Escherichia Coli Proliferation in

Chemically-Induced Tryptic Soy Agar

Tirth Patel and Rafiu Billah

Woburn Collegiate Institute
March 26, 2017
Table of Contents

Abstract 3

Background Research 3-4

Experiment-Related Research 5

Research Questions 5-6

Key Words 6

Purpose 6

Hypothesis 6

Materials 6-7

Procedure 7-8

Observations 8 - 12

Results 13 - 15

Discussion 15 - 17

Conclusion 17 - 18

Application 18 - 19

Bibliography 20 - 21

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Abstract

Diagnosing E.coli-O157:H7 infections in humans can take approximately 48-72 hours. During
this time, the patient can fall ill and experience severe symptoms of the infection. Our
experiment focuses on increasing the growth rates of E.coli-JM101 by inducing tryptic-soy agar
with various chemicals, ultimately enhancing the E. coli diagnosing process.

Background Research
Introduction
Gastrointestinal infections cause bloody diarrhea, nausea and an array of other
symptoms (E. coli, 2017). They are predominantly caused by an influx of bacteria in the
intestines or most notably, through the introduction of a pathogenic strain of bacteria from
uncooked or raw meat (Diseases and Conditions, 2017). Amongst the various strains of
pathogenic intestinal bacteria, Escherichia coli is the most well-known and common type of
strain related to food poisoning (E. coli, 2017). This pathogenic strain of bacteria can even
lead to potential death due to the weakening of the immune system. Additionally, this bacterias
diagnosing process is also symptomatic, as it takes between 48-72 hours to culture the bacteria
prolonging the patients treatment for the bacteria and the accuracy of growing the culture (Dr.
A. Vimal, Personal Communication, 2017). This is a recurring problem not only in Canada, but
in every other country in the entire world that consumes cattle as well. However, this is an even
greater concern within developing countries that have poor hygiene and sanitation conditions
when maintaining livestock (E. coli, 2017).

E. coli
What is it?
E. coli are strains of gram-negative aerobic bacteria commonly found in the
gastrointestinal tracts of humans and animals (Public Health Agency of Canada, E.
coli, 2017). Most strains of E. coli tend to be harmless, however, there are a few strains
that can cause sickness within individuals. For example, E. coli O157: H7 can cause
people to have severe stomach cramps, diarrhea and vomiting (Public Health Agency of
Canada, E. coli, 2017). E. coli infections occur most commonly due to uncooked meat,
raw vegetables or unpasteurized milk. E. coli occurs frequently in countries all over the
world, however they predominantly occur in developing countries where people are more
exposed to contamination (Public Health Agency of Canada, E. coli, 2017).

How often do infections occur?

E. coli infections are widespread throughout the entire world as they can occur anywhere
in which cattle is consumed. In recent years within Canada, an average of 440 cases of
E. coli infections have been reported annually (E. coli Surveillance, 2017). However,
this number does not include many community or provincial outbreaks that unfortunately
have not been reported at the national level (E. coli Surveillance, 2017).

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 Although this graph depicts a decline in E. coli O157: H7 incidences, they have
increased after the years following 2010 (E. coli Surveillance, 2017). Also, in recent
years, non-O157: H7 incidences have also gone up. We are no longer only concerned
about E. coli O157: H7, but are also concerned about other pathogenic E. coli strains as
well. Antibiotic resistance and genetic mutations are causing an increasing difficulty in
eradicating E. coli infection incidences (Koebler, 2013). Therefore, increases in the
number of E. coli infection rates are going to be common in upcoming years. Thus, any
kind of methods or procedures to assist in the process of reducing the harmful impacts
of E. coli are going to be a predominant need in the near future.

How soon do symptoms appear after exposure?

Symptoms typically do not appear until 3 to 4 days after exposure to pathogenic E. coli
(General Information: E. coli, 2015). This period of growth within the human body is
referred to as the E. coli incubation period. During this time, symptoms begin with mild
belly pain and nonbloody diarrhea and as the days progress the symptoms become
worse (General Information: E. coli, 2015). Thus, treatment of E. coli is essential as it
may threaten a persons immune system.

Bacterial Growth? Time?

