Russell - Zabel 1

Introduction

In the United States, ​Escherichia coli ​causes 73,000 illnesses annually (Rangel, et al.​).

Although the bacteria can be so harmful, it is also necessary to daily life. It lives in the lower

intestines of mammals and helps to breakdown food and assist with waste processing

(​Escherichia Coli). The aim of this experiment was to possibly find a way to free foods or water

from harmful ​E.coli ​levels by applying different pH levels and temperatures; or even just

eliminate a way to reduce these levels.

The experiment’s goal was to observe how various pH levels and temperatures affected

the amount of ​E.coli ​that grew. Three different pH levels were used, one of which was applied to

a paper disc; then inserted in a Petri dish prepared with ​E.coli​. After the pH treatment was

applied, the dish would go on to be placed in one of the three incubators.

When stored at 0 °C (32 °F), ​E.coli ​growth completely stops; if temperatures reach -18

°C (0 °F), the bacteria begins to die off. When held between 4 °C (39 °F) and 7 °C (44 °F), ​E.coli

growth drastically slows, but the bacteria is not harmed at all by it. ​E.coli ​grows most quickly at

room temperature, above 10 °C (50 °F); when temperatures reach 70 °C (160 °F), ​E.coli ​begins

to die. Many foods, including meat, are cooked at these high temperatures to insure that it is safe

to eat and free of bacteria (Clay).

E.coli ​grows best around 37 °C (98.6 °F) in the intestines of mammals, and around the

edges of hot springs. Which is why the three temperatures that were chosen were 36 °C, 37 °C,

and 38 °C. It may seem that the band width was too close, but one degree difference can cause

notable changes in the data produced. Think of having a fever, the body's temperature only goes

up a degree and one can find themselves being “sick”.

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Previous experiments show that the bacterium- ​E.coli​, does not grow at all below a pH

level of four, but grow at identical rates at pH levels of six, seven, and eight (Schilling). Six,

seven, and eight were originally the pH levels chosen for this experiment because the standard

would have been great at a neutral pH, but no good data was produced. Instead, a blank disc was

treated with pH levels at ten, eleven, and twelve in attempts to create a zone of inhibition. This

method of treatment was chosen because affecting the pH of the agar did not seem to have an

effect on the bacteria. Also, the amount of ​E.coli ​produced in this case gave numbers that were

good for simply counting the colonies, not measuring the zone of inhibition.

Since this experiment aimed to find how pH and temperature affect ​E.coli ​amounts, the

data was measured in the number of colonies in one Petri dish. One colony of ​E.coli ​is equal to

about 10,000 ​E.coli​. This method of data measurement was chosen to get reliable data, and it was

very simple compared to the other few ways that could have been a possibility for recording

data.
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Problem Statement

Problem

How will the change of temperature and pH of a disc affect the growth of Escherichia

coli to be measured in the number of colonies over a two week period?

Hypothesis

If the pH of the disc is ten and the Petri dishes are stored at 38℃ (-,+), then the number of

E.coli colonies will be the greatest compared to the other trials.

Data Measured

In this experiment, the independent variables that will be tested include pHs at ten,

eleven, and twelve; the other independent variable will be temperatures set at 36 ℃, 37 ℃, and

38 ℃. The dependent variable in this experiment will be the number of E.coli colonies. To

analyze the data, multiple box plots will be used to see the statistical significance. For the pH, the

standard will be eleven, because it is a good base for ​E.coli​. The low for pH is ten, because it's

more acidic than eleven, but if it were any lower, there would be no good data. The high for pH

is twelve, because it’s more basic than the standard, but if it is too high, there would be little to

no growth. For the temperature, the standard will be 37℃, the low will be 36℃, and the high

will be 38℃. All three of these are chosen because that is what the incubators will be preset at.
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Experimental Design

