The Effects pH and UVA Radiation Exposure on the Growth of Escherichia

coli

Brian Le and Justin Schang

Macomb Mathematics Science Technology Center

Biology 1

Section 9A

Mr. Acre/Mr. Estapa/Mrs. Gravel

19 May 2016
 Table of Contents

Introduction ..................................................................................................................... 1

Problem Statement.......................................................................................................... 2

Experimental Design ....................................................................................................... 3

Data and Observations.................................................................................................... 6

Data Analysis and Interpretation ..................................................................................... 9

Conclusion .................................................................................................................... 14

Acknowledgements ....................................................................................................... 17

Works Cited................................................................................................................... 18
 Le – Schang 1

Introduction

Escherichia coli is one of the most common types of bacteria that lives inside of

the human body. E. coli is considered a gut bacteria and one of their functions is taking

up space on the exposed surfaces of internal organs such as the intestines and prevent

the colonization of pathogenic strains of microorganisms. E. coli is optimally grown in an

area with a pH of seven, although it does have a higher resistance to more acidic pH

(Expect). Another thing that affects E. coli growth is UV light exposure. This exposure

damages the bacteria's DNA making it so the E. coli cannot reproduce (Motta et al.).

Finding a way to prevent E. coli growth outside the body is important. Inside our

bodies it is contained in your small intestines. When E. coli enters the body it infects

your digestive track and creates more waster products. One of the most recent E. coli

incidents happened at Chipotle Mexican Grill. This has caused illness to sixty people in

fourteen different states. (Whittyen). Even when precautions are taken, somebosy

could end up sick. Antibiotics can help drop down the populations but eventually the

bacteria will gain immunity. By using a UV light, the E. coli will not be able to reproduce

causing it to not be able to produce a resistance to the UV light.

In this experiment E. coli was grown on Petri Dishes with agar with pH ranging

from six to eight. The dish was then subjected to ultraviolent type A rays (UVA) for one

hour at different distances. Finally, the plate is left to grow overnight in an incubator set

at thirty-seven degrees Celsius and the percentage of E. coli growth was recorded.

Thirty-seven degrees is the optimal growth temperature for E. coli (Mr.Estapa).

 Le – Schang 2

Problem Statement

Problem Statement:

To determine the effect of pH and UVA radiation exposure on the growth of

Escherichia coli.

Hypothesis:

If the pH level is 8 and the plate is placed 22 cm from the UVA light, then the

growth of Escherichia coli will be the lowest value of the data.

Data Measured:

The independent variables are the pH level and the distance away from the UVA

light. The pH levels that will be used will be 6, the negative, 7, the standard, and 8, the

positive. The pH level of 7 was chose for the standard because bacteria grows the best

in 7 pH and the other values were one pH value above and below the standard. The

distance away from the UVA light will be 22 centimeters away for the negative value, 30

centimeters for the standard, and 38 centimeters away for the positive. The optimal

condition for the growth of E. coli of the distance away from a UVA light is unknown, so

30 centimeters away was chosen for the standard. The other values were an eight

centimeters increment above and below that of the standard. The dependent variable is

the percent of coverage on the petri dish after growing the E. coli overnight in agar of

the different pH values and subjecting the bacteria to UVA light at different increments

away from the dish for an hour.

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Experimental Design
Materials:
Agar Mix 33 Petri Dishes
Water Escherichia coli (E. coli)
Sodium Bicarbonate Lego Bricks
Citric Acid Bunsen Burner
21.5 cm x 58 cm x 39 cm box UVA Light
3 80 mL Beakers Wood Blocks
pH Tester Scale
Glass Stir Rod 1 mL Dropper
12 Test Tubes Test Tube Rack
Incubator

Procedure to Prepare the Agar:

1. Add 1 liter of water to a 1-liter beaker.
2. Place on stern hotplate and turn settings to high.
3. Add stirring magnet to 1 liter of water and place magnet setting on #4.
4. Slowly add 23 grams of agar to 1-liter beaker, be careful to not get it on the side
(1-liter batch will fill ~50 petri dishes).
5. Allow the solution to turn from a cloudy liquid to clear like apple juice (the
temperature will be at around 100 degrees Celsius).
6. Remove from heat and allow to cool down so that the agar can be poured.
7. Adjust amount to fit needs for experiment (i.e. 11.5 grams of agar in 500 mL
water makes ~25 plates).
Procedure to Pour the Agar into Plates:
1. Measure out 0.02 grams of citric acid in an 80 mL beaker.
2. Pour 80 mL of agar into the beaker with citric acid.
3. Use a sterile glass stir rod to mix the agar solutions.
4. Pour the agar solution into four plates labeled -, - and -, +.
5. Repeat step 1 through 4, but use 0.5 grams of sodium bicarbonate instead of
citric acid and pour into the four plates labeled +, + and +, -.
6. Pour three plates of agar without the treatment into the plates labeled standard.
7. Allow the agar to cool down and harden.
 Le – Schang 4