According to LifeLabs, the incubation growth period for either non-pathogenic or
pathogenic E. coli is 48-72 hours. At LifeLabs, E. coli is grown in the medium, tryptic-soy
agar. (LifeLabs Ontario, Personal Communication, 2017)

Most Common Pathogenic Type of E. coli:

Escherichia coli (E. coli) normally live within the intestines of people and different kinds
of animals. Majority of E. coli tend to be non-pathogenic, however some E. coli are
pathogenic and can cause illnesses such as diarrhea or other complications outside of
the intestinal tract (General Information: E. coli, 2015). E. coli that causes human
illness are categorized into various pathotypes, and six are associated with diarrhea
(General Information: E. coli, 2015).

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 These six pathotypes also contain the most common type of pathogenic E. coli, which is
the shiga-toxin producing E. coli. The most common strain in North America is E. coli
O157: H7(General Information: E. coli, 2015).

Experiment-Related Research
E. coli Strain
The E. coli strain that we have chosen to experiment with is the non-pathogenic E. coli
strain K12 JM101. This K12 strain itself was sequenced in 1922 at Stanford University because
it approximates wild-type E. coli (Bachman, 1972). The E. coli strain K12 JM101 has fewer point
mutations than other compared strains and is commercially used for laboratory experiments
throughout the microbiology field (Ferenc, 2014). The strain is considered to be an ideal model
organism for E. coli O157:H7 (pathogenic) and is adapted for the laboratory environment
(Ferenc, 2014). Also, unlike other strains, E. coli K12 JM101 has not been mutated or
sequenced for specific purposes (E. coli O157:H7 and Genetic Engineering, 2001). Thus, with
the benefit of being non-pathogenic, the E. coli strain K12 JM101 strain was our ideal choice of
bacteria for studying E. coli growth rate, specifically related to the pathogenic E. coli strain
O157: H7.

Chemicals Chosen:
Ammonium Carbonate:
Ammonium carbonate is a colourless crystalline solid or a white powder that was used
previously as a leavening agent before baking soda, or sodium bicarbonate (Ammonium
Carbonate, 2005). Ammonium carbonate also acts as an acidity regulator, for it is a
base, and is readily soluble in water (Ammonium Carbonate, 2005). Our decision to
use this compound was primarily based on the fact that it contains a source of nitrogen,
hydrogen and phosphorous, which are all essential nutrients for E. coli bacterial growth
(Todar, 2012). Along with the compounds easy availability, it is readily soluble and has
sources of hydrogen, nitrogen and phosphorous (Kaiser, 2006). Therefore, Ammonium
Carbonate was the ideal chemical to use in this experiment.

Disodium Phosphate:
This compound is commonly used in desserts and pudding to quicken cook time and is
also used in Jell-O as a thickening agent (Schrodter and Bettermann, 2008). Disodium
Phosphate is a basic compound, and is a phosphate salt, which is something that is
required for cell growth (Kaiser, 2006). Thus, we decided to induce tryptic soy agar with
this essential and important nutrient for cell growth.

Purpose

The purpose of our experiment is to enhance the speed or accuracy of the E. coli diagnosing
process. The current diagnosing process takes 48 - 72 hours, and during this time the bacteria
uses a secretion system to disrupt host immune responses and infect gut cells (How
Pathogenic E. coli Bacterium Causes Illness, 2011). There is also a margin of error in the
diagnosing process as pathogen growth can occur from airborne diseases (How Pathogenic E.

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coli Bacterium Causes Illness, 2011). Thus, by using chemically induced mediums, we expect
to either increase the speed of the diagnosing process or develop a more accurate medium of
growing E. coli.

Research Questions
What effect will tryptic soy agar induced with ammonium carbonate have on E.coli K12
JM101?
What effect will tryptic soy agar induced with disodium phosphate have on E. coli K12
JM101?
Will E. coli K12 JM101 grow the same way in different mediums?
How will the chemically induced mediums differ from the regular TSA?