Materials

Escherichia coli​ (E.coli) Hot Mitt

(40) Petri Dishes 1 cm x 1 cm x 10 cm Hot Plate
Incubator set at 36 ℃ 10 cm Transfer Loop
Incubator set at 37 ℃ Sharpie
Incubator set at 38 ℃ Bleach 200 ml
Agar Powder 34.5 G Stirring Magnet 5 mm
Sterile Water 1 L Flask 1 L
Eye Dropper 1 ml Spectral 1-14 Plastic pH Indicator Strips
Blank Paper Discs Ruler (mm)
Tweezers

Procedures

Agar preparation

1. Add one liter of water to a one liter flask or one liter beaker.

2. Place on a stirring hotplate. Place the hotplate on high.

3. Add a stirring magnet to the one liter of water. Place the magnet setting on #4

4. Slowly add 23 grams of agar powder to the flask be careful not to get it on the sides (a
full batch 1000 ml or one liter will fill 50 Petri dishes).

5. Allow the solution to turn from a cloudy liquid to clear like apple juice (the temperature
will be nearly 100 Celsius).

6. Remove from the heat and allow to cool so it can be poured.

7. Adjust the amounts to fit the needs of the experiment.( ie. 11.5 grams agar in 500 ml of
water for a half batch about 25 plates).

Without treatment added to agar

1. Follow the preparation directions on the agar container. Do not make a full batch of agar
unless all of it is going to be used (a full batch 1000 ml or one liter will fill 50 Petri
dishes).

2. Check with others in the class when making agar.

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With treatment in the Agar

1. Follow the preparation directions on the agar container. Do not make a full batch of agar
unless all will be used (a full batch 1000 ml or one liter will fill 50 petri dishes).

2. Check with others in the class when making agar.

Agar pouring

1. When pouring the agar into the Petri dish do not open the cover or expose the inside
of the Petri dish to the air.

2. Allow the vessel containing the agar (without any additive) to cool to the touch then add
the treatment. Be sure that the correct amount of treatment is added. Do not add more
than needed to the agar. Stir gently by swirling the vessel.

3. Always open and close the lid without placing the lid on the desk top.

4. Pour just enough to cover the bottom of the dish with a thin layer of agar.

5. Allow to cool without moving the Petri dishes (leave at their location on the desk).
Agar that gets on the lid of the Petri dish will ruin the poor and must be repeated.

6. When cool bacteria may be added. After adding, invert the plates and place them in the
correct incubator for the specific amount of time.

Starter dish

1. Take out three Petri dishes, put them in a row, and label them with the date they were
started with sharpie.

2. Prepare the agar and pour into all three Petri dishes, just enough to fill the bottom. Allow
agar to dry (for about five minutes) and it becomes gel before use.

3. Take out the sterilized water and put one milliliter into the beaker.

4. Grab a sterilized transfer loop and slide the loop across the ​E.coli​ sample, transfer the
E.coli​ into the beaker filled with a mililiter of water; making sure to spin the transfer
loop.

5. Pour the inoculated test tubes into the Petri dish without fully taking off the top of the
dish.
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6. Close the lid after pouring and slightly turn the Petri dish while shaking side to side to
spread the ​E.coli​.

7. After spreading the​ E.coli​ dump out the excess water into a sink without taking the entire
lid off.

8. Store in incubator.

9. Repeat steps 3-8 for two more starter Petri dishes.

Treatment #1 (pH) Standard

1. Take a flask containing ready agar and pour into the bottom of a Petri dish, pour just
enough to fill the bottom.

2. While agar is cooling, add about 20 ml of sterilized water into a beaker.

3. Test the pH of each beaker’s water using pH test strips.

4. To raise the pH to eleven, add seven drops of bleach to the water and mix
thoroughly.

5. Take a sterilized pair of tweezers and grab a blank paper disc out of its container using
the tweezers.

6. Drop the disc into the water in the beaker with pH eleven, leave out so the disc can
saturate with the water.

7. While the blank disc is being saturated, the agar in the Petri dish should be almost or
fully cooled.

8. Take Petri dish, a test tube, and a sterilized transfer loop, and repeat steps 3-7 under
Starter Dish​.

9. Using sterilized tweezers, retrieve the blank disc from beaker with water at pH eleven.

10. Place the disc in the middle of the Petri dish, and push gently down on it, until it stays
stuck in the agar when flipped upside down.