Procedure to Inoculate the Agar:

1. Place 12 test tubes onto a test tube rack.
2. Use a sterile 1 mL dropper to add 1 mL of water into every test tubes.
3. Sterilize an inoculating loop by putting it over a Bunsen burner until red hot and
let cool for twenty seconds.
4. Open the E. coli plate and use the sterilized inoculating loop to scrape off some
E. coli and put the loop into one of the test tubes.
5. Spin the loop around so that the E. coli will spread in the water.
6. Repeat step three.
7. Put the loop in the previously inoculated tube and spin the loop around.
8. Take out the loop and put it into a test tube that has not been inoculated and spin
the loop around.
9. Pour the test tube with the bacteria into each prepared petri dish.
10. While pouring the bacteria in, clam the lid just enough to pour in the liquid.
11. Slowly move the petri dish back and forth, turning the dish slightly every time, to
spread the bacteria evenly throughout the plate.
12. Pour out the excess water into a sink, only opening the lid slightly.
13. Repeat steps six through twelve with the other ten plates.
Procedure to Set up the Experiment:
1. Place the UVA light so that it will be 38 centimeters from the bottom of the box.
2. Take the prepared petri dishes and place them inside the box containing the UVA
light.
3. The positive dishes will be 38 centimeters away from the light, so they will be
placed on the bottom of the box.
4. The standard dishes will be 30 centimeters away from the light, so they will be
placed on two blocks of two by fours and to be raised eight centimeters.
5. The negative dishes will be 22 centimeters away from the light, so they will be
placed on four blocks of two by fours and a layer of Lego bricks to be raised
sixteen centimeters.
6. Seal up the box and make sure that no outside light is getting inside.
 Le – Schang 5

7. Turn the UVA light on for one hour, then put the plates into an incubator set at 37
degrees Celsius and leave it overnight for the E. coli to grow.
Procedure to Record the Data:
1. Take petri dishes out of the incubator and place on lab table.
2. Make a table that includes twelve columns labeled +, +/ +, -/ standard/ -, +/ -, -,
and observations.
3. There will be nine rows, list them with the date of the experiment then skip two
rows after that to leave space for the data.
4. Look at petri dishes and record the percentage of coverage of E. coli in the
corresponding columns.
5. Record any observations made under the observations column.
6. Repeat this experiment two more times, adding onto the previously made chart.
7. Use all of the data to make a DOE to see if any data was significant.
8. If anything in data is statistically significant try to figure out why and possible
errors.

Diagram:

Figure 1: Setup for growing Escherichia coli.

The figure above shows the setup for growing E. coli with the circles representing petri
dishes which are put different distances from the UVA light inside of the box.
 Le – Schang 6

Data and Observations

Data:

Table 1
Design of Experiment Values
pH Distance Away from UVA Light (cm)
- Standard + - Standard +
6 7 8 22 30 38

Table 1 shows that the pH has a range of 2 consisting of 6, 7, and 8. The pH of 7

was chosen to be the standard due to that the optimal growing condition of Escherichia

coli (E. coli) is in a pH of 7. The second factor is the distance away from the UVA light

and has a range of 16 cm, consisting of 22 cm, 30 cm, and 38 cm. The optimal growing

condition of E. coli for distance away from a UV light is unknown, so 30 cm away was

chosen for the standard value.

Table 2
Standard Values
Standard Values (percentage)
DOE 1 DOE 2 DOE 3
98.0 91.0 94.0 100.0 100.0 100.0 95.0 96.0 98.0

Table 2 shows the data recorded from the standard trials.

