2015 Freshman Research Project

The Effect of Hydrogen Peroxide Concentration and Temperature on Escherichia coli

Mahir Ahmed - Dylan Barrick

Macomb Mathematics Science Technology Center

Biology 1 - 9A

Mr. Acre, Mr. Estapa, Mrs. Gravel

May 26, 2015

 Table of Contents

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

Problem Statement ...........................................................................................................................4

Experimental Design ........................................................................................................................6

Data and Observations ...................................................................................................................11

Data Analysis and Interpretation ...................................................................................................16

Conclusion .....................................................................................................................................24

Acknowledgements ........................................................................................................................29

Works Cited ...................................................................................................................................30

 Ahmed Barrick 1

Introduction

Bacteria can cause a variety of different infections and diseases, however this does not

necessarily mean all bacteria is harmful. There are many types of bacteria that live in the human

body, most of them sharing a symbiotic relationship with the organism surrounding them.

Though they are much smaller, bacteria outnumber human cells 10:1 (Statt). A common type of

bacteria is a microbe known as Escherichia coli (E. coli). This bacteria is commonly found in the

human and animal digestive systems. Even within this small group, there are different strains of

Escherichia coli, similar to there being many types of bacteria (E. coli). One of these possibly

harmful strains, or simply too much of Escherichia coli, can make people sick and cause

digestive problems. Because bacteria reproduce at an extremely high rate, if too many bacteria

are present then they will use the nutrients that were originally for human cells, this lack of

nutrients will cause the human cells to die. This bacteria was used in the experiment because,

although usually not, it is known that it can be harmful.

The purpose of the experiment was to identify how well combinations of different

hydrogen peroxide concentrations and varying incubation temperatures inhibited the growth of

Escherichia coli. This was done by incubating the E. coli at 35 C, 36 C, and 37 C, while

exposing it to 2 %, 3 %, and 4 % concentrations of hydrogen peroxide. To determine how well

each of the combinations affected E. coli, the zone of inhibition was measured (in centimeters).

The zone of inhibition is the area in which bacteria did not grow. These zones are roughly round,

so to get the measurements for the zones of inhibition, the diameters of those areas were

measured and then averaged (Effects of Antiseptics on Bacterial Growth). Figure 1. below

shows how the measurements for the zone of inhibitions were acquired.
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Figure 1. Zone of inhibition

Disinfectants and antiseptics are commonly used to prevent bacterial growth on surfaces

and infection in organisms. One common disinfectant that is used to prevent bacterial growth and

infection is hydrogen peroxide (H2O2). Hydrogen peroxide is selective, which means that it

responds to the conditions it is put into (Disinfectants of Hydrogen Peroxide). When hydrogen

peroxide concentration was placed in a new condition (E. coli), it interacted with the Escherichia

coli population, resulting in the death of the bacteria and forming an area where bacteria could

not grow (zone of inhibition). Hydrogen peroxide also works as an antiseptic because of its

reactivity. The oxygen atoms in the molecule are highly reactive and attract electrons from the

cell wall of bacteria. When placed onto the human body, this reaction breaks down the cell wall

of bacteria, leaving the genetic information and organelles of the cell no longer enclosed, and it

is no longer a functioning living cell. This process of the oxygen taking electrons, called

oxidation, is also harmful to human cells, breaking the cell membrane (Melina). But, because
 Ahmed Barrick 3

bacteria are so much smaller than human cells, they will be killed before the hydrogen peroxide

destroys too many human cells (Estapa).

Coming from the human body, E. coli grows at an ideal temperature of 37 C, the

average human body temperature (Nguyen). It is known that hydrogen peroxide works to kill

Escherichia coli, but differing slightly from the normal body temperature in the incubators could

represent a real-world example of someone who has a fever, or lives in a very cold climate.

Bacterial life is supported by the functionality of proteins. The proteins have optimal conditions

at which they function. If the temperature is increased from the optimum level, then they do not

work as well and eventually stop. When the inverse effect, a decrease in temperature, is applied,

the molecules within the bacteria begin to slow down, leading to slower protein activity, nutrient

absorption, and growth (Bisht). In both cases, if the proteins are not functioning correctly, the

bacteria are not supported and it will eventually die.