12. Store at 37 °C.

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Treatment #1 (pH) Positive

1. Take a flask containing ready agar and pour into the bottom of a Petri dish, pour just
enough to fill the bottom.

2. While agar is cooling, add about 20 ml of sterilized water into a beaker.

3. Test the pH of each beaker’s water using pH test strips.

4. To raise the pH to twelve, add nine drops of bleach to the water and mix
thoroughly.

5. Take a sterilized pair of tweezers and grab a blank paper disc out of its container using
the tweezers.

6. Drop the disc into the water in the beaker with pH twelve, leave out so the disc can
saturate with the water.

7. While the blank disc is being saturated, the agar in the Petri dish should be almost or
fully cooled.

8. Take Petri dish, a test tube, and a sterilized transfer loop, and repeat steps 3-7 under
Starter Dish​.

9. Using sterilized tweezers, retrieved blank disc from beaker with water at pH twelve.

10. Place the disc in the middle of the Petri dish, and push gently down on it, until it stays
stuck in the agar when flipped upside down.

11. If the treatment is (+,+), store at 38 °C. If the treatment is (+,-), store at 36 °C.

Treatment #1 (pH) Negative

1. Take a flask containing ready agar and pour into the bottom of a Petri dish, pour just
enough to fill the bottom.

2. While agar is cooling, add about 20 ml of sterilized water into a beaker.

3. Test the pH of each beaker’s water using pH test strips.

4. To raise the pH to 10, add five drops of bleach to the water and mix
thoroughly.
 Russell - Zabel 8

5. Take a sterilized pair of tweezers and grab a blank paper disc out of its container using
the tweezers.

6. Drop the disc into the water in the beaker with pH ten, leave out so the disc can
saturate with the water.

7. While the blank disc is being saturated, the agar in the Petri dish should be almost or
fully cooled.

8. Take Petri dish, a test tube, and a sterilized transfer loop, and repeat steps 3-7 under
Starter Dish​.

9. Using sterilized tweezers, retrieved blank disc from beaker with water at pH ten.

10. Place the disc in the middle of the Petri dish, and push gently down on it, until it stays
stuck in the agar when flipped upside down.

11. If the treatment is (+,+), store at 38 °C. If the treatment is (+,-), store at 36 °C.

Treatment #2 (Temperature) Standard

1. After Petri dish is prepared as a standard, positive, or negative, store dish at 37 °C.

Treatment #2 (Temperature) Positive

1. After Petri dish is prepared as a standard, positive, or negative, store dish at 38 °C.

Treatment #2 (Temperature) Negative

1. After Petri dish is prepared as a standard, positive, or negative, store dish at 36 °C.

Recording Data

1. Looking at the petri dish after treated for 24 hours, place a piece of grid paper under the
Petri dish and find how many colonies are in a certain square. Then multiply that one
square to equal how many colonies will be in the entire dish. Estimate the number to the
nearest ten.
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Diagram

Figure 1. Incubator Setup

The figure above shows the petri dishes that have been inoculated, and are being stored in

an incubator. Flask and test tubes on the right that are used for preparing agar and preparing the

plates.
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Data and Observations

Data:
Table 1
Design of Experiment Values
pH Temperature (° C)
- Standard + - Standard +
10 11 12 36 37 38

Table 1 shows the two variables used in this experiment (pH Level and Incubation

Temperature were used). The pH level of the blank discs were chosen because no significant

change in growth occurred at a lower pH. The incubation temperatures were pre-set at these

temperatures because ​E.coli​ Grows best around these temperatures, and the range of temperature

was kept close because multiple groups used the incubators for their experiment.

