The Effect of Variance in Temperature Due to Physical Activity on the Growth of

Staphylococcus Epidermidis

Nathan Richtarcik and Gabriel Vicencio

Macomb Mathematics Science and Technology Center

Biology

12B

Mr. Estapa/Mrs. Cybulski

16 December, 2019

The Effect of Variance in Temperature Due to Physical Activity on the Growth of

Staphylococcus Epidermidis

The purpose of this experiment was to determine whether 30℃, 33℃, or 36℃
would result in the lowest amount of bacteria grown. The objective, to find the lowest

amount of bacteria growth in relation to temperatures, would be beneficial for those that

normally work out or play sports. The 30°C was chosen to be tested because it was the

average temperature observed after the participants exercised, while 33°C was tested

because it was the average temperature observed before the participants exercised. A

higher temperature at 36°C was included because it is closer to the average core

temperature of the human body (37°C). The hypothesis stated that if the bacteria is grown

in the 36°C incubator, it will result in the largest amount of growth, while the 30°C

incubator will produce the least amount of growth.

Thirty petri dishes were placed in 30℃, 33℃, or 36℃ incubators, and data was

collected by imposing a grid onto the dish and counting the squares that contained

bacteria. Then the number of bacteria squares was taken and divided it by the number of

total squares. The 30℃ dataset produced an average percent coverage of 18.56 percent,

while the 33℃ dataset produced an average percent coverage of 59.33 percent. The 36℃

dataset ended up yielding the highest amount covered, with an average of 87.46 percent.

These results show that this bacteria most likely grows the fastest on 37℃ skin, the

average core temperature of the human body. The skin temperature is very often cooler

than the core temperature, but it shows that the closer the skin temperature is to the core

temperature, the more bacteria will grow.

Table of Contents
Introduction.……………...…………………………………………………………….....1
Review of Literature…………...………………………………………………………....3
Problem Statement……………...………………………………………………………...7
Experimental Design……………………...……………………………………………....9
Data and Observations……………….………………………………………………......11
Data Analysis and Interpretation……………….…………………………………….….17
Conclusion…………………………………………….…………………………………20
Appendix A: Agar Preparation……………….....…………….….…………………..….24
Appendix B: Finding Temperatures to be Used for Bacteria Growth……………….…..25
Appendix C: Creating the Starting Plate of E-coli………………………………………26
Appendix D: E-coli Solution Preparation……………………………………………….28
Appendix E: Professional Contact Email………………………………………………..29
Works Cited……………………………….………………………………………..…....30
 Richtarcik - Vicencio 1

Introduction

As of 2019, there are over three trillion different species of discovered microbes.

About one thousand of these are available for growth on human skin (Nedwell). One of

the most common bacteria found on skin is Staphylococcus epidermidis. This microbe

grows and reacts very differently depending on the climate and temperature of the skin’s

surface (Jung et al.). Failure to eradicate this microbe can cause boils, blisters, infections,

food poisoning, and, in rare cases, death if they enter into the bloodstream. It is crucial

that those who work out often take the necessary precautions to prevent or slow the

growth of this microbe (“Temperature and Microbial Growth”). When a person exercises,

the temperature of the skin changes due to sweat and blood circulation, causing the

bacteria to grow at a different rate. This experiment was performed to investigate exactly

how varying temperatures due to physical activit y affects the growth of bacteria.

To determine the temperatures of the incubators at which the bacteria was grown

in, 15 subjects performed vigorous exercise for five minutes. Their skin temperature was

taken before and after the exercise. It was determined that the average temperature before

exercise was performed was about 33°C. The average after the activity was about 30°C.

These temperatures were used as incubation temperatures for the E.coli bacteria which

mimics the growth of Staphylococcus epidermidis on various skin temperatures. E.coli

has the same favorable growth temperature as Staphylococcus epidermidis so it was able

to be used to mimic the growth of Staph. Finally 36°C was used as it is a temperature

similar to the average core temperature of the human body. The bacteria was allotted

growth over a day for each trial in which nutrient-rich agar fed the bacteria in the petri

dishes. There were 30 trials run for each incubation temperature and the chosen
 Richtarcik - Vicencio 2

incubators were randomized every day.