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Problem Statement

Problem:

To use different concentrations of hydrogen peroxide paired with varying temperatures to

affect Escherichia coli populations. Commented [Ma1]: To find which combination of best
effects.

Hypothesis:

If the Escherichia coli is exposed to 4 % concentration of hydrogen peroxide and

incubated at 37 C then the bacterias zone of inhibition will have the greatest area. Commented [Ma2]: Bacterias zone of inhibition or the
treatments zone of inhibition?

Data Measured:

The zone of inhibition will be measured 24 hours after being incubated in the specific

temperature while being exposed to the certain concentration of hydrogen peroxide. The effect

on the bacteria will be measured by calculating the zone of inhibition surrounding the hydrogen

peroxide (treatment). The zone of inhibition is found by measuring the distance from one end of

the zone to the other. The treatments, which are the independent variables of the experiment, are

hydrogen peroxide which is measured in milliliters (ml) and temperature measured in degrees

Celsius. The dependent variable (response variable) of the experiment is the area of the zone of

inhibition, which is measured in millimeters (mm). This is the response variable because the area

is dependent on the concentration of the hydrogen peroxide and the temperature. The

concentrations of hydrogen peroxide are 2 %, 3 %, and 4 %, because the standard concentration

of hydrogen peroxide used is 3 %.To maintain reliability, the other concentrations were both

deviated by 1 %. As a result, the low value is 2 % concentration, and the 4 % concentration is the

high value. E. coli usually grow in the human body, so 37 C (the normal human body

temperature) was used as one of the temperature values in the experiment. Deviating too much

from that value could have too great of an effect on the bacteria, so all the temperature values
 Ahmed Barrick 5

were within a range of 3 C. The low value for temperature is 35 C, the standard value is 36 C,

and the high value is 37 C, because those were the incubator temperatures provided closest to

the ideal temperature at which Escherichia coli grows. The effects of the two independent

variables, as well as their interaction effect, were measured and analyzed.

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Experimental Design

Materials:

100 x 15 mm Petri dishes 20 pack (4) Vial of Escherichia coli

500 ml of 4% Hydrogen peroxide concentration 30.34 cm ruler
500 ml of 3% Hydrogen peroxide concentration 1 L beaker (2)
500 ml of 2% Hydrogen peroxide concentration Incubator set at 35 C
Transfer loop Incubator set at 36 C
1.5 L of tap water Incubator set at 37 C
Hot plate Stirring magnet (5 cm)
34.5 grams of nutrient agar powder Bunsen burner
Tongs 30 ml test tubes (5)
48 test tube rack Printer paper (8.5 x 11)
Digital scale (measured in grams) Hot mitt
100 ml beaker (3) Masking tape (18 mm x 55mm)
Felt Scissors
Tweezers Refrigerator

Procedures:

Boiling the water:

1. Add 500 ml of tap water to the 1 L beaker.

2. Place the beaker on a hot plate, and set the hot plate to high.

3. Once the water starts to boil (when the water starts bubbling), turn off the hot plate.

Sterilizing the test tubes:

1. Using the tongs, take each of the test tubes and place them within the 500 mL of boiling
water.

2. Hold the test tubes under the surface of the boiling water, allowing water to fill the test
tubes.

3. Pour out the water from the test tubes back into the beaker with 500 ml of boiling water.

4. Place the test tubes into the rack with the opening faced downward.

Preparing the Agar:

1. Take the printer paper and tear into two roughly equal pieces (throw one of the pieces
away).
 Ahmed Barrick 7

2. Turn on the digital scale and press the tare button so the scale does not include the
weight of the paper as part of the weight of the agar.

3. Fold the remaining piece of paper in half (to make it easier to pour the agar later).

4. Unfold the paper and place it on the digital scale.

5. Pour agar powder onto the half sheet of paper that is inside the digital scale until the
digital scale reaches 23 grams.

6. Add 1 L of water into the unused 1 L beaker and place the stirring magnet within the
beaker (set the setting on #4).

7. Slowly add the 23 grams of agar powder to the beaker to make a full batch (will fill 50
Petri dishes). Be careful not to get any powder onto the sides of the beaker.

8. If using different amounts of Petri dishes, change amounts of agar powder and water to fit
specific needs (for example, 11.5 grams of agar powder and 500 ml of water would make
half of a batch, and fill 25 Petri dishes).