Keywords

E. coli O157:H7: most-common North American pathogenic strain of E. coli, causes infections
E. coli K12 JM101: non-pathogenic strain of E. coli, used in this experiment
Tryptic Soy Agar (TSA): medium in which E. coli is grown
Disodium Phosphate: chemical used in this experiment
Ammonium Carbonate: chemical used in this experiment

Hypothesis

If tryptic soy agar is induced with a nitrogen source (ammonium carbonate) and a phosphate
salt (disodium phosphate), then the growth rate of E. coli JM101 will increase. We believe that
there will be a clear difference in the growth rates of the E. coli on the regular tryptic soy agar in
comparison to the growth rate on the chemically induced tryptic soy agar.

Materials/Apparatus
3 Beakers (250 mL)
1 Beaker (1000 mL)
Double Distilled Water (1000 mL)
Graduated Cylinder (100 mL)
2 Graduated Cylinders (10 mL)
3 Petri Dishes with Covers
Hot plate
Magnetic stir bar
Tongs
Weigh boat
Electronic Laboratory Scale
Disodium Phosphate 7.5% solution (10 mL)
Ammonium Carbonate 15% solution (10 mL)
Tryptic Soy Agar (16 grams)
Incubator

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 Isopropyl Alcohol
E. coli JM101 specimen
5 Plastic Inoculating Loops
LabQuest 2 Vernier pH meter
2 Glass Slides
Microscope

Procedure
Sterilization
1. Sterilize countertop with Isopropyl Alcohol.
2. Obtain materials and place them on the sterilized countertop.
3. Sterilize the beakers and graduated cylinders with hot water and alcohol.

Making of Tryptic Soy Agar

1. Place weigh boat on the electronic scale and tear the mass. Weigh 16 grams of TSA on
the weigh boat.
2. Obtain a 1000 mL beaker and measure 384 milliliters of double distilled water
3. Place the measured TSA in the beaker containing DD water.
4. Carefully drop the magnetic stir bar inside the solution. Place the beaker on top of the
hot plate.
5. Using the magnetic stir bar, mix the solution for 2 minutes at a maximum setting of 10.
6. After TSA dissolves in DD water, turn the heat option to a maximum of 10. Using a
thermometer stabilize the temperature at 85 degrees Celsius for 5 minutes.
7. Using tongs, place the hot beaker containing TSA on the countertop.

Making the Different TSA Solutions

1. Pour 70 mL of TSA in a 100-mL graduated cylinder.
2. Using the graduated cylinder, placed 30 mL of TSA into one beaker (250 mL), and 20
mL of TSA into two beakers (250 mL).
3. Measure 10 mL of disodium phosphate and 10 mL of ammonium carbonate with the two
10 mL graduated cylinders.
4. Pour the 10 mL of solution into two different beakers containing 20 mL of TSA,
respectively.
5. Using a stirring rod, mix all three beakers containing TSA.

Preparing TSA Plates

1. Obtain three sterile petri dishes.
2. Pour each beaker of TSA into one petri dish, respectively. Cover the petri dish and let
the TSA solidify for 30 minutes.
3. Label the petri dishes according to their contents and time.

Preparing E. coli TSA Plates

1. Obtain the E. coli JM101 specimen and three plastic inoculating loops.
2. Using the inoculating loop, obtain a specimen of E. coli at one end.

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 3. Using the swabbing technique, make a Z pattern 4 times horizontally and vertically on
the solidified TSA.
4. Dispose the inoculating loop.
5. Repeat Steps 1-4 two times with the remaining TSA petri dishes.
6. There will now be three E. coli TSA plates. Place the three plates inside an incubator set
at 37 degrees Celsius.

Preparing an E. coli Slide for Microscopic Analysis:

1. Gently remove the E. coli bacteria from the middle of the regular TSA plate.
2. Place the E. coli on a clean glass slide and view it under the microscope.
3. Dispose the inoculating loop.
4. Repeat steps 1-3 from the TSA induced with disodium phosphate.