After exercising or working out, people often assume they are riddled with

bacteria. This experiment found that it is not necessarily true as bacteria growth is slowed

during the period of movement. Staying active has many benefits and slowed bacteria

growth is just one more plus. These results may persuade people to want to exercise more

to reap benefits other than staying in-shape. Finally this research furthered the current

known information on how these specific microbes interact in differing temperatures. It

reinforces an evolutionary standpoint that bacteria adapted to grow fastest in the

temperature of the nutrient-rich human epidermis layer. It is apparent that their adaptation

is so specific that even small changes in temperature can vary the growth of the bacteria

by large margins.

Review of Literature

The purpose of this experiment was to determine the effect of differing

temperatures on the growth of Escherichia coli, specifically those observed when the skin
 Richtarcik - Vicencio 3

temperature is changed due to physical activity. Escherichia coli was used as it is acts

similarly to Staphylococcus epidermidis due to both bacteria growing fastest in the same

temperature. The temperatures due to physical activity were chosen for determining if

those who played sports or exercised on a regular basis, thus having a higher surface

temp, had on average less bacteria on the surface layers of the epidermis. It was

determined that investigating their growth in these observed temperatures would be the

best way to find the answer to that question. Staphylococcus epidermidis can cause

infections in open wounds, and it is the major cause of the unpleasant smell associated

with prolonged exercise. Finding the lowest bacteria growth would be beneficial because

it can persuade people to stay active in order to avoid negative effects of more bacteria.

A major effect on the growth of skin bacteria is the temperature of the skin itself.

Skin temperature of the human body acts and is much different from core temperature.

Most people have a core temperature of about 37.06°C, while skin temperatures can vary

greatly based on the environment surrounding the skin. Exercise can affect this surface

temperature greatly. An experiment ran on skin temperatures due to biking found that

during muscular exercise, “...there is an initial tendency for the skin temperature of the

trunk and leg to rise. This rise may be obscured by the cooling effect of the wind caused

by the motion” (Burton). The small increase in temperature can be due to increased

friction in the muscles. As soon as the body starts sweating, a sharp decrease in

temperature is found (Burton). Sweating is the body’s natural way of cooling itself down

in which the precipitation secreted from the eccrine glands evaporates into the

surrounding air, taking energy in the form of heat with it (Newcomb). Burton’s

experiment can be further explained by a similar experiment ran by researchers in Japan.

 Richtarcik - Vicencio 4

They suggested that their findings were not due to sweat entirely but because of

thermoregulatory vasodilation (Torri et al). Thermoregulatory vasodilation is when the

increased heart rate and movement causes blood to circulate more from the core to the

surface (Cyr). The blood reaches the surface and it is cooled due to the cooler

surrounding environment. When the exercise is halted, the blood begins to stop

circulating as it did before. This results in an even greater cooling effect as,

“...evaporation continues and the extra heat is no longer brought up to the skin” (Burton).

These experiments gave important information towards how the temperatures were found

in the research and as to why the temperatures decreased.

Figure 1. Thermoregulatory Vasodilation. Gillam. “Temperature Regulation Strategies.”

Khan Academy, Khan Academy, 2016,

www.khanacademy.org/science/biology/principles-of-physiology/metabolism-and-

thermoregulation/a/animal-temperature-regulation-strategies.

The figure above shows how thermoregulatory vasodilation works under the

superficial epidermis layer. It occurs when increased heart rate and movement causes

blood to circulate more from the core to the surface near the skin. The blood that flows to

the surface loses heat (shown by the red arrows pointing outwards) due to the colder

temperature of the environment. This cools the skin even more.

 Richtarcik - Vicencio 5

Staphylococcus epidermidis is a bacteria strand commonly found on the surface

of human skin. The bacteria flourishes when given a moist, nutrient rich environment,

and it’s most optimal temperature for growth is at 37.06°C (OpenStax). Escherichia coli,

the bacteria used in this experiment, flourishes best under the exact same conditions,

which is why it can be used in the place of Staphylococcus epidermidis (Jung et al). As

explained previously, the surface temperature of the human body varies greatly

depending on the outside temperature and the amount of physical activity done.

Staphylococcus Epidermidis growth decreases as the temperature of the skin decreases

(Schwab et al.). An experiment run by D.B. Nedwell at the University of Essex,

Department of Biological Sciences, Wivenhoe Park, Colchester, says, “As the

temperature drops, the organism's ability to maintain its natural processes drops, and

therefore it has a harder time functioning and reproducing” (Nedwell). Senior lecturer

Andrew Libby at Indiana University, Department of Human Biology, Indiana

Bloomington, further backed Nedwell’s explanation in which he said, “Altering

temperature below the optimum point results in the lipids of the cell membrane to stiffen.