9. Place the beaker containing the water and agar powder onto the hotplate, and set the
hotplate to high.

10. Once the agar resembles apple juice, use a hot mitt to remove the beaker from the
hotplate. Turn off and unplug the hotplate, and allow the beaker to cool for three minutes.

Pouring the prepared agar:

1. Slightly open the lid of the Petri dish without fully exposing the interior to the
surrounding air.

2. Immediately after, using the hot mitt, pour enough agar (around 8-10 ml) into the Petri
dish so the entire bottom has a thin coating (be careful not to get any agar onto the
interior of the lid of the Petri dish, or else the Petri dish cannot be used).

3. Wait for the agar to cool off and solidify (should take around 15 minutes).

(Use steps 1-3 for all Petri dishes before placing bacteria within the dish)

Preparing the starter Petri dish:

1. Take one 30 ml sterile test tube and pour 1 ml of sterile water into it.

2. Turn on the Bunsen burner and sterilize the transfer loop by holding it over the fire (the
transfer loop is sterilized once it has turned red).
 Ahmed Barrick 8

3. Open the vial containing the Escherichia coli (make sure the cap is not set down), and
place the transfer loop into the vial (the loop should be in contact with the Escherichia
coli).

4. Remove the E. coli from the vial using the transfer loop.

5. Cap the vial of the Escherichia coli.

6. Using the transfer loop, place the bacteria into the 30 ml test tube containing 1 ml of
sterile water. Stir the water with the transfer loop to ensure that the Escherichia coli have
been transferred into the test tube.

7. Slightly open the Petri dish, and pour the contents of the test tube into the Petri dish.

8. Slowly slide the Petri dish on a surface in a circular motion to ensure that the E. coli is
spread throughout the Petri dish.

9. Pour the water out of the Petri dish (make sure the lid is on after pouring out the water).

10. Place the Petri dish into an incubator for at least 24 hours.

11. Remove the Petri dish from the incubator and place it in a refrigerator to slow down the
growth rate of the bacteria (the bacteria in this Petri dish will be used for all future trials).

Performing the trials:

1. Remove the starter Petri dish from the refrigerator.

2. Streak the sterilized transfer loop across the starter dish to collect E. coli on the transfer
loop.

3. Repeat steps 6-9 of Preparing the starter Petri dish.

4. Put 30 ml of 2 % concentration hydrogen peroxide into one of the 100 ml beakers. Label
with masking tape as 2 %. Repeat this with the 3 % concentration and the 4 %
concentration.

5. Use the scissors to cut out squares (1 cm x 1 cm) from the felt.

6. Using the tweezers, place the squares of felt into the 100 ml beaker containing the 2%
concentration of hydrogen peroxide. Let the squares soak for at least three minutes.

7. Using the tweezers place the squares of felt into the center of the Petri dishes (only use
one square of felt per Petri dish).

8. Place the Petri dishes into the incubator.

 Ahmed Barrick 9

9. Leave the Petri dishes in the incubator for 24 hours, giving the treatment time to take
effect.

10. After 24 hours, remove the Petri dishes from the incubator.

11. There will be a circular area around each square where there is no bacterial growth
(called the zone of inhibition). Measure the diameter of this area using the ruler and
record it in a data table. (If the zone of inhibition is uneven, measure three axes and
average them to get the diameter.)

12. Repeat all previous steps (beginning at Boiling the water, excluding Preparing the
starter Petri dish) for each combination.

(For the (-,-) Petri dishes use the felt squares soaked in 2 % hydrogen peroxide
concentration and place into the incubator set at 35 C.

For the (-,+) Petri dishes use the felt squares soaked in 2 % hydrogen peroxide
concentration and place into the incubator set at 37 C.

For the (+,-) Petri dishes use the felt squares soaked in 4 % hydrogen peroxide
concentration and place into the incubator set at 35 C.

For the (+,+) Petri dishes use the felt squares soaked in 4 % hydrogen peroxide
concentration and place into the incubator set at 37 C.