Observations

Tryptic Soy Agar 30 mL

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Tryptic Soy Agar 20 mL + Disodium Phosphate 10 mL

Tryptic Soy Agar 20 mL + Ammonium Carbonate 10 mL

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Under the Microscope - Regular TSA

Region A (Large) Region B (Small)

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Under the Microscope - TSA + Disodium Phosphate

Region A (Large) Region B (Small)

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pH of Double Distilled Water:

pH of Ammonium Carbonate:

pH of disodium Phosphate:

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Results
After conducting the experiment and taking photographs after every 24-hour period, we are now
able to analyze the growth of E. coli in the different mediums. The controls in our experiment
include the temperature which was set to 37 degrees Celsius and sterilization of all materials.
We conducted the experiment for each petri dish in the same environment (air quality,
temperature, lighting etc.). Every 24 hours, the TSA plates were removed from the incubator for
2 minutes to observe and take a photograph.

Regular TSA:
0 Hours:
o After initially plating our TSA induced with disodium phosphate, we can see that
there is some condensation under the lid of the petri dish, and the TSA medium
is a very faint clear yellow colour. Condensation indicates that the bacteria is still
increasing in number.
24 Hours:
o After one day, we can see that there is an adequate growth of bacteria in places
where we streaked the plate. However, throughout the dish there are multiple
spots of fungal growth. The colour of the medium has turned a darker yellow.
48 Hours:
o Visually, no further significant growth of bacteria can be seen, however, an
increase in fungal growth is present, especially around the edges of the dish. The
spots of fungus across the plate have gotten larger. The colour of the medium
has become a darker shade of yellow.
72 Hours:
o The colour of the medium is yellow, significant growth of fungus especially
around the edges of the dish. The spots of fungus across the dish have grown
larger, it is now difficult to differentiate between fungal colony and bacterial
colony. Visually, there is no further increase in bacterial growth.
96 Hours:
o Another significant increase in fungal growth, the colonies are now growing on
top of one another. Fungal spots across the petri dish have gotten larger. Fungus
around the dish appear to be moving away from the edges in circular colonies
towards the edge of the dish. The colour of the dish has once again turned a
darker colour of yellow then the previous day. Visually, there is no growth of
bacteria.
120 Hours:
o Fungal colonies appear to be moving out even further onto the dish in circular
colonies. They also retain the space in between them when moving out,
therefore, it is evident that the fungal colonies are aware of each other. There is
no visual increase in bacterial growth. It is very hard to differentiate between
fungus and bacteria.

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Disodium Phosphate:
0 Hours:
o After initially plating the TSA induced with disodium phosphate, we can see that
there is some condensation under the lid of the petri dish, and the TSA medium
is a faint transparent yellow colour.
24 Hours:
o After one day, we can see that there is an adequate growth of E. coli in the
medium. The colour of the medium has become more transparent, and the E. coli
has grown in every place that we streaked the petri dish.
48 Hours:
o After two days, a significant increase in bacterial growth cannot be seen,
however around the edge of the dish, a few colonies of E. coli can be seen. No
evident fungal growth can be seen. Additionally, the colour of the medium in
which the bacteria is grown has turned a darker hue of yellow.
72 Hours:
o After three days, a further increase in bacteria has been observed. The places in
which the plate was streaked with bacteria now has some colonies growing
vertically.
96 Hours:
o Bacterial growth relatively stayed the same, however an increase in
condensation can be seen, yet no fungal growth can be seen.
120 Hours:
o Areas of some fungal growth around the edges of the petri dish. The colour of the
medium is still the same as before. There is no increase in bacterial growth.

Ammonium Carbonate:
0 hours:
o After the initial plating of TSA induced with ammonium carbonate, we can see
that there is some condensation under the lid of the petri dish, and the TSA
medium is a faint transparent yellow colour.
24 hours:
o After one day, the petri dish is still transparent while retaining its faint yellow
colour from before. There are no signs of bacterial or fungal growth.
48 hours:
o After two days, the petri dish is still resembling the same characteristics as
before. However, there is a faint diagonal line visible in the middle of the dish.
This line may be an incision from the inoculating loop when it was used to swab
the E. coli on the TSA.
72 hours:
o After three days, the petri dish remains the same as it is still transparent and
faintly yellow.
96 hours:

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 o After four days, the incision made by the inoculating loop is clearly visible. There
are a few fungi colonies in different areas of the plate. However, there is no sign
of E. coli growth.
120 hours:
o After five days, there is still no sign of E. coli growth. There are a few spots a
fungal growth and the incision made by the inoculating loop is infected. The TSA
induced with ammonium carbonate did not show any potential for E. coli growth

Individual Strains
To compare and contrast the number of individual strains that grew in regular TSA and TSA
induced with disodium phosphate, we took a small sample from the middle of the petri dish. We
put that small sample on a glass slide and observed it underneath the microscope. We took a
photograph of the E. coli underneath the microscope and analyzed the bacterial growth by
counting the number of individual E. coli strains in two distinct parts, region A (4 mm squared)
and region B (1 mm squared). After using a data imaging method, we were able to count each
individual strain in a given area. The results were as follows:

TSA TSA + Disodium Phosphate

Region A (4 mm^2) ~221 ~176

Region B (1 mm^2) ~58 ~56

In Region A, which was measured to be 4 mm squared the total individual strain count for
regular TSA was approximately 221, compared to TSA with disodium phosphate which had 176.
In Region B, which was measured to be 1 mm squared the total individual strain count for
regular TSA was approximately 58 in comparison to TSA with disodium phosphate which had
56.

Therefore, the number of individual strains in regular TSA and TSA with disodium phosphate
are closely related. The smaller regions provided evidence of close relatedness between the
two TSA plates as they were approximately the same number.

Discussion
From our lab data, we have observed that by inducing TSA with ammonium carbonate and
disodium phosphate, the growth rates of E. coli K12 JM101 does not increase. Thus, our
hypothesis was nullified.

As evident in both of our plates of chemically induced TSA, there wasnt a significant
increase in the growth of E. coli JM101. In the case of the ammonium carbonate, the growth of
E. coli was completely inhibited and there was no growth of the bacteria or any fungus.
However, we can confirm that pH was not a factor towards bacterial inhibition, but rather it is
apparent that the ammonium, carbonate or the entire compound itself is responsible for our
results. The ammonium carbonate has a pH of 8.76 and the disodium phosphate has a similar

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pH level of 8.72, a pH that is slightly less basic than the ammonium carbonate. The E.coli
JM101 was able to thrive and grow in the disodium phosphate, however unable to grow in the
ammonium carbonate.

Additionally, ammonium is a naturally occurring compound used in food processing as a

leavening agent. It has been found that ammonium, specifically in ammonium hydroxide, is able
to destroy the acidic compounds in the intestinal tracts of animals and can cause the
environment of the E. coli to be unsuitable for habilitation (Questions and Answers about
Ammonium Hydroxide Use in Food Production, 2009). Also, raising pH levels of the medium in
which the E. coli is grown to 8.5 or 9 can cause the bacteria to die off (Todar, 2012).
Furthermore, carbonates, such as ammonium carbonate, can hasten the growth of bacteria or
even destroy them completely (Park, 2003), however a study by the Department of Microbiology
of Cornell University has shown that this property is recurring only in carbonates, because when
an alternative solution such as ammonium chloride is used to increase the pH of the medium to
8.5, the bacteria is not able to thrive and the compound proves to be ineffective in eliminating
the E. coli (Jarvis, 2001). Therefore, in our experiment, since we combined the use of
ammonium as well as a carbonate, we believe that the inhibiting effects of both ammonium and
carbonate decreased or potentially even eliminated the E. coli in our sample. As a result, we
were unable to witness any E. coli growth in the TSA sample induced with ammonium
carbonate.

In the other sample, we induced TSA with disodium phosphate and were able to see
relatively similar growth rates of E. coli in comparison to the E. coli grown in regular TSA.
Phosphates such as sodium phosphate are typically given to people over the age of 18 before a
colonoscopy in order to clean the colon (Sodium Phosphate, 2016). It acts as a laxative and
helps to empty out the bowels (Sodium Phosphate, 2016). However, phosphates generally
have been found to not inhibit microbial growth by the Institution of Food Microbiologists
(Molins, Kraft and Olson, 1985). Additionally, phosphate pH changes have also been found to
not have an effect on bacterial numbers (Molins, Kraft, and Olson, 1985). Therefore, we
presume that our bacteria was able to thrive on the disodium phosphate because not only are
phosphate salts important for bacterial growth, but at the same time, phosphates have been
found to not affect bacterial growth. The growth rates of E. coli on both the sodium phosphate
and regular TSA was found to be on average 57 individual cells per square millimetre of the
petri dish.