The transport proteins that cycle throughout the membrane are therefore slowed, causing

decreased overall efficiency” (Libby).

Sweat formed due to physical activity comes from the eccrine glands. This sweat

is not very nutrient rich so bacteria takes a much longer time to grow. The smell affiliated

with this sweat is not from the sweat itself but from the bacteria that flourishes in the

sweat (Newcomb). Sweat formed due to being nervous, emotional, or excited comes from

the apocrine glands. This sweat is much more nutrient rich and results in more bacteria to

be formed (Newcomb). The agar used to grow the bacteria in the experiment mimicked
 Richtarcik - Vicencio 6

the sweat from the eccrine glands on a human body.

Overall, the growth of Staphylococcus Epidermidis is affected by the nutrients

available and the temperature of its surrounding environment. Working out and prolonged

exercise causes the temperature of the skin to decrease. This experiment focused on how

the growth of E.coli, which mimics Staph, is affected by the decreased surface

temperatures due to physical activity.

Problem Statement

Problem:

Determine the effect of varying temperatures due to physical activity on the

growth of Staphylococcus epidermidis.

Hypothesis:

If the bacteria is grown in the lowest temperature, it will result in the least amount
 Richtarcik - Vicencio 7

of growth.

Data Measured:

The independent variable measured in this experiment was the temperature at

which the bacteria will be grown in. The temperature was determined by the surface

temperatures of the human subjects. The middle temperature was held at the temperature

of the subject before any exercise was performed at 33°C. The high temperature was held

at 36°C, the most ideal temperature for bacteria growth. Finally, the low temperature was

held at the temperature of the subjects after five minutes of vigorous exercise was

performed (30°C). The dependent variable was the amount of bacteria found at the end of

the day growth period measured through area of inhibition. The amount of exercise each

subject performed remained constant throughout the experiment. Also, the amount of

time at which the bacteria was allocated to grow was held constant at twenty four hours.

In order to analyze the results, the two-sample T test was used. This test was used

because it is a comparative experiment in which the mean difference in bacteria growth

was compared between the three groups, through three test repetitions. There were 90

trials run, 30 trials per test group. Finally, there were 15 human tests run to record the

temperatures.
 Richtarcik - Vicencio 8

Experimental Design

Materials:

Agar
(90) Petri Dishes
Escherichia coli Solution
(3) Incubators
(6) Pipettes

Procedures:

1. Pour agar into 15 petri dishes. See Appendix A for agar preparation.

2. Pour 1 mL of the Escherichia coli (E-coli) solution on top of the agar using a pipe.
Refer to Appendix D for the E-coli solution preparation.

3. Turn on each incubator, set one temperature to 30°C, one at 33°C, and one at 36°C.
 Richtarcik - Vicencio 9

4. Close petri dish containers and randomize incubator placement between 30°C, 33°C,
and 36°C. See Appendix B for temperature findings
5. Place each petri dish inverted in the randomly assigned incubator.

6. Wait 24 hours for the E-coli to grow.

7. Measure the growth of the E-coli through zones of inhibition for each sample.

8. Record data on spreadsheet.

9. Repeat steps 1-8, 6 times.

Diagram:

Figure 1. Adding E-coli to Petri Dish

The figure above shows the 1 mL of E-coli solution being added to the agar inside

of the petri dish. As shown, the Agar had a dark yellow color. The agar solution will

allow for the E-coli to grow as it is filled with nutrients.

 Richtarcik - Vicencio 10

Data and Observations

The experiment chosen was to measure the growth of bacteria in different

temperatures to simulate how the temperature of the skin changes during physical

activity. The bacteria was placed in 30℃, 33℃, and 36℃ incubators to determine if

there was a clear difference in the growth of the bacteria. The following tables and

figures include all of the data collected throughout the experiment, as well as any

observations made, and any photos pertaining to the experiment.