For the (s,s) Petri dishes use the felt squares soaked in 3 % hydrogen peroxide
concentration and place into the incubator set at 36 C.)
 Ahmed Barrick 10

Figure 2. Pouring the prepared agar

Figure 2. above shows the proper method for pouring the agar. The lid of the Petri dish is

slightly open; however the lid is not completely removed. The agar is also held over the edge of

the table and poured slowly into the Petri dish. This is the proper method for pouring the

prepared agar.
 Ahmed Barrick 11

Data and Observation

Data:

Table 1
Design of Experiment Values
Hydrogen Peroxide (% concentration) Temperature C
- Standard + - Standard +
2 3 4 35 36 37

Table 1 above shows the values chosen for the independent variables of the experiment.

The numbers for the values of the two variables were determined by doing research on

Escherichia coli growth and the effect of hydrogen peroxide on Escherichia coli. The average

amount of hydrogen peroxide used on Escherichia coli was 3 % concentration which is used as

the standard. The low value was chosen to be 2 % concentration and the high was chosen to be 4

%, because both of these are close to the standard. The standard temperature of 36 C was

chosen because it is closest to the average body temperature, in which Escherichia coli usually

grows. The other two values were chosen because they are close enough to the standard to not

completely kill all the bacteria.

 Ahmed Barrick 12

Table 2
Results of Trials
Zone of Inhibition (cm)

(Hydrogen peroxide, Temperature)

Trails (-,-) (-,+) (+,-) (+,+) (s,s)

1 5.53 3.13 1.70 3.20 6.53
2 0.00 2.00 2.83 2.96 7.10
3 4.46 5.26 4.63 0.40 6.33
4 5.93 6.40 3.80 0.73 6.56
5 6.46 6.10 4.23 2.63 6.66
6 5.16 5.56 6.30 3.40 7.16
7 3.16 6.16 2.73 0.96 7.33
8 0.00 N/A 1.00 0.23 5.50
9 5.83 1.93 0.40 1.83 6.83
10 2.03 5.83 2.63 2.30 6.76
11 0.00 3.36 3.50 3.30 N/A
12 2.60 4.00 2.76 3.03 N/A
13 2.00 5.46 0.00 2.80 N/A
14 5.00 5.63 4.63 0.43 N/A
15 5.93 5.60 5.46 0.76 N/A
Average: 3.60 4.74 3.10 1.93 6.68

Table 2 above shows the average for each of the combinations of hydrogen peroxide

concentration and temperature. The trails were not completed in a randomized sequence, such

that each combination was completed on a separate day. This can lead to the standards being

unreliable and unable to serve as a basis for comparison.

 Ahmed Barrick 13

Observations:

Table 3
Observations
Date Observation

(-,-) Trials 2, 8, and 11 had no zone of inhibition. The other twelve Petri
3/16/15
dishes for that combination had zones of inhibition.

(-,+) Trial 8 had no zone of inhibition because the water used to transfer the
3/18/15 bacteria to the Petri dish was left in the Petri dish two minutes before being
poured out.

3/18/15 Partner was absent, prepared Petri dishes with substituted partner.

(+,-) This combination had very low zone of inhibition, some had no zone.
3/23/ 15 Bacteria had grown over the treatment in trial 13, and there was no zone of
inhibition.

Bacteria in trials 2, 4 and 5 were a lighter shade than usual. Different starter
3/25/15
Escherichia coli were used. Combination had small zones of inhibition.

As shown in Table 3, some trials did not result as expected. The possibilities for these

errors are unknown, however it is likely the most common problem was human error. All of the

trials with errors and obscure results are mentioned in Table 3.

 Ahmed Barrick 14

(- , -)

(- , -)

(- , -)

Figure 3. (-,-) Error

Figure 3. above shows the three trials that resulted in no zone of inhibition on 3/16/15

during the (-,-) trials. The Petri dishes in this figure were incubated at 35 C and the treatment

that was applied was 2% hydrogen peroxide.

(-, +)

Figure 4. (-,+) Error

Figure 4. above shows that trial 8 of the (-,+) trials conducted on 3/18/15 had no bacterial

growth, and therefore, no zone of inhibition could be recorded. The water within the Petri dish

was not emptied out correctly. This Petri dish was incubated at 37 C and the treatment was 2 %

hydrogen peroxide.
 Ahmed Barrick 15

(+ , -)

Figure 5. (+,-) Error

Figure 5. above shows the error of trial 13 on 3/23/15 during the (+,-) trials. The bacteria

grew over the zone of inhibition so the data was counted as 0 instead of N/A. The Petri dish was

incubated at 35 C and the treatment applied was 4% hydrogen peroxide.