 Le – Schang 7

Table 3
DOE Values
Percentage of Plate Covered (%)
DOE pH, Distance Away from the UVA Light
(+,+) (+,-) (-,+) (-,-)
94.0 91.0 0.0 0.0
1
97.0 93.0 0.0 0.0
78.0 100.0 0.0 0.0
2
83.0 98.0 0.0 0.0
88.0 92.0 0.0 0.0
3
91.0 94.0 0.0 0.0
Average 88.5 94.6 0.0 0.0

Table 3 shows the data collected from the DOE trials. 3 trials were conducted

that consisted of 11 dishes each. Notice that there is no bacteria growth in the negative

pH values.

Observations:

Table 4
Observations
Date Observations
Nothing grew in the negative pH values. The rest of the plates had over
90% coverage, but none had 100% coverage. A no dilution sample was
3-16
used inoculate one of the plates, whereas the rest had a one dilution
sample added to it.
Still nothing grew in negative pH values. The standards and one (+,-)
3-29 had 100% coverage, but the (+,-) plate had a less dense layer of E. coli
growing on it. The (+,-) plates had lower values, 78% and 83%.
Still no growth in the negative pH value. The (+,+) percentage of
coverage went back up to 88% and 91%, whereas the (+,-) plates has
3-31
lower values, 92% and 94%. The standards also had less growth, and
average of 4% less per plate in relevance to the second DOE.

Table 4 shows the observations of each DOE throughout the experiment.

 Le – Schang 8

( S ,S )

(+,+)
(-,-)

Figure 1. Results of DOE 2

Figure 1 shows three samples out of eleven from DOE 2. The density of the

bacteria is obviously different in each dish. The plate on top ( S , S ) is the most dense.

The plate on the bottom right ( - , - ) is showing no growth at all. In every trial, nothing

grew in every negative pH dish. The density of bacteria changes due to the different

treatments. In each DOE, every treatment, except for the negative pH, shows a slightly

different amount of growth.

Recording the percentage of coverage is the method that is used to record the

amount of bacterial growth. The same person recorded the percentage to keep the data

as accurate as possible.
 Le – Schang 9

Data Analysis and Interpretation

Table 5
Design of Experiments Factors
pH (pH) Distance Away (cm)
- Standard + - Standard +
6 7 8 22 30 38

Table 5 shows the values used for the predictor variables. The pH has a range

from 6 to 8 and the distance away from the UVA light has a range from 22 to 38.

Table 6
Averages
Trials
Distance Averages (%)
pH
(cm)
(+) 8 (+) 38 88.5
(-) 6 (-) 22 0
(+) 8 (-) 22 94.67
(-) 6 (+) 38 0
Grand Average 45.791

Table 6 shows the averages of the percentage of growth that were recorded

during the experiment. The (+,-) treatment has the highest average growth, whereas

both (-,-) and (-,+) treatments has no growth shown whatsoever.

Table 7 Effect of a Change in pH

Effect of a Change in pH
100%
pH (%) 90% 91.583%
Percentage of Coverage

6 8 80%
0 94.67 70%
60%
0 88.5 50%
Avg = 0 Avg = 91.583 40%
30%
20%
10% 0%
0%
-1 1
pH

Figure 2. Effect of a Change in pH

 Le – Schang 10

Figure 2 and Table 7 shows that on average, as the pH level increases, the

percentage of the plate covered in Escherichia coli increases by 91.583%. This was

found by subtracting the low average (0%) form the high average (91.583%). Figure 2

shows that as the pH level increases, the growth of E. coli increases as well. 91.583% is

well over 18%, so the effect of pH is statically significant in this experiment.

Effect of the Distance

Away from the UVA Light
100%
Table 8 90%

Percentage of Coverage
Effect of the Distance Away from the UVA Light 80%
70%
Distance Away (%) 60%
22 cm 38 cm 50% 47.33%
40% 44.250%
94.67 88.5
30%
0 0 20%
Avg = 47.33 Avg = 44.25 10%
0%
-1 1
Distance Away from the UV Light

Figure 3. Effect of the Distance Away from the UVA Light

Figure 3 and Table 8 shows that on average, as the distance away from the UVA

light increases, the percentage of the plate covered in E. coli decreases by 3.0833%.

This was found by subtracting the low average (47.33%) form the high average

(44.25%). Figure 3 shows that as the distance away from the UVA light increases, the

growth of E. coli decreases. 3.0833% is under 18%, so the effect of the distance away

from the UVA light is shown to be not statically significant.