Table 1
30℃ Incubation Temperature Bacteria Growth
Trial % Covered Trial % Covered
1 0.15 16 0.2
2 0.14 17 0.15
3 0.24 18 0.14
4 0.21 19 0.18
5 0.2 20 0.23
 Richtarcik - Vicencio 11

6 0.05 21 0.25
7 0.1 22 0.3
8 0.05 23 0.07
9 0.2 24 0.24
10 0.23 25 0.11
11 0.18 26 0.28
12 0.13 27 0.23
13 0.23 28 0.24
14 0.16 29 0.22
15 0.13 30 0.33

Table 1 shows the percent of the petri dishes covered by bacteria when placed in

the 30℃ incubator. The amount of E.Coli added was kept constant throughout these

trials. Three dilutions took place before the E.Coli was placed in the Petri dishes. This

was to help the researchers measure the growth of the bacteria by reducing the amount

grown until there was a clear difference between the different datasets. The average

percentage covered for the 30℃ dataset was about 18.56%. Overall there appears to be a

low range in this data set which shows low variability.

Table 2
33℃ Incubation Temperature Bacteria Growth
Trial % Covered Trial % Covered
1 0.6 16 0.6
2 0.73 17 0.76
3 0.56 18 0.4
4 0.64 19 0.44
5 0.7 20 0.55
6 0.5 21 0.67
7 0.63 22 0.56
8 0.78 23 0.45
9 0.69 24 0.63
10 0.56 25 0.59
 Richtarcik - Vicencio 12

11 0.6 26 0.68
12 0.56 27 0.45
13 0.58 28 0.48
14 0.4 29 0.77
15 0.74 30 0.5

Table 2 shows the percent of the surface covered by bacteria when placed in the

33℃ incubator. The amount of E.Coli added was kept constant throughout these trials.

Three dilutions took place before the E.Coli was placed in the Petri dishes. The average

percentage covered for the 33°C dataset was about 59.33% percent covered. Overall the

data appears fairly consistent, with a law range, and therefore there is low variance.

Table 3
36°C Incubation Temperature Bacteria Growth
Trial % Covered Trial % Covered
1 0.82 16 0.78
2 0.85 17 0.9
3 0.98 18 0.89
4 0.79 19 0.84
5 0.85 20 0.75
6 0.83 21 0.93
7 0.89 22 0.94
8 0.94 23 0.86
9 0.87 24 0.79
10 0.88 25 0.85
11 0.96 26 0.86
12 0.99 27 0.92
13 0.92 28 0.84
14 0.8 29 0.88
15 0.95 30 0.89

Table 3 shows the percent of the surface covered by bacteria when placed in the
 Richtarcik - Vicencio 13

36℃ incubator. The amount of E.Coli added was kept constant throughout these trials.

Three dilutions took place before the E.Coli was placed in the Petri dishes. The average

percentage covered for the 36℃ dataset was about 87.46% percent covered. Overall this

data is consistent with a low range.

Several observations were made for each set of data and for each temperature

tested. These observations can be found below in Figure 4, Figure 5, and Figure 6.

Table 4
30℃ Trial Observations
Trial Observations
The lowest percentage covered observed in all datasets, as well as
6 the 30℃ dataset at 5 percent
Lowest percentage covered observed, tied with trial number 6 at 5
8 percent
21 Highest percentage covered observed at 25 percent.

Table 4 shows the observations from the 30℃ trials. Significant observations

were not recorded for all 30 trials. The highest recorded percentage covered are pictured

below in Figure 7 respectively.

Table 5
33℃ Cover Trial Observations
Trial Observations
The lowest percentage covered observed in the 33℃ dataset at 40
14 percent
Lowest percentage covered observed, tied with trial number 14 at
18 40 percent
29 Highest percentage covered observed at 77 percent.

Table 5 shows the observations from the 33℃ trials. Significant observations

were not recorded for all 30 trials. The highest recorded percentage covered are pictured

in Figure 8.
 Richtarcik - Vicencio 14

Table 6
36℃ Trial Observations
Trial Observations
12 Highest percent covered observed at 99 percent.
16 Second lowest percentage covered observed at 78 percent
20 The lowest percentage covered observed in the 36℃ data at 75%

Table 6 shows the observations from the 36℃ trials. Significant observations

were not recorded for all 30 trials. The highest recorded percentage covered is pictured

below in Figure 9 respectively.

Figure 7. Highest Percentage Covered For 30℃

Figure 7 above displays the highest percentage covered for the 30℃ dataset. It

was recorded as 0.25, which means 25 percent of the surface was covered. This was

measured by counting the squares that included bacteria and dividing that number by the

total number of squares in the petri dish.