(+ , -)

Figure 6. (+,-) Different Shade

The (+,-) trials conducted on 3/23/15 all displayed a lighter shade of Escherichia coli

compared to the other trials. During these trials a different starter Escherichia coli set was used.
 Ahmed Barrick 16

Data Analysis and Interpretation

Table 4
Values
Hydrogen Peroxide (% solution) Temperature (C)
- Standard + - Standard +
2 3 4 35 36 37

Table 4 above shows the values for the high, low, and standard for each factor in the

experiment.

Table 5
Results of Experiment
Trials (-,-) (-,+) (+,-) (+,+)

Averages 3.608888889 4.747619048 3.108888889 1.933333333

Grand Average 3.34968253979

Table 5 above shows the averages of the trials (look in Data and Observations for full

data). The average for the low hydrogen peroxide and low temperature was about 3.608888889

cm in diameter for the zone of inhibition. The average for the low hydrogen peroxide and high

temperature was around 4.747619048 cm. The average for the high hydrogen peroxide and low

temperature was about 3.108888889 cm. The average for the high hydrogen peroxide and high

temperature was around 1.933333333 cm. The grand average (average of all four averages) was

around 3.34968253979 cm. These numbers are used to calculate the effects of the experiment.
 Ahmed Barrick 17

Effect of Hydrogen Peroxide

Zone of Inhibition Diameter (cm)

Table 6
Effect of Hydrogen Peroxide
Hydrogen Peroxide (% solution)
(2) (4)
3.608888889 1.933333333
4.747619048 3.108888889
Avg =4.17825 Avg =2.52111

Figure 7. Effect of Hydrogen Peroxide

Table 6 above shows the averages of the low value and high value for hydrogen peroxide.

The effect of hydrogen peroxide is -1.657. This number means that on average, as hydrogen

peroxide increases, the zone of inhibition decreases by 1.657 cm. This effect was found by

calculating the difference between the high average and the low average. Figure 7. above is the

graph of the hydrogen peroxide as it increases in concentration. It can be clearly seen that as

hydrogen peroxide increases the average zone of inhibition decreases.

 Ahmed Barrick 18

Effect of Temperature

Table 7 Zone of Inhibition Diameter (cm)

Effect of Temperature
Temperature (C)
(35) (37)
3.608888889 1.933333333
3.108888889 4.747619048
Avg =3.35889 Avg =3.34048

Figure 8. Effect of Temperature

Table 7 above represents the average of the low temperature and the average of the high

value for temperature. The effect of temperature in this experiment is -0.018. This states that on

average as temperature increases the bacterial zone of inhibition decreases by around 0.018 cm.

Figure 8. shows the graph of the two average values for temperature, it also shows the decreasing

slope as the temperature increases.

 Ahmed Barrick 19

Interaction Effect

Zone of Inhibition Diameter (cm)

Table 8
Interaction Effect Data
Hydrogen Peroxide
( % solution)
(2) (4)
Solid 4.74761 1.93333
Temperature

(37)
Segment 9048 3333
(C)

Dotted 3.60888 3.10888

(35)
Segment 8889 8889

Figure 9. Interaction Effect

The graph in Figure 9. above implies that the effect of hydrogen peroxide is much greater

than the effect of temperature.

Table 8 above shows the average measurement (cm) for each combination of hydrogen

peroxide and temperature. The interaction effect value is approximately -1.157 (found by

subtracting the slope of the dotted segment from the slope of the solid segment). The dotted line

segment represents the hydrogen peroxide concentration moving from low to high while the

temperature is low, and the solid segment shows the hydrogen peroxide concentration moving

from low to high while the temperature is high.

The low temperature on average yielded a zone of inhibition around 3.358 cm. However,

when the temperature was low and combined with the low concentration of hydrogen peroxide,
 Ahmed Barrick 20

the zone of inhibition had a greater value of 3.608 cm. The low temperature paired with the high

concentration of hydrogen peroxide had an average zone of inhibition of 3.108888889 cm. This

implies that in the lower temperature, a lower hydrogen peroxide concentration will increase the

zone of inhibition, and a higher concentration will lower it.