Although phosphates are essential to E. coli growth, it only constitutes to helping the E.
coli in developing nucleic acids, nucleotides, phospholipids, and teichoic acids (Todar, 2012).
They do not necessarily help the bacteria in all other functions. Therefore, we believe that a
medium filled with only one type of nutrient essential for E. coli growth does not allow the
bacteria to rapidly reproduce. Even if the bacteria is receiving nutrients to help develop one
aspect of the organism's body, it does not necessarily mean it can reproduce rapidly since other
aspects of its body still may be lacking other essential nutrients required for cell growth.

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 However, the sodium phosphate medium was able to successfully inhibit fungal growth
over the course of the 72-hour period and still allowed the E. coli to grow. When we looked at
the sample of E. coli under the microscope at the 72-hour period, it was evident that they were
all alive as we could see the E. coli moving around. According to the University of Hawaii, most
fungi require large amounts of water to grow (Fungal Diversity, n.d.), and according to Roger
Vertefeuille from Legend Technical Services, most fungi prefer a water activity of over 0.9
(Vertefeuille, 2010). Water activity is defined as a measure of the water within a substrate that
an organism can use to support growth, and is measured on a scale from 0 - 1.0 (Vertefeuille,
2010). The closer the number is to 1.0, the more water activity an organism requires
(Vertefeuille, 2010). As disodium phosphate is a salt, Robert LaBudde from the food science
department of Least Cost Formulations comments on how salts remove the available water in a
medium (LaBudde, 1999). Thus, since fungi generally require higher levels of water then
bacteria, and since disodium phosphate is a salt that is proven to not have an imminent effect
on bacterial growth, we believe that the TSA induced with disodium phosphate was able to have
E. coli grow without any fungal growth.

Conclusion
It was hypothesized that ammonium carbonate and disodium phosphate would increase
the growth rate of E. coli K12 JM101. However, the data from our experiment shows that our
hypothesis was nullified. The ammonium carbonate did not allow the E. coli to thrive at all,
whereas the disodium phosphate had similar growth rates compared to the E. coli grown in
regular TSA.

One factor that may have led to no E. coli growth on the TSA induced with ammonium
carbonate is the fact that carbonate appears to have a unique effect on inhibiting or killing off E.
coli. We know that it is the carbonate that has this unique effect and not the ammonium because
past research from Cornell University has shown that ammonium in other compounds such as
ammonium chloride, actually allow for E. coli growth, whereas sodium carbonate was effective
in eliminating the E. coli. In our experiment, we used sodium in the compound disodium
phosphate and we were able to witness E. coli growth. Therefore, we can conclude that
carbonate itself has a unique inhibiting factor on E. coli.

Opposed to the ammonium carbonate, the TSA induced with disodium phosphate was
still able to retain similar E. coli growth rates. Past studies conducted by the Institution of Food
Microbiologists, have shown that phosphates do not influence bacterial numbers. Even if the
phosphate does induce a pH change, it still does not necessarily affect bacterial numbers.
Therefore, we can conclude that the disodium phosphate, although an essential nutrient for
bacterial growth, does not necessarily affect bacterial growth rate.

Additionally, there wasnt a significant growth of E. coli in the disodium phosphate

medium due to the fact that the E. coli was exposed to one type of essential nutrient. Although
the E. coli may have that necessary nutrient to develop specific aspects of their body, they still
may be lacking nutrients for other parts of their body. Thus, since they are unable to develop
their entire body as a whole, none of them actually had a reproductive or survival advantage.

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Therefore, E. coli growth rates in the disodium phosphate did not show a significant increase in
numbers, and resulted in the same growth rate as regular TSA.