 Le – Schang 11

Table 9 Interaction of pH and

Interaction of Distance and pH Distance Away from the
pH (%) UVA Light
(-)6 (+)8 100%

Percentage of Coverage
90% 94.667%
Distance Away (%)

(+) 80%
Solid 70% 88.50%
38 0 88.5 60%
Segment
cm 50%
40%
30% 0%
(-) 20%
Dotted 10%
22 0 94.67 0%
0%
Segment
cm -1 1
pH

Figure 4. Interaction Effect

Figure 4 and Table 9 shows that -3.0833% is the effect of the interaction of a

change in pH and the distance away from the UVA light. This implies that there does not

appear to be much of an interaction. The interaction effect is found by first finding the

slopes of the two lines. The slope of the solid segment is 44.25%, found by subtracting

0% from 88.5% then divide that number by two. The slope of the dashed segment is

47.33%, found by subtracting 0% from 94.67% then divide that number by two. Now

subtract the slope of the dashed segment from the solid segment to get -3.0833% as

the interaction effect. This effect is not statically significant due to the effect not being

more than 18% away from 0%.

 Le – Schang 12

Nine Standard Runs

Percentage of coverage

100%
80%
60%
40%
20%
0%
0 1 2 3 4 5 6 7 8 9 10
Trial

Figure 5. Scatter Plot of Standards

Figure 5 shows a scatter plot of the standard runs during the experiment. All of

the standard plates are over 90% covered in E. coli. Notice that the trials done on the

second DOE were 100% covered in E. coli. Figure 4 shows that there is no significant

pattern over time, meaning that there appears to be no lurking variables that effect the

experiment. The range of standards is 9%, which was found by subtracting the lowest

standard (91%) from the highest standard (100%). Doubling the range of standards gets

18%, which will be used to decide which effect are statistically significant and which are

not.

Interaction -​ 3.0833% Distance -3.0833%​

pH 91.583% ​

-100%-90% -80% -70% -60% -50% -40% -30% -20% -10% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Figure 6. Dot Plot of Effects

Figure 6 shows which effects are statically significant. The two lines are the

fences set at 18%, the amount found by using Figure 5. Any effects that are inside of

the fences are nor statically significantly and any effect that are outside of the fences

are statically significant. In this experiment, the only effect that is significant would be

the effect of a change in pH, which means that it did not happen by chance alone. The
 Le – Schang 13

interaction effect and the effect of the distance away from the UVA light is inside of the

fences, so they are not significant and most likely happened by chance.

Y = 45.79167% + 91.583% / 2 * pH + “noise”

Figure 7. Parsimonious Prediction Equation

Figure 7 shows the parsimonious prediction equation for this experiment. A

parsimonious prediction equation is the grand average (45.79167%) plus half of the

statically significant effects (91.583% / 2) multiplied by their variable (pH) plus “noise”.

The “noise” is the errors that cannot be placed a value on. For example, this experiment

had a small difference in the intervals between the time of the treatment and the time of

recording the results.

Y = 45.79167% + 45.79167% * -0.5 + “noise” ≈ 23%

Figure 8. Interpolation Calculation

Figure 8 shows the result if this experiment were to be done with the pH variable

set at -0.5, which would be a pH of 6.5, halfway between a pH of 6 and 7. The equation

predicts that if the experiment were to be done again with a pH level of 6.5, about 23%

of the plate would be covered in E. coli. This implies that as the pH level increases, so

does the amount of growth in E. coli.

In the data collected the pH showed to be statistically significant. The data

showed that the pH of the agar seemed to affect the E. coli much more than the

distance away from the ultraviolet-A light and the interaction between the two factors.

The effect of the distance away from the ultraviolet-A light and the interaction between

the two factors are not close enough to the fences to show that is has any practical

significant at all.
 Le – Schang 14

Conclusion
The goal of this research was to determine the effect of pH and UVA radiation

exposure on the growth of Escherichia coli. The hypothesis was; If the pH level is 8 and

the plate is placed 22 cm from the UVA light, then the growth of E. coli will be the lowest

value of the data. The pH level of 8 was hypothesized to have the least growth due to E.

coli ideal growth in acidic conditions (Exptec). The distance away from the UVA light set

at 22 cm was hypothesized to cause less growth because the UV ray would lose some

power after a larger distance. The initial hypothesis was rejected and proved to be the

total opposite of what was actually happened.