When the temperature was high the average zone of inhibition was about 3.340 cm, but

when the temperature was high and the hydrogen peroxide was at the low value, the zone of

inhibition increased to 4.747619048 cm. When these variables were both at high values, the zone

of inhibition was 1.933333333 cm. This implies that in the high temperature as well, a lower

hydrogen peroxide concentration increases the zone of inhibition, while a higher concentration

lowers it.

The graph in Figure 9. also implies that a higher temperature increases the range of

effectiveness of the hydrogen peroxide. As it can be seen on the graph, when the temperature is

high, the zone of inhibition measurements ranged from 4.747619048 cm to 1.933333333 cm.

Although when the temperature is low, the range is only 3.608888889 cm to 3.108888889 cm.

Since the slopes of the two effects are not parallel (as it can be seen in Figure 9.) there might be

some significance about the interaction between the two variables, although this interaction

effect was deemed insignificant (refer to Figure 11.).

 Ahmed Barrick 21

Figure 10. Standard Trials

The graph above in Figure 10. represents the standard trials of the experiment. It shows

that there are no clear patterns and no major variations among these standards; which means that

the standards are a good basis for comparison. However, these standards were all conducted on

the same day. Each combination was done on a separate day. This lowers the reliability of these

standards being used as a basis of comparison. The range of standards was calculated to be about

1.833 cm; this was found by subtracting the highest standard (7.333 cm) by the lowest standard

(5.500 cm). Doubling the value of 1.833, is equal to 3.667, this product is used to decide whether

effects are deemed statistically significant or not.

 Ahmed Barrick 22

Legend:
T- Effect of temperature
H- Effect of hydrogen
peroxide
HT- Interaction effect
of hydrogen peroxide
and temperature

Figure 11. Dot plot of Effects

Figure 11. above shows the effects of the experiment plotted on a dot plot. The (H)

represents the effect of hydrogen peroxide, the (T) represents the effect of temperature, and

finally (HT) represents the interaction effect. The fences that are set up (an absolute value of

around 3.667) were used to determine whether each of the effects are significant or not. These

fences are found by doubling the range of standards. If the dots (representing the effects) are past

either of the fences, then those effects are deemed significant. It can be clearly seen that none of

the effects were outside of the fences which deems all three effect values statistically

insignificant, so none of these values can be applied to the parsimonious prediction equation.

Y= 3.34968253979 + noise

Figure 12. Parsimonious Prediction Equation

In Figure 12. the parsimonious prediction equation is given. The parsimonious prediction

equation is the grand average, plus any of the effects deemed statistically significant multiplied

by the corresponding variable and divided by two. In this experiment, none of the effects were

deemed statistically significant so they were not included in the figure above, although they did

play major roles affecting the results.

 Ahmed Barrick 23

Y=3.34968253979 + noise

Figure 13. Interpolated Prediction Equation

The interpolated prediction equation is the same as the parsimonious prediction equation

in this case, the grand average. This is because none of the effects were deemed significant. This

does not allow for any different numbers to be plugged in for the predictor variables. If the

experiment were to be run again with the variables at different values, the average measurement

for the zone of inhibition is expected to be approximately 3.349 cm (grand average).

The hydrogen peroxide did fail the statistical significance test, however it did affect the

outcome of the response variable. As it can be seen with the graph of the interaction effect (in

Figure 9.), the steep slopes of the segments as hydrogen peroxide moves from the low value to

the high value indicate that hydrogen peroxide was a large factor. It can be stated that the

hydrogen peroxide concentration had a key role in the outcome of the zone of inhibition, due to

the downward trend.

 Ahmed Barrick 24

Conclusion

The effects of temperature and hydrogen peroxide concentration on Escherichia coli

acted differently than expected. It was hypothesized that the high concentration of hydrogen

peroxide (4 %) and the high temperature (37 C) would yield the largest zone of inhibition. The

hypothesis was rejected, and instead of the predicted combination, the combination that yielded

the largest zone of inhibition was the low concentration of hydrogen peroxide (2 %) and the high

temperature value (37 C).