Table 2
Data Collected
Number of ​E.coli​ Colonies
DOE (pH, Temperature)
(+, +) (+, -) (-, +) (-, -) Standard
T1 4,000 9,500 10,000 2,000 5,000
T2 6,000 7,800 9,200 1,100 8,000
T3 3,000 550 8,100 6,000 7,900
T4 9,400 6,400 9,850 8,860 8,200
T5 5,400 8,000 9,000 5,600 5,800
T6 7,450 6,800 8,800 4,000 8,100
T7 6,300 1,000 9,750 4,500 7,400
T8 4,700 3,500 9,400 7,900 6,850
Average 5,781.25 5,443.75 9,262.50 4,995.00 7,156.25

Table 2 shows how many DOEs were completed and the exact sequence in which the

trials were executed. The data is measured in number of ​E.coli ​Colonies.

 Russell - Zabel 11

Observations:
Table 3
Observations
Date Observation
3/21/2016 (+, -) and (-, +) contained the most colonies
3/22/2016 Standard increased significantly
(+, -) dropped very far. May be caused by setup error or
3/28/2016
lurking variables. Although the other (+,-) was fine
3/29/2016 Gas from the burner smelled a lot stronger than usual
3/30/2016 The plates were moved slightly around in the incubators

Table 3 shows some notes taken throughout the experiment on anything that was out of

the ordinary, or may have been caused by human error. The trials were all meant to be run for 24

hours, but some days the plates would be ran more or less than 24 hours; due to biology class

being at a different hour everyday.

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Possible error caused by Petri

dish being moved around

Figure 2. Incubation Error

Figure 2 shows a possible way of error. A few of the data points were found to be quite

out of the ordinary, and this may have been the cause for some. It was found that a few times, the

Petri dishes that were being incubated got moved around in the incubator; shown above, this

specific dish got moved a whole shelf up, away from the other dishes.
 Russell - Zabel 13

Data Analysis and Interpretation

Table 4
Table of Factors
pH Temperature (° C)
- Standard + - Standard +
10 11 12 36 37 38

Table 4 shows the two variables used in this experiment (pH Level and Incubation

Temperature were used). The pH level of the blank discs were chosen because no significant

change in growth occurred at a lower pH. The incubation temperatures were pre-set at these

temperatures because ​E.coli​ Grows best around these temperatures, and the range of temperature

was kept close because multiple groups used the incubators for their experiment.

Table 5
Table of Averages
Runs
Averages
pH Temperature
+ + 5,781.25
- - 4,995.00
+ - 5,443.75
- + 9,262.50
Grand Average 6,370.63

Table 5 shows the average number of colonies of each treatment, these averages will be

needed to find a lot of important data in this experiment. Also shown above is the grand average,

another very important number that will be used later in the experiment. The grand average was

calculated by adding all of the data together, then dividing the sum by 40 (number of trials).
 Russell - Zabel 14

Table 6
Effect of pH Effect of pH

pH
(-) 10 (+) 12
4,995.00 5,781.25
9,262.50 5,443.75
Avg= 7,128.75 Avg= 5,612.50

Effect Value= -1516.25

Figure 3. Effect of pH

Table 6 shows the average of both high trials, and both low trials in regard to pH. The

two averages differ a little, but the high trials contained about 1516.25 less colonies than the low

trials, as shown in Figure 3 which shows a negative effect value. This effect value represents the

difference in the number of colonies from the high pH level compared to the low pH level. This

means on average, a Petri dish containing ​E.coli ​that is treated with a disc with pH 10 will

contain 1516.25 more colonies than a dish with a disc of pH 12. The effect value was found by

subtracting the low average from the high average to get -1516.25.
 Russell - Zabel 15

Table 7
Effect of Temperature Effect of Temperature

Temperature (° C)
(-) 36 (+) 38
4,995.00 5,781.25
5,443.75 9,262.50
Avg= 5,219.38 Avg= 7,521.88

Effect Value= 2302.5

Figure 4. Effect of Temperature

Table 7 shows the average of both high trials, and both low trials in regard to

temperature. The averages are further apart from each other compared to the averages of Table 5.