There are multiple things that we could have done in order to ensure that the data
supports our hypothesis. In order to ensure that the compound does not have a unique inhibiting
effect on bacterial numbers, we can conduct further research into the makeup of the compound
itself that we are planning to use. Furthermore, rather than inducing the TSA with only one
essential nutrient, we can add a variety of nutrients that are all essential for E. coli growth. As a
result, we believe that since our bacteria will have all the nutrients required for full body growth,
there will be a significant increase in bacterial numbers on the TSA induced with numerous
nutrients.

Application
Grow bacteria without any fungal growth in TSA
Wrongful diagnosis of an E. coli infection can be detrimental to a patient's health and the
treatment provided. From our experiment, we witnessed the growth of fungi and bacteria in our
normal TSA which is currently being used at LifeLabs. This shows that regular TSA is inefficient
and can lead to numerous growths of airborne diseases. The regular TSA plate that we
prepared was exposed to the same setting as the TSA plate with disodium phosphate, however,
the disodium phosphate did not show any growth of fungi but rather the same amount of E. coli
growth as regular TSA. Thus, the disodium phosphate proved to be more accurate and efficient
as it eliminated growth of fungus and allowed the growth of E. coli. E. coli in both plates was
similar and comparable as the colour, size, and appearance were evidently the same.

This information can be applied to the current diagnosing process of E. coli that
eliminates any pathogens travelling in the air. Airborne diseases are a great concern when
growing TSA cultures as they are caused by pathogenic microbes small enough to be
discharged from an infected person (Infectious Disease Epidemiology Program,
2017). Therefore, by using disodium phosphate not only will the E. coli growth results be more
efficient but they will also decrease the rate of false diagnosis, removing any external growth of
fungus and bacteria, while focusing on growth of E. coli.

Use of carbonate in order to eliminate E. coli in cow fecal matter

One of the main causes for E. coli infections is when humans come into contact with
feces or stool from cattle, humans, and various animals (E. coli Infection From Food and
Water, 2016). In addition, due the presence of E. coli in cattle, the meat must be thoroughly
cooked at a temperature of 71 degrees Celsius (E. coli Infection From Food and Water, 2016).
If this is not done, the bacteria can still survive and inadvertently get into the consumers
intestine, thus causing an E. coli infection. This is the most common way of getting an E. coli
infection in Canada and the United States (E. coli Infection From Food and Water, 2016).
However, this becomes an even greater problem in developing countries where sanitation,
proper cooking materials, and hygiene especially for food preparation isnt readily available.

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 Through our research, we can confirm that previous studies conducted by scientists on
carbonates and its effect on E. coli do result in an inhibiting growth of E. coli. In the study done
by Cornell University, scientists used ammonium chloride and sodium carbonate, similarly we
also used two bases with one of them containing carbonate: one being sodium phosphate, and
the other being ammonium carbonate (Jarvis, 2001). In both of our cases, the carbonate
compound effectively inhibited or presumably destroyed all the bacteria, whereas the other
compound allowed the bacteria to grow regardless of pH. Therefore, by exposing fecal matter of
animals or possibly even raw meat to some form of carbonate, we can readily destroy all the E.
coli on the product. As a result, we can prevent E. coli contamination of water sources that could
have been contaminated by surface runoff that containing E. coli infected fecal matter.

From our calculations, we can conclude that this is also a very cost effective method, in
which the average Canadian grocery store sells baking soda at about $0.89 CAD per 100 grams
of sodium bicarbonate (Baking Powder, 2017). Thus, we believe that our study has a great
implication towards naturally inhibiting E. coli from getting into food and water supplies not only
in Canada, but the entire world as well.

Possibly create a medication that uses ammonium carbonate

According to Mayo Clinic, there is currently no treatment that can cure E. coli infections
(Diseases and Infections, 2014). In fact, according to the Centers for Disease Control and
Prevention, taking antibiotics or diuretics can actually worsen the effects of the infection and
make the infection more difficult to get rid of (General Information: E. coli, 2015). Thus, we
believe that further research into ammonium carbonate or possibly even the carbonate
compound itself can reveal a natural antibiotic in the near future in order to cure an E. coli
infection. In a day and age where antibiotic resistance and genetic mutations are very common
in bacterial strains, including E. coli, it is very important that we discover safe ways of dealing
with such bacterial infections.

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