The purpose of the experiment was to identify which combination of hydrogen peroxide

concentration and temperature had the greatest effect on the area of the zone of inhibition. This

was tested by incubating Escherichia coli in 35 C, 36 C, and 37 C, while being exposed to

hydrogen peroxide solutions with concentrations of 2 %, 3 %, and 4 %. The independent

variables in the experiment were the hydrogen peroxide concentration and temperature. These

variables were paired with one another to interact and affect the response variable, the zone of

inhibition. The zone of inhibition is the roughly round area where bacteria does not grow, and is

found by measuring the diameter of the round area (Effects of Antiseptics on Bacterial

Growth). Escherichia coli was the bacteria used in the experiment because it is commonly

found in the human body.

The average zone of inhibition for the low value of hydrogen peroxide was 4.178253 cm,

while the average for the high value was 2.52111 cm, meaning the effect of hydrogen peroxide

concentration was -1.657 (refer to Data Analysis and Interpretation for exact calculations).

This implies that, on average, as hydrogen peroxide increases, the zone of inhibition decreases

by 1.657 cm. The zone of inhibition had the greatest area when the hydrogen peroxide

concentration was held at a low value of 2 % concentration. One possible reason for the low
 Ahmed Barrick 25

value of hydrogen peroxide yielding the greatest zone of inhibition may be osmosis. If the

hydrogen peroxide has a 2 % concentration, then it is 98 % water. If the value of 98 % water is

too far from the percent of water in the cells, then osmosis could occur, either shrinking or

bloating the cell to the point of death (Osmosis in Prokaryotes). Another reason that the low

value had a greater effect than the high value may be because of the interaction between

hydrogen peroxide and temperature, which could have caused the higher value to result in a

lower area of the zone of inhibition compared to the low value.

The average zone of inhibition for the low value of temperature was 3.358888 cm, and

the average for the high value was 3.340476 cm. These numbers were used to calculate the effect

value for temperature which is -0.018 (refer to Data Analysis and Interpretation for

calculations). This implies that on average as temperature increases the zone of inhibition

slightly decreases. It is clearly shown that the effect of hydrogen peroxide concentration is much

greater than the effect of temperature, which means that the hydrogen peroxide was the

independent variable that led the experiment. On average the zone of inhibition had a higher area

when the temperature was held at a low value of 35 C. A possible reason for the lower

temperature resulting in the higher area is because 35 C is 2 C away from the optimum value at

which Escherichia coli grows (37 C). This could have slowed down the growth rate of the

Escherichia coli (Nguyen). With a slower growth rate, there would be fewer bacteria for the

hydrogen peroxide to kill in the 24 hours before it was recorded, creating a larger zone of

inhibition. Another possible reason for the lower temperature resulting in a larger zone of

inhibition area is because a lower temperature slows down the growth rate of the E. coli, and a

slowed growth rate alone can eventually cause the bacteria to die.
 Ahmed Barrick 26

Using hydrogen peroxide to prevent bacterial growth is one of the many methods that can

be used to kill bacteria. Like disinfectants and antiseptics, antibiotics can also be used to prevent

bacterial growth. One of the most common antibiotics used to kill bacteria is penicillin. Penicillin

is derived from Penicillium fungi, and is used to treat bacterial infections (Penicillin V

Potassium Oral). Hydrogen peroxide and penicillin affect bacteria in a similar way, breaking

apart the cell wall, and causing the bacteria to die due to the leaking of cell contents (Trucco).

Because of the similarity, the results from the experiments involving penicillin can be compared

to the results of this experiment to assure that this experiment yielded feasible results. The

research involving penicillin has shown that a lower dosage of penicillin can be just as lethal as a

higher concentration. In the experiment (refer to Data Analysis and Interpretation) the low

concentration of hydrogen peroxide (2 %) had a greater effect on the response variable (zone of

inhibition) than the higher value of hydrogen peroxide concentration (4 %). These findings are

similar to that of the research involving penicillin. Both research experiments indicate that a high

value does not always result in the best possible outcome.