 Richtarcik - Vicencio 15

Figure 8. Highest Percentage Covered For 33℃

Figure 8 above displays the highest percentage covered for the 33℃ dataset. It

was recorded as 0.77, which means 77 percent of the surface was covered. This was

measured by counting the squares that included bacteria and dividing that number by the

total number of squares in the petri dish.

Figure 9. Highest Percentage Covered For 36℃

Figure 3 above displays the highest percentage covered for the 36℃ dataset. It

was recorded as 0.99, which means 99 percent of the surface was covered. This was

measured by counting the squares that included bacteria and dividing that number by the

total number of squares in the petri dish.

Data Analysis and Interpretation

 Richtarcik - Vicencio 16

In this experiment, E-coli was grown in three different temperatures: 30°C,

33℃, and 36°C in order to determine the effects of varying skin temperatures due to

physical activity on the growth of E.coli bacteria which mimics Staphylococcus

epidermidis. The bacteria growth was calculated using the total percent coverage of each

petri-dish. Each incubation temperature consisted of 30 trials, which means that the data

comes from an approximately normal sampling distribution based upon the Central Limit

Theorem. Reliability was ensured by conducting the experiment over a 6-day time period

which reduced the effects of lurking variables like humidity. Humidity can change from

day to day and it can change the composition of the nutrient rich agar slightly, which

could affect the results. To also reduce the effects of lurking variables, the same person

was in charge of doing each part of the experiment and the same procedure was followed

for each trial. Finally each part of the procedure involved sterilization using heat to avoid

any other bacteria from the lab and test materials contaminating the result.

Randomization is also another key factor to take into consideration when performing an

experiment, randomization reduces bias in order to ensure the data is reliable.

Randomization was ensured throughout the experiment by doing 5 trials of each

incubation temperature and randomly organizing the order in which the temperatures

were tested using a random integer generator on a calculator.

 Richtarcik - Vicencio 17

.99
.92
.875 .875
.78
.84
Percent Coverage of Petri-Dish

.68
.75
.59 .59

.45
.51

.4
.26
.215
.18

.14
.05

Incubation Temperatures

Figure 1. Box Plots and Averages of All Three Incubation Temperature

The box plots for all of the temperatures at which the bacteria were grown in are

shown above. These box plots show little overlap with each other which means that there

is a significant difference in the bacteria growth of each temperature. Each plot is almost

normally distributed with little skewness, besides the 30°C data that is slightly right

skewed. The median and mean are the same for the top two data sets, and there are no

outliers in any of the data as well, which further shows the small variability in data. Each

temperature has a far different mean from one another. Seventy-five percent of the 33°C

data is above all of the 30°C data, and 75% of the 36°C growth is above all of the 33°C

growth.

Interpretation:
 Richtarcik - Vicencio 18

As shown in Figure 1, there was a clear difference between the growth of bacteria

in each temperature. The 36°C on average, resulted in the most bacteria growth compared

to the other temperatures. The 33°C produced less bacteria growth than the 36°C but still

more than the 30°C. The box plot of all three incubation temperatures had very little

overlap. In fact, the only overlap is between the 36°C bottom 25% bacteria growth and

the 33°C top 25% bacteria growth. As well as, the 33°C bottom 25% bacteria growth and

the 30°C top 25% bacteria growth. Even these parts of the plots barely overlap, showing

that there is a clear difference and therefore the incubation temperature can be considered

statistically significant on the growth of E-coli. Because the incubation temperature can

be considered statically significant from the box plots, no statistical test was required.

Conclusion

The purpose of this experiment was to identify which temperature, 30°C, 33°C, or
 Richtarcik - Vicencio 19

36°C, resulted in the highest and lowest growth of epidermis bacteria. These temperatures

were observed from the reading of the skin before, after, and during prolonged periods of

physical activity. Temperature was used as the variable because the goal of the

experiment was to determine if working out and/or playing sports caused more or less

bacterial growth on the epidermis layer. The hypothesis stated that if the bacteria is grown

in the 36°C incubator, it will result in the largest amount of growth, while the 30°C

incubator will produce the least amount of growth. After the experiment was ran, it is

shown clearly that the highest incubation temperature offered the highest growth on

average and therefore the hypothesis was accepted. The 30°C was chosen to be tested

because it was the average temperature observed after the participants exercised, while

33°C was tested because it was the average temperature observed before the participants

exercised. A higher temperature at 36°C was included because it is closer to the average

core temperature of the human body (37°C) which is also the temperature at which most

bacteria thrives in (Newcomb). The experiment was performed by collecting the

temperatures from people exercising, and then placing bacteria to grow in those same

temperatures. The independent variable was the temperature of the incubator that the

bacteria grew in, and the dependent variable was how much bacteria grew. Constant

values included the amount of agar poured into each petri dish, and the amount of the

water and E.Coli added. The maximum and minimum temperature of each trial served to

show that there was a low range of data and therefore each mean can be considered a

reliable predictor of the bacteria growth.