The 38 ℃ trials contained about 2302.5 more colonies than the 36 ℃ trials, as shown in Figure 4

which has a positive effect value. The effect value represents the difference in the number of

colonies from the high temperature compared to the low temperature. This means, on average, a

Petri dish that is stored at 36 ℃ will contain 2302.5 less colonies than a dish that is stored at

38 ℃. The effect value was found by subtracting the low average from the high average to get

2302.5.
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Table 8
Interaction Effect Interaction Effect

Temperature (° C)
(-) 36 (+) 38
Solid
(+) 12 5,443.75 5,781.25
Segment
pH
Dotted
(-) 10 4,995.00 9,262.50
Segment

Interaction Effect= -1965

Figure 5. Interaction of pH and Temperature

Table 8 shows the averages of the trials again, but this time they are being put into a

graph, Figure 5, to show the interaction of pH and Temperature. The interaction effect (-1965) is

the interaction between the two variables (pH and Temperature). The slope of the segment of the

high values (+, -) and (+, +) minus the slope of the segment of the low values (-, -) and (-, +)

gives the effect (pH vs Temperature) = -1965. The slope of the solid segment is 168.75 and

represents the high values. The slope of the dotted segment is 2133.75 and represents the low

values. The two segment intersect and end up slightly far apart on the positive side of the graph;

meaning that the data shows a possible interaction between pH and Temperature in this

experiment. The interaction effect represents how the two variables were affected by one

another. In this case, the graph shows that there was possibly some interaction with pH and

Temperature. The interaction effect was found by subtracting the (+, -) from the (+, +) and
 Russell - Zabel 17

dividing that by two, then repeating the same process using the (-, -) and the (-, +) respectively,

then subtract the slope of the lows from the slope of the highs for the final result.

Figure 6. Eight Standard Runs

Figure 6 shows the results of the eight standards that were ran. The results are in order

from when it was ran. The data shows no specific trend, but there are a couple of points that are

slightly lower than the others. The standards that were ran serve as a basis to the rest of the

experiment; meaning if the range of standards is close to the range of the data, there may have

been some lurking variable that affected the data, or the experimental design itself needed to be

changed.

Dot Plot of Effects

Legend:
I - Interaction Effect
P - Effect of pH
T - Effect of Temperature

Figure 7. Dot Plot of Effects

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Figure 7 shows all three effect values on a dot plot. It also shows the range of standards

multiplied by two, indicated by the “fence” lines. If an effect value is outside of these fences,

then it is deemed as mathematically significant. In this case, it is obvious that none of the effect

values are outside of these fences; therefore the no data in this experiment can be deemed as

significant. This could mean that the effects had little value compared to the standards, the

standards were not conducted properly and there was some error in the experimental design, or it

could have happened by chance alone. This also happened in pre-trials, but the effects had

almost no value compared to the range of standards. The experiment was changed and fixed

multiple times before usable data was being produced; it is thought that the bandwidth of the

variables were not great enough. To test for mathematical significance, the range of standards

was simply multiplied by two then graphed on either side of zero, then the effects were plotted; if

any of the effect points were outside of the range of standards times two, then they would be

mathematically significant.

Parsimonious Prediction:

Y = 6370.63 + “Noise”

Figure 8. Parsimonious Prediction

Figure 8 shows the parsimonious prediction. This prediction equation, instead if using all

effect values, only uses the significant values. In this experiment, nothing was found to be

significant, therefore the equation will only be the grand average plus “noise;” which is any

human error or factors that may have altered the data. This means that for example, if instead of

using pH 12 (one) and 38 ℃ (one), pH 11.5 (0.5) and 37.5 ℃ (0.5) were used, then on average

the Petri dish would contain about 6370 colonies.

