negative bacteria. The catalase test produces a positive result when the colony

inoculated on the glass slide reacts with hydrogen peroxide reagent to produce a by-

product gas reaction (bubbles) instantly [Figure 3]. The oxidase test produces a positive

result when the inoculated colony with a sterile wooden stick produced a blue colour

instantly on the DrySlideTM. For comparison, a positive control was used for the oxidase

test, which was Pseudomonas florescens.

A
B

Figure 3 - The catalase test for yeast (above) Figure 4 - The oxidase test of yeast
and gram-negative bacteria (below). The (A) and the gram-negative bacteria
image above shows bubbles (positive) and (B). Both show no blue production;
the image below show no bubbles (negative). therefore, negative result.

8.4. The Rapid Identification Test Analysis

The yeast and the possible gram-negative colonies were further tested based on

negative reaction on the oxidase test. The Rapid ID kits were used to perform a series

of biochemical testing in one component. The Entero Pluri Test [Figure 5] was to identify

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the possible gram-negative bacteria, which would be a possible Enterobacteriaceae, or

the confirmation of the possible error in the gram-staining procedure. The Yeast Plus

Test [Figure 6] was to identify the type of yeast species that was present.

Figure 5 - The EnteroPluri Test results to test Figure 6 - The RapidID Yeast Plus Test to
the gram-negative bacteria incubated at 35oC identify the yeast species incubated for 4 hours
for 24 hours. at 35oC.

The EnteroPluri Test confirmed negative results for all biochemical tests except

the urea test, which was positive (pink colour production). Glucose is an important

reaction for Enterobacteriaceae species as most produce a positive reaction; therefore,

if it’s negative, then it’s most likely not a Enterobacteriaceae. Due to possible errors, we

kept glucose as a possible positive reaction. The microcode analyzed was 20002, and

concluded species was Yersinia pseudotuberculosis. The Yeast Plus Test confirmed a

positive result for glucose (yellow reaction), urea (pink), histidine (pink), and LGY (pink).

The alpha-glucose and beta galactosidase produce a faint yellow, so we analyzed the

possible species and decided to reject the results. The code was 100046 and conclude

a Candida krusei species.

9. Discussion

The 2.5 mL filtered soda sample was the only one in range, therefore the other

volumes were rejected. In addition, all filter bottle samples gave too low of a range;

hence, also rejected. The “gram positive” microorganism was concluded to be yeast.

Furthermore, the gram-negative bacteria were mostly likely a source of error which
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could have been due too much decolorizer. Media contamination might have occurred

during the plating process or the lack of aseptic technique during the experimental

process, especially the filtration process, may have caused some of the cell growth for

invalid results. The Yeast Plus Rapid ID confirmed the present of a possible Candida

krusei species and a very low possibility of Rhodotorula rubra. Based on the

morphology of R. rubra, the very low possibility was accurate because R. rubra is

uniquely characterized by its orange pigment on TSA or SDA media. The yeast colonies

produced on the TSA plate were white colonies, so the analysis of R. rubra being the

yeasts species were rejected and Candida krusei was kept. The Entero Pluri Test

confirmed the very low possibility of a Yersinia pseudotuberculosis in the sample; or

confirmed the gram staining error to be gram positive instead. As well, the Glucose

reaction was not producing a positive result, which indicates a Enterobacteriaceae

species was not found on either Coca-Cola samples. Based on further research of Y.

pseudotuberculosis, there weren’t any articles indicating the presence of any

contamination in carbonated drinks; therefore, the Y. pseudotuberculosis could not have

been the bacteria isolated. Moreover, the fountain Coca-Cola sample contained more

microorganisms than the Coca-Cola bottle sample.

The adjustment of the experimental methodology can be produced, if repeating

another trial of the procedure such as, using EMB plates as the membrane filtration

media to get more accurate coliform isolation or gram-negative bacteria and inhibiting

the gram-positive bacteria or use other differential media (e.g., MSA, etc.) to isolate the

coccus species isolated in the gram-staining procedure.

10. Conclusion

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 In conclusion, hypothesis 1 states that the Coca-Cola obtained from the self-

serve fountain dispenser will contain a higher concentration of coliforms, yeast, and/or

Listeria due to the microbiological build-up of flora in the machine, and the soda fountain

sample did produce a higher yeast concentration compared to the bottle Coca-Cola

sample, but not Listeria bacteria; therefore, the hypothesis was not rejected, but a

second trial can be implemented. The second hypothesis states that the standard plate

count of the self-serve soda fountain sample will have more colony growth than the

bottled sample and the hypothesis was not rejected as the soda fountain sample did

contain more colonies than the bottled sample.

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