The decreased temperatures due to intense physical activity found in the

preliminary part of the experiment can be explained by two reasons: thermoregulatory

 Richtarcik - Vicencio 20

vasodilation and sweat evaporation from the skin. Thermoregulatory vasodilation is when

increased heart rate and movement causes blood to circulate more from the core to the

surface (Cyr). The blood reaches the surface and it is cooled due to the cooler

surrounding environment, causing a decreased skin temperature. When the subjects began

to sweat their skin cooled further. Sweating is the body’s natural way of cooling itself

down in which the precipitation secreted from the eccrine glands evaporates into the

surrounding air, taking energy in the form of heat with it (Burton). Both of these effects

contributed to the average skin temperature after physical activity of 30°C.

E-coli bacteria was used in the experiment to mimic Staphylococcus epidermidis

bacteria, a common strand found on the skin (Jung et al.). These bacteria flourish and

grow best in a temperature close to 37°C (“Temperature and Microbial Growth”). This

can be explained from an evolutionary standpoint. Mammals offer a large amount of

nutrients to bacteria. Many strands of bacteria live entirely off of mammals and they must

adapt to the mammal’s environment. Therefore as humans evolved to have a stable core

temperature of 37°C, the bacteria evolved to grow most efficiently at this temperature

(Schwab et al.). “This effect may be because of stiffening of the lipids of the membrane

below the temperature optimum, leading to decreased efficiency of transport proteins

embedded in the cell membrane” (Nedwell). As the temperature drops, the organism's

ability to maintain its natural processes drops, and therefore it has a harder time

functioning and reproducing. This explains the experiment’s large difference in bacteria

growth as the temperature dropped. As shown in the Data Analysis and Interpretation, the

36°C dataset produced the highest average coverage out of the three temperatures at

87.46 percent coverage. The data was measured in percent coverage, which was
 Richtarcik - Vicencio 21

measured by imposing a grid over the petri dishes and counting the squares that contained

bacteria and the ones that did not. Then the number of squares that contained bacteria and

the total number of squares were divided to get the percent coverage for that dish. The

33°C dataset had the second highest average at 59.33 percent, and lastly the 30°C dataset

had the lowest average at 18.56 percent. Both of these results were observed due to the

further distance from the bacteria’s favored growth temperature of 37°C.

There were a few errors made over the course of this experiment. The largest

would be the incubator control. Often times the incubators were set slightly too high or

too low. Even though the temperature was at the desired number originally, the next day,

after several hours of small decimal jumps, the incubator would sometimes have a

slightly different temperature. This could have skewed the results slightly but not a large

amount as the temperatures only differed by half of a degree. Another error in the

experiment that occurred was contamination of the test materials by minimal talking over

the testing area in which bacteria from saliva could have infiltrated the sterile

environment. This error was written off as a confounding variable because it was mostly

out of the researchers control and it did not affect the overall strong consistency of the

data. If this experiment were to be redone, it would be run in a closed sterile environment

and a better attempt to keep each test material away from contamination would be made.

The experiment also required three dilutions of the original E-coli due to the large

spanning coverage of the petri-dishes without the dilutions. In a rerun, larger petri dishes

could be used to avoid dilutions that offered more time of exposure to contamination.

Further research into the topic of how skin temperature affects the growth of

microbes can be done. To investigate this, incubation temperatures higher than 37°C and
 Richtarcik - Vicencio 22

lower than 30°C could be used to see how the bacteria grows in more extreme

environments. Other types of bacteria could be used as well to see how they interact

compared to E-coli and Staphylococcus epidermidis. This experiment showed that

prolonged physical activity can slow the growth of bacteria on the skin due to decreased

temperatures. These results add on to the benefits of exercise as well as furthers the

scientific community's understanding of how bacteria behaves at human body

temperatures.