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Interpretation:

None of the data collected in this experiment was deemed to be significant. The data does

however, show that the independent variables had some effect on how ​E.coli​ grew. This is

specifically shown in the (- +) trials, all of these trials were higher than the rest by about 4,000

colonies. This shows that either pH and/or temperature may have had an effect on the bacteria.

Temperature however, played the largest role on the number of ​E.coli ​colonies that grew.
 Russell - Zabel 20

Conclusion

It was hypothesized that when ​Escherichia coli ​is treated with a blank disc with a pH of

ten, and stored at 38 °C (- +), the number of colonies would be the greatest compared to the other

trials. The hypothesis was accepted after all of the trials were ran. For every five trials, there was

one (- +); forty trials were ran, and all eight of the (- +) trials contained the highest amount of

E.coli ​colonies (10,000; 9,200; 8,100; 9,850; 9,000; 8,800; 9,750; 9,400).

This experiment was planned to possibly find a way to free foods or water from ​E.coli ​by

applying different pH levels and temperatures. Even though the experiment wasn’t conducted to

kill ​E.coli ​off, it eliminated a way to solve the issues that the bacteria causes. The two variables

used were pH and temperature. The purpose of the pH variable was to test how high the pH can

be while ​E.coli ​can survive with it. The purpose of the temperature variable was to test if a

difference of just one degree could have a significant effect on the ​E.coli ​amounts. To test these

variables, a Petri dish containing agar was inoculated with ​E.coli​, then a blank disc with a pH

level of ten, eleven, or twelve, was placed in the dish; the disc would then be placed into one of

the three incubators (36 °C, 37 °C, 38 °C). The “- +” trials yielded the highest amount of bacteria

compared to the rest of the trials with an average of 9262.50 colonies. The “- -” trials yielded the

least amount of bacteria compared to the other trials with an average of 4995.00 colonies. This

suggests that temperature had more of an effect than the pH level. Although the data showed that

the variables had almost no significance, it did show that temperature played the largest role. A

high pH level will cause the bacteria to have elevated levels of transporters and enzymes that

promote proton capture and retention, metabolic changes that lead to increased acid production,

and changes in the cell surface layers that contribute to cytoplasmic proton retention (Padan, et
 Russell - Zabel 21

al.). The higher pH levels would result in less amounts of ​E.coli​, rather than the Petri dishes that

were ran in pre-trials that were full of bacteria.

Out of all the research being conducted with ​E.coli​, very few have to do with pH or

temperature variables. One experiment showed that cold temperatures (below 5 °C) had a

statistically significant effect on the log reductions of ​E.coli​. A single log reduction is a decrease

in the amount of live bacteria by 90% (​Marois-Fiset, et al.). Another experiment having to do

with pH treatment found that ​E.coli​ grows at identical rates around pH levels of six, seven and

eight, but there’s almost no growth below five (Schilling). This experiment was to show that

adding acids to foods, such as vinegar, will help to keep the food safe and free of bacteria. Some

research being conducted also has to do with how ​E.coli ​adapts to changing acidities. It was

found that when the bacteria is exposed to one environment, it shows fitness gains when

transitioning into their preferred environment. On the other hand, when it was exposed to

multiple acidic environments, the bacteria showed no significant fitness gain or loss when

transitioning between environments. The ​E.coli ​had adapted to multiple acidic environments

(​How E. Coli Evolves To Adapt To Changing Acidity). This experiment took the ideas of these

past experiment by using a pH variable and a temperature variable, and the data was measured in

E.coli ​amounts.

Throughout the experiment there were three flaws in the experimental design. The first of

these flaws was the band width of the pH that was tested. The levels should have varied more or

have been further apart to get more noticeable differences in the trials. Second, the plates were

prepared in the same order every trial that was ran; meaning the randomization was missing. The

plates were all prepared within minutes of each other in one trial, but this could have made a
 Russell - Zabel 22

difference. The final flaw was the placement of the dishes in the incubators. After the plates were

prepared, there were a few plates that went into the same incubator. Sometimes these plates

would be stacked on top of each other randomly, or placed on the bottom of the incubator

separately.