There are multiple factors that can be ruled as design flaws within the experiment. The

largest flaw was that the experiment was not conducted as a two-factor Design of Experiment

(DOE), although it should have been. The experiment was not a DOE because the order in which

each of the trials were executed was not randomly selected. This flaw could have deemed the

averages of the combinations as less reliable, because each set of trials was exposed to the same

environment. The standard trials were also not randomized, which decreases the reliability of all

the data gathered, since the standard trials served as the basis of comparison for the other

combinations, and were used to determine which effects were statistically significant. The final

design flaw is the streaking of the bacteria. When streaking the bacteria from the Petri dish
 Ahmed Barrick 27

containing the starter E. coli, the amount of E. coli being streaked and placed into the test tubes

was not measured. Not measuring the amount of Escherichia coli in each test tube could have

caused some trails to start with more bacteria than others, affecting the end result, because the

independent variable may react slower to larger amounts of bacteria. Because this variability

may have influenced the outcome of the trials, this could make the data less reliable.

During the experiment, several errors occurred while conducting the procedures (refer to

Experimental Design for proper procedures). One error is that the test tubes were not sterilized

directly before each set of trials were conducted. Instead, test tubes were sometimes sterilized the

day before conducting trials. Altering the procedure this way could have caused the test tubes

and other sterilized objects to become unsanitary overnight. Another error that occurred was,

during the set of low hydrogen peroxide concentration (2 %) and a high value of temperature (37

C), the water from the test tube was poured into the Petri dish, but instead of being poured out

directly afterward, the water remained in the Petri dish for approximately one minute. The result

for this trial was unavailable because there were no bacteria within the Petri dish (refer to Figure

4.), and the assumption was made that because of the error, there was no bacterial growth within

the Petri dish.

There are some changes that could be made to improve and enhance the experiment. The

temperature values of 35 C, 36 C, and 37 C were used because they were the only incubator

temperatures provided. It is recommended that the values would be changed if the experiment

were to be conducted again, because the temperatures were so close to each other that they

yielded very similar results, causing the temperature to have a small effect on the bacteria. If the

differences of these temperatures were increased, there could be a greater effect from the

temperature, and a possible interaction effect could be identified more clearly. Another change
 Ahmed Barrick 28

that could be made if this experiment were to be conducted again is randomizing the trials and

conducting a Design of Experiment (DOE). One final aspect that could be enhanced is allowing

for trials to be incubated for longer periods of time while observing the trials more frequently.

The findings of the research support a widely used method for preventing bacterial

growth and preventing infection from certain bacteria that are harmful to the human body. The

hydrogen peroxide concentration of 2 % resulted in a larger zone of inhibition, showing that a

high concentration of hydrogen peroxide is not needed when getting hydrogen peroxide to treat

cuts with. This clears up the possible misconception that higher concentrations work better. In

the case of hydrogen peroxide, if the concentration is too high, it can prove to be more harmful

than helpful, killing human cells and slowing the healing process. It is advised that hydrogen

peroxide is used in moderation as too much hydrogen peroxide can harm the human cells.

Though the hydrogen peroxide was tested on bacteria, it can be assumed that it can destroy

human cells as well, because of the similarity of the structure between the two cells. Usually,

when using hydrogen peroxide, it is strong enough to kill off the bacteria, but not the larger

human cells. However it was identified in the experiment that hydrogen peroxide is slightly more

effective at a lower temperature. This means it is possible that the hydrogen peroxide could be

powerful enough to destroy more human cells at a lower temperature. Therefore it is advised that

people with problems that cause a lower body temperature, such as diabetes, should use

hydrogen peroxide with more caution.

From this experience, it was derived that one must conduct research on every aspect of

the experiment before the results can be comprehended. Once one has complete background

knowledge on the experiment, it can be understood why things happened, as opposed to what

happened.
 Ahmed Barrick 29

Acknowledgements

We would like to thank our IDS teacher, Mrs. Gravel, for supporting us and making sure

our paper was perfectly formatted and understandable. Another individual we would like to

thank is our biology teacher, Mr. Estapa, for helping us conduct our experiments properly,

making sure our science was perfect, and explaining the scientific concepts behind our

experiment. Mr. Acre, our GAT teacher, helped us as well by going over our math and

explanations in detail. We would also like to thank our classmates, especially Noah and Martin,

for providing us the competitive drive to do better and making this project a fun experience.

Finally, we want to show appreciation for our parents. They gave us the confidence and

encouragement that we needed, they asked questions with genuine interest, and listened to us

work on the project during many late nights over the course of four months.
 Ahmed Barrick 30

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