The idea of the experiment was to find how the growth of E. coli is effected by a

change in pH and the distance away from a UVA light. To change the pH, treatment

was applied to the nutrient agar before E. coli was added. To lower the pH, citric acid

was used, and to raise the pH, sodium bicarbonate was added. To change the distance

away from the UVA source, the plates were placed on blocks of different sizes per

treatment and left under the light for one hour. After the treatment, the plate was left in

an incubator set at 37 degrees Celsius overnight. Finally, the percent of the plate

covered in E. coli was recorded.

The results were completely different from expected. The (+,-) plate, or the plate

with a pH of 8 and was placed 22 cm away from the UVA light, had the highest average

growth, at 94.67% coverage, rather than the lowest like hypothesized. The plates that

had the treatment to lower the pH to 6 had no E. coli growth at all. The results

compared to an experiment that had E. coli growing in acidic conditions up to a pH of

4(Conner and Kotrola), and an experiment that had no growth at all in a pH of 3.7, but
 Le – Schang 15

did in a pH of 4 (Presser, Ratkowsky, and Ross). This anomaly most likely happened in

this experiment due to lack of experience, causing multiple small mistakes, and not

exact pH testers.

The distance away from the UVA light had a very small effect, at 3.0833%

decrease as the distance increased. This was also opposite of what was hypothesized

that if the plate is placed closer to the UVA light, then less growth will occur, but instead

the UVA light helped the E. coli grow slightly more. UVA rays is supposed to damage

the DNA of the E. coli, “the broad wavelength spectrum of UVA can harm the cells in

many different ways, such as membrane damage, DNA damage, or indirect damage by

reactive oxygen species. Cells from chemostats run at higher dilution rates also

exhibited initial first-order inactivation kinetics when the cells were irradiated with UVA

light” (Berney, Michael et al). Comparing this data to another experiment with UVA

which had the UVA rays effecting and lowering the growth of E. coli, the experiment had

the opposite effect, having UVA irradiation increase the growth of E. coli. This was most

likely caused by inexperience and not removing the lids of the dishes before the UVA

irradiation.

The information found in this experiment could be used to pasteurize foods that

cannot be heated to prevent food poisoning. This information could also be used to

prevent an incident like the E. coli breakout at Chipotle Mexican Grill (Whittyen).

Some errors that should be pointed out is that timing was not exact, the

procedures were not followed exactly, and the procedure was flawed. The timing was

not constant between the time of placing the plates into the incubator and the time of

recording the percentage of growth. The procedures were not followed completely due
 Le – Schang 16

to tweaking of the amount of citric acid and sodium bicarbonate to change the pH along

with not making detailed observations. The procedure did not include removing the lid

and placing it back on before and after UVA irradiation. If this experiment were to be

done again, the procedure should be modified to remove the lid of the plate before UVA

irradiation, have the time in-between placing the plates into the incubator and recording

the percentage of growth be constant, use a better pH tester, and take more detailed

observations.
 Le – Schang 17

Acknowledgments

We would like to acknowledge Mr. Estapa for helping us with the scientific

concept and providing most of the materials, Mrs. Gravel for checking our formatting to

make sure it is correct, and Mr. Acre for going over our DOE and data and analysis to

make sure that the math is correct.

 Le – Schang 18

Citations

Berney, Michael et al. “Specific Growth Rate Determines the Sensitivity of Escherichia

Coli to Thermal, UVA, and Solar Disinfection.” Applied and Environmental

Microbiology 72.4

Conner, D E, and J S Kotrola. “Growth and Survival of Escherichia Coli O157:H7 under

Acidic Conditions.” Applied and Environmental Microbiology61.1 (1995): 382–

385. web.

Exptec. Expression Technologies Inc., n.d. Google Scholar. Web. 12 May 2016.

Motta, Ellen S. et al. “Endonuclease IV Is the Main Base Excision Repair Enzyme

Involved in DNA Damage Induced by UVA Radiation and Stannous

Chloride.” Journal of Biomedicine and Biotechnology 2010 (2010): 376218.PMC.

Web. 10 May 2016.

Presser, K A, D A Ratkowsky, and T Ross. “Modelling the Growth Rate of Escherichia

Coli as a Function of pH and Lactic Acid Concentration.” Applied and

Environmental Microbiology 63.6 (1997): 2355–2360. Print.

Whittyen, Sarah. "CDC declares Chipotle-linked E. coli outbreak over." CNBC. NBC

Universal, 1 Feb. 2016. Web. 10 May 2016.