There were a few human errors while following the experimental design. One of these

being how the agar was poured; sometimes it would get messy and end up on both sides of the

Petri dish, or on the lab table. Another error was being lazy when it came to sterilizing all the

instruments that were used. Near the end there was a rush to finish trials and the sterilization

process became less and less, maybe causing the amounts of ​E.coli ​to change slightly.

These flaws and errors however, can be used for future reference when dealing with

experimental design and handling ​E.coli​. The difference between the pH levels should be much

greater; if more variability was used, perhaps the data would have been changed greatly and may

have actually been significant. Also the experimental design should be followed much more

precisely. This could have likely changed the data slightly, but most likely not much. Taking

these flaws into consideration can be a way to conduct better, more significant research on how

pH and temperature affect the growth of ​E.coli​.

By conducting this experiment, much experience and knowledge was gained. Most

importantly, how to conduct research and write a lab report. Also, the research that was

conducted can be used for later research dealing with ​E.coli​. The purpose was to maybe find a

way to keep foods and water free of ​E.coli​. Science however, is a lot of failure and process of

elimination by learning from these failures. This experiment was not a failure in the fact that it
 Russell - Zabel 23

eliminated a possibility to solving ​E.coli ​issues, and future researches can save valuable time by

not repeating this process.

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Works Cited

Clay, Charles. "The Effects of Temperature on E. Coli." ​EHow​. N.p., n.d. Web.

<​http://www.ehow.com/info_8515155_effects-temperature-coli.html​>.

"Epidemiology of Escherichia Coli O157:H7 Outbreaks, United States, 1982–2002 - Volume 11,

Number 4-April 2005 - Emerging Infectious Disease Journal - CDC."Rangel,

Josefa M., Phyllis H. Phyllis, Collen Crowe, Patricia M. Griffin, and David L.

Swerdlow. Epidemiology of Escherichia Coli O157:H7 Outbreaks, United

States, 1982–2002 - Volume 11, Number 4-April 2005 - Emerging Infectious Disease

Journal - CDC​. Centers for Disease Control and Prevention/ National Center for Emerging

and Zoonotic Infectious Diseases, 23 May 2011. Web.

<​http://wwwnc.cdc.gov/eid/article/11/4/04-0739_article​>.

"Escherichia Coli." Federation of European Biochemical Societies. ​Microbe Wiki​. Ed. Nicole

Jacques and Nancy Ngo. European Journal of Biochemistry, 2004. Web.

<​https://microbewiki.kenyon.edu/index.php/Escherichia_coli​>.

"How E. Coli Evolves To Adapt To Changing Acidity." University of Chicago Press Journals.

ScienceDaily​. ScienceDaily, 29 May 2007. Web.

<​https://www.sciencedaily.com/releases/2007/05/070529101015.htm​>.

Marois-Fiset, Jean-Thomas, Anne Carabin, Audrey Lavoie, and Caetano C. Dorea. "Effects of

Temperature and PH on Reduction of Bacteria in a Point-of-Use Drinking Water

Treatment Product for Emergency Relief."​Applied and Environmental Microbiology​.

American Society for Microbiology, Mar. 2013. Web.

<​http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3592257/​>.
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Padan, Etana, Eitan Bibi, Masahiro Ito, and Terry A. Krulwich. "Alkaline PH Homeostasis in Bacteria:

New Insights." ​Alkaline PH Homeostasis in Bacteria: New Insights​. N.p., 30 Nov. 2005. Web.

http://www.sciencedirect.com/science/article/pii/S0005273605002865​>

Schilling, Andrew T. "The Effect of PH on the Bacterium E. Coli." ​Usc.edu​. N.p., 2 Apr. 2008.

Web. <​https://www.usc.edu/CSSF/History/2008/Projects/J1429.pdf​>.