Appendix A

Agar Preparation

Materials:

Purified Water (1L)

2 L Beaker
Hot Plate
Stirring Magnet
Agar Powder 23 g

Procedures:

1. Add 1 L of water to a 2 L beaker.

 Richtarcik - Vicencio 23

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

3. Add a stirring magnet to the beaker of water. Place the magnet setting on #4 spinning
speed.

4. Slowly add 23 grams of agar powder to the beaker.

5. Allow the solution to turn from a cloudy liquid to clear similar to apple juice.

6. Remove from heat and allow to cool.

Appendix B

Finding Temperatures to be Used for Bacteria Growth

Materials:

(15) Human Subjects

Surface temperature probe

Procedures:

1. Gather test subjects to an inside area with a large space where the temperature is
controlled.

2. Record subject’s sitting temperatures at the forehead, cheek, and back of the neck
using the surface temperature probe.
 Richtarcik - Vicencio 24

3. Have the subjects run for five straight minutes in the large space.

4. Record subject’s new temperatures at the forehead, cheek, and back of the neck using
the surface temperature probe.

5. Repeat 15 times till all subjects have their before and after surface temperatures
recorded.

6. Average all temperatures between forehead, cheek, and back of the neck over the 15
trials. The before and after total averages will be the testing temperatures at which the
bacteria will be grown in.

Appendix C

Creating the Starting Plate of E-coli

Materials:

Pipette
Purified Water
Test Tube 15 mL
Aluminum Foil
Bunsen Burner
Striker
Inoculation Loop

Procedures:

1. Using the pipette, extract 1 mL of purified water.

 Richtarcik - Vicencio 25

2. Transfer water into the test tube. Make sure to squeeze all the water out and limit as
much contact with the side of the tube as possible.

3. Repeat steps 1-2, two more times. There should now be a total of 3 mL of water in the
test tube.

4. Rip off a small piece of the aluminum foil sheet, and use it to cover the test tube.

5. Turn the Bunsen Burner on, and using a striker, light the flame.

6. Run the inoculation loop through the flame to sterilize it. Allow for every part to glow
orange, signaling that it is sterile.

7. Remove from heat. Continue to hold the loop and let it cool for about 5 seconds.

8. Insert inoculation loop into E-coli.

9. Place inoculation loop with bacteria on it in water and swirl around for 5 seconds.
Make sure to avoid loop contact with the side of the tube as it is being moved in and
out. This will limit extra bacterial exposure. The water will begin to turn cloudy,
signaling that the bacteria is in the water.

10. Pour the test tube on the agar in a Petri dish, keeping the lid clamped. Refer to
Appendix A for agar preparation.

11. Spread out mixture by sliding the dish back and forth on a flat surface. Do not swirl
because this will spray mixture on lid. Make sure that the mixture fully covers the
surface of the agar.

12. Empty any extra liquid in the dish over the sink. Make sure to keep lid clamped. This
will not affect cover of the mixtures.

13. Invert dish and place in the incubator set at 37°C for 24 hours. After 24 hours, the
Petri dish can be placed in the fridge. This plate will be used throughout the duration
of the experiment to take E-coli from.
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Appendix D

E-coli Solution Preparation

Materials:

Pipette
Purified Water
Test Tube 15 mL
Inoculation Loop
Bunsen Burner
E-coli Starter Plate (refer to Appendix C)

Procedures:

1. Using pipette, extract 1 mL of purified water.

2. Transfer water into the test tube. Make sure to squeeze all the water out and limit as
much contact with the side of the tube as possible.

3. Repeat steps 1-2, two more times. There should now be a total of 3 mL of water in the
 Richtarcik - Vicencio 27

test tube.

4. Turn the Bunsen Burner on, and using a striker, light the flame.

5. Run the inoculation loop through the flame to sterilize it. Allow for every part to glow
orange, signaling its sterile.

6. Remove from heat. Continue to hold the loop and let it cool for about 5 seconds.

7. Insert inoculation loop into E-coli starter plate (refer to Appendix C) and
scrape against the top of the agar to gather bacteria.

8. Place inoculation loop with bacteria on it in the test tube with water and swirl around
for five seconds. Make sure to avoid loop contact with the side of the tube as it is being
moved in and out. This will limit extra bacterial exposure. The water will begin to turn
cloudy, signaling that the bacteria is in the water.

9. Repeat step five to sterilize the loop.

10. Repeat steps 1-9 for each petri dish.

Appendix E

Professional Contact Email

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